KR101911398B1 - Mass isolation method of hemolymph from cricket - Google Patents

Abstract

The present invention relates to a method for massively separating cricket hemolymph, and it is necessary to conduct research for diagnosis of cricket disease, development of new drugs and development of new food materials by separating blood lymphedema in a large amount while preventing oxidation during hemolymph separation process. It is expected that blood lymph is secured.

Description

[0002] Mass isolation method of hemolymph from cricket [

The present invention relates to a method for massive separation of cricket blood lymph.

Modern food problems are caused by the imbalance in grain supply due to climate change and the increase in grain production, distribution and consumption, as well as rising animal protein prices and imbalance in demand. As an alternative, there is growing interest in insects that emit less greenhouse gases and ammonia than livestock and do not require extensive breeding grounds (Huis A.V., et al., 2013; Kim M.A., et al., 2014).

Insects are a group of organisms that account for 70% of the existing animal kingdom, with more than 800,000 known species. Insects grow rapidly and form large populations, making them an attractive research material not only in agriculture but also in various fields such as medicine, environment, chemistry, biotechnology, and biotechnology (biomimetics) (Choi, Young Chul, 2011 ). Especially, the habit of eating insect as a food, which is called food insecticide, has come to present as an important resource for solving human food problems and hunger problems. Materials and researches on raw materials and pharmaceutical products have been conducted.

With the development of the insect industry, mass rearing of insects is required, and large-scale damage caused by diseases caused by mass rearing of insects is occurring chronically. However, accurate diagnosis and treatment methods for insect diseases have yet to be developed.

The circulatory system of insects is an open blood vessel system, and there are no blood vessels. In mammals, blood and lymph are present. Insects have a combined form of hemolymph. Insect hemolymph has antimicrobial proteins whose synthesis is induced or quantitatively increased due to the invasion of pathogens. The hemolymph also has nutritional components necessary for the growth of animal cells including insect cells.

Through various molecules present in the hemolymph, it is possible to diagnose insect diseases that can occur in the mass rearing of insects, thereby preventing the death of insects, and also to develop new drugs using antimicrobial proteins and various active molecules in hemolymph And research on hemolymph is necessary to develop new food materials through nutrition.

However, insect hemolymphs are low in quantity, high in viscosity, and are likely to be denatured in hemolymph due to rapid oxidation due to contact with air. Therefore, it is necessary to develop a method for stably separating a large amount of hemolymph.

Cricket is a generic term for Orthoptera and Gryllidae insects (Ahn MY, et al., 2000). About 800 species are known globally and about 40 species are known in Korea. Crickets are known as emotional insects, but they are used as feed for ornamental fish and pet animals worldwide (Fast PG, 1967). In addition, in Korea, a pair of crickets ( Gryllus bimaculatus ) has been certified as an edible insect in Korea through research for the edible use of insects.

Gryllus bimaculatus contains unsaturated fatty acids and essential fatty acids, and its high protein content is recognized as a food value (Kim EM, et al., 2015). It has also been used as a stone therapy and diuretic. In Japan, it has been used to treat diarrhea, dysentery and typhoid fever (Park KT, 2001) and has also been studied as an effective source of increased immunity (Seo DH, et al., 2004).

Thus, the present inventors have succeeded in accomplishing the present invention by preventing the oxidation of blood lymphedema and separating a large amount of hemolymph in the process of studying cricket blood limb.

On the other hand, US Pat. No. 5,866,317 discloses a method for separating insect bloom, but the insect is a lepidopteran insect, and a method such as centrifugation and dialysis is not described, which is different from the construction of the present invention. Korean Patent No. 0999858 and Korean Patent No. 0530506 disclose collecting body fluids of larvae or silkworms, but the separation process such as the antioxidant treatment, centrifugation and dialysis of the present invention is not described.

U.S. Patent No. 5,866,317, Method for collecting hemolymph of insects, 1999. 02. 02. Disclosure. Korean Patent No. 0999858, a method for separating peptidoglycan-recognizing proteins from body fluids of brown goat larvae, Registered on Mar. 13, 2010. Korean Patent No. 0530506, fractions of silkworm body fluid inhibiting apatosis, and a method for separation and purification thereof, November 16, 2005, registration.

Choi, Young Chul et al., New value of insects, RDA Interrobang, 4, 2011. Ahn M. Y., et al., Effect of Cricket Supplements on Chicken Meats and Their Eggs, Korean J. Poult. Sci., 27 (3), 197-202, 2000. Fast P. G., An analysis of the lipids of Gryllus bimaculatus (DeGeer) (Insecta. Orthoptera), Can. J. Biochem., 45, 503-505, 1967. Huis A. V., et al., Edible insects: future prospects for food and feed security, FAO Forestry paper, 171, 1-185, 2013. Kim E. E., et al., Physicochemical properties of edible cricket (Gryllus bimaculatus) in different districts, Korea J. Food Preserv., 22, 831-837, 2015. Kim M. A., et al., Ediable & medicinal insects, RDA Interrobang, 119, 5-6, 2014. Park KT, Insect resources, World science publishing company, Seoul, Korea, 202, 2001. Seo D.H., et al., Immune-enhancing activity screening on extracts from two crickets, Gryllus bimaculatus and Teleogryllus emma, Entomol. Res., 34, 207-211, 2004).

It is an object of the present invention to provide a method for massively separating cricket blood lymph.

The present invention provides a method for purifying crickets comprising: a first step of anesthetizing and then washing crickets; A second step of removing the washed cricket neck and placing the trunk in a 15 ml tube having a hole at the bottom thereof; A third step of adding an antioxidant to a 50 ml tube; A fourth step of placing the 15 ml tube of the second step into a 50 ml tube of the third step and centrifuging to obtain a hemolymph mixture containing hemolymph and antioxidant; A fifth step of treating the hemolymph mixture solution of the fourth step at 56 DEG C for 25 minutes to 35 minutes to inactivate the antioxidant and then centrifuging to obtain hemolymph supernatant; And a sixth step of dialyzing the hemolymph supernatant of step 5 with a phosphate buffer solution and then filtering to obtain hemolymph; To a method for mass separation of cricket blood lymph.

The cricket may be an adult.

The crickets Cricket applicable ssangbyeol cricket (Gryllus bimaculatus), wanggwitturami (Teleogryllus emma), alrak cricket (Loxoblemmus arietulus), fed crickets (Nigrogryllus sibiricus), fat cricket (Duolandrevus ivani), drops worm (Homoeogryllus japonicus, Meloimorpha japonica), ink alrak drop worm (Dianemobius nigrofasciatus), pool Skylark (Paratrigonidium bifasciatum), can be selected from the group consisting of Skylark (Trigonidium japonicum), brush cricket (Xenogryllus marmoratus), Chongsol cricket (Truljalia hibinonis) and slender cricket (Euscyrtus japonicus).

The anesthesia may be an anesthetic using carbon dioxide gas.

The diameter of the bore in the 15 ml tube with the hole at the bottom may be between 1 mm and 1.5 mm.

The antioxidant may contain 8% to 15% of the total hemolymph volume based on obtaining hemolymph of 30 내지 to 40 당 per cricket.

The antioxidant may be selected from the group consisting of erythorbic acid sodium salt, butyl hydroxyanisole (BHA), dibutyl-hydroxytoluene (BHT), alpha-tocopherol, N N-phenylthiourea, and N-propylthiouracil may be added. Preferably N-propylthiouracil.

The filtration in the third step may use a filter having a pore diameter of 0.2 탆 to 0.5 탆.

Hereinafter, the present invention will be described in detail.

The hemolymph is an insect body fluid circulating in the open vasculature of insects. It is a combination of blood and lymph in mammals. It is an antimicrobial agent that is synthesized or increased quantitatively by pathogen invasion. Protein and nutrients necessary for the growth of animal cells. Therefore, hemolymph research is a necessary process for the diagnosis of diseases of insects, and it is a necessary process for developing new drugs and food materials in the future because it contains various efficacies required for the development of new drugs and necessary nutrients for food development.

Since blood lumps of crickets are highly viscous and easily oxidized and hardened when they come into contact with air, blood lym- phs are sequentially collected one by one. Generally, however, by using the centrifugal separation method of the present invention, Blood lymphs can be isolated from dozens of crickets.

The crickets may be all the insects contained in the cricket and are preferably selected from the group consisting of Gryllus bimaculatus , Teleogryllus emma , Loxoblemmus arietulus , Nigrogryllus sibiricus , Duolandrevus ivani , drop worm (Homoeogryllus japonicus, Meloimorpha japonica), alrak drop worm (Dianemobius nigrofasciatus), pool Skylark (Paratrigonidium bifasciatum), ink Skylark (Trigonidium japonicum), brush cricket (Xenogryllus marmoratus), Chongsol cricket (Truljalia hibinonis) and slender cricket (Euscyrtus japonicus ) is an adult.

The anesthesia can utilize carbon dioxide gas, and when the carbon dioxide gas is inhaled into the cricket, the anesthesia can be instantaneously performed, and a large amount of crickets can be anesthetized without damaging the cricket. In addition, if the crickets are placed on ice after carbon dioxide gas anesthesia, the anesthesia is maintained without releasing. At this time, it is undesirable that anesthetizing by putting on ice from the beginning without carbon dioxide gas anesthesia can kill crickets.

The 15 ml tube of the second step is prepared by drilling a hole in the bottom of the tube so that the hemolymph from the cricket can be collected in a 50 ml tube during centrifugation. Put the 15 ml tube into a 50 ml tube, You can cut the top of the 15-ml tube to close the lid when it closes.

The hole of the 15 ml tube may have a diameter of 1 mm to 1.5 mm. If the diameter of the hole exceeds 1.5 mm, other components coming from the cricket may be mixed in the blood lymph, making it difficult to obtain a pure blood lymph.

The crickets of the second stage can be put into 10 to 20 grains in a 15 ml tube. If more than 20 animals are placed in a 15-ml tube, it is not desirable because adults may be infested and micro-organisms present in the intestine may contaminate the blood lymph.

In the third step, the antioxidant is used to prevent the oxidation of the hemolymph of the cricket, which is easily oxidized when it comes into contact with air. The antioxidant is selected from the group consisting of erythorbic acid sodium salt, butyl hydroxyanisole (BHA) One selected from the group consisting of dibutyl-hydroxytoluene (BHT), alpha-tocopherol, N-phenylthiourea and N-propylthiouracil Or more, and preferably N-propylthiouracil can be added.

The hemolymph mixture in the fourth step may be a mixture of hemolymphs that have been removed from crickets in a 15 ml tube through centrifugation with an antioxidant contained in a 50 ml tube.

The antioxidant may contain 8% to 15% of the total blood lymph volume, preferably 10%, based on obtaining hemolymph of 30 ㎕ to 40 당 per cricket.

The centrifugation in the fourth step can collect hemolymph from a large number of crickets at a time.

The treatment of the hemolymph mixture solution of the fifth step at 56 ° C for 25 minutes to 35 minutes is for deactivating the antioxidant.

The dialysis of the sixth step may be performed by placing the hemolymph supernatant in a phosphate buffer solution at 4 ° C for 10 hours to 15 hours.

The dialysis removes low molecular weight metal ions, minerals and the like contained in the hemolymph and has the same osmotic pressure as that of the phosphate buffer solution, so that the dialysis can be directly used for in vivo experiments.

The filtration in the sixth step may use a filter having a pore diameter of 0.2 탆 to 0.5 탆.

In the process of separating the cricket's hemolymph, a hemolymph of about 10 mu l per cricket can be obtained when the centrifugation method is not used, whereas when using the mass separation method of the present invention, 30 mu l per cricket To about 40 [micro] l of hemolymph can be obtained.

The present invention relates to a method of massively separating cricket blood lymph, comprising: a first step of anesthetizing and then washing crickets; A second step of removing the washed cricket neck and placing the trunk in a 15 ml tube having a hole at the bottom thereof; A third step of adding an antioxidant to a 50 ml tube; A fourth step of placing the 15 ml tube of the second step into a 50 ml tube of the third step and centrifuging to obtain a hemolymph mixture mixed with hemolymph and an antioxidant; A fifth step of treating the hemolymph mixture solution of the fourth step at 56 DEG C for 25 minutes to 35 minutes to inactivate the antioxidant and then centrifuging to obtain hemolymph supernatant; And a sixth step of dialyzing the hemolymph supernatant of step 5 with a phosphate buffer solution and then filtering to obtain hemolymph; .

By separating blood lymphedema in large quantities while avoiding oxidation during the hemolymph separation process by using such a separation method, it is expected to be able to secure hemolymph necessary for diagnosis of disease of cricket, development of new drug and development of new food material do.

1 shows materials and devices (1A, 1B, 1C) used in cricket hemolymph separation of the present invention and cricket blood lymph collection results (1D) using the same.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1: Bulk separation of cricket blood lymph <

The equipment of Figure 1 was used to massively isolate the cricket's hemolymph.

The adult crickets were anesthetized with carbon dioxide gas and then cleaned 2 ~ 3 times with cold distilled water. After washing, twenty birds were placed in a 15-ml tube (Fig. 1A) in which about 10 orifices were drilled at the bottom of the trunk with the neck of the cricket removed. A 15 ml tube containing crickets was placed in a 50 ml tube (Fig. 1B), the lid was closed, and centrifugation was carried out at 4 ° C for 3 minutes at 1,000 rpm for collection of hemolymphs (Fig. 1C, 1D). If the lid of the 50 ml tube is not closed at this time, cut the top of the 15 ml tube. In addition, N-propylthiouracil of about 10% (v / v) of Cricket Blood Lymph (40 μl of prothrombin count per adult) was placed in a 50 ml tube before centrifugation. The collected blood was treated for 30 minutes at 56 ° C, centrifuged at 20,000g for 30 minutes at 4 ° C, and dialyzed against phosphate buffered saline (PBS) for 12 hours at 4 ° C. ). After hemodialysis, the hemolymph was sequentially filtered with a filter having pore diameters of 0.45 mu m and 0.22 mu m, and stored at -70 DEG C.

As a result of checking the volume of hemolymph obtained by centrifugation and without centrifugation, 10 쨉 l hemolymph was obtained per cricket adult in case of not centrifuging, whereas in case of centrifugation, per 1 cricket adult 40 쨉 l of hemolymph was obtained.

Thus, it was found that a large amount of hemolymph can be separated by using the hemolymph separation method of the present invention.

Claims (9)

A first step of anesthetizing crickets and then washing;
A second step of removing the washed cricket neck and placing the trunk in a 15 ml tube having a hole at the bottom thereof;
A third step of adding 8% to 15% of antioxidant to the total blood lymph volume on the basis of obtaining a hemolymph of 30 占 퐇 to 40 占 퐇 per insect on a 50 ml tube;
A fourth step of placing the 15 ml tube of the second step into a 50 ml tube of the third step and centrifuging to obtain a hemolymph mixture mixed with hemolymph and an antioxidant;
A fifth step of treating the hemolymph mixture solution of the fourth step at 56 DEG C for 25 minutes to 35 minutes to inactivate the antioxidant and then centrifuging to obtain hemolymph supernatant; And
A sixth step of dialyzing the hemolymph supernatant of the fifth step with a phosphate buffer solution and then filtering to obtain hemolymph;
Wherein the cricket blood lymph is selected from the group consisting of: The method according to claim 1,
Wherein the cricket is an adult. 3. The method according to claim 1 or 2,
The crickets can be classified into two groups according to the number of crickets: Gryllus bimaculatus , Teleogryllus emma , Loxoblemmus arietulus , Nigrogryllus sibiricus , Duolandrevus ivani , Homoeogryllus japonicus, Meloimorpha japonica , (Dianemobius nigrofasciatus), pool Skylark (Paratrigonidium bifasciatum), ink Skylark (Trigonidium japonicum), brush cricket (Xenogryllus marmoratus), Chongsol cricket (Truljalia hibinonis) and slender cricket blood, characterized in that selected from the group consisting of (Euscyrtus japonicus) A method for massive isolation of lymph. The method according to claim 1,
Wherein said anesthesia is carbon dioxide gas anesthesia. The method according to claim 1,
Wherein a 15-ml tube having a hole at the bottom has a diameter of 1 mm to 1.5 mm. delete The method according to claim 1,
The antioxidant may be selected from the group consisting of erythorbic acid sodium salt, butyl hydroxyanisole (BHA), dibutyl-hydroxytoluene (BHT), alpha-tocopherol, N Wherein at least one selected from the group consisting of N-phenylthiourea and N-propylthiouracil is added. 8. The method of claim 7,
Wherein the antioxidant is N-propylthiouracil. 2. The method of claim 1, wherein the antioxidant is N-propylthiouracil. The method according to claim 1,
Wherein the filtration in the third step uses a filter having a pore diameter of 0.2 탆 to 0.5 탆.

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