Method of producing dry earthworm powder

FIELD: chemistry.

SUBSTANCE: method of producing dry earthworm powder includes: contacting live earthworms with chloride(s) of at least one metal selected from a group consisting of potassium, sodium, magnesium and calcium; subsequently contacting the live earthworms with powder of a hydroxycarboxylic acid(s) (or aqueous solution) and diluting the obtained mixture with water to adjust pH to 2-5, followed by leaving the live earthworms to stand for 3-180 minutes, washing the live earthworms with water, grinding the washed live earthworms and freeze-drying the obtained ground product (version).

EFFECT: method enables to obtain powder with high enzymatic activity.

6 cl, 8 tbl, 2 ex

 

The technical FIELD

The present invention relates to a method of obtaining a dry powder of earthworms, in particular to a method of obtaining a dry powder of earthworms, through which can be obtained dry powder of earthworms containing a high titer of enzymes, with the elimination of toxic substances contained in the body of earthworms.

PRIOR art

Extracts of earthworms and dry powder of earthworms used since ancient times, mainly in Eastern countries as a prophylactic and therapeutic agents for the treatment of various diseases, and examples to date include the use as a means to reduce bladder stones, and means of ensuring the urinary bladder stones, remedies for the treatment of jaundice, tools, enabling clan activities, tonics, tools, improves hair growth, aphrodisiacs, antipyretics, the means for the treatment of convulsions, amplifiers blood circulation, for the treatment of hemiplegia, indirect analgesics, diuretics, funds for the treatment of asthma and for the treatment of hypertension.

However, earthworms are kept and fed in cultivation substrates, contain toxic elements such as mercury, Cadmus is th, lead and arsenic and pathogens, even if rain worms give carefully selected feed. If these toxic substances are absorbed by earthworms and accumulate in their bodies during the cultivation, the receiving therapeutic agent derived from living earthworms, might adversely affect the human body.

Consequently, when the tool for oral administration obtained using living bodies of earthworms as a raw material, such toxic substances must be eliminated, and this has been proposed many methods. Examples of methods proposed thus include methods where the living earthworms are immersed in an aqueous solution of an alkaline salt such as sodium salt or potassium salt, in order to cause the excretion of mass in the digestive tract, with subsequent wet grinding of earthworms and vacuum lyophilization of the resulting suspension to obtain a dry powder of earthworms applicable as a treatment for diabetes, antihyperlipidemic means or tools for the regulation of blood pressure (see Patent documents 1-4); and a method where the living earthworms leave to stand in an aqueous acid solution, when stored at 6-26°C for 0.1 to 5 hours to remove waste from the digestive tract is, with subsequent grinding of earthworms, degassing the resulting milled product, and then vacuum drying degassed product with step-by-step raising the temperature to obtain a therapeutic agent for patients suffering from thrombosis (see patent document 5).

In addition, a method has been proposed, where in order to remove or reduce the content of heavy metals and substances that inhibit fibrinolytic activity and predecessors of platelet activating factor, the dry powder of earthworms get in aqueous solution and from it removed a thick components to obtain an aqueous solution of earthworms, having a turbidity of not more than 1.5 in relation to the absorption at a wavelength of 700 nm (see patent document 6).

DOCUMENTS RELATED technical FIELD

Patent documents:

Patent document 1: publication of Japanese unexamined patent application No. N1-47718

Patent document 2: Japanese publication of unexamined patent application No. N1-47719

Patent document 3: Japanese publication of unexamined patent application No. N1-47720

Patent document 4: Japanese publication of unexamined patent application No. N1-268639

Patent document 5: Japanese publication of unexamined patent application No. H3-72427

Patent document 6: Japanese publication of unexamined is atentos application No. 2006-96673

The INVENTION

PROBLEMS RESHENIE USING INVENTIONS

However, immersion of live earthworms in fresh water, an aqueous solution of an alkaline salt or an aqueous solution of acid for a long period of time can cause physical exhaustion earthworms, resulting in denaturation of proteins contained in a living body, and lower enzymatic action, contributing to the deterioration of the pharmacological effects of the obtained powder of earthworms. In addition, if the earthworm dies in the presence of water, the body of an earthworm is quickly dissolved because of the action of fibrinolytic enzymes existing in the rain worm and rot. Therefore, the processing in aqueous solution should be fast that problematic.

Therefore, the present invention aims to provide a way to obtain a dry powder of earthworms, through which you can get the dry powder of earthworms containing a high titer of enzymes, with the elimination of toxic substances contained in the body of an earthworm.

The authors of the present invention conducted intensive studies to solve the above problems and found that the above problems can be solved by contact a live earthworm chloride(s) of metal(s) and then contact a live earthworm with g is toxicarol acid(s), thus, carrying out the present invention.

TOOLS FOR PROBLEM SOLVING

Thus the method according to the present invention for obtaining a dry powder of earthworms include:

contact live earthworms chloride(AMI) at least one metal selected from the group consisting of potassium, sodium, magnesium and calcium; and

subsequent contact of live earthworms powder hydroxycarbonate acid(s) and the dilution of the mixture with water and bring the pH from 2 to 5 subsequent abandonment of a live earthworm to stand for 3 to 180 minutes, rinse living earthworms water, grinding the washed earthworms and freeze-drying the resulting milled product.

Furthermore, the method of the present invention to obtain a dry powder of earthworms include:

contact live earthworms chloride(s) of metal(s) selected from the group consisting of potassium, sodium, magnesium and calcium;

subsequent immersion of live earthworms in aqueous solution hydroxycarbonate acid(s), the pH of which is set at 2-5 with subsequent separation of live earthworms to stand for 3-180 minutes, rinse living earthworms water, crushing the living earthworms and freeze-drying the resulting milled product.

Preferably in the method according to us is oedema invention to obtain a dry powder of earthworms living earthworms leave to stand in a bright place for 10-50 hours and then exfoliate dirt, adhering to the body surface prior to contact with chloride(s) of metal(s).

In addition, in the method according to the present invention for obtaining a dry powder of earthworms the lyophilization is preferably carried out by freezing the ground product at -18°C -35°C for 20-240 hours and then freeze-drying the obtained product in a vacuum.

In addition, in the method according to the present invention for obtaining a dry powder of earthworms chloride of the metal is preferably sodium chloride.

In addition, in the method according to the present invention for obtaining a dry powder of earthworms hydroxycarbonate acid(s) is at least one selected from the group consisting of acetic acid, malic acid, citric acid, lactic acid, malonic acid and succinic acid.

The EFFECT of the INVENTION

By the present invention it is possible to provide a method for obtaining a dry powder of earthworms, which can be obtained dry powder of earthworms containing a high titer of enzymes, with the elimination of toxic substances contained in the body of an earthworm.

WAYS of carrying out the INVENTION

In the method according to the present invention for obtaining a dry powder of earthworms, live earthworms in contact with chloride(AMI at least one metal, selected from the group consisting of potassium, sodium, magnesium and calcium, then there is

contact live earthworm powder hydroxycarbonate acid(s), dilution of the mixture with water to bring the pH from 2 to 5 and leaving live worms to stand for 3 to 180 minutes; or

immersion of live earthworms in aqueous solution hydroxycarbonate acid(s), the pH is increased to 2-5, and leaving the living earthworms to stand for 3 to 180 minutes;

these worms then washed with water and crushed, followed by lyophilization of the obtained ground product.

By contact of live earthworms with a predetermined chloride(AMI) metal and then hydroxycarbonate acid(s) before processing of earthworms is formed environment unfavorable for earthworm, and resulting in earthworms egest food from the digestive tract to adapt to the environment and at the same time excretiruyutza toxic substances such as mercury, cadmium and lead contained in the body.

Known methods, where the environment is unfavourable for earthworms, is created for the earthworm monster faeces and similar masses containing toxic substances in the body of an earthworm, with subsequent grinding of an earthworm. Now is sabreena was done on the basis of the opening, the combination of specific methods among the above methods, to obtain an unfavorable environment, and carrying out such methods in a certain order, allows to obtain a dry powder of earthworms, having superior enzymatic activity. That is, it is important that the chloride(s) of metal (osmotic stress) and hydroxycarbonate acid(s) (pH stress) was applied in that order.

Although earthworms have not been developed molecular biological analysis and, therefore, the detailed mechanisms are not fully clear, it is known that osmotic stress activates the transcription of various stress response genes, such as genes for heat shock proteins, in studies in other model organisms such as yeast, nematodes, and plants. Therefore, one reason may be the effect of the present invention may be, for example, the fact that HSP genes and such first activated by osmotic stress and subsequently earthworms additionally subjected to different stress, pH stress, which causes subsequent activation pathways of gene expression, stress response, leading to a marked increase in the produced quantities applicable enzymes.

In the method according to the present invention using the living earthworms, i.e. earthworms that live. Live dogdave the worms are not limited and examples include Lumbricus rubellus, Lumbricus terrestris, Eisenia foetida, Allobophora caliginosa, Dendrobaena octaedra, Allobophora japonica Michaelsen, Drawida hattamimizu Hatai, Pheretima divergens Michaelsen, Pheretina communissima, Pheretima agrestis, Pheretima sieboldi Horst, Pheretima hilgendorfi, Pontodrilus matsushimensis Iizuka, Tubifex hattai Nomura and Limnodrilus gotoi Hatai (=L. Socialis Stephenson).

In the method according to the present invention prior to contact of live earthworms chloride(AMI) metals of living earthworms is preferably placed in a flat container such as the container for the bread and leave to stand in a bright place for 10-50 hours, followed by removing dirt adhered to the surface of the phone, the Length of time during which earthworms leave to stand in a bright place, is more preferably from 12 to 24 hours. The number of earthworms preferably represents a number, in which earthworms are grouped to a thickness of approximately 30 to 60 mm, preferably from about 40 to 50 mm So flat container make free from foreign substances, such as sand and silt, and inside the container preferably support the light the night by the light processing or similar, because earthworms are the night and in a dark place is their daily activity, leading to physical exhaustion. Through this procedure, earthworms are the instinct of self-defense and egest digestive waste remaining in the digestive t the act, which cover the whole body to prevent water evaporation and thereby maintaining their living environment. Therefore, by repeated removal of this covering of dirt, that is excrement, by an appropriate method, the wastes of the digestive tract and dirt adhered to the surface of the body, can be completely removed.

The dirt adhered to the surface of the body of earthworms, can be exfoliated by, for example, coverage of live earthworms non-woven product for adsorption to him dirt. By combining this abandonment of earthworms to stand in the bright place with the subsequent removal of dirt stuck to the surface of the body, and contact earthworms chloride(AMI) metals and hydroxycarbonate acid(s), you can also expect the excretion and removal of toxic substances from the body of earthworms.

Chloride(AMI) metals that are applicable in the present invention is(are) chloride(s) at least one metal selected from the group consisting of potassium, sodium, magnesium and calcium. I.e. chloride(s) of the metal is(are) at least one selected from the group consisting of potassium chloride, sodium chloride, magnesium chloride and calcium chloride. In addition, chloride(s) of the metal may be, or their mixture, or a mixture thereof and other harmless whom is onenow, which can be added to food. Examples of such mixtures include table salt, rock salt and sea salt. The above chloride(s) of metals can be used by spraying them powder on the live earthworms, which causes the contact chloride(s) of metals with earthworms.

After exposure chloride(s) of metals with live worms, live worms are put in contact with hydroxycarbonate acid(AMI). Contact with hydroxycarbonate acid(s) can also be carried out by spraying powder hydroxycarbonate acid(s) on the live earthworms. Contact with hydroxycarbonate acid(AMI) was carried out directly after contact with the above chloride(AMI) metals. Also, before you contact the living earthworms with hydroxycarbonate acid(s), earthworms preferably washed with water. Remove chloride(s) of metals by washing with water followed by contact of live earthworms with hydroxycarbonate acid(AMI) allows the production of dry powder of earthworms with high enzymatic activity. When earthworms were washed with water before contact with hydroxycarbonate acid(s), wash earthworms water carried out preferably within 30 minutes, more preferably within 20 minutes, the donkey began contact with chloride(AMI) metals. The method of washing with water is not limited, and can be used known methods.

If you live earthworms kept in contact with the powder hydroxycarbonate acid(s) in a long time, their bodily functions are lost and digested waste in the digestive tract not excretiruyutza. Therefore, hydroxycarbonyl acid(s) should be diluted with water as fast as possible, preferably within 30 seconds, more preferably for 20 seconds to obtain a pH of from 2 to 5.

As hydroxycarbonate acid(s) form(s) the environment uncomfortable for earthworms, live earthworms are trying to improve their environment by excretion of body fluids and excrement due to the instinct of self-defense. In addition, since hydroxycarbonate acids have antibacterial properties, expect that they play a role not only in ensuring the excretion of digestive waste and the like remaining in the digestive tract, as described above, but also in the destruction of bacteria in the rain worms.

Crystal hydroxycarbonate acid, applicable in the method according to the present invention is not limited to the number of hydroxyl groups and carboxyl groups, while it is in the form of crystalline bodies under operating conditions. That is, the crystal is hydroxycarbonate acid can be any of monohydroxybenzene acid, monoperoxyphthalic acid, polyhydroxyalkanoates acid and polyhydroxyalkanoic acid.

Examples hydroxycarbonate acid(s) applicable in the present invention include glycolic acid, lactic acid, acetic acid, β-hydroxypropionic acid, α-hydroxy-n-butyric acid, β-hydroxy-n-butyric acid, α-hydroxy-n-valeric acid, β-hydroxy-n-valeric acid, malic acid, α-methyl-malic acid, α-hydroxyglutaric acid, β-hydroxyglutaric acid, citric acid, malonic acid and succinic acid. Among them, lactic acid, acetic acid, malic acid, citric acid, malonic acid and succinic acid are preferred in light of the fact that they can be used in food and easily obtained. One type hydroxycarbonate acid can be used alone or may be used a mixture of 2 or more types.

Water makes up 65% of all tissue components a live earthworm. Although the protective functions of the living earthworm are effective for a certain length of time, the death of a live earthworm triggers the action of enzymes, so the length of time during which a live earthworm placed in an adverse environment, think the mo carefully controlled. The length of time varies depending on conditions and is typically in the range from 3 to 180 minutes.

In the present invention the living earthworms, processed hydroxycarbonate acid(AMI), washed with water and then crushed in the crushed product in the form of liquid or paste. Washing is preferably carried out with clean water. Method of leaching is not limited and can be used a known method for washing with water. In addition, the total duration of time stages before grinding, i.e. the total duration of time stages from spraying chloride(s) of metal on the live earthworms to the final rinse hydroxycarbonate acid(s) with water is preferably not more than 240 minutes.

The method of the above-described grinding is not limited and, for example, chopping, make use of a homogenizer, blender, homemaker, grinder, French press or the like, usually when 1-25°C. In light of suppression of degradation of the components of earthworms grinding is preferably carried out at a low temperature, preferably at a temperature of from 2 to 15°C.

The crushed product obtained by grinding the earthworms, placed in a cell made of stainless steel and subjected to lyophilization. Although the enzymes in a living body of an earthworm, not on setout on living cells, they immediately act on dead cells. Therefore, in the above process there is a risk of formation of septic gases. To prevent the formation of septic gases, the crushed product is preferable to suppress the action of enzymes immediately subjected to freezing by rapid cooling to -18°C -35°C, followed by lyophilization.

Therefore, spraying earthworms without loss of pharmacological action needs to be quick freezing, but, on the other hand, too rapid freezing is not preferable, because when earthworms freeze too quickly, impurities, existing together with proteins that are essential components of the paste from earthworms, can form unfrozen areas and cannot be separated. Therefore, freezing is carried out at a low temperature of -18°C To -35°C for preferably 20-240 hours, more preferably is 50-170 hours.

For lyophilization, it is important to choose the conditions in which impurities can be removed without residue along with the water. Therefore, the lyophilization is preferably performed under the control of the pressure not more than 50 PA at a temperature of from -60°C to +90°C temperature increase incrementally within 10-60 hours.

Examples of the method of lyophilization including the up method, when the crushed product is frozen as described above at a temperature of -18°C To -35°C for 20-240 hours, and then the temperature was raised at several stages in the range from -60°C to +90°C, and the pressure is reduced in several stages in the range of 25-40 PA, with lyophilizer product in vacuum for 10 to 60 hours, thereby obtaining a sterile, pale yellow dry powder of earthworms.

Thus obtained dry powder of earthworms contains 100 g of powder 70-120 mg arginine, 110-150 mg lysine, 35-60 mg histidine, 55-80 mg phenylalanine, 50-75 mg of tyrosine, 100-150 mg of leucine, 60-90 mg of isoleucine, 25-40 mg methionine, 70-105 mg valine, 85-135 mg alanine, 75-105 mg glycine, 60-85 mg Proline, 210-300 mg glutamic acid, 80-110 mg serine, 75-110 mg threonine, 150-220 mg aspartic acid, 15-30 mg of tryptophan and 20-35 mg of cystine, although the content varies depending on the type of earthworms and the place and time of collection of earthworms.

EXAMPLES

The present invention will be described hereinafter in more detail. The present invention is not limited by the examples presented below.

Obtaining a dry powder of earthworms

(Example 1)

30 kg of live Lumbricus rubellus was left to stand for 24 hours in the bright place, and dirt adhered to the surface of the bodies otslushival, with subsequent distribution of earthworms on a flat Cup with a thickness of about 5 cm and about the popular spray on them 250 g of sodium chloride. After 20 minutes of earthworms were washed with water.

Subsequently, 250 g of citric acid was sprayed on earthworms in a similar way and 15 seconds after that, the obtained was diluted by adding 30 liters of clean water. At this point, the pH immediately after adding water amounted to 2.25 and pH after full dilution amounted to 2.74.

When sprayed powder, citric acid, earthworms immediately released biological yellow liquid. After dilution with water earthworms were left to stand in this state for 20 minutes.

Subsequently, the living earthworms were removed from a contaminated aqueous solution of citric acid and washed with water and then crushed using a homogenizer at 10°C to obtain a paste of earthworms. After that, the resultant paste from earthworms were degirolami by aspiration to remove the gases contained therein, and transferred into a cuvette made of stainless steel, with subsequent immediate and rapid cooling to -35°C, at this temperature, pasta earthworms were kept at 50 hours for slow freezing.

Frozen pasta earthworms kept at -35°C at a pressure of 0 PA for 2 hours and then the temperature was raised to 25°C followed by lyophilization under vacuum at 40 PA for 10 hours at 40°C when the pressure is AI 35 PA for 14 hours. at 65°C at a pressure of 35 PA for 12 hours; and , finally, at a temperature of 80°C at a pressure of 25 PA for 6 hours. Through such processing was obtained a pale yellow dry powder of earthworms, having a moisture content of 8 wt.%.

Comparative example 1

30 kg of live Lumbricus rubellus was left to stand for 24 hours in the bright place, and dirt adhered to the surface of the bodies otslushival, with subsequent distribution of earthworms on a flat Cup with a thickness of about 5 cm and add 30 litres of water on them. After that earthworms were left to stand in this state for 20 minutes.

Subsequently, the living earthworms were removed from the water and washed with water and then crushed using a homogenizer at 10°C to obtain a paste of earthworms. After that, the resulting paste of earthworms was degirolami by aspiration to remove the gases contained therein, and transferred into a cuvette made of stainless steel with subsequent immediate and rapid cooling to -35°C, at this temperature, pasta earthworms were kept at 50 hours for slow freezing.

Frozen pasta earthworms maintained at -35°C at a pressure of 0 PA for 2 hours and then the temperature was raised to 25°C followed by lyophilization under vacuum at 40 PA for 10 hours at 40°C when d is the pressure of 35 PA for 14 hours. at 65°C at a pressure of 35 PA for 12 hours; and , finally, at a temperature of 80°C at a pressure of 25 PA for 6 hours. Through such processing was obtained a pale yellow dry powder of earthworms, having a moisture content of 8 wt.%.

Comparative example 2

30 kg of live Lumbricus rubellus was left to stand for 24 hours in the bright place, and dirt adhered to the surface of the bodies otslushival with subsequent distribution of earthworms on a flat Cup with a thickness of about 5 cm and spray them 250 g of citric acid. 15 seconds after it received for cultivation were added to 30 liters of clean water.

When sprayed powder, citric acid, earthworms immediately released biological yellow liquid. After dilution with water earthworms were left to stand in this state for 20 minutes.

Subsequently, the living earthworms were removed from a contaminated aqueous solution of citric acid and washed with water and then crushed using a homogenizer at 10°C to obtain a paste of earthworms. After that, the resulting paste of earthworms was degirolami by aspiration to remove the gases contained therein, and transferred into a cuvette made of stainless steel with subsequent immediate and rapid cooling to -35°C, at this temperature, pasta earthworms kept during the 50 hours for slow freezing.

Frozen pasta earthworms maintained at -35°C at a pressure of 0 PA for 2 hours and then the temperature was raised to 25°C followed by lyophilization under vacuum at 40 PA for 10 hours at 40°C at a pressure of 35 PA for 14 hours at 65°C at a pressure of 35 PA for 12 hours; and , finally, at a temperature of 80°C at a pressure of 25 PA for 6 hours. Through such processing was obtained a pale yellow dry powder of earthworms, having a moisture content of 8 wt.%.

Comparative example 3

30 kg of live Lumbricus rubellus was left to stand for 24 hours in the bright place, and dirt adhered to the surface of the bodies otslushival with subsequent distribution of earthworms on a flat Cup with a thickness of about 5 cm and spray them 250 g of citric acid. Then, to the obtained for dilution was added to 30 liters of clean water. When sprayed powder, citric acid, earthworms immediately released biological yellow liquid. After dilution with water earthworms were left to stand in this state for 20 minutes. Subsequently, the living earthworms were removed from a contaminated aqueous solution of citric acid and washed with water followed by spray 250 g of sodium chloride and left to stand in this state for 20 minutes.

Subsequently, the live earthworms were washed with water and crushed with COI is whether the homogenizer at 10°C to obtain a paste of earthworms. After that, the resulting paste of earthworms was degirolami by aspiration to remove the gases contained therein, and transferred into a cuvette made of stainless steel, with subsequent immediate and rapid cooling to -35°C, at this temperature, pasta earthworms were kept at 50 hours for slow freezing.

Frozen pasta earthworms maintained at -35°C at a pressure of 0 PA for 2 hours and then the temperature was raised to 25°C followed by lyophilization under vacuum at 40 PA for 10 hours at 40°C at a pressure of 35 PA for 14 hours at 65°C at a pressure of 35 PA for 12 hours; and , finally, at a temperature of 80°C at a pressure of 25 PA for 6 hours. Through such processing was obtained a pale yellow dry powder of earthworms, having a moisture content of 8 wt.%.

Comparative example 4

30 kg of live Lumbricus rubellus was left to stand for 24 hours in a bright place and dirt adhered to the surface of the bodies otslushival with subsequent distribution of earthworms on a flat Cup with a thickness of about 5 cm, was mixed with 250 g of citric acid with 250 g of sodium chloride and uniformly sprayed the mixture on earthworms. 15 seconds after it received for cultivation were added to 30 liters of clean water.

When sprayed powder citric acid and sodium chloride, rain is the new worms immediately released biological yellow liquid. After dilution with water earthworms were left to stand in this state for 20 minutes.

Subsequently, the living earthworms were removed from a contaminated aqueous solution of citric acid and washed with water and then crushed using a homogenizer at 10°C to obtain a paste of earthworms. After that, the resulting paste of earthworms was degirolami by aspiration to remove the gases contained therein, and transferred into a cuvette made of stainless steel with subsequent immediate and rapid cooling to -35°C, at this temperature, pasta earthworms were kept at 50 hours for slow freezing.

Frozen pasta earthworms maintained at -35°C at a pressure of 0 PA for 2 hours and then the temperature was raised to 25°C followed by lyophilization under vacuum at 40 PA for 10 hours at 40°C at a pressure of 35 PA for 14 hours at 65°C at a pressure of 35 PA for 12 hours; and , finally, at a temperature of 80°C at a pressure of 25 PA for 6 hours. Through such processing was obtained a pale yellow dry powder of earthworms, having a moisture content of 8 wt.%.

Titration of the dry powder of earthworms

Get sample to measure

To 1 g of each dry powder of earthworms, obtained as described above was added 20 ml of physiological what about the salt solution, the resulting mixture was shaken at 1500 rpm for 1 hour. Then the mixture was centrifuged at 10000×g at 4°C for 15 minutes and the resulting supernatant was used as a sample for measurement.

Method for quantitative assessment of protein

In terms of quantitative protein estimation the calculation was performed in accordance with the method of Bradford (M. Bradford, Anal. Biochem., 72-248-254, 1976).

The sample for measuring the mass of the protein was obtained for the above sample for measurement using the kit for protein research (Bio-Rad Laboratories, Inc.,) and he measured the absorption at 595 nm. Using the calibration curve obtained separately using bovine serum albumin (Bovine, Sigma-Aldrich Co.), the measured value was converted into the mass of the protein.

Table 1
Example 1Comparative
example 1
Comparative
example 2
Comparative
example 3
Comparative
example 4
1st dimension1,912,66,26,13,2
2nd dimension2,011,16,15,62,9
3rd dimension1,111,85,85,93,2
Average1,711,86,05,93,1

Method of degradation of the substrate of synthetic amide

In accordance with the method described in non-patent document (Sumi, H., Okamoto T. And Ishii, Y., Titration method for lumbrokinase (Eathworm enzyme) - Fibrinolytic and synthetic amidolytic activities - Clinical Pharmacology and Therapy, 20: 347-351, 2010), we measured the degradation activity of the synthetic substrate amide.

The substrate is a synthetic amide was obtained by dissolving pyroGlu-Gly-Arg-pNA (BIOPHEN CS-61(44), COSMO BIO Co., Ltd), which is a synthetic substrate for urokinase, in dimethyl sulfoxide (DMSO) to a concentration of 5×10-3M

To 0.1 ml of sample for measurement, obtained as described above was added to 0.8 ml of borate saline buffer (BSB), and the resulting solution was incubated for 2 minutes followed by the addition of 0.1 ml of substrate a synthetic amide to a solution and gave the opportunity for the ity of the reaction to progress at 37°C for 5 minutes. Subsequently, we measured the absorption at 405 nm and the amount of released pNA was calculated based on the maximum slope (original speed) per minute with a coefficient of absorption 10,79 mm-1·cm-1. The results obtained are shown in table 2 below.

Moreover, the activity of degradation of the substrate of synthetic amide divided by the total quantity of protein to obtain the specific activity, which is shown in table 3 below.

Table 2
Example 1Comparative
example 1
Comparative
example 2
Comparative
example 3
Comparative
example 4
1st dimension59,256,372,854,959,9
2nd dimension59,553,679,256,0of 60.5
3rd dimension59,554,2 77,755,661,7
4th dimension63,056,582,565,866,0
5th dimension62,154,780,665,865,5
6th dimension64,953,879,763,0to 66.3
Average61,454,978,860,263,3

Table 3
The activity of degradation of synthetic amide (nmol/ml/min)The amount of protein
(mg/ml)
Specific activity (nmol/ml/min/mg)
Example 161,41,736,
Comparative example 154,911,8the 4.7
Comparative example 278,86,013,1
Comparative example 360,25,910,2
Comparative example 463,33,120,4

Method fibrin tablet

In accordance with the method described in non-patent document (T. Astrup & Mullertz, S., The fibrin plate method for estimating fibrinolytic activity, Arch. Biochem. Biophys., 40: 346-351, 1952), received fibrin pad, and a comparison of the activity of dissolution was performed by the method fibrin tablet.

Fibrinogen (Bovine Fraction I-S, Sigma-Aldrich Co.) was dissolved in borate buffered saline (pH 7,8, BSB) to a final concentration of 0.5%. To 10 ml of the resulting solution was added 0.5 ml of thrombin (for clinical application, Fuji Pharma Co., Ltd) to obtain fibrin tablet.

Measuring the area of dissolution was performed by placing 30 µl of each sample for measurement on the tablet and area measurement of dissolved zones, which appeared after 4 hours of Incubus the AI at 37°C. In addition, the received calibration curve for trypsin (Bovine, Sigma-Aldrich Co.), which is a protease, and conversion of units was conducted to determine the specific activity based on the amount of protein in each sample for measurement. The results are shown in table 4 below.

Table 4
Dissolved area (mm2)Activity in units of trypsin (IU/ml)The amount of protein
(mg/ml)
Specific activity (U/mg)
Example 1223,3287,21,7168,9
Comparative example 1211,3256,511,821,7
Comparative example 2272,5462,86,077,1
Comparative example 3259,7level of 414.25,9 to 70.2
Comparative example 4171,6156,03,150,3

Using the method according to the present invention can be obtained dry powder of earthworms high quality, from which you have removed toxic substances. In addition, as is evident from example 1, the dry powder of earthworms obtained by the method of the present invention, contains a high titer of enzymes.

On the other hand, as apparent from comparative examples 1 and 2, the enzymatic activity when conducted only handle citric acid, was only 2-4 times higher than that in the case when the processing carried out by the water. In addition, as is evident from the results of comparative examples 2 and 3, when first performed the processing of citric acid and then spent processing the chloride of the metal, the enzymatic activity was almost the same as when performed processing only citric acid.

Example 2

30 kg of live Lumbricus rubellus was left to stand for 24 hours in a bright place and dirt adhered to the surface of the bodies otslushival with subsequent distribution of earthworms on a flat Cup with a thickness of about 5 cm and spray them 250 g of magnesium chloride of agnor the bottom. After 20 minutes, earthworms were washed with water.

Subsequently, 250 g of lactic acid was sprayed on earthworms thus obtained was dissolved in 15 seconds by adding 30 liters of clean water. When sprayed powder, lactic acid, earthworms immediately released biological yellow liquid. After dilution with water earthworms were left to stand in this state for 20 minutes.

Subsequently, the living earthworms were removed from a contaminated aqueous solution of lactic acid and washed with water and then crushed using a homogenizer at 10°C to obtain a paste of earthworms. After that, the resulting paste of earthworms was degirolami by aspiration to remove the gases contained therein, and transferred into a cuvette made of stainless steel with subsequent immediate and rapid cooling to -35°C, at this temperature, pasta earthworms were kept at 50 hours for slow freezing.

Frozen pasta earthworms kept at -35°C at a pressure of 0 PA for 2 hours and then the temperature was raised to 25°C followed by lyophilization under vacuum at 40 PA for 10 hours at 40°C at a pressure of 35 PA for 14 hours at 65°C at a pressure of 35 PA for 12 hours; and , finally, at a temperature of 80°C when the pressure is 25 PA for 6 hours. Through such processing was obtained a pale yellow dry powder of earthworms, having a moisture content of 8 wt.%.

Comparative example 5

30 kg of live Lumbricus rubellus was left to stand for 24 hours in a bright place and dirt adhered to the surface of the bodies otslushival with subsequent distribution of earthworms on a flat Cup with a thickness of about 5 cm, spray on them 250 ml of lactic acid in the same manner and breeding received after 15 seconds by adding 30 liters of clean water. When sprayed with lactic acid, earthworms immediately released biological yellow liquid. After dilution with water earthworms were left to stand in this state for 20 minutes.

Subsequently, the living earthworms were removed and washed with water followed by spraying them 250 g of magnesium chloride uniformly, and left earthworms to stand in this state for 20 minutes.

Subsequently, the living earthworms were removed and washed with water and then crushed using a homogenizer at 10°C to obtain a paste of earthworms. After that, the resulting paste of earthworms was degirolami by aspiration to remove the gases contained therein, and transferred into a cuvette made of stainless steel, with subsequent immediate and rapid cooling to -35°C, when such fact is the temperature value pasta earthworms were kept at 50 hours for slow freezing.

Frozen pasta earthworms maintained at -35°C at a pressure of 0 PA for 2 hours and then the temperature was raised to 25°C followed by lyophilization under vacuum at 40 PA for 10 hours at 40°C at a pressure of 35 PA for 14 hours at 65°C at a pressure of 35 PA for 12 hours; and , finally, at a temperature of 80°C at a pressure of 25 PA for 6 hours. Through such processing was obtained a pale yellow dry powder of earthworms, having a moisture content of 8 wt.%.

Titration of the dry powder of earthworms

Get sample to measure

To 1 g of each of the dry powder of earthworms, obtained as described above in example 2 and comparative example 5, was added 20 ml of physiological solution, and the resulting mixture was shaken at 1500 rpm for 1 hour. Then the mixture was centrifuged at 10-000×g at 4°C for 15 minutes and the resulting supernatant was used as a sample for measurement.

Method for quantitative assessment of protein

In relation to quantitative protein estimation, calculation conducted in accordance with the method of Bradford (M. Bradford, Anal. Biochem., 72: 248-254, 1976).

The sample for measuring the mass of the protein was obtained for the above sample for measurement using the kit for protein analysis (Bio-Rad Laboratories, Inc.) and he measured the absorption at 595 nm. Using the calibration curve, recip is authorized separately using bovine serum albumin (Bovine, Sigma-Aldrich Co.,) the measured value was converted into the mass of the protein.

Table 5
Example 2Comparative example 5
1st dimension6,67,1
2nd dimension7,07,8
3rd dimension7,07,0
Average6,97,3

Method of degradation of the substrate of synthetic amide

In accordance with the method described in non-patent document (Sumi, H., Okamoto T. And Ishii, Y., Titration method for lumbrokinase (Eathworm enzyme) - Fibrinolytic and synthetic amidolytic activities - Clinical Pharmacology and Therapy, 20: 347-351, 2010), we measured the degradation activity of the synthetic substrate amide.

The substrate is a synthetic amide was obtained by dissolving pyroGlu-Gly-Arg-pNA (BIOPHEN CS-61(44), COSMO BIO Co., Ltd), which is a synthetic substrate for urokinase, in dimethyl sulfoxide (DMSO) to a concentration of 5×10-3M

To 0.1 ml of sample for measurement, obtained as described above was added to 0.8 ml of borate salt is of buffer (BSB) and the resulting solution was incubated for 2 minutes followed by the addition to the solution of 0.1 ml of substrate a synthetic amide and allowed the reaction to progress at 37°C for 5 minutes. Subsequently, we measured the absorption at 405 nm, and the number of released pNA was calculated based on the maximum slope (original speed) per minute with a coefficient of absorption 10,79 mm-1·cm-1. The results obtained are shown in table 6 below.

Moreover, the activity of degradation of the substrate of synthetic amide divided by the total quantity of protein to obtain the specific activity, which is shown in table 7 below.

Table 6
Example 2Comparative example 5
1st dimension71,141,5
2nd dimension73,741,4
3rd dimension72,042,3
4th dimension73,644,4
5th dimension73,743,8
6th dimension70,641,6
Average72,542,5

Table 7
The activity of degradation of synthetic amide (nmol/ml/min)The amount of protein (mg/ml)Specific activity (nmol/ml/min/mg)
Example 272,56,910,5
Comparative example 542,57,35,8

Method fibrin tablet

In accordance with the method described in non-patent document (T. Astrup & Mullertz, S., The fibrin plate method for estimating fibrinolytic activity, Arch. Biochem. Biophys., 40: 346-351, 1952), received fibrin tablet, and were compared to the activity of dissolution through the fibrin pad.

Fibrinogen (Bovine Fraction I-S, Sigma-Aldrich Co.) was dissolved in borate buffered saline (pH 7,8, BSB) to a final concentration of 0.5%. To 10 ml of the resulting solution was added 0.5 ml of thrombin (for clinical application, Fuji Pharma Co., Ltd) to obtain fibrin tablet.

Measuring the area of dissolution was performed by placing 30 μl of each the sample for measurement on the tablet and area measurement of dissolved zone, which appeared after 4 hours incubation at 37°C. in Addition, the received calibration curve for trypsin (Bovine, Sigma-Aldrich Co.), which is a protease, and carried out the conversion of the units to determine specific activity based on the amount of protein in each sample for measurement. The results are shown in table 8 below.

Table 8
Dissolved area (mm2)Activity in units of trypsin (IU/ml)The amount of protein (mg/ml)Specific activity (U/mg)
Example 2243,4351,76,951,0
Comparative example 5207,4246,67,3to 33.8

As evident from table 7 and table 8 and in the degradation of the substrate of synthetic amide and method fibrin tablet, the specific activity of the enzyme was almost two times higher in cases when the environment is unfavorable for earthworms, obtained by adding the Oia of magnesium chloride (chloride of the metal) and lactic acid (hydroxycarbonate acid) in this order, compared with the cases when they were added in reverse order. Therefore, it can be confirmed that, even when using chloride(s) metal other than sodium chloride and hydroxycarbonate acid(s), other than citric acid, can be obtained powder of earthworms with high enzymatic activity by adding chloride(s) of metal and hydroxycarbonate acid(s) in that order.

INDUSTRIAL APPLICABILITY

The dry powder of earthworms obtained by the method according to the present invention is applicable as a means for regulation of blood pressure, antihyperlipidemic agents; agents used to treat diabetes, thrombolytic means and/or the like, and similar to the dry powder of earthworms obtained in the usual way. In addition, by extraction of the obtained powder of pure water, alcohol or similar and centrifugation of the resulting solution, followed by fractionation of the supernatant liquid in accordance with the molecular weight, the resulting product can be used as an effective component of a pharmaceutical, cosmetic or AIDS.

1. The way to obtain a dry powder of earthworms, including:
contact live earthworms chloride(AMI) at the ore of the same metal, selected from the group consisting of potassium, sodium, magnesium and calcium; and
subsequent contact of these live earthworm powder hydroxycarbonate acid(s) and the dilution of the mixture with water to bring the pH to between 2 and 5, with the subsequent abandonment of these earthworms to stand for 3-180 minutes, rinse these living earthworms water, grinding the washed live earthworms and lyophilization the resulting milled product.

2. The way to obtain a dry powder of earthworms, including:
contact live earthworms chloride(s) of metal(s) selected from the group consisting of potassium, sodium, magnesium and calcium; and
subsequent immersion of these living earthworms in aqueous solution hydroxycarbonate acid(s), with a pH in the range of 2-5, with the subsequent abandonment of these earthworms to stand for 3-180 minutes, rinse these living earthworms water, grinding the washed live earthworms and lyophilization the resulting milled product.

3. The way to obtain a dry powder of earthworms under item 1 or 2, where these living earthworms leave to stand in a bright place for 10-50 hours and dirt adhered to the surface of the bodies exfoliate before contact with the specified chloride(s) of metal(s).

4. How to get dry what about the powder of earthworms according to any one of paragraphs.1-3, where specified lyophilization is carried out by freezing the specified ground product at -18°C to -35°C for 20-240 hours and subsequent freeze-drying the obtained product in a vacuum.

5. The way to obtain a dry powder of earthworms according to any one of paragraphs.1-4, where the specified metal chloride is sodium chloride.

6. The way to obtain a dry powder of earthworms according to any one of paragraphs.1-5, where the specified hydroxycarbonate acid(s) is at least one selected from the group consisting of acetic acid, malic acid, citric acid, lactic acid, malonic acid and succinic acid.



 

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