Method for detecting degradation of micro-plastics in spotted-brown chickens, nematodes and mouse living bodies

Abstract

The invention discloses a method for detecting degradation of micro-plastics in living bodies of spotted-brown chickens, nematodes and mice, and belongs to the technical field of biology of degradation of micro-plastics in living animal bodies. According to the characteristic that tenebrio molitor intestinal microorganisms degrade plastics, multiple groups of contrast experiments are carried out, the optimal tenebrio molitor feeding amount for degrading the microplastics in vivo is obtained, and the purpose of obviously degrading the microplastics in animal bodies is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biology, more specifically, it relates to an ecological technology for reducing plastic in euhadroma. BACKGROUND

[0002] Microplastics refer to particles or fibers composed of high-molecular polymers with a diameter of less than 5 microns that cannot be filtered by sewage treatment plants. Plastic waste, washed synthetic fiber textiles, moving vehicle tires, and skin exfoliating products are the main sources of microplastics. In 2017-2018, the University of Arizona Institute of Earth and Environmental Sciences in the United States conducted sampling and testing on tap water in 14 countries around the world and 11 brands of bottled water around the world, and found that 83% of tap water samples and 92% of bottled water were contaminated with microplastics. On November 30, 2018, Vice Director Wang Juying of the National Marine Environmental Monitoring Center said that microplastics have been found in seawater, seabed and seabed sediments. In March 2022, scientists first detected microplastic pollution in human blood. In nearly 80% of experimental subjects, such small particles were found. In April, British scientists first found microplastics in the deep part of the human lung. In June, according to a new study published in the journal "Ice Cap", microplastics were first found in freshly fallen snow in Antarctica. In June 2023, a new study found that when heated in a microwave oven, plastic containers used to package baby food release a large number of microplastic particles. In January 2024, a study by Columbia University found that one liter of water from three best-selling plastic bottled water brands on the US market contains about 110,000 to 370,000 plastic particles, of which 10% are microplastics and the remaining 90% are smaller nanoscale plastics.

[0003] The impact of microplastics on the physiology of aquatic animals mainly focuses on the digestive system, respiratory system, and reproductive system, which in turn affects digestion, respiration, and reproduction in organisms. Relevant results have been found in fish, shellfish, crustaceans, and echinoderms.

[0004] The impact of microplastics on human health involves multiple systems, including the nervous system, endocrine system, and immune system. The latest discovery of microplastics in the human body is in arterial plaques, which may be the core of arterial plaque composition. Therefore, if we are exposed to a large amount of microplastics in our daily life, it may cause cardiovascular and cerebrovascular diseases. This may also be one of the reasons for the high incidence of modern cardiovascular and cerebrovascular diseases.

[0005] Huainan partridge chicken (hereinafter referred to as partridge chicken) is a high-quality local breed chicken for meat and egg in Anhui Province, which has the appearance characteristics of green shins, green beaks, and all-red feathers of male chickens and partridge feathers of female chickens. The central production area is Anhui and Henan and other areas along the Huaihe River, which has the advantages of thin skin, tender meat, delicious meat, yellow and many eggs, unique flavor, and is deeply loved by the masses. Poultry animals such as partridge chickens will inevitably ingest some microplastics during free-range or intensive farming, which not only harms the health of the chickens themselves, but also eventually enriches the human body through the food chain, thereby affecting human health.

[0006] Tenebrio molitor, commonly known as breadworm, is an insect of the Coleoptera family, Tenebrio genus, which is the most ideal feed insect for artificial breeding. It has the advantages of short growth cycle, strong reproductive capacity, wide distribution, rich source, low artificial breeding cost, and rich nutritional value. The protein nutritional composition of Tenebrio molitor is the highest in animal protein feed, and it is also known as "the treasure of protein feed". Elahi et al. found that Tenebrio molitor can improve the growth performance of broilers by adding it to their daily diet. Benzertiha et al. showed that the addition of Tenebrio molitor can improve the body weight and feed intake of broilers, and has a certain improvement effect on the growth performance of broilers.

[0007] More and more researchers have verified that Tenebrio molitor has good degradation effect on plastic polymers in the natural environment. Tenebrio molitor larvae produced in Asia and North America can eat plastic, and eating plastic does not affect their activity. Carbon-13 isotope tracing experiments have shown that polystyrene can be completely degraded and mineralized into carbon dioxide by Tenebrio molitor in the body, and a part of it can also be converted into fat in the body. Researchers have successfully isolated a polystyrene-degrading bacterium, Exiguobacterium sp. YT2, from Tenebrio molitor, which can grow using polystyrene as the sole carbon source. This strain is the first polystyrene-degrading bacterium reported in the international strain center. This study provides scientific and strong evidence that microorganisms can effectively degrade polystyrene. SUMMARY

[0008] 1. Technical problems to be solved by the invention

[0009] The present invention aims to solve the following problems:

[0010] Although it has been reported that microorganisms in Tenebrio molitor can degrade microplastics in nature, or that Tenebrio molitor can eat and degrade plastic products (polystyrene), whether Tenebrio molitor can track "ingested" microplastics in the animal body and effectively degrade them is still rarely reported. If it can be achieved, how to operate and how much is the most appropriate amount?

[0011] 2. Technical solutions

[0012] To solve the above technical problems, the specific technical solutions provided by the present application are as follows:

[0013] The Tenebrio molitor selected has a body length of 15-20 mm, and the 40-60-day-old larvae with strong vitality are fed with wheat bran, and at the same time, the selenium-enriched lettuce leaves are fed to the Tenebrio molitor at a ratio of 10:1 based on the body weight of the Tenebrio molitor, and the Tenebrio molitor is continuously fed for one week to obtain selenium-enriched Tenebrio molitor. 50% of the Tenebrio molitor with strong vitality of the selenium-enriched Tenebrio molitor is used for subsequent experiments. The specific selection principles are as follows: first, the appearance characteristics: exclude brown and dark varieties, and select golden yellow, shiny body surface, and obvious white abdomen; second, the movement characteristics: fast crawling speed, can quickly climb up to feed when feeding lettuce, and strong light reaction, can quickly crawl to the dark environment when there is light.

[0014] The selenium-enriched Tenebrio molitor selected according to the above principles is fed to the same-age healthy Ephedra chickens. Before feeding the Ephedra chickens, the Tenebrio molitor is starved for 2 days to ensure that all the organic selenium in the Tenebrio molitor is obtained. Then, 10 Tenebrio molitors per 500g of selenium-enriched Tenebrio molitors are fed to the Ephedra chickens and other poultry per day to achieve the goal of selenium enrichment and plastic reduction of Tenebrio molitor.

[0015] The methods and steps for testing the selenium enrichment effect and plastic reduction effect of Tenebrio molitor are different, and are described as follows:

[0016] The selenium-enriched Tenebrio molitor can be directly fed at a rate of 10 live Tenebrio molitors per 500g of selenium-enriched Tenebrio molitors per day for 6 consecutive days. If further selenium enrichment is required, 3-5 live Tenebrio molitors per 500g can be fed per day, or the Tenebrio molitors can be washed, boiled, dried, and ground into powder and added to the feed according to the body weight ratio. After 30 days, the selenium-enriched effect of the fed poultry can be achieved.

[0017] Before the experiment of Tenebrio molitor degrading microplastics in Ephedra chickens, experiments of Tenebrio molitor degrading microplastics in lower model organisms nematodes and higher model organisms mice have been carried out to ensure the feasibility and scientificity of the experiment, and the optimal form and amount of Tenebrio molitor for degrading microplastics in Ephedra chickens have been found. Some of the contents have been embodied in the form of examples, which will not be described here.

[0018] For the detection of Tenebrio molitor plastic reduction experiment, 10 live Tenebrio molitors per 500g of Tenebrio molitors per day are fed 4 days before feeding microplastics, and the Tenebrio molitors are continuously fed for 4 days to make the Ephedra chickens have a constant amount of plastic-reducing microorganisms. Then, the microplastics are gavaged according to the proportion, and the Tenebrio molitors are fed for another 2 days. The effect of plastic reduction is investigated by comparing the Ephedra chickens with and without Tenebrio molitors before and after gavaging microplastics.

[0019] Specific test experimental steps: 3 groups of ephedra chickens were set up: the negative control group consisted of 3 healthy 9-month-old chicks without any treatment; the 3 positive control groups were chicks of the same age, and polystyrene green fluorescent microspheres with a concentration of 200 mg / kg and a particle size of 100 nm were added at one time, and the volume was fixed in 0.5 mL of normal saline. The ephedra chickens chose to feed them by active swallowing without damage; the experimental group was selected mealworms, and 3 healthy ephedra chickens of the same age were fed with 10 mealworms per 500 g of ephedra chicken body weight per day. After feeding for 4 consecutive days, the positive control group experimental operation was carried out, and then the chickens were fed again at 10 / 500 g of selenium-enriched mealworms per day for 2 days; subsequently, relevant experiments were used to verify whether the experimental group and the negative control group had a selenium-enriched effect, and relevant experiments were used to verify whether the experimental group and the positive control group had a plastic reduction effect.

[0020] The microplastic content detection experiment was carried out according to the method of Zhang Baigang et al. The liver and kidneys of the animals were quickly frozen, fixed, and sliced, and the prepared slices were placed under a fluorescence microscope for photography. Poultry blood was collected from the subwing vein using a 1mL syringe, then smeared on a slide and directly observed under a fluorescence microscope. The excitation light wavelength of the fluorescent sample was set to 460nm, and the corresponding filter block was selected. Under the eyepiece, adjust the magnification of the objective lens used. A 10x objective lens was used in this experiment. Find the sample under the microscope, change the light source to the excitation light, and then take a photo. The photo was saved and processed.

[0021] In order to achieve ecological selenium enrichment and plastic reduction, poultry farming companies can feed 3-5 mealworms per 500g of poultry for 30 days one month before selling them, which can also achieve the effect of ecological selenium enrichment and plastic reduction.

[0022] As for plastic reduction technology, this technology has been used to carry out plastic reduction experiments in animals such as Huainan Ephedra chickens, mice, nematodes and fish. Therefore, in addition to being applied to Ephedra chickens, this technology can also be applied to other poultry and other farmed animals that are directly consumed by humans, such as fish, livestock, flying birds, cattle and sheep, etc.

[0023] 3. Beneficial effects

[0024] Compared with the prior art, the technical method provided by the present invention has the following significant effects:

[0025] (1) Taking advantage of the fact that mealworms are rich in selenium and can degrade microplastics, poultry can be fed directly with selenium-rich mealworms, which will achieve the dual purpose of enriching selenium and reducing plastics. At the same time, adding mealworms to feed can also improve the meat quality of livestock and poultry.

[0026] (2) In order to verify that the Tenebrio molitor can degrade microplastics in vivo, the present technology has carried out microplastic degradation experiments on nematodes, mice, Huainan Ephedra chickens and the like. The experiments have proved that the intestinal flora in the Tenebrio molitor, such as Morganella, Hafnia, bacteria KHJ-1 and Citrobacter, can partially degrade the microplastics in the above-mentioned animals and mineralize them into carbon dioxide, and a part of them can also be converted into fat in the worm body, so as to achieve the purpose of ecological degradation of microplastics in the Tenebrio molitor. Considering that there is no similar technology at present, the present method can effectively degrade the microplastics in the animal body, reduce the content thereof, and is simple to operate, and can be applied to different animal microplastic degradation by large livestock and poultry manufacturers and individual farmers. The use of the present method also reduces the intake of microplastics by people when eating poultry animals containing microplastics, and protects the health of people.

[0027] (4) The present application explores whether the Tenebrio molitor can degrade microplastics in vivo. Research shows that the flora in the Tenebrio molitor can effectively degrade the microplastics in the biological body, so the flora or the extract in the Tenebrio molitor can be added to the feed of animals in the form of live bacteria to prepare a microplastic-degradable feed. The present method can mass-produce the microplastic-degradable feed to achieve the goal of effectively and quickly degrading microplastics in the animal body, and has a good development prospect in the feed production industry and the breeding industry. BRIEF DESCRIPTION OF DRAWINGS

[0028] ATTACHED DRAWINGS Figure 1 Patent flowchart of selenium-enriched Huaiyang chicken microplastic degradation

[0029] Figure 2 Selenium content column chart of Tenebrio molitor in different soil added with sodium selenite in the lettuce leaves and two selenium-enriched methods of Example 1 (NTT is the selenium content of the lettuce leaves cultivated in the soil mixed with peat soil and fine sand soil and added with 60 mg / kg sodium selenite, PTT is the selenium content of the lettuce leaves cultivated in the ordinary soil added with the same dose of sodium selenite, YFX is the selenium content of the Tenebrio molitor fed with the sodium selenite sprayed lettuce leaves, TFX is the selenium content of the Tenebrio molitor fed with the sodium selenite added lettuce leaves, and SWL is the selenium content of the Tenebrio molitor fed with the sodium selenite added soil and lettuce leaves)

[0030] Figure 3 Selenium content column chart of each organ of the selenium-enriched Huaiyang chicken in the food chain and the control group in Example 1 (the sample T is the head of the experimental group, and the sample C is the head of the control group, XY: blood, XZ: heart, GZ: liver, WB: stomach, JR: muscle, SZ: kidney, and FB: lung)

[0031] Figure 4 Distribution diagram of fluorescent microplastics in the nematode in Example 2

[0032] Figure 5Fluorescent microplastics distribution in the control group of nematodes after 1 h in Example 2

[0033] Figure 6 Fluorescent microplastics distribution in the experimental group of nematodes after 1 h in Example 2

[0034] Figure 7 Fluorescent microplastics distribution in the control group of nematodes after 3 h in Example 2

[0035] Figure 8 Fluorescent microplastics distribution in the experimental group of nematodes after 3 h in Example 2

[0036] Figure 9 Fluorescent microplastics distribution in the blood of mice in the experimental group in Example 3

[0037] Figure 10 Fluorescent microplastics distribution in the blood of mice in the control group in Example 3

[0038] Figure 11 Fluorescent microplastics distribution in the kidney sections of mice in Example 3

[0039] Figure 12 Fluorescent microplastics distribution in the liver sections of mice in Example 3

[0040] Figure 13 Fluorescent microplastics distribution in the blood of ephedrine chickens in the experimental group in Example 4

[0041] Figure 14 Fluorescent microplastics distribution in the blood of ephedrine chickens in the control group in Example 4

[0042] Figure 15 Fluorescent microplastics distribution in the kidney sections of ephedrine chickens in the control group in Example 4

[0043] Figure 16 Fluorescent microplastics distribution in the kidney sections of ephedrine chickens in the experimental group in Example 4 Detailed implementation method

[0044] In order to further understand the content of the present application, the present application will be described in detail in combination with the drawings and implementation cases.

[0045] Example 1

[0046] In this implementation case, the situation of polystyrene entering the nematodes and the degradation effect of the Tenebrio intestinal extract on the polystyrene plastic in the nematodes will be introduced. The specific content includes:

[0047] The L4 wild-type nematodes (N2) used in this example are active and are cultured in a 25°C incubator. The Tenebrio molitor larvae used in this example are about 60 days old and have just molted, with a body length of about 15-20 mm and strong activity. NGM medium is used for nematode growth medium. M9 buffer is used to clean the surface of the nematodes with fluorescent microspheres. A body microscope is used to pick nematodes. A fluorescence microscope is used to take pictures and observe. 75% alcohol is used for sterilization. 0.9% sterile sodium chloride solution is used for dilution of the Tenebrio molitor intestinal extract. An autoclave is used to sterilize the mortar. 2% agar is used to make agar pads. The polystyrene green fluorescent microspheres used in this example have a particle size of 100 nm, an excitation wavelength of 460 nm, and an emission wavelength of 505 nm.

[0048] (1) Observe the distribution of polystyrene microplastics in the body of nematodes

[0049] Design a high concentration of 50ug / mL polystyrene fluorescent microspheres is directly added to the NGM medium containing synchronized nematodes. After 1h, the nematodes washed with M9 buffer are picked out under a body microscope and placed on a glass slide. The slide is placed under a fluorescence microscope and observed with a 10x lens. It can be seen that after the nematodes ingest the fluorescent microspheres, the microspheres enter the pharynx, intestine, rectum, and some are excreted through the anus (such as Figure 4 ).

[0050] (2) Preparation of Tenebrio molitor larval intestinal extract

[0051] Two groups of experiments were designed for comparison. Twenty Tenebrio molitor larvae of the same age were selected and repeatedly washed with sterile water in a beaker. The washed Tenebrio molitor larvae were then soaked in 75% alcohol for a period of time. The sterilized Tenebrio molitor larvae were again washed with sterile water and placed in a culture dish. The head of the Tenebrio molitor larvae was cut off with scissors heated on an alcohol lamp, and the intestinal contents were slowly squeezed out from the tail of the Tenebrio molitor larvae with a glass rod. After all the intestinal contents were removed, they were transferred to a sterile mortar. The mortar was ground on ice. 2mL of sterile 0.9% NaCL solution was added. After grinding, the mixture was uniform and no visible flocculation was observed. This was the Tenebrio molitor larval intestinal extract required for the experiment.

[0052] (3) Exposure of nematodes to Tenebrio molitor intestinal extract and polystyrene microplastics

[0053] Take 20 L4 stage experimental group nematodes and transfer them to new NGM medium. Use a pipette to evenly spread the yellow mealworm homogenate on the experimental group medium. The control group does not add anything. Treat for 7 hours. Prepare fluorescent polystyrene nanospheres with E. coli OP50 solution, with an exposure concentration of 50 μg / mL. Spread them on the NGM medium as the experimental group, and spread the same volume of E. coli OP50 solution on the medium as the control group. Place synchronized L1 stage nematodes in the experimental and control groups of medium, and cultivate until L4 stage for experiments.

[0054] (4) Observation of the effect of yellow mealworm intestinal extract on microplastics in nematodes

[0055] Take L4 stage nematodes from the experimental group described above and place them on new NGM medium. Allow the nematodes to crawl for a period of time, then rinse them with M9 buffer three times. Use a nematode picking needle to pick the three most active nematodes from each group and place them on three glass slides with agar pads, labeled. Repeat three times. The control group is treated the same as the experimental group, and the distribution of fluorescent polystyrene nanospheres in the nematodes is observed under a fluorescence microscope for 1 hour and 3 hours.

[0056] After 1 hour of adding an equal amount of polystyrene fluorescent microspheres, the control group nematodes without yellow mealworm intestinal extract have a bright "fluorescent band" visible throughout the body of the nematodes. Figure 5 The experimental group nematodes with added yellow mealworm intestinal extract have the same fluorescent distribution as the control group, but the fluorescence is not as clear and bright as the control group. The overall brightness is dim, and the fluorescence in the hindgut and anus is brighter than in other parts. Figure 6 After 3 hours, the "fluorescent band" in the control group nematodes is less bright than the 1 hour control group, and the fluorescence intensity in the nematode throat is reduced. The fluorescence is concentrated in the hindgut of the nematodes. Figure 7 In the 3 hour experimental group pictures, the overall fluorescence of the nematodes is weak, and the outline of the nematodes is almost invisible. Compared to the 1 hour experimental group, the fluorescence intensity is lower. Figure 8

[0057] Example 2

[0058] In this embodiment, we will introduce a kind of yellow mealworm that can reduce the effect of polystyrene microplastics in mice, and 100 nm polystyrene fluorescent microspheres can enter the mouse organs through blood circulation after gavage. The specific content includes:

[0059] ​The mice used in this example were six 7-week-old clean-type male mice, weighing approximately 30-33g. They were healthy, active, and had no adverse reactions. They were kept in an animal room with a room temperature of (23±2)°C, a relative humidity of 45%-60%, and a 12-hour day-night cycle. After acclimating to the environment for one week, the mice had free access to food and water. This example selected mealworm larvae that had just molted and grown to about 60 days old, which was conducive to the mice's chewing. They were approximately 15-20mm long and highly active. This example used a 1000mL beaker to feed the mealworms to the mice; a No. 12 gavage needle was used for gavage of the experimental animals; a freezing microtome was used to prepare kidney and liver slices; an anticoagulant prepared with sodium citrate; and polystyrene green fluorescent microspheres with a particle size of 100nm were used in this example. The excitation wavelength of the fluorescent microspheres was 460nm and the emission wavelength was 505nm. The dosage for mice was 200mg / kg polystyrene fluorescent microspheres.

[0060] (1) Feeding mice with mealworms as a protein supplement

[0061] Mealworms were used as a dietary protein supplement for the experimental mice. Three mice in the experimental group were separated and each placed individually in a large beaker. Two mealworms were placed at the bottom of the beaker. The beaker was placed in a quiet environment and the mice were encouraged to eat. If the mice did not actively eat, the mealworms were picked up with tweezers and passed to the mouse's mouth to encourage them to eat. The mice were observed until they had consumed the mealworms and then returned to their cages. The experimental group mice were fed mealworms continuously for one week, while the control group mice were not fed mealworms.

[0062] (2) After 7 days of feeding with mealworms, mice were deprived of food and water for 6 hours before gavage. The mice were then lifted by their tails and secured. The prepared drug was then delivered into the mouse stomach using a gavage device connected to a syringe. This example employed a single oral gavage. The drug administered to the mice was 200 mg / kg polystyrene fluorescent microspheres, diluted to 0.5 mL with normal saline. The same dose was used in both the experimental and control groups.

[0063] (3) By observing the fluorescence content in the blood of mice, it was determined that mealworms have a degrading effect on microplastics in the mice's bodies.

[0064] Blood was collected from the tail of the mice 0.5 h after gavage. In this experiment, tail cutting was used for blood collection. The blood samples collected from the tail of the mice were mixed with an anticoagulant made of sodium citrate in a ratio of 1:1 and then made into glass slides for observation under a fluorescence microscope. There were fewer fluorescent spots in the glass slide samples of the experimental group, and the fluorescent spots were scattered and the fluorescence intensity was low, with almost no visible fluorescence ( Figure 9 The number of fluorescent spots in the slide samples of the control group was larger, the fluorescent spots were more concentrated, and the fluorescence intensity was stronger than that of the experimental group ( Figure 10 ).

[0065] (4) By observing the liver, kidney slices of mice to determine that microplastics can enter the organs through blood circulation.

[0066] After gavage, the liver and kidney of mice were quick-frozen, fixed and sectioned. Kidney and liver slices of mice were made and observed under a fluorescence microscope. Green fluorescence was observed in the kidney slices of mice, and the distribution was uniform Figure 11 ). The fluorescence distribution in the liver slices of mice was uneven, and the fluorescence in some areas was more obvious Figure 12 ).

[0067] Example 3

[0068] In this embodiment, the degradation of polystyrene microspheres microplastics in the blood of Huainan ephedra chickens by tenebrio molitor and the degradation of polystyrene microspheres microplastics in the kidney of Huainan ephedra chickens by tenebrio molitor are specifically included.

[0069] In this embodiment, 1 mL syringe is used for wing vein blood sampling of chickens; freezing microtome is used for making kidney slices; fluorescence microscope is used for photographing observation of kidney slices and blood of Huainan ephedra chickens; polystyrene green fluorescent microspheres with a particle size of 100 nm are used in this example. The excitation wavelength of the fluorescent microspheres is 460 nm, the emission wavelength is 505 nm, the dose is 200 mg / kg for Huainan ephedra chickens, and the gavage is used for Huainan ephedra chickens; sodium citrate is used to make anticoagulant; this embodiment selects tenebrio molitor larvae which have just molted and grow to about 60 days, with a body length of about 15-20 mm and strong activity; 6 Huainan ephedra chickens, weighing about 300-500 g, are healthy and active without adverse reactions.

[0070] (1) Feeding tenebrio molitor to ephedra chickens and gavage operation of ephedra chickens

[0071] The feeding method of ephedra chickens is slightly changed based on example 2, 10 tenebrio molitor are fed to each of the 3 ephedra chickens in the experimental group every day, and the feeding is continuous for 4 days. After gavage, tenebrio molitor is continuously fed for 2 days. The gavage method of ephedra chickens is slightly changed based on example 2, the ephedra chickens are deprived of water and food for 6-8 hours before gavage, the gavage needle is inserted into the ephedra chicken's beak to 1 / 5, and then the liquid in the needle is slowly pushed to soak the ephedra chicken's tongue, and the ephedra chicken's habit of drinking water is used to swallow the liquid to achieve the purpose of gavage.

[0072] (2) By observing the blood samples of ephedra chickens to determine that tenebrio molitor has a degradation effect on microplastics in the blood of ephedra chickens.

[0073] Blood was collected from the subwing vein of the chicken 0.5 hours after gavage. After the chicken was fixed, the right hand used a syringe to wait for it to calm down and then inserted the needle into the skin at a 45-degree angle. The needle was inserted parallel to the blood vessel for 0.2 to 0.4 cm and then into the vein. Blood collection stopped when blood returned. The collected blood was mixed with an anticoagulant made of sodium citrate in a 1:1 ratio to prepare a glass slide. The glass slide was placed under a fluorescence microscope for observation. The blood test results showed that in the blood samples of the experimental group, the microspheres gathered together to form clumps, and the fluorescence of the microspheres was weak and difficult to see clearly ( Figure 13 In the blood samples of the control group, the microsphere fluorescence was clear and bright, forming a sharp contrast with that of the experimental group ( Figure 14 ).

[0074] (2) By observing the kidney sections of Ephedra chicken, it was determined that mealworms have a degrading effect on microplastics in the kidneys of Ephedra chicken.

[0075] After gavage, the rats were fed mealworms for 2 days, and then the kidneys of the Huainan Ephedra chickens in the experimental and control groups were quickly frozen, fixed, and sliced. The kidney slices of the Ephedra chickens were made and observed under a fluorescence microscope. The kidney slices of the Ephedra chickens in the experimental and control groups were compared. The results showed that there were significant differences between the kidney slices of the control group and the kidney slices of the experimental group. Multiple clear and bright fluorescent spots were seen in the slices of the control group ( Figure 15 ), while there were no clear and bright fluorescent spots in the slices of the experimental group, and the fluorescence in some kidney slices of the experimental group was weak and almost invisible ( Figure 16 ).

Claims

1. A method for detecting microplastic degradation in living ephedra chickens, characterized in that: The ephedra chickens were divided into a negative control group, a positive control group, and an experimental group; The negative control group consisted of three healthy 9-month-old chicks without any treatment; The positive control group consisted of three chicks of the same age, and polystyrene green fluorescent microspheres with a concentration of 200 mg / kg and a particle size of 100 nm were added at one time, and the volume was adjusted to 0.5 mL of normal saline. Ephedra chickens chose to actively swallow the food without any damage. The experimental group was fed with 3 healthy Ephedra chickens of the same age at a rate of 10 mealworms per 500g of Ephedra chicken body weight per day. After 4 consecutive days of feeding, the positive control group was treated with experimental manipulations. After that, the chickens were fed with 10 selenium-enriched mealworms per 500g of Ephedra chicken body weight per day for 2 days. Subsequently, relevant experiments were used to verify whether the experimental group and the negative control group had a selenium-enriching effect, and experiments were used to verify whether the experimental group and the positive control group had a plastic-reducing effect.

2. A method for detecting microplastic degradation in living ephedra chickens according to claim 1, characterized in that: Breeding companies should feed 3-5 mealworms per 500g per day for 30 days to achieve the effect of selenium-enriched plastic reduction within one month.

3. A method for detecting microplastic degradation in living nematodes, characterized in that: The nematodes were set as a control group and an experimental group; The control group was fed with nematodes containing a high concentration of 50 μg / mL polystyrene fluorescent microspheres; After the experimental group added the mealworm larvae intestinal extract homogenate and evenly spread it on the nematode experimental group culture medium, the effect of the mealworm intestinal extract on the microplastics in the nematodes was observed. Compared with the control group, the overall fluorescence of the nematodes became weak, and the mealworm intestinal extract had a degrading effect on polystyrene plastics in the nematodes.

4. A method for detecting microplastic degradation in living mice, characterized in that: The mice were set as a control group and an experimental group; The control group was orally administered with 200 mg / kg of polystyrene fluorescent microspheres to mice once; The mice in the experimental group were first gavaged with mealworms 7 days in advance, and then orally gavaged with polystyrene fluorescent microspheres in the same dosage as the control group. Mice were fed mealworms continuously for 7 days and then deprived of water and food for 6 hours before gavage. Then, they were gavaged with 100nm polystyrene fluorescent microspheres at a concentration of 200mg / kg. Compared with the control group, the experimental group had fewer fluorescent spots in the blood and organ slide samples, and the fluorescent spots were scattered and the fluorescence intensity was lower.

5. A method for detecting microplastic degradation in living chickens, nematodes and mice according to any one of claims 1, 3 and 4, characterized in that: The mealworm or the extract from the mealworm is added to the animal feed in the form of live bacteria to prepare the plastic-reducing feed. This method can be used to mass-produce the plastic-reducing feed to achieve the goal of effectively, quickly and simply reducing plastic in the animal body.

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