KR102305089B1 - Manufacturing process of body fluid extract of sea squirt - Google Patents

Abstract

The present invention relates to a method for producing a sea squirt bodily fluid extract having the effect of inhibiting the synthesis of body fat and preventing and improving obesity by inhibiting the activity of malic enzyme in vivo. A method for producing a sea urchin body fluid extract comprising the steps of obtaining only a liquid component by solid-liquid separation, adding ethanol to the liquid component to form a precipitate in which proteins are aggregated, and separating and washing the precipitate will be.

Description

Manufacturing process of body fluid extract of sea squirt

The present invention relates to a method for producing a sea squirt bodily fluid extract having the effect of inhibiting the synthesis of body fat and preventing and improving obesity by inhibiting the activity of malic enzyme in vivo. A method for producing a sea urchin body fluid extract comprising the steps of obtaining only a liquid component by solid-liquid separation, adding ethanol to the liquid component to form a precipitate in which proteins are aggregated, and separating and washing the precipitate will be.

Sea urchins are creatures that belong to the chordoptera group, seaweeds, and are widely used as food because they are native to the oceans around the world and have a unique flavor and good edible taste. In Korea, mass production is carried out through aquaculture in the South Sea and East Sea. However, only the inner flesh of sea squirts is used for food, and most of the body fluids, skins and intestines generated during meat production are discarded.

These sea squirts have the characteristic of accumulating and protecting heavy metals, especially vanadium, in the body. Vanadium deficiency in sea squirts is known to cause growth and reproductive disorders, and metabolic and functional disorders of the kidneys, heart and brain. Vanadium is present in seaweed body fluid in the form of a salt (vanadate) and an ion (vanadyl ion), and vanadyl ion (vanadyl ion) binds to transferrin (transferrin) or albumin (albumin) protein and accumulates in the tissue.

On the other hand, as an essential nutrient for humans and mammals, vanadium is known to have anti-diabetic, anti-obesity, anti-hypertension, anti-hyperlipidemia, and the like effects in the human body. Therefore, even in the prior art, efforts have been made to secure a functional material containing vanadium useful for the human body from sea squirt.

For example, in Korean Patent No. 10-1998301 (July 03, 2019), a composition containing vanadium-binding protein isolated from the plasma or intestines of sea squirt can be used as a functional food having antioxidant and antidiabetic activity. It has been reported, and in Korean Patent No. 10-2002962 (July 17, 2019), it is reported that a composition containing the vanadium-binding protein can be used as a functional food having anti-obesity and therapeutic effects.

Patent No. 10-1998301 (July 03, 2019) Patent No. 10-2002962 (July 17, 2019)

As described above, it has been reported that vanadium present in the body of sea squirts has anti-diabetic and anti-obesity effects in the human body. And, in the Examples of Patent Nos. 10-1998301 and 10-2002962, sea squirt body fluid and A method for extracting and purifying purified vanadium binding protein from the intestine is described.

However, the method described in the above example is a method carried out on a laboratory scale to test the efficacy of the vanadium-binding protein, and since it requires the use of an expensive ion exchange resin and special purification equipment to operate it, this method is industrially price competitive. It is not possible to prepare a vanadium extract with Therefore, a method for mass production of vanadium-containing extracts from sea squirt body fluid on an industrial scale has not yet been developed.

Accordingly, it is an object of the present invention to provide a new manufacturing method capable of mass-producing sea squirt bodily fluid extract containing a large amount of vanadium useful for the human body from sea squirt fluid at low cost.

According to the present invention, a method for producing a sea squirt bodily fluid extract comprises the steps of: A) separating the sea squirt bodily fluid from solid and liquid at a temperature of 2 to 8° C. to obtain only liquid components; B) forming a precipitate by adding high-concentration ethanol to the liquid component so that the concentration of ethanol becomes 70-80%, and leaving it at a temperature of 2-8° C. for 16-20 hours; C) centrifuging the precipitate to obtain a wet body, washing the wet body with ethanol having a concentration of 80 to 90%, separating and drying the wet body; It is characterized in that it is manufactured including.

In step A), the sea squirt body fluid is centrifuged at a temperature of 2 to 8° C. to obtain a supernatant, or the sea squirt body fluid is filtered under pressure with a filter press coated with diatomaceous earth at a temperature of 2 to 8° C. It is characterized in that the solid-liquid separation is performed by taking the filtrate.

In addition, the sea squirt body fluid extract prepared according to the present invention has a vanadium concentration of 10 to 25 ppm and inhibits the synthesis of body fat by inhibiting the activity of malic enzyme in vivo, and has the effect of preventing and improving obesity. characterized in that

The method for producing a sea squirt bodily fluid extract according to the present invention has the effect of producing an industrially usable sea urchin bodily fluid extract containing a large amount of vanadium useful for the human body because it can be mass-produced and has high price competitiveness.

In addition, the sea urchin body fluid extract prepared according to the present invention inhibits the synthesis of body fat by inhibiting the activity of malic enzyme in vivo, and has the effect of preventing and improving obesity, so it can be usefully used as a functional food material. is expected to

1 is a graph showing the change in blood concentration of malic enzyme in obese rats administered with a sea urchin body fluid extract of the present invention compared to a control group administered with vanadium sulfate;
2 is a graph showing the change in the concentration of NEFA (Non-esterified fatty acid, free fatty acid), which is a functional biomarker for body fat reduction, in obese mice administered with the sea urchin body fluid extract of the present invention compared with the control group administered with vanadium sulfate; ,
3 is a graph showing the change in the concentration of Tg (Triglyceride), a functional biomarker for body fat reduction, in obese mice administered with the sea urchin body fluid extract of the present invention compared with the control group administered with vanadium sulfate;
4 is a graph showing the change in body weight of obese and normal mice administered with a sea urchin bodily fluid extract of the present invention compared with a control group administered with vanadium sulfate.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. However, even if it is a necessary configuration for carrying out the present invention, a detailed description of the matters introduced in the prior art or can be easily implemented by those skilled in the art from the known technology will be omitted.

The method for producing a sea urchin bodily fluid extract according to the present invention includes A) a step of collecting liquid components, B) a step of forming a precipitate, and C) a step of drying the precipitate.

First, in step A), fresh sea squirt body fluid generated in the process of separating the flesh of sea squirt is separately collected, the sea squirt body fluid is separated into solid and liquid at a temperature of 2 to 8 ° C. Then, the solid component is removed and only the liquid component is obtained.

At this time, as a method of separating the sea squirt body fluid from solid and liquid, for example, a method of centrifuging the sea squirt body fluid to obtain a supernatant may be used. You can also use the method you take. The solid-liquid separation of the sea squirt body fluid is preferably performed at a low temperature of 2 to 8° C. in order to prevent the transformation of proteins and active ingredients present in the sea squirt body fluid.

In addition, the diatomaceous earth coated on the filter for filtering the sea squirt body fluid is mainly composed of silicic acid (SiO ₂ ) and is white or grayish-white porous soil. It has the function of removing pigments and peculiar odors.

For reference, it is preferable to use a large-capacity centrifugal separation method when the amount of sea squirt body fluid is greater than 100 liters, and when the amount of sea squirt body fluid is relatively small, pressure filtration can be operated at a relatively low cost. It is preferable to use the method.

Next, in step B), high-concentration ethanol is added to the liquid component obtained in step A), and is added little by little while checking the concentration of ethanol. And when the concentration of ethanol reaches 70-80%, it is left in this state at a temperature of 2-8 ℃ for 16-20 hours. In this way, due to the salting out phenomenon, the effective protein contained in the liquid component aggregates to form a precipitate.

In general, the concentration of salt or ethanol directly affects the solubility of the protein. When the concentration of salt or ethanol is lowered, the solubility of the protein increases. In an aqueous solution with low ionic strength, the protein molecules are surrounded by ions, and the chemical activity of the protein is reduced. In this way, the electrostatic interaction between the protein molecules is reduced, thereby increasing the solubility. This condition is called salting in.

This salting-in effect occurs due to the dissociation of ions present in the protein, but when the concentration of ions is increased even more, the solubility of the protein is rather decreased. Therefore, when a large amount of salt or ethanol is added to increase the concentration of ions, the protein is almost completely dissociated from the solution and precipitation occurs. This phenomenon is called salting out.

That is, when the concentration of ions increases, these ions attract water molecules distributed in the hydrophobic portion of the protein surface. In this case, the hydrophobic portion of the protein surface is exposed to the outside, and precipitation occurs due to hydrophobic force between proteins.

According to a preferred embodiment of the present invention, alcohol having a concentration of 95% is used as the high-concentration ethanol in step B), and 2 to 4 times the volume of alcohol is added to the liquid component in a volume ratio to increase the concentration of ethanol It is preferable to set it to 70-80%.

In step B), if the concentration of the ethanol is less than 70%, only some proteins that easily precipitate among the milk protein components are precipitated, and there is a problem that the remaining many effective proteins do not precipitate. Conversely, when the concentration of ethanol exceeds 80%, there is a problem in that a lot of impurities are generated while all proteins are precipitated.

In addition, if the working temperature of step B) is less than 2° C., there is a problem that the operation is difficult because it is too low. On the contrary, if it exceeds 8° C., it is not preferable because there is a possibility that the protein component may be denatured.

In the last step C), the precipitate is centrifuged to obtain a wet body, and the wet body is washed with ethanol having a concentration of 80 to 90%, separated and dried to obtain the sea urchin body fluid extract of the present invention.

Here, if the concentration of ethanol is less than 80%, the effective protein contained in the wet body is dissolved and there is a fear that the yield may decrease. It is undesirable because it only rises.

The sea urchin body fluid extract prepared by the above method was obtained in powder form, and it was confirmed that the concentration of vanadium was 10 to 25 ppm. In addition, it was confirmed that the sea squirt body fluid extract inhibits the activity of malic enzyme in vivo, thereby inhibiting the synthesis of body fat, and preventing and improving obesity.

Hereinafter, the present invention will be described in detail according to preferred embodiments. However, since the following examples illustrate the present invention in more detail, the protection scope of the present invention is not limited by these examples.

Fresh sea squirt body fluid was collected and centrifuged to obtain 30 liters of a supernatant from which solids were removed. To the supernatant, 90 liters of 95% alcohol was added dropwise while stirring slowly to adjust the ethanol concentration to 75%, and it was left at 4° C. for 16 to 20 hours to confirm that a precipitate was formed.

Then, the precipitate was centrifuged (8xg, 20min, 4°C) to obtain 872 g of a wet body. The wet body was washed with 300 ml of 80% ethanol at a temperature of 4° C. for 30 minutes, and centrifuged again (8×g, 20 min, 4° C.) to obtain 781 g of a wet body from which impurities were removed. The wet body was vacuum-dried at a temperature of 50° C. for 5 hours to obtain 285 g of a powdery sea urchin body fluid extract.

The fresh sea urchin body fluid was filtered through a filter press coated with diatomaceous earth to obtain 30 liters of a filtrate from which solids were removed. To the supernatant, 90 liters of alcohol (95%) was added dropwise while stirring slowly to adjust the concentration of ethanol to 75%, and it was confirmed that a precipitate was formed by allowing it to stand at 4°C for 18 hours.

Then, the precipitate was centrifuged (8xg, 20min, 4°C) to obtain 912 g of a wet body. The wet body was washed with 300 ml of 80% ethanol at a temperature of 4° C. for 30 minutes, and centrifuged again (8×g, 20 min, 4° C.) to obtain 826 g of a wet body from which impurities were removed. The wet body was vacuum-dried at a temperature of 50° C. for 5 hours to obtain 331 g of a powdery seaweed extract.

[Test Example]

1) Determination of vanadium content

The content of vanadium contained in the sea urchin bodily fluid extract obtained according to Examples 1 and 2 was analyzed and measured using ICP-OES (Inductively Coupled Plasm Optical Emission Spectrometry, Perkin Elmer/OES-8300). A certain amount (0.1~0.5g) of each sample is collected from the sea squirt body fluid extract, put into a microwave digestion system (Mars/ Mars 5), nitric acid (HNO ₃ ) 7mL and hydrogen peroxide 1mL are added, and the Digestion program (5min, 80 °C, 1000W → 5min, 50°C, 1000W → 15min, 190°C, 1000W → 20min, 190°C, 1000W).

After cooling the decomposed sample, a certain amount is taken into the mass flask, and then the vanadium element is ionized by inductively coupled plasma using ICP-OES (Inductively Coupled Plasm Optical Emission Spectrometry, Perkin Elmer/OES-8300), and the ionized element The separated ion spectra and its intensity were analyzed according to the mass/charge ratio (m/z) of

Vanadium standards were prepared at concentrations of 5, 1, 0.5, and 0.1 mg/L, respectively, to prepare standard solutions for calibration curves (Quality control standard 21/Perkin Elmer/99%). The standard solution for the calibration curve was analyzed by ICP-MS, and the peak data was plotted to prepare a standard calibration curve for quantitative analysis. The concentration value diffused with respect to the standard calibration curve is obtained, and the content (g) and concentration (ppm) of vanadium for the samples are calculated by multiplying the dilution factor (50 to 100), and then the results are listed in Table 1 did.

division Vanadium concentration (content) of sea squirt body fluid Yield of sea squirt body fluid extract Vanadium Concentration (Content) of Sea Crab Body Fluid Extract of vanadium
Acquisition yield Example 1 0.32 ppm (9.6 mg) 285g 11.8 ppm (3.36 mg) 35.0% Example 2 0.53 ppm (15.9 mg) 331g 20.0 ppm (6.62 mg) 41.6%

In [Table 1], the 'vanadium yield (%)' is the value obtained by dividing the 'vanadium content (mg) of the sea squirt body fluid extract' by the 'vanadium content (mg) of the sea squirt bodily fluid (mg)' is expressed as a percentage (%). . And the reason that the vanadium concentration of the sea squirt body fluid used in Example 1 and the sea squirt body fluid used in Example 2 is different is presumed to be because the body fluids of sea squirt harvested in different seasons in Examples 1 and 2 were used. .

2) Malic enzyme activity inhibition efficacy test

If you refer to the “Help in reducing body fat” section of “Health functional food functional evaluation guide (2012.03)” issued by the Ministry of Food and Drug Safety, it is known that malic enzyme is involved in the synthesis of body fat. That is, body fat synthesis undergoes fatty acid synthesis and triglyceride synthesis, and at this time, fatty acid synthesis mainly proceeds in the cytoplasm of the liver. Acetyl CoA is produced from glucose by the action of malic enzyme in mitochondria, and the Acetyl CoA combines with oxaloacetate to form citrate in order to move to the cytoplasm where fatty acid synthesis occurs.

After the citrate is transported to the cytoplasm, it is again decomposed into oxaloacetic acid and Acetyl CoA. The Acetyl CoA produces malonyl CoA by the action of Acetyl CoA carboxylase, and then synthesizes fatty acids by the action of the Fatty acid synthase complex. Therefore, when the activity of malic enzyme acting on the mitochondria is inhibited, the synthesis of fatty acids can be inhibited.

Based on this metabolic mechanism, the following test was conducted to determine how the sea squirt body fluid extract prepared according to the present invention affects the activity of malic enzyme. 100 mg/kg of the sea urchin body fluid extract obtained in Example 2 was orally administered once to obese zucker rats, and for the control group, 10 mg/kg, 30 mg/kg, and 90 mg/kg of vanadium sulfate (VS) was administered. Each was orally administered once.

At 24 hours and 120 hours after administration, the blood of the obese rats was collected, respectively, and the change in the concentration of malic enzyme in the blood was measured, and the results were recorded in the graph of FIG. 1 attached. In the graph of FIG. 1, the blue bar graph shows the inhibitory effect (%) after 24 hours, and the orange bar graph shows the inhibitory effect (%) after 120 hours. In addition, 'sea squirt extract' is a test group administered with 100 mg/kg of the sea squirt body fluid extract of the present invention, and 'VS 10 mg/kg, VS 30 mg/kg, and VS 90 mg/kg' is vanadium sulfate 10 mg/kg and 30 mg/kg, respectively. and a control group administered with 90 mg/kg.

As a result of the test, in the control group, it was found that the inhibitory effect on malic enzyme increased as the dose of vanadium sulfate (VS) increased. And when the dose of vanadium sulfate (VS) was small, 10 mg/kg, the inhibitory effect was maintained until 24 hours after administration, but when the dose was increased to 30 mg/kg and 90 mg/kg, the inhibitory effect was maintained up to 120 hours. appeared to be maintained.

In addition, the test group administered with 100 mg/kg of the sea urchin bodily fluid extract of the present invention showed overall better efficacy than the control group, and in particular, the inhibition of malate enzyme at the time of 120 hours rather than the time of 24 hours. It has been shown to further increase the efficacy.

3) Obesity prevention efficacy test

In order to confirm the effect of the sea urchin bodily fluid extract prepared according to the present invention on body fat synthesis and weight gain, the sea urchin bodily fluid extract of Example 2 was administered to lean zucker rats and obese zucker rats, respectively. After a single oral administration of 100 mg/kg, the concentrations of non-esterified fatty acid (NEFA) and triglyceride (Tg), which are functional biomarkers for reducing body fat, and the change in body weight of mice were measured for 5 days (120 hours).

To the control group, 10 mg/kg, 30 mg/kg, and 90 mg/kg of vanadium sulfate (VS) were orally administered once, respectively, and changes in NEFA and Tg and changes in body weight were measured in the same manner. Attached Figure 2 is to record the concentration change of NEFA (Non-esterified fatty acid) for obese mice, Figure 3 is to record the concentration of Tg (Triglyceride) for obese mice.

2 and 3, 'fatty-seaweed' is a test group of obese rats administered 100mg/kg of the sea squirt body fluid extract of the present invention, and 'fatty-10, fatty-30, fatty-90' is vanadium sulfate 10mg/kg, respectively. A control group of obese rats administered with kg, 30 mg/kg, and 90 mg/kg.

As a result of the test, the concentrations of NEFA and Tg were significantly decreased until 12 hours after sample administration in obese rats (fatty_sea squirt) administered once 100 mg/kg of the sea squirt body fluid extract of the present invention, and then increased again after 12 hours. appeared to do The control group (fatty_10/fatty_30) administered with 10 mg/kg and 30 mg/kg of vanadium sulfate showed a pattern almost similar to that of the test group administered with 100 mg/kg of the sea urchin bodily fluid extract of the present invention, but 90 mg/kg of vanadium sulfate was administered. The single-administered control group (fatty_90) kept the concentrations of NEFA and Tg low until 120 hours had elapsed.

On the other hand, Figure 4 is a graph showing the change in body weight of obese and normal rats, 'fatty-sea squirt extract' is a test group of obese rats administered with 100 mg/kg of the sea squirt body fluid extract of the present invention, and 'lean- sea squirt extract' is This is a normal mouse test group administered with 100 mg/kg of the sea urchin body fluid extract of the present invention. And, 'fatty-VS10, fatty-VS30, and fatty-VS90' were control groups of obese rats administered 10 mg/kg, 30 mg/kg, and 90 mg/kg of vanadium sulfate, respectively, and 'lean-VS10, lean-VS30, lean-VS90' ' is a control group of normal rats administered 10 mg/kg, 30 mg/kg, and 90 mg/kg of vanadium sulfate.

As shown in FIG. 4 , when 100 mg/kg of the sea urchin body fluid extract of the present invention was administered once, weight gain was very low in both fatty and lean rats until the 3rd day, but after the 4th day, the body weight rapidly increased. increased. A control group (VS 10mg/VS 30mg/VS 90mg) administered with 10 mg/kg, 30 mg/kg and 90 mg/kg of vanadium sulfate similarly suppressed weight gain in rats.

Claims (5)

A) solid-liquid separation of sea squirt body fluid at a temperature of 2 to 8° C. to obtain only liquid components;
B) adding high-concentration ethanol to the liquid component so that the concentration of ethanol is 70-80%, and leaving it at a temperature of 2-8° C. for 16-20 hours to form a precipitate in which proteins are aggregated;
C) centrifuging the precipitate to obtain a wet body, washing the wet body with ethanol having a concentration of 80 to 90%, separating and drying the wet body;
A method for producing a sea squirt body fluid extract, comprising:
The method according to claim 1, wherein in step A), the sea squirt body fluid is centrifuged at a temperature of 2 to 8° C. and the supernatant is separated from solid and liquid.
[Claim 2] The method of claim 1, wherein in step A), the sea squirt body fluid is filtered under pressure using a filter press coated with diatomaceous earth at a temperature of 2 to 8° C. Method for producing sea squirt body fluid extract.
According to claim 1, wherein the high-concentration ethanol in step B) uses alcohol having a concentration of 95%, and 2-4 times the volume ratio of the liquid component is added to increase the concentration of ethanol to 70-80%. A method for producing a sea squirt body fluid extract, characterized in that it is matched.
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