KR102685908B1 - Method for breeding millworm using styrofoam waste - Google Patents

Abstract

The present invention relates to a method of raising mealworms using Styrofoam waste, which includes an egg-laying step (S100) of feeding adult mealworms in the egg-laying module 11 of the breeding module 10 to lay eggs for one week; The eggs spawned in the spawning step (S100) are collected together with the excrement of adult mealworms and grown in the first growth module (12-1) of the growth modules (12-1, 12-2, 12-3, 12-4) composed of 4 zones. ) and mixing with wheat bran (S200); After hatching and growing for one week in the mixing step (S200), they are transferred (shifted) to the second growth module (12-2), and grown for one week in the second growth module (12-2) and then transferred to the third growth module. A growth step (S300) of transferring to (12-3), growing for one week in the third growth module (12-3), and sequentially transferring to the fourth growth module (12-4); The larvae that grew for 4 weeks in the growth stage (S300) are sequentially transferred to the feeding modules (20-1 to 20-10) and grow into larvae of the desired size while eating the Styrofoam cake (110) for 10 weeks. Feeding step (S400); In the feeding step (S400), the larvae that grew in the feeding module for 10 weeks are transferred to the stabilization module (30) and fed only with wheat bran for 5 days to completely eliminate Styrofoam in the body, and are fasted for 2 days to empty the intestines of the wheat source. Stabilization step (S500); And in order to check whether there is any Styrofoam residue in the body of the mealworm that has gone through the stabilization step (S500), a sample or all samples are examined using ultrasound, In addition, it is characterized by including an inspection step (S600) of completely removing residues by fasting.

Description

Method for breeding millworm using styrofoam waste}

The present invention relates to a method of raising mealworms, and more specifically, to a method of rearing mealworms using Styrofoam waste.

Recently, due to the rapid growth of the delivery business since the COVID-19 incident, the use of expanded polystyrene (EPS: EXPANDED POLYSTYRENE), also called Styrofoam, has rapidly increased, and environmental pollution due to Styrofoam waste is becoming more serious.

Technologies for processing Styrofoam used to solve this problem include reprocessing it into ingots, pellets, etc., and simple disposal methods such as heat generation through incineration and landfill. However, due to the price reduction of reprocessed Styrofoam products, simple disposal methods are mainly used. When used in landfill, it does not naturally decompose for more than 500 years, and when incinerated, environmental problems are raised due to the generation of carcinogens such as dioxin, so the world is demanding a solution to dispose of such Styrofoam.

As a means to solve this problem, a method of causing biological organic transformation to decompose Styrofoam using mealworms (mealworm larvae), a living organism that can decompose Styrofoam by eating it, has been introduced. However, research on the safety of mealworms that grew up eating Styrofoam has been introduced. is needed.

Republic of Korea Patent Publication No. 10-2017-0109470 (Title: Experimental teaching method using a Styrofoam decomposition tester for mealworm larva test specimens, Publication date: September 29, 2017, Applicants: Jo Ik-hyung, Kim Won-mok) Republic of Korea Patent Publication No. 10-2020-0051812 (Title: Insect larvae rearing, Publication date: May 13, 2020, Applicant: Entomix Biosystems Limited)

The purpose of the present invention to solve the above problems is to provide a method of raising mealworms using Styrofoam waste, which can reduce environmental pollution by rearing mealworms using Styrofoam waste as feed.

To achieve the above object, the method of raising mealworms using Styrofoam waste of the present invention includes an egg-laying step (S100) in which adult mealworms are fed for one week in the egg-laying module 11 of the breeding module 10 to lay eggs; The eggs spawned in the spawning step (S100) are collected together with the excrement of adult mealworms and grown in the first growth module (12-1) of the growth modules (12-1, 12-2, 12-3, 12-4) composed of 4 zones. ) and mixing with wheat bran (S200); After hatching and growing for one week in the mixing step (S200), they are transferred (shifted) to the second growth module (12-2), and grown for one week in the second growth module (12-2) and then transferred to the third growth module. A growth step (S300) of transferring to (12-3), growing for one week in the third growth module (12-3), and sequentially transferring to the fourth growth module (12-4); The larvae that grew for 4 weeks in the growth stage (S300) are sequentially transferred to the feeding modules (20-1 to 20-10) and grow into larvae of the desired size while eating the Styrofoam cake (110) for 10 weeks. Feeding step (S400); In the feeding step (S400), the larvae that grew in the feeding module for 10 weeks are transferred to the stabilization module (30) and fed only with wheat bran for 5 days to completely eliminate Styrofoam in the body, and are fasted for 2 days to empty the intestines of the wheat source. Stabilization step (S500); And in order to check whether there is any Styrofoam residue in the body of the mealworm that has gone through the stabilization step (S500), a sample or all samples are examined using ultrasound, In addition, it is characterized by including an inspection step (S600) of completely removing residues by fasting.

According to these characteristics, the present invention can raise mealworms using Styrofoam as feed, and can reduce environmental pollution by using Styrofoam as feed.

Additionally, mealworms reared by the method of the present invention are non-toxic and can be used for food.

Figure 1 is a diagram illustrating each step of raising mealworms by recycling Styrofoam (EPS) waste according to an embodiment of the present invention.
Figure 2 is a diagram of a Styrofoam cake in which wheat bran is applied to a perforated Styrofoam slit to raise mealworms by recycling Styrofoam (EPS) waste according to an embodiment of the present invention.
Figure 3 is a flowchart showing a method of raising mealworms by recycling Styrofoam (EPS) waste according to an embodiment of the present invention.

Below, with reference to the attached drawings, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. In order to clearly explain the present invention in the drawings, parts that are not related to the description are omitted, and similar parts are given similar reference numerals throughout the specification.

Then, with reference to the attached FIGS. 1 to 3, the method of raising mealworms by recycling Styrofoam (EPS) waste according to the present invention will be described in detail.

Mealworms are the only living organisms discovered to date that can decompose Styrofoam. Approximately 3 million mealworms can organically decompose 1 kilogram (kg) of Styrofoam within 24 hours. At the same time, mealworms are high in fat and high in protein and fat. It is evaluated as a food group with high nutritional value.

After successfully ingesting Styrofoam (ESP), it usually takes at least 24 hours to be able to excrete excrement, and it is completely excreted within 48 hours.

Mealworms are edible insects that have been officially registered as edible food with food safety recognition from the Food and Drug Administration. Mealworms are based on bran, a by-product of wheat milling, so they consume less grain, require less breeding space compared to livestock farming, and especially produce greenhouse gases (Green House). It has the advantage that the amount of gas generated is very small compared to existing livestock farming.

However, since Styrofoam is not a food preferred by mealworms, they cannot eat it well and eat it slowly, and removal of Styrofoam residues from the mealworm's body was also a problem, making it impossible to raise mealworms by recycling Styrofoam.

Therefore, in the present invention, the contaminated portion of Styrofoam waste was removed, processed into a slit shape with a thickness of 5 mm to 10 mm, a perforation was formed in the slit, and wheat bran, which mealworms like, was attached to the perforated Styrofoam to produce a Styrofoam cake. .

In the present invention, the adhesive used to bond wheat bran, etc. to Styrofoam is a weakly viscous adhesive made from wheat gluten, but any material that is not harmful to mealworms can be used instead.

Figure 1 shows a mealworm breeding device 100 using Styrofoam (EPS) waste according to an embodiment of the present invention, which includes a breeding module 110, a feeding module 120, and a stabilization module 130, and the breeding The module 110 is further divided into an egg-laying module 111 and a growth module 112. In the egg-laying module 111, adult mealworms are introduced and discarded fruits, etc. are provided as food to excrete and lay eggs. At this time, they lay eggs. The eggs and excrement are allowed to naturally fall below the mesh net (not shown), and the eggs and excrement of adult mealworms are mixed with bran and grown in the growth module 112 for 4 weeks.

The mealworms grown for 4 weeks in the growth module 112 are transferred to the feeding module 120 and grow into larvae while consuming the Styrofoam cake 110 for 10 weeks.

After the eggs hatched for 14 weeks as described above become larvae, the stabilization module 130 supplies regular wheat bran instead of the Styrofoam cake 110 for 7 days to provide a stabilization period to completely decompose the remaining Styrofoam present in the body of the mealworm. After letting it go through the process, it fasts for at least two days to empty the intestines of the mealworm and make it edible.

In addition, in order to confirm the presence of Styrofoam residues in the body of mealworms reared by the method of the present invention, samples or all samples are examined using ultrasound, It is fasted for an additional day to completely remove residues so that it can be shipped or processed.

In the present invention, the optimal embodiment is a 4-week growth process in the breeding module 110, a 10-week breeding process in the feeding module 120, and a 7-day period in the stabilization module 130, but the temperature of the country or region Depending on the temperature and humidity, the breeding period may be shortened or extended.

Now, if the operating method of the mealworm breeding device 100 of the present invention is explained in detail using FIGS. 1 and 3, first, adult mealworms are secured in the spawning module 11 of the breeding module 10 of FIG. A spawning stage (S100) in which eggs are fed by feeding them for one week, and the eggs spawned in the spawning stage (S100) are collected together with the excrement of adult mealworms and grown into a growth module (12-1, 12-2, 12-) consisting of 4 zones. 3, 12-4) is transferred (shifted) to the first growth module (12-1) and mixed with wheat bran (S200), hatched and grown for one week in the mixing step (S200), and then grown for the second time. Transferred (shifted) to the module 12-2, grown for one week in the second growth module 12-2, then transferred to the third growth module 12-3, and grown in the third growth module 12-3. The growth stage (S300) involves growing for one week and sequentially transferring to the fourth growth module (12-4). The larvae that grew for 4 weeks in the growth stage (S300) are fed to the feeding modules (20-1 to 20-10). ) and are sequentially transferred to the feeding stage (S400), where they grow into larvae of the desired size while consuming the Styrofoam cake (110) for 10 weeks. The larvae grown in the feeding module for 10 weeks in the feeding stage (S400) are A stabilization step (S500) in which the wheat worm is transported to the stabilization module (30) and fed only wheat bran for 5 days to completely eliminate Styrofoam in the body, and then fasted for 2 days to empty the intestines of the wheat worm. Meal worms that have gone through the stabilization step (S500) In order to check for the presence of Styrofoam residues in the mealworm's body, a sample or entire sample is inspected using ultrasound, The final desired edible larvae can be secured by going through an inspection step (S600) to completely remove them.

When reared according to the embodiment of the present invention, edible larvae can be obtained only 16 weeks after the first egg laying, but after 16 weeks, at least 100 million edible mealworms can be secured every week.

In order to mass produce such edible mealworms, by additionally installing the mealworm breeding device 100 of FIG. 1 of the present invention, a desired amount of edible mealworms can be obtained.

Edible mealworms secured as described above are generally sold after going through a sterilization process and drying process, or can be sold in powder form after additionally going through a grinding process.

A prototype made from powdered mealworms raised by recycling Styrofoam (EPS) waste in the same manner as above was provided to the College of Industrial Sciences at Kongju University, and the safety of mealworm powder fed with Styrofoam was confirmed through cell and animal experiments. As a result, it appears that there is no effect of estrogen on cells, and the results show that it is not toxic to male rats.

To prove this, the important parts of the research conducted by the College of Industrial Sciences at Kongju National University are described in detail as follows.

[Main parts of the research]

2.1. Research content

In order to evaluate the safety of mealworms used to decompose Styrofoam as feed, this is an experiment to determine whether there is a hormonal effect on cells and whether there is toxicity when administered to experimental animals.

2.2. Study Purpose

The purpose of this test is to determine the safety of Styrofoam-fed mealworm powder after administration or feeding to estrogen-dependent cells and white rats in cell and animal test models.

3. Experimental materials and methods

3.1 Experimental materials

The mealworm powder used in this experiment was provided and used.

3.1.1 Cell line: MCF-7

MCF-7 is a human-derived breast cancer cell that is often used in experiments related to endocrine disorders, especially those related to estrogen. It has an estrogen receptor and thus responds to cell proliferation in the presence of estrogen. Confirm.

17-β estradiol is used as a positive control in estrogen induction assays, and exposure to 17-β estradiol increases the incidence and proliferation of breast cancer.

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3.2 Experimental method

3.2.1 Preparation of extract from mealworms

The active ingredient is extracted from mealworm powder using a reduced pressure concentration method.

This is a test that uses the ability of mitochondria to reduce the yellow water-soluble substrate MTT tetrazolium to bluish-purple non-water-soluble formazan cystal by the action of mitochondrial dehydrogenase in living cells.

3.2.2 MTT assay

MTT assay is an experimental method that measures cell toxicity and survival rate.

The active ingredient is extracted from mealworm powder using a reduced pressure concentration method.

3.2.3 Experimental animals

Experimental species and strain: Rat, Sprague-Dawley (Crl:CD(SD)), male, 4 weeks old (4wk)

Producer: Nara-Biotech INC., Korea

Reasons for selecting the experimental system

In previous studies, the safety of mealworm powder has been evaluated using rats (SD rats), and rats have been widely used in subacute toxicity tests so far, so there is a lot of physiological, anatomical, and toxicological basic data to compare. I chose it because it was there.

Quarantine and Purification

Upon acquisition, a 4-week-old rat is inspected externally, its weight is measured using an electronic scale, and general symptoms are observed once a day after an acclimatization period of 6 to 7 days. Upon acquisition, the tail of the rat is marked with a black permanent marker, and when separating into groups, the individual is marked with a blue permanent marker.

military separation

Among the purified animals, those with no abnormalities in weight or general symptoms are selected and separated into 5 groups with 5 animals per group.

Breeding conditions

The breeding box uses a stainless steel wire mesh breeding box of 260W The study was conducted in the animal room (A323) of Kongju University, which was set at 300 lux and ventilation frequency of 10 to 15 times/h. All tests were conducted in accordance with Kongju National University IACUC (KNU_2021-08).

Administration method

The route of administration was oral administration, which is the route expected for clinical application. It is administered once a day for 5 weeks for a total of 35 days. The administration time is 10:00-11:00 am and is administered after fasting for 12 hours to minimize the burden on the stomach.

Observation Item

(1) General symptom observation: Conducted once a day during the test period. The observation method records the severity depending on the presence or absence of general clinical symptoms (anorexia, salivation, diarrhea, polyuria, anuria, fecal change, etc.).

(2) Body weight measurement: All animals are measured twice a week before the start of administration, after the start of administration, and until the end of the test.

(3) Measurement of feed intake: The total amount fed and the remaining amount for each breeding box are measured twice a week for 5 weeks after the start of administration.

(4) Measurement of water intake: Measure the total amount and remaining amount of water in each breeding box twice a week for 5 weeks after the start of administration.

(5) Ophthalmological examination: During the administration period, an ophthalmological examination using the naked eye and an ophthalmoscope is performed once on five animals in each group at random.

(6) Hematological and serum biochemical tests: General hematological tests and serum biochemical tests are performed.

(7) Organ weight measurement: Absolute organ weight and relative organ weight are measured for the body weight of the test animal, including kidneys (left and right), spleen, heart, adrenal glands (left and right), testes (left and right), lungs, and thymus.

(8) Histopathological examination: All organs whose weights have been measured are made into paraffin blocks and then stained with H&E (Hematoxylin & Eosin) for observation.

3.2.4 Experimental design and group composition

experimental design

In vitro and in vivo experiments were planned to confirm the safety of mealworm powder.

In the in vitro stage, the cell proliferation rate was compared between mealworms (w/o sty) that were not fed Styrofoam and those treated with mealworm powder (w/sty) that were fed Styrofoam.

In the in vivo stage, mealworms not fed Styrofoam and mealworm powder fed were orally administered daily for 5 weeks.

The administered dose was selected as a percentage of the most recently measured feed intake. After 5 weeks, an autopsy was performed and samples were collected and analyzed.

Animal test group and administration dose settings (the test group was set to 5 groups in total, with 5 animals per group.)

4. Experiment details

4.1 Extract preparation from mealworms

The active ingredients were leached with collected mealworm powder and 70% ethyl alcohol at a ratio of 3:7 for 3 days, then concentrated under reduced pressure for one day at 45-50°C using a rotary vacuum concentrator (EYELA N-1000). The concentrated sample was prepared into the final material using dimethylsulfoxide (DMSO).

4.2 Cell experiment

4.2.1 Setting up the cell experiment application method of the material

After selecting a solvent that can dissolve the substance, the formation of foreign substances was checked when mixed with the medium. If foreign substances were observed, a sterilization method for future cell experiments was selected.

4.2.2 Positive control (17-βestradiol) measurement

The concentration affecting MCF-7 survival rate after estradiol treatment was applied as a positive control for subsequent cell experiments.

4.2.3 Confirmation of proliferation effect of MCF-7 when treated with mealworm powder

The effect on cell viability was confirmed when MCF-7 cells were treated with mealworm (w/o sty) powder not fed Styrofoam and mealworm (w/ sty) powder fed with Styrofoam.

4.3 Animal testing

4.3.1 Weekly weight measurement

The test substance was orally administered to rats every day, and changes in body weight, food intake, and water amount were measured twice a week to evaluate the toxic response caused by the test substance administration. General symptoms were observed for all animals once a day, and the presence or absence of dead animals was checked twice a day.

4.3.2 Organ weight measurement

After oral administration of experimental substances to rats every day for 5 weeks, an autopsy was performed on the last day to determine the absolute organ weight of kidneys (left and right), spleen, heart, adrenal glands (left and right), testes (left and right), lungs, and thymus. and relative organ weights were measured.

4.3.3 Histopathological examination

All organs whose organ weights were measured were fixed in 10% neutral formalin solution, and all organ tissues that had been sufficiently fixed for more than 2 weeks were embedded in a paraffin embedder (SAKURA, Tissue-Tek VIP, vaccum infiltration processor) and microtome (Microm, HM340E). ), 2-3μm sections were made, stained with Hematoxylin & Eosin, and observed to check for toxicity.

4.3.4 Hematological and serum biochemical tests

General hematological tests include red blood cell count (RBC), total white blood cell count (WBC), hemoglobin, hematocrit, mean corpuscular volume (MCV), mean (red) hemoglobin (MCH), and average (red) hemoglobin. ) Blood hemoglobin concentration (MCHC), platelets, lymphocytes, neutrophils, blood coagulation time (Prothrombin time (PT), Partial thromboplastin time (PTT), etc.) were tested.

For the serum biochemical test, a portion of the collected blood was left at room temperature for 30 minutes, coagulated, and then centrifuged (3,000 rpm, 30 min). The serum obtained was tested for alanine transaminase (ALT) and aspartate aminotransferase. (aspartate transaminase (AST)), alkaline phosphatase (ALP), albumin, total bilirubin, blood urea nitrogen (BUN), total cholesterol, creatinine , glucose, total protein, blood fat (triglyceride), calcium (Ca), etc. were measured.

4.4 Statistical processing

The results obtained from the experiment were expressed as mean ± standard deviation (S.D) and statistical analysis was performed using the method below. One-way analysis of variance (ANOVA) was performed to observe significance (significance level: ??0.05), and F-test was performed to test variance. If equal variance (significance level: 0.05) was recognized, A T-test (significance level: 0.05) was performed and P<0.05 was judged to indicate a statistically significant difference.

5. Research results

5.1 Preparation of extract from mealworms

The active ingredients were leached out by reacting the collected mealworm powder with 70% ethyl alcohol in a 3:7 ratio at room temperature for 3 days.

The leached sample was concentrated under reduced pressure at 45-50°C using a rotary vacuum concentrator (EYELA N-1000, EYELA, Tokyo, Japan).

The final material was prepared from the concentrated sample using dimethylsulfoxide (DMSO).

The final material was diluted step by step according to the treatment concentration.

5.2 Result of cell test application method of material

The solvent used was DMSO, which can dissolve the substance.

When mixed with medium, foreign substances were found regardless of sterilization.

No foreign substances were found when mixed with the medium after using the filter.

No foreign substances or contamination were found when applied to cells after using the filter.

5.3 Cell experiments

5.3.1 Positive control (17-βestradiol) measurement

When treated with the positive control (E2), the proliferation rate increased from concentrations above 10 ^-9 M.

The concentration of E2 was selected at 10 ^-8 M, which showed the greatest increase in cell proliferation rate compared to the control group.

5.3.2 Confirmation of proliferation effect of MCF-7 when treated with mealworm powder

E2 was set as a positive control for estrogen-dependent breast cancer cells, and then treated with mealworm extract fed with Styrofoam to evaluate the cell proliferation rate.

The mealworm extract was filtered and applied to cells, and the maximum amount that could be dissolved in DMSO was selected as the high concentration.

When treated with E2, the proliferation rate of cells increased, confirming the effectiveness of the experiment.

When two types of mealworm extracts were treated with or without Styrofoam, no significant increase or decrease was seen in both groups compared to the control group.

No difference in proliferation rate was observed between the two groups with or without Styrofoam.

5.4 Animal testing

5.4.1 Weekly weight measurement

After the start of the experiment, the body weight, food intake, and water volume of all animals were measured twice a week and immediately before necropsy.

When observing clinical symptoms, it was confirmed that no individual showed abnormal signs or died in any group.

A decrease in body weight was observed in the group fed mealworm powder compared to the control group administered sterilized distilled water.

Compared to the regular mealworm group (w/o sty 10%), the mealworm group that ate Styrofoam (w/sty 10%) did not show significant weight loss.

5.4.2 Weekly feed intake measurement

During the experiment, the control group showed higher feed intake compared to the mealworm powder-treated group except for days 4 and 10.

A decrease in feed intake was observed in all groups on the 14th day after administration, but it tended to increase again thereafter.

On the 31st day of administration, a significant decrease in feed intake was observed in the w/sty 10% group compared to the control group, but an increase was observed again thereafter.

There was no significant difference in feed intake between the general mealworm group (w/o sty 10%) and the Styrofoam-fed mealworm group (w/sty 10%).

5.4.3 Measurement of drinking water volume per week

A decrease in drinking water was observed in all groups on the 17th day after administration, but it tended to increase again thereafter.

On days 17, 28, 31, and 35, the w/sty 5% group showed lower drinking water volume compared to other groups.

On the 31st day after administration, the amount of drinking water significantly decreased in the w/sty 5, 15% group compared to the control group, but recovered again later.

There was no significant difference in water volume between the w/o sty 10% group and the w/ sty 10% group.

5.5 Organ weight measurement

After completing the experiment on the 35th day of administration, an autopsy was performed and absolute and relative organ weights were measured.

In the case of organs excluding the heart and thymus, the absolute organ weight was found to be reduced in the mealworm-administered group compared to the control group, but when this was confirmed by relative organ weight, no significant difference was found.

In the case of the heart and thymus, not only the absolute organ weight but also the relative organ weight tended to increase in the mealworm-administered group compared to the control group.

In the case of the thymus, both absolute and relative organ weights tended to increase in the w/sty 5, 10, and 15% groups compared to the w/o sty 10% group.

In the case of lung and spleen, the w/o sty 10% group showed the lowest absolute and relative organ weights.

In the case of testes, absolute organ weight tended to decrease in the group administered mealworm powder compared to the control group, but there was no difference in relative organ weight.

When comparing the W/o sty 10% group and the w/ sty 10% group, no significant differences were observed in both absolute and relative organ weights.

5.6 Histopathological examination

Hematoxylin stains basophilic substances such as ribosomes and cell nuclei in dark blue.

Eosin stains eosinophilic substances such as cytoplasm and cell membranes red.

Changes in cells and tissues of the liver and kidney were confirmed through hematoxylin and eosin (H&E) staining.

Compared to the control group, no abnormalities such as enlargement or reduction of glomerular size or liver hemorrhage were found in the group administered mealworm powder.

When comparing the w/o sty 10% group and the w/ sty 10% group, no significant differences were observed in both liver and kidney.

5.7 Hematological and serum biochemical tests

5.7.1 Hematological tests

The w/ sty 5% group and the w/ sty 15% group showed a tendency for WBC to be higher compared to the control group, but there was no significant increase.

Compared to the w/o sty 10% group, there was no significant difference in all items except neutrophils and lymphocytes in the w/ sty 10% group.

Eosinophils tended to be lowest in the w/sty 5% group.

In the case of basophils, the mealworm-treated group showed a tendency to decrease compared to the control group, but there was no significant difference.

In the case of platelets, the lowest measurements were made in the w/o sty 10% group, and conversely, the highest measurements were made in the w/ sty 15% group.

When comparing the w/o sty 10% group and the w/ sty 10% group, there was no significant hematological difference.

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5.7.2 Serum biochemical tests

In the case of total bilirubin, the control group and w/ sty 5, 10% group showed values below the measurement range.

Aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, which are widely used as indicators of liver levels, all showed a tendency to decrease compared to the control group, but there was a significant decrease. There was no difference.

Compared to the control group, sodium (Na) significantly increased in the w/sty 10% and w/sty 15% groups.

When comparing the w/o sty 10% group and the w/ sty 10% group, there was no significant difference in serum biochemistry.

6. Conclusion and Discussion

The purpose of this experiment is to evaluate the toxicity of estrogen-dependent cells and male rats (SD rats) treated with mealworm powder fed to Styrofoam.

First, the proliferation of MCF-7 cells was confirmed using mealworm powder extract, and the safety was evaluated by orally administering mealworm powder to SD rats for 5 weeks.

For cell experiments, 17-βestradiol (E2), which is mainly used in estrogen induction tests, was selected as a positive control.

The mealworm powder extract was extracted from the group not fed Styrofoam (w/o sty) and the mealworm powder group fed Styrofoam (w/sty), and the concentration of the extract was 250 mg/ml, which is the maximum capacity that can be dissolved in the solvent DMSO. After selection, cell experiments were conducted.

As a result, compared to the control group, the cell proliferation rate tended to increase in the positive control group treated with E2, but there was no significant difference in the treatment group.

Additionally, no difference in proliferation rate was observed between w/o sty and w/ sty. These results suggest that the mealworms fed Styrofoam did not show any toxicity to the cells, and furthermore, did not show an estrogen-inducing response.

The animal test consisted of a total of 5 experimental groups: a control group (sterilized saline solution), a group administered regular mealworm powder (w/o sty 10%), a low concentration group administered mealworm powder fed Styrofoam (w/ sty 5%), and a medium concentration group (w/ sty 10). %), was set at a high concentration (w/sty 15%), and oral administration was conducted daily for 5 weeks. The concentration of mealworm powder was selected at 5%, 10%, and 15% of the recently measured feed intake.

After administration, general symptoms were observed every day, and body weight, food intake, and drinking amount were measured for all rats twice a week. At the 5th week, the rat's organ weight was measured through an autopsy, and hematoxylin and eosin were administered. Histological analysis was performed through (and Eosin (H&E)) staining, and hematological and serum biochemical tests were performed using blood collected from the abdominal aorta during the autopsy.

No animals died during the test period and no animals showed unusual clinical symptoms.

In case of weight change, no difference was observed between the w/o sty 10% group and the w/ sty 10% group. However, compared to the control group, there was a tendency to decrease in all groups administered mealworm powder, but there was no significant difference.

These results suggest that the difference in body weight occurred because it took a long time to calibrate the rats during the process of administering mealworm powder, whereas the control group fed sterilized saline solution experienced low stress due to the short calibration time.

In the case of feed intake, the average consumption was found to be lower in the mealworm powder administration group than the control group during the experimental period. It tended to decrease in all experimental groups on the 14th day, but then tended to gradually increase. Since feed intake did not show a consistent trend before 14 days, this is thought to be due to the period of adaptation to administration.

In case of drinking water volume, no significant difference was observed between the w/o 10% group and the w/ 10% group. When observing histopathological changes, no liver or kidney abnormalities were found in the group administered mealworm powder compared to the control group.

Moreover, when comparing the w/o sty 10% group and the w/ sty 10% group, no significant differences were observed in both liver and kidney.

After an autopsy at the 5th week of mealworm administration, the liver, kidneys, adrenal glands, heart, thymus, testes, lungs, and spleen were removed. Organ weight (g) and relative organ weight (%) were measured.

When comparing the mealworm-administered group with the control group, the absolute organ weights of organs except the heart and thymus tended to decrease, but when viewed in terms of relative organ mass, there was no significant difference.

These results are believed to be dependent on the rat's body weight. When comparing the organs of the w/o sty 10% group and the w/ sty 10% group, there was no significant difference in organs except the thymus.

In the case of the thymus, both organ weights tended to increase in the w/sty 10% group, but there was no significant difference.

In addition, in the case of testes, absolute organ weight tended to decrease in the group administered mealworm powder compared to the control group, but no significant difference was observed when measured by relative organ weight.

Considering these results, it is judged that mealworms fed with Styrofoam did not cause toxicity in living organisms.

Blood collected from the abdominal aorta during autopsy was tested for blood components through hematological and serum biochemical tests.

In the case of neutrophils, there was a tendency to increase when comparing the w/o sty 10% group with the w/ sty 10% group, but this was within the normal range (6.2-26.7%) and is thought to be due to individual differences rather than disease. do.

Similarly, in the case of lymphocytes, a tendency to decrease was seen when comparing the w/o sty 10% group with the w/ sty 10% group, but since this was within the normal range (66.6-90.3%), the difference due to the presence or absence of Styrofoam was It is judged not to be so.

Basophils tended to decrease in the mealworm-treated group compared to the control group, but there was no significant difference.

Additionally, biochemical tests were performed using serum collected from blood. Serum was tested using biochemical tests.

As a result, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, which are widely used as indicators of liver levels, tended to decrease compared to the control group.

In the case of total bilirubin, the control group and the w/sty 5% and 15% groups showed values below the measurement range. As a result, no significant difference was observed between the w/o sty 10% group and the w/ sty 10% group in the serum test.

Overall, when this experimental material (Styrofoam-fed mealworm powder) was applied to estrogen-dependent cells, it did not increase the proliferation rate of the cells, suggesting that it does not show an estrogen-inducing response.

In addition, as a result of animal testing, there was a decrease in body weight in both groups administered mealworms compared to the control group, but no significant difference was found when comparing the w/o sty 10% group and the w/ sty 10% group.

These results are believed to be due to stress caused by an increase in correction time due to the viscosity of meal worm powder rather than the effect of feeding Styrofoam.

Additionally, the w/ sty 10% group compared to the w/o sty 10% group. The absolute organ weights of organs except the lungs and spleen were found to be reduced, but when this was calculated as relative organ weights, no differences were observed between the two groups.

Additionally, when comparing the liver and kidneys through H&E staining, no difference was found between the w/ sty 10% group and the w/o sty 10% group. In hematological and serum biochemical tests, differences were observed between the w/o sty 10% group and the w/ sty 10% group, but the values were within the normal range.

Therefore, as a result of confirming the safety of mealworm powder fed with Styrofoam through cell and animal experiments, it is believed that there is no effect of estrogen on cells and it is judged not to be toxic to male rats. do.

As described above, it was confirmed through experiments that mealworms raised by the method of recycling Styrofoam (EPS) waste of the present invention are non-toxic and can be used as food.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims are also possible. It falls within the scope of rights.

100: Mealworm breeding device 10: Breeding module
11: Spawning module 12-1~12-4: Growth module
20-1~20-10: Feeding module 30: Stabilization module

Claims (2)

A spawning step (S100) in which adult mealworms are fed for one week to lay eggs in the spawning module 11 of the breeding module 10;
The eggs spawned in the spawning step (S100) are collected together with the excrement of adult mealworms and grown in the first growth module (12-1) of the growth modules (12-1, 12-2, 12-3, 12-4) composed of 4 zones. ) and mixing with wheat bran (S200);
After hatching and growing for one week in the mixing step (S200), they are transferred (shifted) to the second growth module (12-2), and grown for one week in the second growth module (12-2) and then transferred to the third growth module. A growth step (S300) of transferring to (12-3), growing for one week in the third growth module (12-3), and sequentially transferring to the fourth growth module (12-4);
The larvae that grew for 4 weeks in the growth stage (S300) are sequentially transferred to the feeding modules (20-1 to 20-10) and grow into larvae of the desired size while eating the Styrofoam cake (110) for 10 weeks. Feeding step (S400);
In the feeding step (S400), the larvae that grew in the feeding module for 10 weeks are transferred to the stabilization module (30) and fed only with wheat bran for 5 days to completely eliminate Styrofoam in the body, and are fasted for 2 days to empty the intestines of the wheat source. Stabilization step (S500); and
In order to check whether there is any Styrofoam residue in the body of the mealworm that has gone through the above stabilization step (S500), a sample or all samples are examined using ultrasound, A method of raising mealworms using Styrofoam waste, comprising an inspection step (S600) of completely removing residues by fasting. delete