KR20190118823A - Health functional food for protecting liver and preparing method thereof - Google Patents

Abstract

The present invention relates to a hepatoprotective health functional food for liver protection, which comprises chymotrypsin of beta-casein and trypsin hydrolyzate, thereby having excellent antioxidant activities for hepatocytes, not exhibiting cytotoxicity and exhibiting an excellent hepatocellular protective effect.

Description

HEALTH FUNCTIONAL FOOD FOR PROTECTING LIVER AND PREPARING METHOD THEREOF}

The present invention relates to a dietary supplement for liver protection and a method of manufacturing the same.

In addition to nutritional value, milk has recently attracted much attention on bioactive substances with various functionalities contained in milk components. Especially, various functional peptides that can be derived from milk proteins casein and whey proteins are used for human metabolism. It has been found to play a very important physiological role in functioning and maintaining health. Milk-derived peptides have been studied to have a variety of biologically active functions such as antioxidant activity, antimicrobial activity, and immune enhancement. Peptides that exhibit physiological activity among the functional components of milk are naturally produced during the digestion of milk proteins in the body, and microorganisms in the case of artificially decomposing milk proteins by adding proteolytic enzymes in the manufacturing process of food and during fermentation of dairy products This secretion is also produced by the action of proteolytic enzymes.

The liver is a key organ in the body that is responsible for metabolism, secretion, storage and detoxification, and liver damage means this impairment of liver function. Liver diseases are mainly caused by toxic compounds, excessive alcohol intake, infections and autoimmune diseases. Reactive oxygen species (ROS), such as nitric oxide, hydroxyl, and superoxide, are continuously produced as a byproduct of metabolic processes in vivo and are unstable and highly reactive. It has antioxidant enzymes such as catalase, glutathione peroxidase, glutathione S-transferase and superoxide dismutase. Cells or tissues are damaged when excessive free radicals are generated or the balance is broken in vivo. Unbalanced free radicals oxidize lipids, denature proteins, destroy cell membranes, damage DNA, and cause cancer, as well as cataracts, diabetes, hepatitis and nephritis.

As mentioned above, as the risk of liver disease is raised by the harmful environment, there is a growing interest in food materials having a small amount of side effects and protecting liver cells derived from natural products which may have a positive recognition.

Korean Patent Publication No. 10-2017-0113809

It is an object of the present invention to provide a dietary supplement for liver protection having excellent antioxidant activity against the liver.

1. A dietary supplement for liver protection comprising chymotrypsin and trypsin hydrolyzate of beta-casein.

2. In the above 1, wherein the hydrolyzate is a liver functional health functional food is a fraction of 1000Da to 3000Da.

3. Method of producing a dietary supplement for liver protection comprising treating the beta-casein solution with chymotrypsin and trypsin to hydrolyze beta-casein.

4. The method according to the above 3, wherein the hydrolyzate is first ultrafiltered with a molecular weight of 3000 Da, the ultrafiltration of the filtrate to a second molecular weight of 1000 Da further comprising the step of producing a dietary supplement for liver protection.

The beta-casein hydrolyzate according to the present invention has excellent antioxidant activity against hepatocytes, but does not exhibit cytotoxicity. Thus, it can exhibit an excellent hepatocyte protective effect.

1 is an ABTS radical scavenging activity of a casein hydrolyzate. ad was statistically significant at p <0.05 and each value was expressed as mean ± standard deviation (n = 3).
Figure 2 shows the reducing power according to the FRAP measurement of casein hydrolyzate in Trolox equivalents. Each value is expressed as mean ± standard deviation (n = 3), and the other characters indicate statistical significance at p <0.05.
3 shows the effect of hydrolyzate on the viability of HepG2 cells. Each value is expressed as mean ± standard deviation (n = 3), and the other characters indicate statistical significance at p <0.05.
Figure 4 shows the effect of hydrolyzate on superoxide dismutase-1 (SOD-1) gene expression in HepG2 cells. Each value is expressed as mean ± standard deviation (n = 3), and the other characters indicate statistical significance at p <0.05.
Figure 5 shows the effect of hydrolyzate on superoxide dismutase-2 (SOD-2) gene expression in HepG2 cells. Each value is expressed as mean ± standard deviation (n = 3), and the other characters indicate statistical significance at p <0.05.
6 shows the effect of hydrolyzate on catalase (CAT) gene expression in HepG2 cells. Each value is expressed as mean ± standard deviation (n = 3), and the other characters indicate statistical significance at p <0.05.
Figure 7 shows the effect of hydrolyzate on the expression of glutathione peroxidase (GPx) gene in HepG2 cells. Each value is expressed as mean ± standard deviation (n = 3), and the other characters indicate statistical significance at p <0.05.

Hereinafter, the present invention will be described in detail.

The present invention relates to a dietary supplement for liver protection.

The hepatoprotective dietary supplement of the present invention comprises chymotrypsin and trypsin hydrolyzate of beta-casein.

Beta-casein (beta-casein) is a protein contained in the milk, the beta-casein according to the present invention is not limited in its origin, it may be obtained or synthesized from milk by a method known in the art.

Hydrolysates according to the invention are chymotrypsin and trypsin hydrolysates of beta-casein.

Chymotrypsin may be alpha-chymotrypsin.

The hydrolyzate of beta-casein is hydrolyzed to chymotrypsin and trypsin, hydrolyzed to chymotrypsin and trypsin, or hydrolyzed to trypsin and then to chymotrypsin, or hydrolyzed in the presence of both enzymes It may be.

The hydrolyzate according to the invention may be a fraction. For example, it may be a fraction having a molecular weight of 1000 Da to 3000 Da. The fraction may be obtained by ultrafiltration, but is not limited thereto.

In addition, the hydrolyzate according to the present invention may be in the form of lyophilized powder, but is not limited thereto.

The functional health food of the present invention has been newly defined through the "2002 Act on Health Functional Food", and the health functional food raw material prescribed in the KFDA notice No. 2004-12 has been fully established. Or health foods listed in the Inventory of Regulations for Ingredient Recognition.

The health functional food of the present invention may be various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, health functional foods, etc., to which the hydrolyzate is added.

The health functional food of the present invention includes the hydrolyzate, and further includes at least one of a carrier, a diluent, an excipient, and an additive, and is selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations. It can be formulated as one.

Specific examples of the carrier, excipient, diluent and additives include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium phosphate, calcium Silicates, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate And one or more selected from the group consisting of mineral oils.

In formulating the health functional food of the present invention, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.

The content of the extract according to the present invention as an active ingredient in the above-described formulations can be appropriately adjusted according to the use form and purpose, patient condition, type of symptom and severity, etc., and 0.001 to 99.9 wt% based on solids weight, preferably 0.01 to 50% by weight, but is not limited thereto.

The present invention also relates to a method for producing a dietary supplement for liver protection.

The method for preparing a dietary supplement for liver protection of the present invention comprises the step of hydrolyzing beta-casein by treating beta-casein with chymotrypsin and trypsin.

Beta-casein (beta-casein) is a protein contained in the milk, the beta-casein according to the present invention is not limited in its origin, it may be obtained or synthesized from milk by a method known in the art.

The reaction can be carried out in a solvent, the solvent can for example be water. The water may be, for example, primary, secondary or tertiary distilled water, but is not limited thereto.

The solvent may have a pH of, for example, 7 to 8 for promoting chymotrypsin and trypsin activity, and more specifically, a pH of 7.2 to 7.6.

Chymotrypsin may be alpha-chymotrypsin.

Chymotrypsin and trypsin are treated with beta-casein, which can be processed sequentially or simultaneously, the order of which is not limited.

The hydrolysis may be performed at, for example, 30 to 40 ° C., for example, 32 to 38 ° C., for example, but is not limited thereto.

The hydrolysis time may be appropriately performed in consideration of beta-casein, enzyme amount, solvent amount, and the like, for example, 1 hour to 48 hours, but is not limited thereto.

The method of the present invention may further comprise ultrafiltration of the hydrolyzate after the hydrolysis.

Ultrafiltration can be first filtered to 3000 Da after ultrafiltration, and the filtrate can be ultrafiltered to 1000 Da second. Thereby a fraction of molecular weight 1000 Da to 3000 Da can be obtained.

The filtrate may be lyophilized as necessary to obtain a powder.

The method of the present invention may further comprise the step of formulating the filtrate obtained by the above method by a known method, or adding it to a food.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example

material

The β-casein (From bovine milk), α-chymotrypsin (From bovine pancreas), and Trypsin (From bovine pancreas) used in this study were purchased from Sigma-Aldrich (USA).

Way

1. Enzymes from β-casein Hydrolyzate  Separation

150 mL of distilled water is adjusted to pH 7.4 using a pH meter (pp-15, satorius), and β-casein is prepared at a concentration of 2 mg / mL. Thereafter, 3 mg of α-chymotrypsin and 3 mg of trypsin are dissolved in the prepared solution, and then hydrolyzed with stirring at 37 ° C. for 16 hours. UF treatment was performed using a Stirred Ultrafiltration Cell (Model 8200, Millipore). Membrane filter size was separated once with a filter of 3000 Da or less, and then separated by a filter of membrane filter size 1000 or less to obtain a hydrolyzate having a molecular weight of 1000 Da to 3000 Da. The hydrolyzate was lyophilized (fd-1000, Eyela) to obtain a hydrolyzate.

2. ABTS ⁺  Radical Scavenging power

Preparation of Potassium persulfate (Daejung, Korea) with 140 mM of 7 mM 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, sigma Chemical Co., St. Louis, MO, USA) After mixing 5 mL of ABTS solution and 88 μL of Potassium persulfate and reacting for 12 to 16 hours in the dark, a working ABTS + solution was prepared. The absorbance was diluted with ethanol to a absorbance value of 0.7 ± 0.02 at 734 nm and used as a reagent. 100 μL of the hydrolyzate by concentration and 100 μL of the working ABTS ⁺ solution were mixed in a 96-well-plate for 30 minutes, and then absorbance (Epoch, BioTek, USA) was measured at 734 nm to indicate radical scavenging rate (%). It was.

3. FRAP ( Rerric  Reducing Ability of Plasma Measurement

300 mM acetate buffer (1) was prepared using sodium acetate (C ₂ H ₃ NaO _{2 ·} 3H ₂ O, Riedel-de Haen, Germany) and acetic acid, and the pH was adjusted to 3.6 to 10 mM dissolved in 40 mM HCl. TPTZ (2,4,6-tripyridyl-s-triazine, sigma Chemical Co., St. Louis, MO, USA) (2) and 20 mM Ferric chloride (FeCl ₃ .6H ₂ O) (3) : (2): (3) = 10: 1: 1 ratio was mixed and stabilized for 30 minutes at 37 ℃ used as a reagent. 60 μL of hydrolyzate by concentration and 140 μL of FRAP were mixed in 96 well-plate, reacted at 37 ° C. for 30 minutes, and the absorbance (Epoch, BioTek, USA) was measured at 593 nm. Reducing power was expressed as Trolox equivalent (μM) using the Trolox equivalent antioxidant capacity (TEAC) method.

4. MTT  Cell survival rate

96-well plate using DMEM culture medium in the received pre-sale from the ATCC The supernatant was removed and incubated for 24 hours at 5 × 10 ⁴ cells / well of the HepG2 cells by dividing the 37 ℃, 5% CO ₂ conditions and 2 in PBS Wash twice. After dispensing the hydrolyzate by concentration in each well and incubated for 24 hours at 37 ℃, 5% CO ₂ conditions. After removing the culture solution, 20 μL of 5 mg / mL MTT solution and 180 μL of PBS were dispensed and reacted at 37 ° C. and 5% CO ₂ for 4 hours. After the reaction, all the supernatant was removed, and 200 μL of DMSO was added to dissolve the produced formazan, which was then measured at 550 nm using an ELISA reader. The cell survival rate (%) was used as follows.

[Equation 1]

% Cell viability = (absorbance of sample treated group / absorbance of control group) × 100

5. Total RNA Extraction and cDNA Synthesis

Dispense 5 × 10 ⁵ cells / well HepG2 cells in a 24-well plate using DMEM medium, incubate for 24 hours at 37 ° C, 5% CO ₂ , and add hydrolysates and 5% Ethanol to each well. 24 hours treatment. Since the supernatant was removed and the collected cells were RNA extracted using RNeasy Mini Kit (Qiagen), RNase-free DNase set (Qiagen) was used to remove genomic DNA. The extracted total RNA was prepared cDNA using qPCR RT Master Mix (Toyobo) according to the provided protocol. PCR reaction conditions were carried out in order of maintaining at 4 ℃ 15 minutes at 37 ℃, 5 minutes at 50 ℃, 5 minutes at 98 ℃.

6. Real-time PCR  Gene expression analysis using

To examine the effects of hydrolyzate on gene expression of antioxidant enzymes, superoxide dismutase-1 (SOD-1, Cu-Zn SOD), Superoxide dismutase-2 (SOD-2, Mn SOD), catalase (CAT) and glutathione peroxidase MRNA expression levels of (GPx) were analyzed using real-time PCR. Primer sequences of antioxidant enzymes are shown in Table 1. Real-time PCR reaction solution was used with the cDNA extracted after mixing Rotor-Green SYBR Green Master mix (QIAGEN), inflammatory response genes -F, -R primer (10 pmol / μL), H ₂ O. Real-time PCR used Qiagen's Rotor-Gene Q 2 plex, and the reaction conditions were repeated 40 cycles of 5 seconds at 95 ℃ and 10 seconds at 60 ℃ based on the two step cycling protocol, and from 60 ℃ to 95 ℃. The melting step was followed by increasing melting for 3 minutes. The product amplified by real-time PCR was quantified using the Delta delta CT method, each sample was corrected by the expression level of β-actin.

primer SEQ ID NO: order Length
(bp) SOD-1 One 5'-ACGGTGGGCCAAAGGATGAA-3 ' 151 2 5'-TCATGGACCACCAGTGTGCG-3 ' SOD-2 3 5'-AGAAGCACAGCCTCCCCGAC-3 ' 152 4 5'-GGCCAACGCCTCCTGGTACT-3 ' GPx 5 5'-TCGGTGTATGCCTTCTCGGC-3 ' 150 6 5'-CCGCTGCAGCTCGTTCATCT-3 ' CAT 7 5'-CCAACAGCTTTGGTGCTCCG-3 ' 180 8 5'-GGCCGGCAATGTTCTCACAC-3 '

-actin

-actin 9 5'-CGGGAAATCGTGCGTGACAT-3 ' 198 10 5'-GGACTCCATGCCCAGGAAGG-3 '

7. Statistical Analysis

The experimental results were analyzed by ANOVA using SPSS 22.0 (SPSS Inc., Chicago, IL, USA), and the post hoc analysis for comparison between groups was verified by Tukey's b, and it was considered statistically significant only when P <0.05 or more. . All analysis items were expressed as mean ± standard deviation of the results obtained by repeating the test three times or more.

result

One. ABTS ⁺  Radical Scavenging power

ABTS assay is a method of measuring the antioxidant power by measuring the radical scavenging ability of the sample after generating radicals by reacting ABTS with Potassium persulfate. As a result of measuring ABTS radical scavenging ability of peptide using this principle, the concentration of hydrolyzate was 26.27% at 17.5 μg / mL, 46.62% at 25 μg / mL, 67.44% at 50 μg / mL, and 84.55% at 100 μg / mL. The scavenging rate appeared and significantly increased the radical scavenging rate (see Fig. 1).

2. FRAP ( Rerric  Reducing Ability of Plasma Measurement

The FRAP assay measures the reducing power to the extent that the sample reduces Fe ₃ to Fe _{2. The} FRAP assay measures the degree to which the reagent turns to blue as the reagent close to yellow is reduced. Trolox is a vitamin E family and has a strong antioxidant power and is widely used as a standard for antioxidant experiments. As a result of measuring the reducing power of the hydrolyzate and expressed as Trolox equivalent (μg / mL), the peptide showed a significant increase in reducing power in a concentration-dependent manner, and showed the highest reducing power at 100 μg / mL (see FIG. 2).

3. MTT  Cell survival rate

To determine the cytotoxicity of the hydrolyzate, cell viability was analyzed using HepG2 cells. As a result, the cell viability of 95% was observed at the concentration of 17.5-100 μg / mL of the hydrolyzate, which did not significantly affect cytotoxicity (see FIG. 3). Therefore, the experiment was conducted at a concentration that the hydrolyzate does not affect the cell viability of HepG2 cells.

4. HepG2  Effect on antioxidant enzyme gene expression

HepG2, a hepatic cancer cell line, plays the same functional role as some normal hepatocytes and can be used for both normal hepatoprotective effect analysis and hepatocellular growth inhibition experiments. Therefore, the expression levels of four kinds of antioxidant enzymes were analyzed by real-time PCR in order to investigate the hepatocellular protective effect from ethanol-induced oxidative stress and liver damage using HepG2 cell line. In the case of SOD-1, there was a significant difference of about 20% at the treatment concentration of 25 μg / mL of the hydrolyzate than the control treated with ethanol alone. In addition, it was confirmed that the expression level of SOD-1 significantly increased with increasing concentration (Fig. 4). In the case of SOD-2, the expression level was not significantly different from that of the control at the concentration of peptide 100 μg / mL, and the expression level was increased as the concentration of hydrolyzate was increased similarly to SOD-1 (FIG. 5). CAT showed no significant difference between the control treated with ethanol and the treated concentration of 25 μg / mL of the hydrolyzate, but the expression level was significantly higher than that of the ethanol-treated control at the concentration of 100 μg / mL. Can be. In addition, when compared with the control group, there was no significant difference with the hydrolyzate 100 μg / mL (Fig. 6). In GPx, the mRNA expression was increased in proportion to the treatment concentration, and 50 μg / mL and 100 μg / mL of the hydrolyzate did not show a significant difference (FIG. 7).

<110> KANGWON NATIONAL UNIVERSITY INDUSTRY COOPERATION FOUNDATION <120> HEALTH FUNCTIONAL FOOD FOR PROTECTING LIVER AND PREPARING METHOD          THEREOF <130> 2017-110 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOD-1 <400> 1 acggtgggcc aaaggatgaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOD-1 <400> 2 tcatggacca ccagtgtgcg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOD-2 <400> 3 agaagcacag cctccccgac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOD-2 <400> 4 ggccaacgcc tcctggtact 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GPx <400> 5 tcggtgtatg ccttctcggc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GPx <400> 6 ccgctgcagc tcgttcatct 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAT <400> 7 ccaacagctt tggtgctccg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAT <400> 8 ggccggcaat gttctcacac 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta actin <400> 9 cgggaaatcg tgcgtgacat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta actin <400> 10 ggactccatg cccaggaagg 20

Claims (4)

Hepatoprotective dietary supplement comprising beta-casein chymotrypsin and trypsin hydrolyzate.
The liver functional health functional food of claim 1, wherein the hydrolyzate is a fraction having a molecular weight of 1000 Da to 3000 Da.
Method of producing a dietary supplement for liver protection comprising the step of hydrolyzing beta-casein by treating beta-casein chymotrypsin and trypsin.
The method of claim 3, further comprising ultrafiltration of the hydrolyzate with 3000 Da and second ultrafiltration of the filtrate again with 1000 Da.

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