A biological breeding method for ephedra chicken to enrich selenium and reduce plasticity

Abstract

The present invention discloses a biological breeding method for selenium enrichment and plastic reduction in Ephedra chickens, belonging to the biological technology field of poultry breeding. The method converts inorganic selenium into organic selenium through the food chain transmission of selenium from soil to lettuce, then to mealworms, and finally to Huainan Ephedra chickens. The conversion rate of inorganic selenium is improved by controlling the biochemical properties of the soil and the growth environment of the lettuce. Based on the characteristics of the intestinal microorganisms of mealworms that degrade plastics, multiple groups of comparative experiments were conducted to determine the optimal mealworm feeding amount for in vivo microplastic degradation. This method achieves the goals of both effectively enriching selenium and significantly degrading microplastics in the body.

Description

Technical Field

[0001] The present invention belongs to the technical field of poultry biological breeding, and more specifically, relates to an ecological breeding technology for ephedra chickens that is rich in selenium and reduces plasticity. Background Art

[0002] Selenium is an essential trace element for the human body. Studies have found that selenium deficiency can cause dysfunction in certain vital organs and lead to a number of serious illnesses. Studies have shown that for people with low or deficient selenium, adequate selenium supplementation can not only prevent the development of tumors and liver disease, but also enhance the body's immune system, maintain the normal function of vital organs such as the heart, liver, lungs, and stomach, and prevent the onset of cardiovascular and cerebrovascular diseases in the elderly.

[0003] Selenium in nature is divided into two types: plant-active selenium and inorganic selenium. Inorganic selenium generally refers to sodium selenite and sodium selenate, including yeast selenium and malt selenium, which contain large amounts of inorganic selenium residues. These are obtained from metal mine byproducts. Inorganic selenium is highly toxic and poorly absorbed, making it unsuitable for human and animal use. Plant-active selenium is formed through biotransformation and combines with amino acids, generally in the form of selenomethionine. Plant-active selenium is a permitted source of selenium for human and animal use. Compared to inorganic selenium supplements, biological organic selenium is safer, less likely to cause poisoning, easily absorbed and utilized by the human body, and highly effective in supplementing selenium.

[0004] Microplastics are particles or fibers smaller than 5 microns in diameter that are made of high molecular weight polymers and cannot be filtered out by sewage treatment plants. The main sources of microplastics include plastic waste, washed synthetic textiles, tires from vehicles, and skin exfoliating products.

[0005] The impact of microplastics on the physiology of aquatic animals is mainly concentrated in the digestive system, respiratory system and reproductive system, which in turn affects the digestion, respiration and reproduction of organisms. Relevant results have been found in fish, shellfish, crustaceans, echinoderms, etc.

[0006] The impact of microplastics on human health involves multiple systems, including the nervous system, endocrine system, and immune system. Microplastics have recently been found in arterial plaques, suggesting they may be the core component of these plaques. Therefore, excessive exposure to microplastics in our daily lives may contribute to cardiovascular and cerebrovascular diseases.

[0007] Huainan Partridge Chicken (also known as Partridge Chicken) is a high-quality, local Anhui Province-bred chicken bred for both meat and eggs. Characterized by green shanks and beaks, red feathers for roosters, and yellow feathers for hens, the chicken is primarily produced in Anhui and Henan provinces along the Huai River. Partridge Chicken is beloved by the public for its thin skin, tender meat, delicious texture, fragrant, richly yolk eggs, and unique flavor. However, during both free-range and intensive farming, partridge chickens and other poultry inevitably ingest microplastics. These microplastics pose a health risk to the chickens themselves and can also accumulate in humans through the food chain.

[0008] Tenebrio molitor, commonly known as mealworms, is an insect of the order Coleoptera, family Tenebrionidae, and genus Tenebrio. It is an ideal feed insect for artificial cultivation, characterized by a short growth cycle, strong reproductive capacity, wide distribution, abundant sources, low artificial cultivation costs, and high nutritional value. Mealworm protein has the highest nutritional value among animal protein feeds, earning it the nickname "a treasure trove of protein feed." Elahi et al. found that adding mealworms to broiler diets improved growth performance. Benzertiha et al. also showed that adding mealworms increased broiler weight and feed intake, significantly improving growth performance.

[0009] Currently, a growing number of researchers have verified that mealworms are effective at degrading plastic polymers in the natural environment. Mealworm larvae from Asia and North America will consume plastic, and this consumption does not affect their activity. Carbon-13 isotope tracing experiments have demonstrated that polystyrene can be completely degraded in mealworms and mineralized as carbon dioxide for excretion, with some of it converted into body fat. Researchers have successfully isolated a polystyrene-degrading bacterium, Exiguobacterium sp. YT2, from mealworms that can grow using polystyrene as its sole carbon source. This strain is the first polystyrene-degrading bacterium reported internationally and deposited in a strain center. This study once again provides strong scientific evidence that microorganisms can directly and effectively degrade polystyrene. Summary of the Invention

[0010] 1. Technical problem to be solved by the invention

[0011] The present invention will solve the following problems:

[0012] (1) How to enrich poultry such as Ephedra chicken with selenium in a safe and effective way, and improve the quality of chicken meat and the content of organic selenium.

[0013] (2) Although it has been reported that the microorganisms in mealworms can degrade microplastics in nature, or that mealworms can eat and degrade plastic products (polystyrene), there are still few reports on whether mealworms can track "eaten" microplastics in the body and effectively degrade them. If it can be achieved, how to do it, and what is the most appropriate amount to add?

[0014] (3) In order to reduce the number of steps in the actual operation process, how to achieve selenium enrichment and plastic reduction at the same time, how to obtain the best selenium enrichment concentration and the appropriate number of feed for plastic reduction, so as to avoid the yellow mealworms eating selenium-rich lettuce leaves and affecting the content of plastic-reducing microorganisms in their intestines, resulting in a decrease in the plastic reduction effect.

[0015] 2. Technical solution

[0016] In order to solve the above technical problems, the specific technical solutions provided by the present invention are as follows:

[0017] In view of the fact that inorganic selenium is not as safe as organic selenium, this technology can completely convert inorganic selenium into organic selenium through two transformations in the food chain, and then enrich it in animals twice in the middle to detect their activity and mental state, so as to avoid the potential harm that abnormal enriched selenium content may cause to animals and humans.

[0018] When plants grow naturally, they absorb nutrients from the soil and convert inorganic selenium into organic selenium through photosynthesis and biotransformation. The selenium produced in this process is phyto-active selenium. Adding inorganic selenium, sodium selenite, to the soil directly converts the inorganic selenium into phyto-active selenium through absorption by plant roots. This type of artificially cultivated phyto-active selenium has a high absorption rate in the human body and is considered a safe and effective method of selenium supplementation.

[0019] Considering that lettuce is a selenium-rich plant, reportedly containing 1.15 mg of selenium per 100 grams, using lettuce for selenium enrichment is easier than using other plants. Lettuce prefers moisture, which facilitates the transfer and transport of inorganic selenium within the soil. Experiments have shown that soil selenium enrichment and foliar selenium spraying are two highly effective methods for enriching lettuce. This technology utilizes a two-pronged approach, primarily soil selenium enrichment and supplemented by foliar selenium enrichment, to maximize selenium enrichment efficiency.

[0020] Sodium selenite is added to the soil for growing lettuce at one time, supplemented by spraying the lettuce leaves directly with a sodium selenite solution. The general range of sodium selenite added to the soil is 30-300 mg / kg. This technology adds 60 mg / kg, achieving the best selenium-enriched effect. Lettuce that has grown to a certain size, with leaves 6-12 cm long, is selected to improve its transplant survival rate and disease resistance, and to aid in the absorption of inorganic substances. The leaf spray concentration is 30 mg / L, with 0.2 ml sprayed per plant per day, once in the morning and evening, 0.1 ml each time, for 6 consecutive days, ultimately yielding lettuce enriched with organic selenium.

[0021] To facilitate the transport and transfer of inorganic selenium in the soil, multiple experiments have shown that slightly acidic soils, well-ventilated soils, soils with strong reducing properties, and soils with high moisture content are more conducive to plant root absorption of minerals in the soil. Therefore, the soil is prepared in a 3:1 ratio of peat soil to fine sand. Peat soil is processed from the mud at the bottom of a pond with abundant fallen leaves near the forest edge. This type of soil has strong reducing properties. Fine sand is added to make the soil loose and breathable. Ferrous sulfate is appropriately added to adjust the soil pH to the range of 6.0-6.5, while ensuring that the soil has reducing properties.

[0022] A large temperature difference between day and night is conducive to the rapid growth of lettuce, but the growth temperature of lettuce cannot exceed 25 degrees Celsius. Excessively high temperatures will cause the lettuce to produce more lactucin, making it bitter and not conducive to subsequent mealworm consumption. Therefore, during the experiment, the lettuce's natural growth temperature range was 18-20 degrees Celsius during the day, or the artificial climate chamber was set to 20 degrees Celsius during the day, and the lowest nighttime temperature was lowered to 6-8 degrees Celsius, ultimately reducing the daytime temperature difference to less than 10 degrees Celsius. Lettuce cultivation requires at least 6 hours of sunlight per day or at least 10 hours of incandescent light exposure per day. Lettuce was grown continuously in sodium selenite soil for at least 7 days. The soil needed to be watered once a day to keep it moist.

[0023] Mealworm larvae, 15-20 mm long and around 40-60 days old, were selected for vigor. They were fed wheat bran and then, at a 10:1 ratio of body weight, selenium-enriched lettuce leaves washed and dried with clean water. This diet was continued for one week to obtain organically selenium-enriched mealworms. The 50% of mealworms with the highest vigor compared to the selenium-enriched mealworms were used for subsequent experiments. The selection criteria were: first, appearance: brown and dark varieties were excluded, while those with a golden, shiny body and a prominent white abdomen were selected. Second, locomotor characteristics: fast crawling speed, the ability to quickly climb onto lettuce to feed, and strong light reactivity, the ability to quickly move towards darkness when exposed to light.

[0024] Select selenium-enriched mealworms prepared according to the above principles and feed them to healthy ephedra chickens of the same age. Before feeding the chickens, starve the mealworms for two days to allow them to excrete their feces, ensuring that all the selenium in the mealworms is organic. Then, feed the chickens and other poultry 10 mealworms per 500g of selenium-enriched mealworms daily to achieve both selenium enrichment and plastic reduction.

[0025] There are different methods and steps for testing the selenium-enriched effect and plastic-reducing effect of mealworms, which are described in detail as follows:

[0026] Mealworms can be directly fed to poultry at a rate of 10 live mealworms per 500g per day for 6 consecutive days. If selenium enrichment is still needed in the future, live mealworms can be fed at a rate of 3-5 per 500g per day, or the mealworms can be washed, scalded, dried, ground into powder and added to the feed according to the weight ratio. The selenium-enriched effect can be achieved by feeding poultry for 30 days.

[0027] Before the experiment on mealworms degrading microplastics in ephedra chickens, in order to ensure the feasibility and scientificity of the experiment, experiments on mealworms degrading microplastics in lower model organisms nematodes and microorganisms in higher model organisms mice were carried out. The optimal form and amount of mealworm addition for plastic reduction in ephedra chickens were found. Some of the content has been reflected in the form of examples and will not be repeated here.

[0028] The plastic degradation test for mealworms requires feeding 10 live mealworms per 500g of food daily for four days before microplastics are introduced. This is done to allow the ephedra chickens to establish a fixed number of plastic-degrading microorganisms in their bodies. Microplastics are then gavaged in the stomachs of the chickens according to a specific ratio, followed by two days of mealworm feeding. The plastic degradation effect is then compared to the ephedra chickens fed without mealworms before and after microplastics gavage.

[0029] 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.

[0030] Selenium content was tested using the inductively coupled plasma mass spectrometry (ICP-MS) method, the first method in the National Food Safety Standard for the Determination of Multiple Elements in Food. The primary instrument used was an inductively coupled plasma mass spectrometer. Microwave digestion was used, but unlike the standard, this patent added 5 mL of 1% nitric acid solution to the digestion vessel. Simultaneously, 1 mL of 30% hydrogen peroxide solution was added to prevent foaming.

[0031] 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.

[0032] 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.

[0033] As for selenium-enrichment technology, this technology has been used in Huainan Ephedra chickens for selenium enrichment and detection experiments. Therefore, in addition to being applied to Ephedra chickens, this technology can also be applied to other poultry, including poultry from other places, such as Taihe black-bone chickens, Wanxi white geese, etc.

[0034] 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.

[0035] 3. Beneficial effects

[0036] Compared with the prior art, the technical method provided by the present invention has the following significant effects:

[0037] (1) The present invention converts selenium in the soil into plant-active selenium through the food chain, thereby improving the safety of selenium enrichment. In addition, the addition of mealworms to the food chain helps to prevent potential damage to animals caused by high concentrations of organic selenium. The ability of plants to enrich selenium is enhanced by improving the physical and chemical properties of the soil. By combining soil selenium enrichment with leaf selenium enrichment, the selenium enrichment effect of food and vegetables is enhanced.

[0038] (2) 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.

[0039] (3) In order to verify that living mealworms can degrade microplastics in animals, this technology has carried out experiments on microplastic degradation in nematodes, mice, Huainan ephedra chickens, etc. The experiment confirmed that the intestinal flora of the mealworms, such as Morganella, Hafnia, KHJ-1 and Citrobacter, can partially degrade and mineralize the microplastics in the above-mentioned animals into carbon dioxide and discharge them, and a part of them can also be converted into body fat. In this way, the purpose of ecological degradation of microplastics in animals by mealworms is achieved. Considering that there is no similar technology at present, this method can effectively degrade microplastics in animals and reduce their content. It is simple to operate and can be applied to major livestock manufacturers and individual farmers to reduce plastic in different animals. The use of this method also reduces the consumption of poultry containing microplastics by people, reduces the intake of microplastics, and protects people's health.

[0040] (4) The present invention explores whether mealworms can reduce plastic in vivo. Studies have shown that the bacterial flora in mealworms can effectively degrade microplastics in the body. Therefore, the bacterial flora or the extract from mealworms can be added to animal feed in the form of live bacteria to make plastic-degradable feed. This method can be used to mass-produce plastic-degrading feed to achieve the goal of effectively, quickly and simply reducing plastic in animals, and has good development prospects in the feed production industry and the breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Simple diagram of the process of enriching selenium and reducing plastic in Ephedra chicken.

[0042] Figure 2 This is a bar graph of the selenium content of lettuce leaves in different soils with sodium selenite added and two selenium-enriched methods for mealworms in Example 1 (NTT is the selenium content of lettuce leaves cultured in a mixture of peat soil and fine sand with 60 mg / kg sodium selenite added, PTT is the selenium content of lettuce leaves cultured in ordinary soil with the same dose of sodium selenite added, YFX is the selenium content of mealworms fed on lettuce leaves sprayed with sodium selenite solution, TFX is the selenium content of mealworms in the control group, i.e., the lettuce leaves fed with sodium selenite added to the soil, and SWL is the selenium content of mealworms fed on lettuce leaves with sodium selenite added to both the soil and leaves).

[0043] Figure 3 This is a bar graph of the selenium content in various organs of selenium-enriched food chains and the selenium-enriched control group of Ephedra chicken in Example 1 (samples starting with T represent the experimental group, and samples starting with C represent the control group, XY: blood; XZ: heart; GZ: liver; WB: stomach; JR: muscle; SZ: kidney; FB: lung)

[0044] Figure 4 This is the distribution diagram of fluorescent microplastics in nematodes in Example 2

[0045] Figure 5 This is the distribution diagram of fluorescent microplastics in the nematodes of the control group 1 hour after the experiment in Example 2.

[0046] Figure 6 This is the distribution diagram of fluorescent microplastics in the nematodes of the experimental group 1 hour after the experiment in Example 2.

[0047] Figure 7 The distribution of fluorescent microplastics in the nematodes of the control group 3 hours after the experiment in Example 2

[0048] Figure 8 The distribution of fluorescent microplastics in the nematodes of the experimental group 3 hours after the experiment in Example 2

[0049] Figure 9 The distribution of fluorescent microplastics in the blood of mice in the experimental group in Example 3

[0050] Figure 10 The distribution of fluorescent microplastics in the blood of mice in the control group in Example 3

[0051] Figure 11 The distribution diagram of fluorescent microplastics in mouse kidney slices in Example 3

[0052] Figure 12 The distribution of fluorescent microplastics in mouse liver slices in Example 3

[0053] Figure 13 The distribution diagram of fluorescent microplastics in the blood of the experimental group of Ephedra chicken in Example 4

[0054] Figure 14 The distribution diagram of fluorescent microplastics in the blood of Ephedra chickens in the control group in Example 4

[0055] Figure 15 This is the distribution diagram of fluorescent microplastics in the kidney slices of Ephedra chicken in the control group in Example 4

[0056] Figure 16 The distribution of fluorescent microplastics in the kidney slices of the experimental group of Ephedra chicken in Example 4 Specific implementation methods

[0057] In order to further understand the content of the present invention, the present invention is described in detail with reference to the accompanying drawings and implementation examples.

[0058] Example 1

[0059] This case study will describe how inorganic selenium is gradually enriched and transferred to Ephedra chickens through the food chain, ultimately achieving selenium enrichment in Ephedra chickens. Specific content includes:

[0060] First, prepare the soil for the lettuce. Choose a mixture of peat moss and fine sand in a 3:1 ratio. Mix thoroughly and continue adding ferrous sulfate powder. Use a soil pH meter to adjust the pH to around 6.0. Add sodium selenite to the soil at a concentration of 60 mg / kg at a time, stirring thoroughly. Figure 2 It is shown that compared with general soil, the peat soil + fine sand soil of this patent is easier for lettuce to enrich organic selenium.

[0061] After the seedling stage, 20 well-growing lettuces were selected for transplanting. Plants were approximately 10 cm tall and planted in prepared potting soil. The plants were placed in an artificial climate chamber with a controlled light-dark cycle of 10:14. The light temperature was set at 20 degrees Celsius, and the dark temperature was set at 10 degrees Celsius for 5 hours, 8 degrees Celsius for 4 hours, and 10 degrees Celsius for 5 hours. A 30 mg / L sodium selenite solution was sprayed daily, 0.2 ml per plant, once in the morning and evening, 0.1 ml each time, for 6 consecutive days. This resulted in lettuces rich in organic selenium. Watering was also performed daily to keep the soil moist. Figure 2 It also shows that compared with single soil selenium enrichment and leaf selenium enrichment, the selenium enrichment effect is better when the soil is mainly enriched with leaves as a supplement.

[0062] Next, we selected mealworm larvae that were 40-60 days old, 15-20mm in length, and relatively active. We then fed wheat bran and, at a weight ratio of 10:1, washed and dried selenium-enriched lettuce leaves. This diet was continued for one week to obtain mealworms rich in organic selenium. Highly active mealworms with a bright golden color and rapid movement were selected for subsequent experiments. Before feeding mealworms to ephedra chickens, we starved them for two days to allow for the elimination of feces, ensuring that all of the mealworms contained organic selenium.

[0063] Nine healthy ephedra chickens weighing about 300g were selected and divided into three groups, with three chickens in each group. The negative control group consisted of three chickens that did not receive any treatment. The three positive control groups were chicks of the same age and weight, and polystyrene green fluorescent microspheres with a concentration of 200mg / kg and a particle size of 100nm were added at one time, and the volume was fixed to 0.5mL of normal saline. The ephedra chickens chose to feed them by active swallowing without damage. The experimental group was fed with selected mealworms at a rate of 10 mealworms per 500g of body weight per day to three healthy ephedra chickens of the same age. After four consecutive days of feeding, the positive control group was subjected to a fluorescent microplastic gavage experiment, and then the ephedra chickens were fed again at the same dose for two days.

[0064] Finally, the selenium content in the blood, kidney, liver, heart, lung and muscle tissues of lettuce, mealworms and Ephedra chickens was determined using the first method of inductively coupled plasma mass spectrometry (ICP-MS). The results of selenium enrichment in lettuce and mealworms are shown in the attached Figure 2 The results of selenium enrichment in various organs of Ephedra chicken are as follows Figure 3 As shown. Figure 3 It can be seen that the effect of selenium enrichment in the food chain is significantly better than that of the control group.

[0065] Example 2

[0066] This case study will describe how polystyrene enters the body of nematodes and how Tenebrio molitor intestinal extract can degrade polystyrene plastic in nematodes. Specific details include:

[0067] The wild-type L4 nematodes (N2) used in this example have good activity and are cultured in a constant temperature chamber at 25°C. This example uses mealworm larvae that have just molted and are about 60 days old, with a body length of approximately 15-20 mm and strong activity. NGM medium is used as a nematode growth medium; M9 buffer is used to clean the fluorescent microspheres on the surface of the nematodes; a stereo microscope is used to select the nematodes; a fluorescence microscope is used for photographic observation; 75% alcohol is used for sterilization; 0.9% sterile sodium chloride solution is used for dilution of the mealworm intestinal extract; an autoclave is used for sterilization of the mortar; and 2% agar is used to prepare the agar pad. This example uses polystyrene green fluorescent microspheres with a particle size of 100 nm, an excitation wavelength of 460 nm, and an emission wavelength of 505 nm.

[0068] (1) Observing the distribution of polystyrene microplastics in nematodes

[0069] The design is to directly add 50ug / mL high-concentration polystyrene fluorescent microspheres to the NGM medium containing synchronized nematodes. After 1 hour, the nematodes washed with M9 buffer are picked out under a stereomicroscope and placed on a slide. The slide is placed under a fluorescence microscope and observed with a 10x microscope. It can be seen that after the nematodes ingest the fluorescent microspheres, they enter the pharynx, intestine, rectum through the mouth, and some are excreted through the anus (such as Figure 4 ).

[0070] (2) Preparation of Tenebrio molitor larvae intestinal extract

[0071] Two sets of experiments were designed for comparison. Twenty mealworms of the same age were selected and placed in a beaker, rinsed repeatedly with sterile water. The rinsed mealworms were then disinfected by soaking them in 75% alcohol. The disinfected mealworms were rinsed again with sterile water and placed in a petri dish. The mealworms' heads were cut off using scissors heated over an alcohol lamp. A glass rod was used to slowly squeeze the mealworm's intestinal contents from the tail forward. After removal, the contents were transferred to a sterile mortar and pestle. The mortar was ground on ice. 2 mL of sterile 0.9% NaCl solution was then added. Grind thoroughly until no flocs were visible. This was the mealworm larvae intestinal extract required for the experiment.

[0072] (3) Exposing nematodes to Tenebrio molitor intestinal extract and polystyrene microplastics

[0073] Twenty L4-stage nematodes were transferred to fresh NGM medium. A homogenate of mealworms was pipetted and evenly spread across the experimental medium. No homogenate was added to the control medium for 7 hours. Fluorescent nanoparticles of polystyrene were prepared using an E. coli OP50 solution at an exposure concentration of 50 μg / mL. These were plated onto NGM medium as the experimental group, while the same volume of E. coli OP50 solution was plated onto the medium as the control group. Synchronized L1-stage nematodes were cultured in the experimental and control mediums, respectively, until the L4 stage and used for experiments.

[0074] (4) Observe the effect of Tenebrio molitor intestinal extract on microplastics in nematodes

[0075] L4 nematodes from the experimental groups were selected and placed on fresh NGM medium. After allowing the nematodes to crawl for a while, they were rinsed three times with M9 buffer. Using a pick, the three most active nematodes from each group were picked and placed on three glass slides with agar pads, marking them. This was repeated three times. The control group was treated identically to the experimental group, and the distribution of fluorescent polystyrene nanoparticles in the nematodes was observed under a fluorescence microscope after 1 and 3 hours.

[0076] One hour after adding an equal amount of polystyrene fluorescent microspheres, a bright "fluorescent band" was visible in the nematodes of the control group without the addition of the mealworm intestinal extract, and the fluorescence was almost all over the nematodes ( Figure 5 The distribution of fluorescence in the nematodes in the experimental group with added mealworm intestinal extract was consistent with that in the control group, but the fluorescence was not as clear and bright as that in the control group, and there was no clear "fluorescent band". It was darker overall, and the fluorescence in the hindgut and anus was brighter than that in other parts ( Figure 6 ). After 3 hours, the brightness of the "fluorescent band" in the nematodes in the control group ( Figure 7 ) is darker than the 1h control group, the fluorescence intensity of the nematode throat is weakened, and the fluorescence is more concentrated in the hindgut of the nematode. It can be seen in the pictures of the 3h experimental group ( Figure 8 ), the fluorescence of the nematode as a whole became weak, and the outline of the nematode was almost invisible. Compared with the 1-hour experimental group, the fluorescence intensity was lower.

[0077] Example 3

[0078] This case study demonstrates the plastic-reducing effect of a mealworm on polystyrene microplastics in mice, and demonstrates how 100nm polystyrene fluorescent microspheres can enter the mouse organs through the bloodstream after oral administration. Specific details include:

[0079] 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.

[0080] (1) Feeding mice with mealworms as a protein supplement

[0081] 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.

[0082] (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.

[0083] (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.

[0084] 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 ).

[0085] (4) By observing the liver and kidney slices of mice, it was determined that microplastics can enter the organs through the blood circulation.

[0086] After gavage, the liver and kidney of the mice were quickly frozen, fixed and sliced. The kidney and liver slices of the mice were made and observed under a fluorescence microscope. Green fluorescence was visible in the kidney slices of the mice and was evenly distributed ( Figure 11 The fluorescence in the mouse liver slices was unevenly distributed, with some areas showing more pronounced fluorescence ( Figure 12 ).

[0087] Example 4

[0088] In this implementation case, the degradation effect of mealworms on polystyrene microspheres and microplastics in the blood of Huainan Ephedra chickens and the degradation effect of mealworms on polystyrene microplastics in the kidneys of Huainan Ephedra chickens specifically include:

[0089] In this example, a 1mL syringe was used for blood collection from the subwing vein of chickens; a cryostat was used to prepare kidney sections; and a fluorescence microscope was used to photograph and observe kidney sections and blood from Huainan Ehuang chickens. Polystyrene green fluorescent microspheres with a particle size of 100 nm were used. The fluorescent microspheres had an excitation wavelength of 460 nm and an emission wavelength of 505 nm. The dosage was 200 mg / kg for oral administration to Huainan Ehuang chickens. Sodium citrate was used to prepare the anticoagulant. This example used mealworm larvae, approximately 60 days old and recently molted, with a body length of approximately 15-20 mm and high activity. Six Huainan Ehuang chickens, weighing approximately 300-500 g, were healthy, active, and showed no adverse reactions.

[0090] (1) Feeding Tenebrio worms to Ephedra chickens and gavage operation of Ephedra chickens

[0091] The feeding method for Ephedra chickens was slightly modified from that in Example 2. Each of the three Ephedra chickens in the experimental group was fed 10 mealworms daily for four consecutive days. Mealworms were then fed for two consecutive days after gavage. The gavage method for Ephedra chickens was slightly modified from that in Example 2. Prior to gavage, the chickens were deprived of food and water for 6-8 hours. A gavage needle was inserted one-fifth of the way along the chicken's beak, and the liquid in the needle was slowly pushed to soak the chicken's tongue. This achieved the purpose of gavage by utilizing the chicken's habit of drinking water and swallowing the liquid with its head tilted back.

[0092] (2) By observing the blood samples of Ephedra chickens, it was determined that mealworms have a degrading effect on microplastics in the blood of Ephedra chickens.

[0093] 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 ).

[0094] (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.

[0095] 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 biological breeding method for selenium-enriched and plastic-reducing ephedra chickens, characterized in that A food chain selenium enrichment process is proposed, in which inorganic selenium is transferred from soil to lettuce, to mealworms, and then to chickens, so as to achieve the transfer and enrichment of inorganic selenium into organic selenium; the food chain converts selenium in the soil into plant-active selenium, which improves the safety of selenium enrichment, and adding mealworms to the food chain helps to avoid potential damage to animals caused by high concentrations of organic selenium; selenium-enriched lettuce is prepared by adding sodium selenite to the soil used to grow lettuce at one time, and supplemented by directly spraying the lettuce leaves with a sodium selenite solution, wherein the soil is added with sodium selenite at a ratio of 60 mg / kg, and the addition amount ranges from 30 to 300 mg / kg; the lettuce leaves are selected to be in the range of 6 to 12 cm in length, and the leaf spraying concentration is 30 mg / L. By spraying 0.2 ml per plant per day for 6 consecutive days, lettuce enriched with organic selenium was finally obtained; the soil for growing lettuce was peat soil: fine sand in a ratio of 3:1, and the soil pH was adjusted to the range of 6.0-6.5 by adding ferrous sulfate; lettuce cultivation requires at least 6 hours of sunlight exposure per day or at least 10 hours of incandescent lamp exposure, and lettuce is grown continuously in sodium selenite soil for at least 7 days; the natural growth temperature of lettuce is 18-20 degrees Celsius during the day, and the lowest temperature at night is lowered to make the temperature difference between day and night less than 10 degrees Celsius; the soil needs to be watered once a day to keep the soil moist; at the same time, selenium-rich mealworm larvae are used to degrade microplastics in chickens, thereby realizing biological breeding that enriches selenium in the food chain and reduces plastic.

2. A biological breeding method for selenium-enriched and plastic-reducing ephedra chicken according to claim 1, characterized in that Select mealworms with a body length of 15-20mm and strong vitality of 40-60 days old. Feed them with wheat bran and selenium-enriched lettuce leaves washed and dried with clean water at a weight ratio of 10:

1. Feed the mealworms continuously for one week to obtain mealworms enriched with organic selenium. The specific principles for selecting strong mealworms are: First, appearance characteristics: Eliminate brown and dark varieties and choose those with golden color, shiny body surface and obvious white belly; Secondly, movement characteristics: fast crawling speed, able to quickly climb up to eat lettuce when fed, strong light reaction, able to quickly crawl to a dark environment when there is light; based on the appearance and vitality of the mealworms, 50% of the mealworms with dominant characteristics were selected, and they were starved for 2 days until the feces in their bodies were discharged to ensure that the mealworms contained organic selenium.

3. A biological breeding method for selenium-enriched and plastic-reducing ephedra chicken according to claim 1 or 2, characterized in that The microplastics degraded by mealworms are polystyrene, polyethylene, polypropylene, and polyvinyl chloride.

4. A biological breeding method for selenium-enriched and plastic-reducing ephedra chicken according to claim 1, characterized in that This method can also be applied to Taihe black-bone chickens and Wanxi large white geese.

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