KR20190005594A - Rapid separation and purification of Trichuris suis eggs from feces using artificial gastric juice - Google Patents

Abstract

The present invention relates to a method to separate Trichuris suis eggs from pig feces and a method to mature Trichuris suis separated from the pig feces. According to the methods of the present invention, Trichuris suis eggs in an immature state can be rapidly separated in bulk without a loss of eggs by treating feces containing Trichuris suis eggs and making the same react with ethyl acetate and modified simulated gastric fluid (SGF) and separating the eggs to remove bacteria and residue in the feces. Also, infectious eggs can be rapidly manufactured and produced for treating inflammatory bowel disease such as crohn′s disease by maturing immature eggs using an acid stimulus of ethyl acetate which is not harmful to human body.

Description

Technical Field [0001] The present invention relates to a method for rapidly separating and purifying pork knuckle eggs from feces using artificial gastric juice,

The present invention relates to a method for separating porcine zygomycetes from pig feces and a method for maturing porcine zygomycetes isolated from pig feces.

Inflammatory Bowel Disease (IBD), a type of autoimmune disease, has not been developed yet despite its many studies. In order to obtain a large amount of pig egg-laying eggs used as such a therapeutic agent, it is necessary to establish a method for quickly and massively separating the egg-laying eggs of the adult pigs.

This is because it is difficult to isolate the female adult in the pigs infected with the swine, and it is difficult to obtain sufficient amount of the ovary even when the ovaries existing in the uterus are separated, and the ovaries are directly extracted, . The method of separating the egg from the already established feces requires a small amount of feces and is mainly used for autopsy, so a large amount of time, effort, and separation reagents are needed to treat large amounts of feces. In addition, there is a method of using stainless steel meshes that is used to secure intestinal parasite eggs. However, when there is a large amount of debris in the feces because the capacity for treating the feces of the mesh is limited, Is often clogged. So, the distilled water is poured on the debris to extract the eggs that are present in the clogged mesh, and the time and effort to enter the distilled water is considerable. Recently, a method of securing parasitic eggs in the soil has been reported, but it is actually difficult to treat a large amount of feces.

Therefore, there is a strong need for a method for rapidly securing a large amount of piglets that are used as therapeutic agents.

Therefore, the inventors of the present invention have been studying a method for quickly and massively separating swine egg whites from pigs by separating ethyl acetate, modified simulated gastric fluid (SGF), and PluriStrainer set The present inventors confirmed that the larval egg can be separated quickly and in a large amount, and completed the present invention.

Accordingly, it is an object of the present invention to provide a method for rapidly separating porcine zygomatic follicles in feces of a pig, and a method for maturation of porcine zygomycetes isolated from pig feces.

In order to accomplish the above object, the present invention provides a method for purifying a feces, comprising the steps of: (a) (b) adding ethyl acetate to the filtrate and mixing and centrifuging; (c) reacting the precipitated pig knifalogia with a modified simulated gastric fluid (SGF); And (d) filtering the reactants in step (c) using a filter to obtain pig knock-in eggs.

(A) filtering a dilution of the feces of the pig; (b) adding ethyl acetate to the filtrate and mixing and centrifuging to react the precipitated porcine excising egg with a modified simulated gastric fluid (SGF); And (c) filtering the swine flotation floats of step (b) using a filter to obtain swine flounder. The method of maturation of swine flounder isolated from swine feces is provided.

According to the method of the present invention, the feces containing swine egg whites are treated with ethyl acetate and a simulated gastric fluid (SGF) to remove the egg whites, Immature pig swarms can be rapidly and rapidly isolated without loss of eggs. In addition, it is possible to mature immature embryos by acid stimulation using ethyl acetate which is not harmful to the human body, and thus it is possible to rapidly produce and produce infectious embryos for the treatment of inflammatory bowel disease such as Crohn's disease.

FIG. 1 is a graph showing the results obtained by treating feces containing porcine larvae with ethyl acetate and SGF and then examining them.
Fig. 2 is a graph showing the result of examining pig stomatode larvae in feces by classical method (Comparative Example 1).
FIG. 3 is a graph showing the result of examining the oviposition (Comparative Example 2) by separating the oviposition with the Ritchie concentration method and treating it with pH 2.
4 is a schematic diagram showing the difference between Comparative Example 1 and the separation method of the present invention in a simplified manner.
FIG. 5 is a view showing a result of inspection (FIG. 5A) of a suspended oyster separated according to the separation method of the present invention (FIG. 5A), and a shape of an egg obtained by filtering a suspension (FIG.
FIG. 6 is a diagram showing an egg incubated for 60 days at 25 ° C.
FIG. 7 is a chart comparing the degree of maturity of the larvae isolated by Comparative Example 1, Comparative Example 2, and the method of the present invention.

(A) filtering a dilution of the feces of the pig; (b) adding ethyl acetate to the filtrate and mixing and centrifuging; (c) reacting the precipitated pig knifalogia with a modified simulated gastric fluid (SGF); And (d) filtering the reactants in step (c) using a filter to obtain pig knock-in eggs.

Hereinafter, the present invention will be described in more detail.

The swine larvae ( Trichuris suis ) is genetically similar to the human parasite T. trichiura, and can be used without restriction as long as it can be used in the treatment of inflammatory bowel disease using swine parasites . Characteristics of porcine parasites that can be used in this way are defined to be porcine parasites which are confined to the intestines, do not multiply in the host, can not spread by direct contact, and can be obtained from pigs that have grown in an environment free of special pathogens.

Refers to an egg state in the life history of a swine parasite. The egg white of the present invention can be obtained from various samples of host having feces such as feces, sputum and urine, or commercially available ones can be purchased and used But can be obtained in the feces of pigs infected with swine parasites as a preferred example.

The present invention can provide a method of concentrating pig oviposition using weakly acidic ethyl acetate, unlike the conventional Ritchie concentration method, which uses formaldehyde and ethers known to be toxic.

The ethyl acetate in the step (b) can be 100 (v / v)% of ethyl acetate with a pH of 4 to 6, but preferably a weakly acidic ethyl acetate having a pH of 5 to 5.9 can be used. Ethyl acetate (MW 88.11, 31055-0380, Junsei) may be used, but is not limited thereto.

The modified simulated gastric fluid (SGF) of step (c) comprises 1% Pepsin (P7000-100G, Sigma) and 1% hydrochloric acid (HCl, 35.0-37.0% , H0519, SAMCHUN) in an appropriate ratio (v / v). The modified artificial gastric juice may be a mixture of pepsin and hydrochloric acid at a ratio of 1: 5 to 1:10, preferably 1: 7 to 1: 9.5, more preferably 1 : 9 (pellet 5 ml: 1% Pepsin + 1% HCl 45 ml).

In step (c), the transformed artificial gastric juice and the deposited pig zygote can be reacted at 37 ° C for a time sufficient to mature the egg to infectious egg, preferably for 5 minutes to 1 hour , More preferably from 7 minutes to 30 minutes, and still more preferably from 10 minutes to 20 minutes. By treating the above solution, proteins and wastes in the feces other than egg can be dissolved and the particle size can be reduced. In addition, bacteria present in the feces can be removed, and the antibacterial effect can be exhibited.

The filtration material of step (d) may be used without limitations as long as it has a particle size smaller than the longitudinal size of the egg, and may be made of a mesh made of weaves. Preferably, the filtration material is a stainless steel mesh or a PluriStrainer set Can be used. The PluriStrainer set can be used as a mesh alone, and can be used to quickly dispense residual floats present on the mesh by making a set including a 50 ml syringe. With the PluriStrainer set, particles smaller than the egg size can be filtered out during the purification process, which separates the egg from the feces.

The PluriStrainer mesh can also be a woven mesh material, different from the stainless steel mesh. A soft mesh can be used to safely isolate the weakened egg wall after treatment with the SGF solution, which is composed of components similar to gastric juice.

The reaction product is filtered using a woven mesh having a size of 50 to 56 μm when the reaction product is filtered, followed by filtration of the reaction product using a woven mesh having a size of 22 to 28 μm and 17 to 23 μm in order to obtain pig knitting.

The method for separating porcine ovine porcine feces in the swine feces of the present invention may further include (e) mixing the porcine ovine porcine porcine with sucrose and centrifuging. When the sucrose solution treatment is carried out, the lumps having a larger specific gravity than that of the eggs are submerged down, and only the ovules remain in the middle layer of the sucrose solution and the egg shell suspension Only the eggs collected in the middle layer can be collected with a pipette to obtain purified eggplants.

Purified egg and distilled water collected by pipette can be mixed in a ratio of 1: 8 to 1:15 (v / v), preferably in a ratio of 1:10 to 1: 14.5, Can be mixed in a ratio of 1:14 (3 ml of a mixture of sucrose sucrose: 42 ml of DD.W). The mixed solution may be centrifuged at 3220 g at 25 ° C for 1 to 10 minutes, preferably 3 to 7 minutes.

With this method, it is possible to quickly isolate pig egg whites and obtain mature eggs.

(A) filtering a dilution of the feces of the pig; (b) adding ethyl acetate to the filtrate and mixing and centrifuging to react the precipitated porcine excising egg with a modified simulated gastric fluid (SGF); And (c) filtering the swine flotation floats of step (b) using a filter to obtain swine flounder. The method of maturation of swine flounder isolated from swine feces is provided.

That is, the present invention can induce the immature state of the ovules to mature into the infectious type of ova by applying pH stimulation of ethyl acetate and modified artificial gastric juice to mature the porcine oviposition eggs into infectious ovules.

Hereinafter, the present invention will be described in detail with reference to examples. These embodiments are for illustrative purposes only and do not limit the scope of the present invention.

Example  1. Ethyl acetate and modified Artificial gastric juice (SGF)  And the pig swine Trichuris suis ) Pussy  Separation of eggs and foreign matter

Infection was induced by artificially ingesting pig swine (TSO2500, OVAMED GmbH) to pigs (approximately 23 kg, 12 weeks old, Yorkshire) in order to isolate porcine larvae. The feces from the infected pigs were examined and the presence of porcine oocytes was confirmed under an optical microscope (Zeiss Axioskop2). Egg per gram (EPG) was used to calculate the number of egg whites when the egg whites were observed in the feces. The feces were collected and used daily until the presence of the egg was not observed in the feces. More specifically, the following process was performed. 82.5g (x2 = 165g) of water or solid waste from the pigs was recovered and diluted 3.3 times by adding 275ml of the third distilled water. The diluted mixture was filtered using a double layer of gauze with water and collected in a centrifuge (3141-0500, 500 ml, Nalgene (TM) PPCO Centrifuge Bottles with Sealing Closure) tube.

100% (v / v) ethyl acetate (MW 88.11, 31055-0380, Junsei) at pH 5.8 was added to 1 / 3.7 volume of the filtrate and shaken until all the sediment was floated. After centrifugation at 3,220 g at room temperature of 20 to 25 ° C for 5 minutes, the layer formed between the third distilled water and ethyl acetate was separated and the supernatant was discarded. 25-40 ml of third distilled water was added to the precipitated egg white gave. Transferred into a 50 ml conical tube (SPL, # 50050), and centrifuged at 3,220 g at room temperature (25 캜) for 5 minutes. Thereafter, the supernatant was discarded, 30 to 45 ml of distilled water was added thereto, and the mixture was thoroughly mixed. The mixture was centrifuged at 3,220 g for 5 minutes at room temperature (25 DEG C). Subsequently, the supernatant was discarded and 1% pepsin (P7000-100G, Sigma) and 1% hydrochloric acid (HCl, 35.0-37.0% (T) 1 kg, H0519, SAMCHUN) isolated from the pre- : 9 (pellet 5 ml: 1% Pepsin + 1% HCl 45 ml) at 37 ° C for 15 minutes. The microscopic particles of the separated egg between ethyl acetate and SGF method are shown in Fig.

As can be seen in FIG. 1, separation of the egg from the ethyl acetate and the SGF can be separated into a high degree of purification without a large residue, efficiently dissolving residual particles in the feces, and in fact, It was confirmed that pollen was dissolved. Using ethyl acetate and SGF did not only extinguish the foreign protein in the feces but also removed the debris in the feces to enhance the purity of the egg. And to be able to recover it. In addition, when SGF was used, it could be proved that the acid stimulates the immature embryo to induce the mature embryo, and shows the antibacterial effect of eliminating the bacteria.

Comparative Example  : Depending on the separation method Pork chopper  Comparison of separation effect

The method of the present invention and the classical method of separating eggs from a sieve without treating the prior art chemicals (Comparative Example 1) and the acid solution disclosed in the previously disclosed patent (Publication No. 10-2016-0104182 A) (Comparative Example 2), respectively. The method used in the previously disclosed patents used acidic solutions of pH 2 in combination with citric acid and ethyl acetate. 2 and 3 show the results of microscopic observation of the larvae isolated by the method according to Comparative Examples 1 and 2.

As shown in Figs. 2 and 3, the particles of the residual suspension containing the oyster (Fig. 2) isolated in Comparative Example 1 had a particle size smaller than that of the residual oyster particles (Fig. 1) separated by ethyl acetate and the SGF method The big one was confirmed. As shown in FIG. 3, when the method of treating only the acidic solution according to Comparative Example 2 is used, it can not be confirmed that the coarse particles such as floating matters become small. Therefore, it was confirmed that by using the separation method using ethyl acetate and SGF according to the present invention, it is possible to remove the debris in the feces, thereby increasing the recovery rate of the egg in the subsequent separation process.

Example  2. Stainless-steel mesh, PluriStrainer  Using set and sucrose Adulterous  Pure separation

As a control group, as a control group, a method of separating eggs separated from an experimental group not treated with a chemical agent and a separation method in which only ethyl acetate was treated was used as a control. After the procedure of Example 1, 53 탆 and 25 탆 (Ø 200 mm × 45 mm, CHUNG GYE SANG GONG SA, KOREA) was poured into the stainless-steel meshes poured. Stainless steel meshes were washed with 1 L of tertiary distilled water. The feces suspended in large particles were separated by using 53 ㎛ and 25 ㎛ stainless steel meshes. After separating the upper 53 ㎛ stainless steel mesh, 25 ㎛ (stainless steel mesh) After washing with 50 ml to 500 ml of distilled water, the washed 25 μm stainless steel mesh was turned upside down at an angle of 60 ° and placed on a 50 ml conical tube (SPL, # 50050). It was sprayed with distilled water from the opposite side of the washed surface to collect the oysters below the stainless steel mesh. The oyster was less soluble and the remaining particles larger than the larvae were filtered out. In the 53 μm stainless-steel meshes, The particles were filtered and the eggs were allowed to escape and settled on the 25 μm stainless-steel meshes below.

That contains moving the egg - gathered in Stainless-steel mesh bottom to 50ml conical tubes, and then, the set PluriStrainer Connector Ring (41-50000-03), 20μm Woven mesh (PluriStrainer ®, 43-50020-03, green), Funnel (42 -50000-03, 24 ml) was combined with a new 50 ml conical tube, and the funnel was poured in a stainless steel mesh at an angle of 60 degrees to pour water into the funnel to flow through the funnel inlet. The suspension did not pass through the 20 μm woven mesh.

After that, 50ml syringe is connected to the connector ring, and the air is sucked into the vacuum to make all the solution in the funnel down. In this way, the remaining solution in the 50ml conical tube is filled with 20μm woven mesh I poured it until it was repeated. The 20 μm woven mesh with the collected eggs was turned upside down on a new 50 ml conical tube and washed with 1-5 ml of tertiary distilled water to separate the eggs from the mesh. At this time, 20 ml of a 100% solution of sucrose (Saccharose, Junsei, C12H22O11, M.W: 342.30, 500 g) was filled in a 50 ml conical tube.

The buccal suspension was placed in the upper layer at a ratio of 1: 4 (v / v) (5 ml of the oyster suspension: 20 ml of sucrose) to the filled sucrose solution (100%) and centrifuged at 2520C for 5 minutes at 3,220 g. The larvae present in the sucrose boundary layer in the floated egg-bearing solution (approximately 25 ml) were sucked up with sucrose in a 1 ml pipette. The mixture was transferred into a 50 ml conical tube, diluted with a ratio of 1:14 (3 ml of mixed egg yolk: DD.W 42 ml), and centrifuged at 25 ° C for 5 minutes at 3,220 g. Then, And the egg white was suspended in 1 ml to 5 ml of PBS and stored in a 15 ml conical tube (SPL, # 50015) at 25 ° C. After the treatment time, the ovules of the feces reacted with ethyl acetate were centrifuged at 3,220 g for 5 minutes at room temperature of 25 DEG C and washed three times with distilled water. The general experimental procedure is shown in FIG. 4, and the shape and the time-dependent shape of the larvae obtained according to the method of the present invention are shown in FIG. 5 and the recovery rate of the larvae is shown in Table 1.

Comparison of the recovery rate and the treatment time according to the difference of separation method Method EPG (Stool, g) EPG
(Recovered eggs,%) Times (min)  Classical 641 (153) 365 (56.9) 120-240 Ethyl acetate 5,023 (74) 2,513 (50) 30 Ethyl acetate & SGF 3,286 (165) 3,003 (91.4) 60

As shown in Fig. 5A, only pure eggs were separated from the feces after the pure separation of eggs using ethyl acetate and SGF and using stainless steel mesh, PluriStrainer set and sucrose. As shown in FIG. 5B, when the present invention was separated by the method of the present invention, it was confirmed that it can be separated immediately after the separation (FIG. 5B) without damage to the egg.

As shown in Table 1, in the classic method of control, the recovery rate of the egg is low at 56.9%, and even if the sucrose 100% step is performed thereafter, there is almost no change in the recovery rate and the operation time of 120 to 240 minutes or more is required Respectively. Also, it was confirmed that the recovery rate of the larvae was lowered to 50% when suspended in 100% of sucrose without applying SGF because of the residual residue in the ethyl acetate - treated pupa. Therefore, we confirmed that the intermediate stage of SGF treatment plays a role in inhibiting the growth of other microorganisms. On the other hand, it was confirmed that the method of separating eggs by separating ethyl acetate and SGF, which is a separation method of the present invention, with a short treatment time of about 60 minutes, showed a high recovery rate of 91.4% .

Example  3. Detached Adulterous  Maturation due to acid stimulation

All of the separated eggs from Example 2 were stored in phosphate buffered saline (PBS) for 2 to 3 months at a temperature of 25 ° C. Eggs stored at intervals of one month were taken out and the maturity of the eggs was measured by an optical microscope Respectively. The morphological pattern according to the maturity of the larvae is shown in Fig. 6, and the comparison of the degree of maturation of the larvae obtained by the separation method of the present invention and Comparative Examples 1 and 2 is shown in Fig.

As shown in Fig. 6, it was confirmed that the eggs grew effectively for 60 days. In addition, as shown in FIG. 7, compared with the control group isolated by the classical method (Comparative Example 1), the oviposition of the swine of the pigs stimulated by the ethyl acetate and the SGF method was 22.46% . On the 90th day, induction of maturation of 42.29% of the eggs was confirmed (* p <0.0332). No deaths were observed in the ethyl acetate and SGF groups. These results indicate that even if exposed for a long period of time, they do not mature in an infectious form unless given an appropriate external stimulus. That is, pH stimulation can be a very important inducer in inducing maturation of porcine oyster larvae, and it has been confirmed that acid stimulation can induce maturation as a suitable stimulus source. Using the method of the present invention, At the same time, it was confirmed that mature adult eggs can be matured and mature eggs can be collected. However, in the case of the acid stimulation of Comparative Example 2 and the separation method of the present invention, the treatment group containing acetate and SGF showed a maturation difference of about 2% after 60 days, but a difference of about 10% , It can be confirmed that when using the separation method of the present invention, immature embryo can be effectively obtained by mature embryo.

 Therefore, in the case of using porcine larvae for the study of inflammatory bowel disease and the like, the acidic stimulation such as ethyl acetate and SGF is applied to the immature porcine larvae to induce maturation in a short period of time due to infectious embryo, .

Claims (7)

(a) filtering a dilution of the pig's feces;
(b) adding ethyl acetate to the filtrate and mixing and centrifuging;
(c) reacting the precipitated pig knifalogia with a modified simulated gastric fluid (SGF); And
(d) filtering the reactants of step (c) using a filter to obtain pig knock-in eggs. The method according to claim 1, wherein the modified artificial gastric juice of step (c) is a mixture of pepsin and hydrochloric acid in a ratio of 1: 5 to 1: 10 (v / v) Way. The method according to claim 1, wherein the modified artificial gastric juice of step (c) is reacted for 5 minutes to 60 minutes. The method of claim 1, wherein the step (d) comprises the steps of: (d-1) filtering the reactant of step (c) with a filter having a size of 50 to 56 μm; (d-2) filtering the reactant of step (c) with a filtrate having a size of 22 to 28 mu m; And (d-3) filtering the reactant of step (c) with a filter medium having a particle size of 17 to 23 μm to obtain pig knock-in eggs. The method according to claim 1,
(e) mixing the porcine zygomycota with sucrose and centrifuging the porcine zygote. 2. The method of claim 1, wherein the porcine zygomatic egg is matured egg. (a) filtering a dilution of the pig's feces;
(b) adding ethyl acetate to the filtrate, mixing and centrifuging to react the precipitated pig knifalogia with a simulated gastric fluid (SGF); And
(c) filtering the suspension of swine flounder in step (b) using a filter to obtain pig knock-in eggs.

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