KR20130130114A - Rice peptide for liver protection - Google Patents

Abstract

The present invention relates to a rice peptide which includes a peptide, glutamic acid, and branched amino acid, has liver protection activity against chemicals or toxic substances showing liver cell toxicity, has improved water solubility, reduces bitter tastes, and can be used as health functional food or pharmaceutical compositions for preventing or treating liver toxicity.

Description

Rice peptides with liver protection {Rice peptide for liver protection}

The present invention relates to rice peptides having liver protective function, more specifically rice peptides produced from rice syrup foil, which are excellent in water solubility and bitter taste, and have a liver protecting activity from chemical substances or toxic substances exhibiting hepatocyte toxicity ≪ / RTI >

According to the National Statistical Office (NSO) survey, domestic rice production increased by 4.7% in 2008 compared to 2007, but domestic consumption of rice decreased by 2.4% in 2007 from 76.9 kg in 2007.

Since the consumption of rice is decreasing every year while the amount of rice is increasing, the remaining rice is used to make and sell Takju, etc. The development and production of edible oil and phytoremediation using rice are gradually increasing, Is generated. Processing of byproducts of rice is costly and environmental problems are followed, so it is mostly used for feed or compost.

Therefore, there is a great need for research that can reuse by-products to develop new high value added materials and products and utilize byproducts.

Rice proteins are now attracting worldwide attention because they are free of allergenic problems and are rich in nutritional value compared to soy protein. In the case of soybean-based materials currently in widespread use, there is a problem related to GMO, while in the case of rice, there is no problem of GMO.

Although a number of overseas companies including Sunrich USA are conducting research on rice proteins, there have been no specific developmental achievements so far, and researches using byproducts have been actively conducted in various countries including Southeast Asia where rice is a main source, Research on rice-derived proteins is limited to the United States and Pakistan, and there is a limit to commercialization due to technical limitations.

In addition, considering the present domestic market situation, the commercial rice protein products have problems related to the water-solubilization of proteins, problems in raw material supply and taste, and therefore consumers are very concerned about the use of materials. However, In fact, it has not been revived in its demand.

Meanwhile, the liver is located between the internal digestive system and the systemic circulatory system, and plays an important role in protecting the human body from harmful substances introduced from the outside and taking charge of the metabolism of the exogenous substance. Since the in vivo substance entering the living body once passes through the liver, the liver is exposed to various harmful substances in addition to the nutrients, and the liver itself is more likely to be diseased than other organs.

Recently, as the level of pollution caused by industrialization becomes serious, the human body is exposed to various harmful substances such as various drugs and volatile organic compounds, and these various harmful substances are introduced into the body during the metabolic process, Most of the harmful substances that are infiltrated are decrypted through the liver. However, when the harmful substances are introduced into the body and then transferred to a strong free radical, the transferred free radicals combine with oxygen to form highly toxic peroxides. Therefore, the peroxides have serious adverse effects on the liver and cause liver damage And is involved in various pathological conditions.

As the risk of liver disease caused by harmful substances is raised, there is a growing interest in pharmaceutical compositions or health foods for the cure of liver diseases using various natural materials having small side effects or rejection reactions to the human body.

For the food or medicinal composition for liver protection using natural materials, it is disclosed in Korean Patent Laid-Open No. 10-2010-0106880, which is based on the extract of Hodguswiensis japonica extract, Korean Patent Laid-open No. 10-2010-0074881, And Japanese Patent Laid-open Publication No. 10-2009-0095134, and Korean Patent Laid-Open No. 10-2008-0091627, , Korean Patent No. 10-0661481, and Korean Patent No. 10-0661487. However, rice, rice protein, and hydrolyzate thereof are also disclosed. It is not known that rice peptides exhibit activity to inhibit hepatotoxicity against chemicals or toxic substances exhibiting hepatocellular toxicity.

It is an object of the present invention to provide a rice peptide having hepatoprotective activity from a chemical substance or a toxic substance exhibiting hepatocellular toxicity.

The present invention relates to a peptide comprising a peptide having a molecular weight of 300 to 700 Da of at least 80% by weight of the entire peptide, glutamic acid comprising 15 to 25% by weight of the total amino acid and 12 to 20% There is provided a rice peptide having liver protecting activity from a chemical substance or a toxic substance exhibiting hepatocellular toxicity.

The rice peptide having hepatoprotective activity from a chemical substance or a toxic substance exhibiting hepatocyte toxicity of the present invention is characterized in that the content of amino nitrogen is 10 to 1000 mg%.

The rice peptide having hepatoprotective activity from a chemical substance or a toxic substance exhibiting hepatocyte toxicity of the present invention is characterized in that the water solubility at pH 4.6 to pH 12 is 90% or more.

The rice peptides having hepatoprotective activity from a chemical substance or toxic substance exhibiting hepatocyte toxicity according to the present invention include 9 to 12 parts by weight of aspartic acid, 3 to 5 parts by weight of threonine, 5 to 7 parts by weight of serine, 19 to 22 parts by weight of glutamic acid, 3 to 5 parts by weight of proline, 4 to 6 parts by weight of glycine, 5 to 7 parts by weight of alanine, 5 to 7 parts by weight of valine, 1 to 3 parts by weight of methionine, 3 to 5 parts by weight of isoleucine, 4 to 6 parts by weight of tyrosine, 5 to 7 parts by weight of phenylalanine, 2 to 4 parts by weight of lysine, 1 to 3 parts by weight of histidine and 7 to 10 parts by weight of arginine.

The rice peptide having hepatoprotective activity from a chemical substance or a toxic substance exhibiting hepatocyte toxicity of the present invention is characterized in that it has a fatigue recovery activity after exercise.

The rice protein of the present invention is characterized in that the rice protein is derived from rice syrup foil in a rice peptide having hepatoprotective activity from a chemical substance or a toxic substance exhibiting hepatocellular toxicity.

The present invention also relates to a process for the treatment of alpha-amylase which comprises reacting a rice syrup foil with an alpha-amylase; Removing the water-soluble material to wash the rice syrup foil treated with the alpha-amylase; And a proteolytic enzyme treatment step of reacting the washed rice syrup foil with a proteolytic enzyme to produce the rice peptides.

The present invention also provides a food composition for liver protection comprising the rice peptide as an active ingredient.

The liver protecting food composition of the present invention is characterized by being a health functional food in a beverage form.

The present invention also provides a pharmaceutical composition for preventing and treating hepatotoxicity comprising the rice peptide as an active ingredient.

The present invention can be used as a health functional food for liver protection or a pharmaceutical composition for the prevention or treatment of hepatotoxicity because the rice peptides are those having hepatoprotective activity from chemical substances or toxic substances exhibiting hepatocellular toxicity and excellent in water solubility and bitter taste .

Fig. 1 is a MS chromatogram obtained by confirming the rice peptides of the examples in Example 8 by molecular weight fraction. Fig.
Fig. 2 is a mass spectrum of a rice peptide having a molecular weight of 300 to 1000 Da in Experimental Example 8. Fig.
Fig. 3 is a graph showing the water solubility of rice peptides according to Examples in Experimental Example 9 according to pH. Fig.
4 is a graph showing the water solubility of the rice peptides according to the heating temperature and time according to Examples in Experimental Example 9. FIG.
FIG. 5 is a graph showing the cell viability according to the treatment concentration when the rice peptides of the examples of the present invention were treated with the primary cultured hepatocytes (10 ~ 1000 / / mL) in Experimental Example 11 through MTT analysis.
FIG. 6 is a graph showing the cytotoxicity of t-BHP on primary hepatocytes cultured in Experimental Example 12 when the rice peptide of Example was inhibited by cell viability.
FIG. 7 is a graph showing the increase in AST and APT of t-BHP against primary hepatocytes cultured in Experimental Example 12 when rice peptides of Example were added.
FIG. 8 is a graph showing the increase in LDH of t-BHP against primary hepatocyte cultured in Experimental Example 12 when the rice peptide of Example was added.
FIG. 9 is a photograph of liver tissue obtained by hematoxylin & eosin staining to confirm subacute toxicity of rice peptide administration in Example 13 in Experimental Example 13. FIG.
FIG. 10 is a photograph of the renal tissue obtained by periodic acid staining to confirm subacute toxicity of the rice peptide administration of Example 13 in Experimental Example 13. FIG.
FIG. 11 is a photograph showing the liver tissue of each experimental group through hematoxylin & eosin staining in order to confirm whether the liver damage caused by t-BHP is restored by administration of the rice peptide in Example 14 in Experimental Example 14. FIG. B is the 200-fold magnification of the t-BHP group, C is the 400-fold magnification of the white squares of B, and D is the SRP70 0.5 g / kg group E is the 200-fold magnification of the SRP70 1 g / kg group, and F is the 200-fold magnification of the SRP70 2 g / kg group.

The present invention relates to a peptide comprising a peptide having a molecular weight of 300 to 700 Da of at least 80% by weight of the entire peptide, glutamic acid comprising 15 to 25% by weight of the total amino acid and 12 to 20% There is provided a rice peptide having liver protecting activity from a chemical substance or a toxic substance exhibiting hepatocellular toxicity.

The rice peptides of the present invention can be obtained by treating a rice protein with a proteolytic enzyme such that a majority of the peptide, for example, 70% by weight or more, preferably 80% by weight or more, more preferably 90% When the peptide having a molecular weight of 300 to 700 Da is 80% by weight or more of the total peptide, the liquid portion can be used as it is in the enzyme reaction solution of the protease. However, when the amount is more than 80% by weight, For example, an ultrafiltration membrane capable of removing a peptide having a molecular weight of more than 1000 Da can be further passed through the ultrafiltration membrane to concentrate the peptide having a desired molecular weight range.

The rice peptide of the present invention has a molecular weight of at least 80% by weight, preferably at least 90% by weight, more preferably at least 95% by weight, of the peptide of 300 to 700 Da.

The content of amino nitrogen of rice peptides of the rice peptides of the present invention is 10 to 1000 mg%, preferably 800 mg% or less, more preferably 700 mg% or less. Amino nitrogen content is advantageous as it contains less amino acid or short residue peptide causing bitter taste and bad odor, but is usually about 400 to 700 mg% in the peptide of the present invention.

The content of glutamic acid in the rice peptides of the present invention is 15 to 25% by weight of the total amino acids, and the content of branched amino acids is 12 to 20% by weight of the total amino acids, and the content of branched amino acids known to help improve the raw material and exercise capacity of glutathione high.

The rice peptides of the present invention are similar to the amino acid composition of rice or rice protein, but differ in composition from rice bran protein, preferably 9 to 12 parts by weight of aspartic acid, 3 to 5 parts by weight of threonine, 5 to 7 parts by weight of serine 3 to 5 parts by weight of proline, 4 to 6 parts by weight of glycine, 5 to 7 parts by weight of alanine, 5 to 7 parts by weight of valine, 1 to 3 parts by weight of methionine, 3 to 5 parts by weight of isoleucine, 7 to 9 parts by weight of leucine, 4 to 6 parts by weight of tyrosine, 5 to 7 parts by weight of phenylalanine, 2 to 4 parts by weight of lysine, 1 to 3 parts by weight of histidine and 7 to 10 parts by weight of arginine.

The rice peptide of the present invention has a water solubility of 90% or more, preferably 95% or more, at pH 4.6 to pH 12, and is extremely water-soluble even at pH 4.6 which is an acidic region unlike the conventionally available rice protein. Can be easily applied.

The rice peptides of the present invention can be used for the production of hepatocyte toxins such as alanine aminotransferase released from blood upon liver injury caused by liver damage caused by administration of a chemical substance or a toxic substance such as t- BHP ( tert - butylhydroperoxide) Blood plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are restored to normal levels, and cell necrosis caused by t- BHP administration is restored to restore normal hepatocyte survival. The activity of lactase dehydrogenase (LD) is restored to normal level, especially in the animal test results, while the activity of catalase and glutathione peroxidase is restored to normal level and the glutathione content is restored to the original level Liver protection effect.

In addition, the rice peptides of the present invention exhibit increased endurance exercise capacity or exercise fatigue suppressing activity by increasing blood glucose concentration and reducing plasma ammonia concentration upon oral ingestion.

The rice peptides of the present invention include alpha-amylase treatment step of reacting rice syrup foil with alpha-amylase; Removing the water-soluble material to wash the rice syrup foil treated with the alpha-amylase; And a proteolytic enzyme treatment step of reacting the washed rice syrup foil with a proteolytic enzyme.

In the present invention, the rice peptides are preferably prepared from syrup foil. As a byproduct of rice processing, it is possible to use remaining skim milk, for example, makgeolli and remaining skim milk, remaining rice bran, or rice bran oil remaining therefrom, but the protein content Is preferably at least 40% by weight of rice syrup. As a by-product of the rice syrup-containing rice syrup-containing rice syrup-producing process, the rice syrup itself can be used as a raw material to produce rice peptides or hot air dried at 60 to 70 ° C at a moisture content of 10% It is preferable to use them.

In the present invention, first, alpha-amylase treatment is performed to react rice syrup foil, which is a raw material, with alpha-amylase.

In the present invention, when the rice syrup foil is directly reacted with the proteolytic enzyme for the production of rice peptides, browning and insoluble precipitates can be formed by the starch or its decomposition products remaining in the rice syrup foil. If the protein content exceeds 80 wt% High purity products can not be produced.

As the alpha-amylase in the present invention, Termamyl of novozyme is preferred. In the case of tamarimel, the starch decomposition ability is excellent within a short time, and the optimum reaction temperature is 93 ° C. At the reaction at 80 to 100 ° C, the starch decomposition and sterilization of the raw material can proceed simultaneously.

Next, in the present invention, alpha-amylase treatment is performed to react rice syrup foil with alpha-amylase, and rice silage foil treated with alpha-amylase is washed to remove water-soluble substances.

A rice syrup foil treated with alpha-amylase is a liquid reaction mixture in which rice syrup bulk solids, enzyme-degraded water-soluble carbohydrates (mainly monosaccharides, disaccharides or oligosaccharides), and enzymes are mixed. This liquid reaction mixture is centrifuged, Dehydrated using a decanter or the like.

However, since the water-soluble carbohydrate can not be sufficiently removed by simply dehydrating the enzyme reaction solution, the α-amylase treated rice syrup foil is dehydrated and the amount of the dehydrated rice syrup solids is 2 to 30 times, preferably 5 To 15 times of water is mixed and sufficiently stirred, followed by dewatering is repeated 1 to 5 times, preferably 3 times or more, so that the water-soluble carbohydrate is removed and the protein content is increased.

Next, in the present invention, a proteolytic enzyme treatment step of reacting washed rice syrup foil with a proteolytic enzyme is performed in order to remove a water-soluble substance.

The protease of the present invention is preferably novozyme Alcalase or Neutrase.

In the present invention, proteolytic enzyme is added in an amount of 0.01 to 1% by weight, preferably 0.1 to 0.5% by weight, and the mixture is reacted at 50 to 70 ° C for 0.5 to 3 hours.

When the concentration of the protease is less than the lower limit, the protein degradation efficiency is not sufficient. Therefore, the yield of the water-soluble rice peptide is insufficient relative to the amount of rice syrup used and the protein degradation efficiency is increased up to 0.1 wt% Do not increase anymore.

In addition, the longer the reaction time of the proteolytic enzyme, the higher the protein degradation rate, but the increase in the degradation rate after 3 hours is not clear. On the contrary, if the pH is decreased over 3 hours, Loses.

In addition, when the pH is adjusted to 8 to 9 at the initial stage of the enzymatic degradation by the protease, and the reaction is completed at pH 7 to 7.5, there is less concern about the occurrence of side patches and no need for neutralization after completion of the reaction, The salt content of the rice peptides can be prevented from increasing.

In the present invention, in order to obtain the water-soluble rice peptides in the enzymatic reaction solution, the enzyme reaction solution is first subjected to a proteolytic enzyme treatment step, and then the enzyme reaction solution is first heated at 75 DEG C or higher, preferably 85 to 100 DEG C for 10 minutes to 1 hour, After the enzyme is inactivated by heating for 15 to 30 minutes, the solids are removed by centrifugation or filter press in the enzyme reaction solution, and the liquid portion is concentrated and dried to prepare a water-soluble rice peptide.

The present invention provides a food composition for liver protection comprising the above rice peptide as an active ingredient.

In the present specification, "containing as an active ingredient" means that rice peptides are contained in an amount sufficient to achieve the liver protection effect. In one embodiment of the invention, the rice peptides in the composition of the present invention have a concentration of at least 0.001 mg / kg, preferably at least 0.1 mg / kg, more preferably at least 10 mg / kg, even more preferably at least 1 g / kg or more, even more preferably 10 g / kg or more, and most preferably 100 g / kg or more. The rice peptide of the present invention is a natural product obtained by digesting protein derived from rice, which is consumed in large quantities as a stock, by enzymatic decomposition.

When the extract according to the present invention is used as an active ingredient additive for a health functional food or a general food, the extract according to the present invention can be used as it is or can be used together with other food or food ingredients and can be suitably used according to a conventional method . The amount of the active ingredient to be mixed may be suitably determined according to each use purpose such as prevention, health, or treatment.

Generally, the rice peptides according to the present invention can be added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, when the food or beverage is produced. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range. Further, since the present invention is a peptide derived from natural products, there is no problem from the viewpoint of safety, Can also be used.

There is no particular limitation on the type of the food, and examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, , Various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include foods in a conventional sense.

The beverage food of the health functional food according to the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the functional food according to the present invention. Examples of beverage foods include carbonated beverages, fruit juices, vegetable beverages, protein fortified beverages, sports drinks, ionic beverages, beverages, soy milk or rice milk containing rice saccharified liquid as main ingredients.

In addition to the above, the food composition for protecting the liver according to the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, Glycerin, alcohols, and carbonating agents used in carbonated beverages. In addition, the liver protecting food composition of the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. The ratio of such additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the food composition of the present invention.

The present invention provides a pharmaceutical composition for preventing or treating hepatotoxicity comprising the above rice peptide as an active ingredient.

The pharmaceutical dosage form of the pharmaceutical composition for the prevention or treatment of hepatotoxicity can be used in the form of a pharmaceutically acceptable salt thereof and can be used alone or in combination with other pharmacologically active compounds as well as in a suitable aggregate form.

In addition, the pharmaceutical composition for preventing or treating hepatotoxicity according to the present invention may be in the form of an oral preparation, an external preparation, a suppository and a sterilized injection solution such as a powder, granule, a preparation, a capsule, a suspension, an emulsion, a syrup, And may contain suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions for formulation.

The carrier or the excipient or diluent includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In the case of formulation, a diluent or excipient such as a commonly used filler, a weight agent, a binder, a wetting agent, a disintegrant or a surfactant may be used.

Solid preparations for oral administration may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the extract. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition for the prevention or treatment of hepatotoxicity according to the present invention varies depending on the condition of the patient, the body weight, the degree of the disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, it may be administered at 0.0001 to 2,000 mg / kg, preferably at 0.001 to 2,000 mg / kg. Administration may be once a day or may be divided several times. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition for the prevention or treatment of hepatotoxicity according to the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

Hereinafter, the present invention will be described more specifically with reference to the following examples. However, the following examples are provided only to aid understanding of the present invention, and the scope of the present invention is not limited to the following examples.

Experimental Example 1: Analysis of general components of rice processing by-products

As a byproduct of rice processing, sake bran, takju bran, skim rice bran and rice syrup bran were used. Cheongju, takju and rice syrup were used after drying.

However, in the case of the sake oil (moisture content 91.56 wt%) having a relatively high moisture content, when the hot air drying was performed, the sample could not be analyzed due to the bad smell and the mold could not be used because of the bad odor. Wt%) was dried at 50 캜 for 72 hours because hot air drying at a temperature of 60 캜 or higher failed to use the sample due to carbonization.

The components were analyzed by protein, fat, ash, water, and carbohydrate. The components were analyzed by AOAC method. The results were analyzed by Kjeldahl Nitrogen Analysis, Soxhlet method, Dry ashing and Air oven method. Carbohydrates were calculated as the fraction excluding protein, fat, ash and water.

 division rice Takju Defatted rice bran Rice syrup foil Crude protein (% by weight) 6.12-8.67 25.3 ± 0.6 18.7 ± 0.1 45.1 ± 0.9 Crude fat (% by weight) 0.35-0.45 4.3 ± 0.0 4.3 ± 0.0 8.1 ± 0.2 Views min (% by weight) 0.59-0.63 0.4 ± 0.0 11.2 ± 0.2 0.8 ± 0.0 Water (% by weight) 13.22-14.23 6.4 ± 0.2 7.2 ± 0.3 6.3 ± 0.3 Carbohydrates (% by weight) 76.6-79.12 63.6 ± 0.4 58.6 ± 0.3 36.7 ± 0.1

In the case of takju bean, the carbohydrate fraction decreased by yeast degradation, and that of the defatted rice bran increased compared to that of whole rice. Also, in the case of rice syrup leaves, the carbohydrate fraction was markedly decreased due to the carbohydrate saccharification reaction.

Experimental Example 2: Determination of Optimum Reaction Time by Enzyme

In order to further decompose carbohydrates remaining in the rice syrup foil, the optimal reaction time was checked for carbohydrase such as starch, cellulose, and hullulan. The properties, composition, activity, density, optimum pH and optimum temperature of the six enzymes used in the experiments are summarized in Table 2.

division Appearance Constitutive enzyme Minimal activity density Optimum pH Optimum temperature
(℃) Celluclast Brown liquid cellulase 700 EGU / g 1.22 g / mL 4.5-6.0 (4.5) 50-60 (60) Fungamyl 800L brown liquid α-amylase 800 FAU / g 1.25 g / ml 5 (5) 55-60 (60) Liquozyme supra amber liquid α-amylase 135 KNU / g 1.26 g / ml 5.5 to 7.0 (6.5) 50 (50) Promosy D2 brown liquid pullulanse 400 PUN / ml 1.25 g / ml 5 (5) 60 (60) Termamyl type L brown liquid α-amylase 120 KNU / g 1.20 to 1.25 g / ml 6.5 (6.5) 93 (90) Viscozyme L brown liquid β-glucanase
arabanase, cellulase, hemicellulase, xylanase 120 FBG / ml 1.2 g / ml 4.6 (4.6) 44 (44)

To establish the optimal reaction time for each enzyme, 5 g of rice syrup was added to distilled water (28.3 mL) to prepare a 15% suspension, and the environment was adjusted to the optimum pH and optimum temperature of each enzyme. Were reacted at 1.5, 3, 4, 5, 6, 7.5, 9, 10.5, 12 and 24 hours respectively. The optimum reaction time for each enzyme was determined by using the weight loss amount and it is shown in Table 3.

division Celluclast Fungamyl 800L Liquozyme supra Promosy D2 Termamyl type L Viscozyme L Optimum reaction time (hrs) 24 9 4.5 9 1.5 24

Among the α - amylases, the optimum reaction time was the shortest for the tammaril, so it was selected as the enzyme for starch decomposition of rice syrup. In addition, Celluclast and Viscozyme, a complex enzyme, were selected as a cellulolytic enzyme which is expected to have a synergistic effect when used together with tamarind, and their carbohydrate decomposing effects were confirmed by their respective and combined use.

Experimental Example 3: Confirmation of carbohydrate degradation by alpha-amylase treatment

The weight loss of carbohydrates was calculated as the amount of degradation due to the water-solubilization of the carbohydrate fractions using the weight difference between the precipitates before and after the enzyme reaction. The addition amount of the enzyme was 1 wt% of the content of rice syrup solids. In the case of single enzyme treatment, the optimum pH and temperature of the enzymes used in Table 2 were 1.5 hours for teramylase, 24 hours for viscose, And in the case of complex enzyme treatment, the enzyme treatment was performed sequentially from the highest to the lowest temperature. The reaction solution was subjected to centrifugation (3000 g) for 20 minutes, and the weight and precipitate of the precipitate thus obtained were analyzed to compare and analyze changes in protein content.

 division Carbohydrate decomposition amount
( mg / g) Crude protein
(weight%) Rice syrup foil ^- 43.06 Termamyl 85.69 ± 0.60 ^A 70.68 Viscozyme 61.74 ± 7.12 ^C 74.01 Celluclast 68.43 ± 5.13 ^BC 75.00 T + V 68.03 ± 1.54 ^BC 73.69 V + C 64.03 ± 2.40 ^BC 74.62 T + C 69.70 ± 0.90 ^BC 72.40 T + V + C 72.04 + - 4.75 ^B 72.11

In Table 4, T stands for Termamyl, V stands for Viscozyme, and C stands for Celluclast.

It was found that termamyl (85.69 mg / g) had the greatest effect on weight change with single enzyme. This is a larger change in weight than the treatment of all three enzymes (72.04 mg / g). In addition, the protein content of syrup oil increased from 43.06% by weight to more than 70% by treatment with tamarisk.

In addition, it was confirmed that the starch was decomposed in the carbohydrates through the treatment of tammaril, and the protein content was relatively increased due to the weight reduction.

On the other hand, the amount of carbohydrate degradation was lower in biscozyme or cellulase than that of teramil, but the crude protein content could be increased. However, the increase in the crude protein content was small compared to the increase in the reaction time for the enzyme treatment.

Experimental Example 4: Determination of Protein Content Changes by Washing Number after Alpha-Amylase Treatment

0.1% by weight of turmeric was added to the rice syrupy solid content, and the mixture was allowed to react at 90 DEG C for 1.5 hours. Then, the supernatant was removed and dehydrated through a horizontal decanter to obtain a solid product (Production Example 4-1) 10 times the amount of water was added and mixed with stirring for 20 minutes. Then, again dehydrated solid (Preparation Example 4-2: once washing) and 10 times water were further added to Sample 2 and mixed with stirring for 20 minutes (Example 4-3: twice of washing) and Production Example 4-4 of this washing three times, Production Example 4-5 of four times of washing, Production Example 4-6 of five times of washing And the increase of the protein content was confirmed. The results are shown in Table 5.

division Production Example 4-1 Production example 4-2 Production Example 4-3 Production Example 4-4 Production Example 4-5 Production Example 4-6 Crude protein
(weight%) 67.6 72.69 74.14 78.01 78.45 78.61

After the enzymatic reaction, the protein content of the dehydrated solid was 67.6% by weight, and the protein content was increased with each washing of the water soluble substance to 78.01% by weight and the protein content was increased by about 10% by weight. However, even after washing 4 times or more, no increase in protein content could be confirmed.

Experimental Example 5: Determination of Protein Degradation Rate and Bitter Taste According to Protease

In order to confirm the proteolytic enzyme suitable for proteolysis of rice syrup fiber, protein degradation rate using the proteolytic enzyme shown in Table 6 was confirmed.

 division manufacturer Optimum pH Optimum temperature (℃) Protamex novozyme 5.5-7.5 (7) 35-60 (40) Neutrase novozyme 5.5-7.5 (7) 45-55 (45) Flavorzyme novozyme 5-7 (7) 50 (50) Alcalase novozyme 6.5-8.5 (7) 60 (60) Protease M amano 4.5 (4.5) 50 (50) Protease N amano 7 (7) 55 (55) Protease A amano 7 (7) 50 (50) Protease F kikoman 3 (3) 50 (50)

 To 5 g of the washed rice syrup oil of Preparation Example 4-4 was added distilled water (28.3 ml) to prepare a 15% suspension. Using 1 N HCl or 1 N NaOH, The pH was adjusted, and 0.1% enzyme was added to the solid content of the sample at the optimum temperature, followed by reaction for 4 hours. The reaction suspension was centrifuged (3000 g) for 20 minutes, and the protein content of each of the obtained supernatants was compared with each other.

 division protein (%) Protamex 75.43 + 1.81 Flavorzyme 78.60 + - 0.56 Neutrase 32.73 + - 1.75 Alcalase 82.46 ± 1.24 Protease M 83.32 + - 0.33 Protease N 86.43 + - 2.12 Protease A 62.73 + - 0.35 Protease F 58.07 ± 0.56

The sensory evaluation was performed on the sensory evaluation of bitterness and acceptability of the 17 sensory test panels, which were obtained by centrifuging the supernatant containing the water-soluble rice peptides obtained in the above reaction, using the 9-point rating method. The results are shown in Table 8 Respectively.

 division bitter Likelihood Protamex 7.2 ± 0.5 2.4 ± 0.6 Flavorzyme 5.7 ± 0.4 3.4 ± 0.5 Neutrase 4.1 ± 0.2 4.2 ± 0.3 Alcalase 3.6 ± 0.4 5.5 ± 0.5 Protease M 7.7 ± 0.4 1.9 ± 0.3 Protease N 8.0 ± 0.4 1.3 ± 0.2 Protease A 7.4 ± 0.4 2.3 ± 0.3 Protease F 4.4 ± 0.4 5.1 ± 0.5

From the above results, it has been found that when a water-soluble rice peptide is prepared from rice syrup foil, it is possible to produce a rice peptide having high palatability by using a nutrient or an alkalase. In particular, when alkalase is used, The higher the yield of the water soluble peptide, the higher the bitter taste and the lower the preference.

Experimental Example 6: Reaction conditions of proteolytic enzyme

The protein degradation rate, pH, and sensory properties of the protease selected in Experimental Example 5 and the protein content of the enzymolytic degraded product according to the addition amount of protease were analyzed according to the reaction time of the alkaline protease.

In order to determine the optimum reaction time of the alcalase, distilled water (28.3 ml) was added to 5 g of the washed rice syrup foil of Production Example 4-4 of Experimental Example 4 to prepare a 15% suspension, and 1 N HCl or 1 N NaOH was used to adjust the pH to 7, and 1% of enzyme was added to the solid content of the sample at an optimum temperature of 60 ° C. The protein content of the enzyme hydrolyzate at 1, 2, 3, The pH was measured and the strength of the noodle patch was examined by sensory evaluation using the 9-point scale method for 17 sensory panels.

Reaction time (h) protein (%) pH Strength of side patch One 71.5 6.5 2.4 2 77.3 6.3 2.6 3 81.5 6.1 2.7 5 85.2 5.4 4.2 24 90.3 4.5 5.8

Protein degradation rate gradually increased with reaction time. However, after 3 hours, the pH rapidly decreased and the patches started to occur, and it was determined that there was a quality problem, and the optimum reaction time was determined to be 3 hours.

In order to determine the optimum amount of enzyme to be added to the alkaline protease, the washed rice syrup foil of Production Example 4-4 of Experimental Example 4 was determined to be 0.005, 0.01, 0.2 and 1% by weight based on the substrate. After fixed for 3 hours, the protein was quantified by the lowry method.

Amount of enzyme added (% by weight) 0.005 0.01 0.1 0.5 One protein (%) 42.5 71.2 82.2 83.4 83.5

When the content of protease was more than 0.01 wt%, it was enough to decompose the rice syrup. At 0.1 wt%, the protein degradation rate was increased with increasing amount of enzyme, but the protein decomposition rate was not increased any more.

Example:

0.1% by weight of Termamyl SC was added to a rice syrup foil having a moisture content of 45% by weight and a moisture content of 45% by weight in dry solid matter, and the mixture was reacted at 90 캜 for 1.5 hours. The reaction supernatant was removed and dehydrated through a horizontal decanter. Water was added to the solid material 10 times as much as the solids content, mixed with stirring for 20 minutes, and then dehydrated again. The dehydrated solid obtained by repeating the washing process three times was added with a solid amount 10 times of water was mixed and adjusted to pH 8.5. Then, 0.1 wt% of alkalase was added to the solid amount, and the mixture was reacted at 60 ° C for 3 hours. The final pH of the enzyme reaction solution was 7.2, and the enzyme was inactivated by heating at 85 ° C for 15 minutes. The inactivated enzyme reaction solution was dehydrated through a horizontal decanter, and the liquid phase was concentrated to 20 wt% Dried at 60 ° C for 12 hours, and pulverized to obtain a water-soluble rice peptide powder.

Experimental Example 7: General composition and amino acid composition of the water-soluble rice peptide powder

Crude protein, crude fat, crude protein, and carbohydrate content of the water-soluble rice peptide powder of the above Example were analyzed in the same manner as in Experimental Example 1. [

 division Water-soluble rice peptide powder Crude protein (% by weight) 82.1 Crude fat (% by weight) 2.2 Views min (% by weight) 3.1 Water (% by weight) 6.4 Carbohydrates (% by weight) 6.2

A quantitative test was conducted on 16 kinds of constituent amino acids of the water - soluble rice peptides. The analysis was carried out by ninhydrin HPLC analysis and the results are shown in Table 12.

Constituent amino acid Brown rice
(Korean Journal of Agricultural Chemistry, 1977) Soluble rice peptides
(%) Aspartic acid 9.5 10.45 Threonine 3.9 3.41 Serine 5.2 5.72 Glutamic acid 19.4 21.64 Proline 4.6 4.34 Glycine 4.7 5.10 Alanine 6.5 5.91 Valine 6.2 5.71 Methionine 2.1 1.86 Isoleucine 4.0 3.68 Leucine 8.5 7.59 Tyrosine 4.4 4.98 Phenylalanine 5.7 5.73 Lysine 3.6 2.90 Histidine 2.5 2.38 Arginine 9.3 8.60 total 100.1 100 * BCAA 18.7 16.98

The constituent amino acids of the rice peptides were almost identical to those of the starting rice, and it was confirmed that the content of glutamic acid was contained in a high proportion of about 20%. It was also confirmed that the content of Branched Chain Amino Acid (BCAA), which has been reported to improve exercise performance and fatigue recovery, is as high as 16.98%.

Experimental Example 8: Molecular weight distribution and amino nitrogen content of water-soluble rice peptide powder

HR-MS analysis was performed to confirm the molecular weight distribution of the water-soluble rice peptides of the above examples. The concentration of the sample was 2 mg / mL. Distilled water and acetonitrile were used as solvents, and formic acid was used as a reagent. Experimental methods and instrument conditions are shown in Tables 13 and 14, respectively.

One Chromatography Thermo Accela UPLC 2 Mass spectrometry Thermo LTQ-Orbitrap XL 3 Colum ACQUITY BEH130C18, 1.7um, 100 * 2.1mm 4
Solvent
A: DW (0.1% formic acid) B: Acetonitrile (0.1% Formic acid) 5






Elution condition






Time A B 0.0 99 One 3.0 99 One 53.0 50 50 53.5 0 100 56.0 0 100 56.5 99 One 60.0 99 One 6 Flow rate 0.3 ml / min 7 Injection 3 μl

One Detection ion mode Positive ion ((M + H] +) 2 Scan range MS: m / z 300-1600 3 Spray voltage 4.5 kV 4 Capillary voltage 35 V 5 Capillary Temp 300 6 CID energy 35 7 Software Xcalibur

MS chromatogram confirmed by molecular weight fractions under the above conditions is shown in Fig. According to FIG. 1, most of the molecular weight is 1000 Da or less, and among these, the molecular weight of about 300 to 700 Da is 95% or more.

A mass spectrum of a molecular weight of 300 to 1000 Da is shown in Fig.

Amino nitrogen content was also measured to determine the degree of decomposition of the water soluble rice peptides of the examples into amino acids. According to the Formol titration method, 2 ml of the eluate was taken in a 100 ml measuring flask and mixed with 100 ml of distilled water. 25 ml of the diluted eluate was taken in a 100 ml beaker and 0.1 N NaOH was added and the pH was adjusted to 8.4 with a pH meter. To this, 20 ml of 35% formaldehyde solution (v / v) (Samchun Pure Chemical) was added and neutralized to pH 8.4 with 0.1 N NaOH. Amino nitrogen value was calculated as "[(A-B) x1.4xFxDx100] / S". A is the consumption of 0.1 N NaOH, B is the coarse experimental consumption of 0.1 N NaOH, F is the factor value of 0.1 N NaOH, D is the dilution factor, S is the sample weight (g) (Mg) equivalent to 1 ml.

The amino nitrogen content of the water-soluble rice peptides of the examples was 600 mg%, indicating that the degree of decomposition into amino acid units was not large.

EXPERIMENTAL EXAMPLE 9: Change of Solubility According to pH, Heat and Storage of Water-soluble Rice Peptide Powder

The protein content of the aqueous solution was measured at each pH in order to confirm the stability of the water-soluble rice peptide powder of the examples according to pH. The results are shown in FIG. In general, proteins exhibit an isoelectric point near pH 4 and precipitate. Albumin, a representative milk protein, exhibits an isoelectric point at pH 4.6. The water soluble rice peptides were very stable in aqueous solutions ranging in pH from 5 to 12 and exhibited gradual precipitation from pH 4.8 and maximum precipitation at pH 4.2. and the water solubility was 95% or more at pH 4.6 or higher.

In order to confirm the thermal stability, the water-soluble rice peptide powder was diluted 100 times with distilled water, and then 10 ml of the solution was dispensed into a 15 ml test tube and treated at 60, 80, 100 and 120 ° C for 0.5 h and 1 h, respectively. 4. As a result, the decrease of protein started gradually at 80 ℃ and 1h treatment condition, and loss of about 2% at 100 1h and loss of protein of about 6% at 120 1h were observed. Therefore, it was confirmed that the water-soluble rice peptide powder of the examples was a relatively stable substance in heat, and it was confirmed that it could be sterilized in the usual general beverage manufacturing process and stable at the process temperature.

Water soluble rice peptides were prepared by dissolving 3% by weight of water in water, and then sterilized at 100 ℃ for 15 minutes. In order to confirm the hygienic stability according to the storage period, the microorganisms were tested simultaneously during the storage period after sterilization. There was no significant difference in protein content between 0, 1, 2, 3, 4, 8, and 12 weeks after the sterilization. After 12 weeks from the sterilization, general bacteria, E. coli and E. coli were all not detected and sterilized for 12 weeks .

Experimental Example 10: Fatigue recovery activity after exercise of water-soluble rice peptide powder

Continued exercise reduces the glycogen content of the muscle and increases the amount of blood glucose into the muscle, which reduces blood glucose and results in fatigue.

In addition, aspartic acid is a major constituent of the purine nucleotide circuit in muscle, which regenerates the ATP pool during extreme high-intensity exercise, providing energy to the muscle and forming free ammonia during this process. It is known that the ammonia produced as a result of the decomposition of amino acids during exercise is released from the muscles into the blood, and the blood ammonia is transmitted to the brain to increase the central fatigue. Ammonia accumulated in the muscle cells stimulates the afferent nerves associated with muscle pain sensation, inhibits the TCA cycle and your biological function, and causes muscle fatigue by causing lactic acid production.

Therefore, in order to confirm whether or not the water-soluble rice peptides of the present invention can affect the exercise-restoring fatigue activity, the rats were orally administered for 4 weeks, and then the blood glucose and plasma ammonia concentrations were changed.

The experiment was carried out in Chemon, a GLP (Good Laboratory Practice) certification body, and the blood analysis of rats was conducted through Green Cross Medical Foundation. The water soluble rice peptides of the present invention were administered at low dose, medium dose and high dose, respectively. As a comparative group, isolated soybean protein was administered at an intermediate dose, and negative control was administered only with distilled water. Each dose was 67 mg / kg / day, 335 mg / kg / day, and 670 mg / kg / day, respectively, based on the protein content of the low dose group.

No deaths were observed for 4 weeks, and no significant weight change was observed in all groups compared to the control group.

The blood glucose concentrations after 4 weeks in each experimental group are shown in Table 15. [

division Average( mg / Dl) Standard Deviation Control group 70.80 11.81 Example (Low Capacity) 88.00 12.30 Example (intermediate capacity) 91.71 12.15 Example (high capacity) 98.80 10.52 Isolated soy protein (medium dose) 87.37 11.60

It was found that the concentration of glucose in blood gradually increased as the dose of the water soluble rice peptide of the Example was increased.

Plasma ammonia concentrations after 4 weeks in each experimental group are shown in Table 16.

division Mean (/ / ㎗) Standard Deviation Control group 753.57 199.33 Example (Low Capacity) 694.29 160.32 Example (intermediate capacity) 638.00 140.19 Example (high capacity) 600.71 67.27 Isolated soy protein (medium dose) 705.43 192.78

The plasma ammonia concentration of the control group was 753.57 / / dl. As the concentration of the water soluble rice peptides of the present invention was increased, the plasma ammonia concentration was decreased and the plasma ammonia reduction effect was superior to that of the comparative soybean protein isolate.

Therefore, the water soluble rice peptides of the present invention can be interpreted as having an effect on the fatigue lowering effect of rats after exercise.

Experimental Example 11: Determination of cytotoxicity by concentration of rice peptides in primary cultured hepatocytes

1. Experimental animals and pretreatment

Seven-week-old Sprague-Dawley rats were used. One week after quarantine and purification, 220 ± 5 g of those determined to be healthy were reported in the test. Experimental animals were maintained in a laboratory cage (42 × 8 cm) at a laboratory temperature of 22-24 ° C and a humidity of 60 ± 5%. Day and night cycles (12 hours light / 12 hours dark) Respectively.

2. Surface treatment of Tissue culture well plate

To cultivate hepatocytes, collagen was dispensed evenly on tissue culture well plate and dried for 12 hours.

3. Isolation of hepatocytes

First, the rat was anesthetized with anesthesia. The needle was inserted into the portal vein, then the Hank's 1 solution was perfused, and the blood was perfused with Hank's 2 solution. The temperature of the perfusion fluid was maintained at 37 ° C and oxygen was continuously supplied.

After perfusion, the liver was removed, washed, filtered and centrifuged. Percol solution was added to hepatocytes, centrifuged, and the cells were separated and washed. The number of acquired cells and cell viability were estimated by trypan blue staining.

4. Culture conditions

Hepatocyte cultures are made with cell density 2 × 10 ⁵ cell / mL to the William's Medium E medium Complete, seeded on collagen coating The Tissue culture plate well CO ₂ The culture was started in an incubator (37, 5% CO ₂ , 95% air), and after 4 hours, the culture medium was removed by tilting the tissue culture well plate.

5. MTT analysis

In the MTT assay, the yellow soluble tetrazolium salt is reduced to a blue insoluble formazan product by the mitochondrial succina dehydrogenase of living cells. The absorbance of formazan is maximized at a wavelength of 540 nm and measured at this wavelength The absorbance reflects the concentration of living and metabolically vigorous cells and is used as an indicator of mitochondrial activity.

When the rice peptides of the examples of the present invention were treated with the primary cultured hepatocytes (10-1000 占 퐂 / mL), there was no toxicity in comparison with the untreated cells in the treated concentration (FIG. 5).

Experimental Example 12: Confirmation of liver protective effect of rice peptides in primary cultured hepatocytes

1. Restoration of cytotoxicity by t-BHP

The rice peptides (SRP70) of the example of the present invention were treated with distilled water (control), 1.5 mM t-BHP (t-BHP) and 1.5 mM t-BHP, respectively, in the primary cultured hepatic cells obtained in Experimental Example 11, After the passage of time, galactasamine was removed after 24 hours, and MTT reagent and William's E medium were mixed and placed in a 37 ° C incubator. After 3 hours, 20% SDS was added and incubated at 37 ° C in an incubator. The cells were allowed to stand for 20 hours and then assayed at 540 nm for cytotoxicity by MTT assay. The results are shown in FIG.

In the case of t-BHP treatment, the cell survival rate was lowered to 82.9%, but it was confirmed that the treatment of BHP and the sample simultaneously had the protection activity from 50 ㎍ / mL. In the case of 250 ㎍ / mL, .

2. Restoration effect of AST and APT increase by t-BHP

AST is a hepatocellular leukocyte that increases during damage or necrosis of hepatocytes. APT is an enzyme that exists in cytoplasm and mitochondria. It is increased when liver or muscle cells are damaged.

When rice peptides of the present invention were added together with t-BHP, it was confirmed that the rice peptides had the effect of reducing AST and APT increased from 100 μg / mL to t-BHP (FIG. 7).

3. Restoration effect of LDH increase by t-BHP

LDH is a related enzyme and is widely distributed in various tissues in the body. Especially, it is present in heart, liver, kidney and muscle. LDH in serum increases in heart disease, liver disease malignant tumor and leukemia but has low specificity.

When the rice peptide of the present invention was added in combination with t-BHP, it was confirmed that the rice peptide had an LDH reduction effect from the concentration of 50 μg / mL to that of t-BHP (FIG. 8).

Experimental Example 13: Subacute toxicity of rice peptides in animal experiments

1. Blood biochemical test

The rats were fed in the same manner as in Experimental Example 11. In the control group, 0.5 g / kg of rice peptide (SRP70) and 1.0 g of rice peptide (SRP70) of the Example after administration of physiological saline were orally administered to give the same stress as the experimental group / kg, and 2.0 g / kg, respectively. Oral administration was repeated once a day for 5 days. Each subject was orally administered with 1 mL / day of margarine dissolved in physiological saline according to the dose corresponding to the average weight of the corresponding group.

The control and SRP70-treated experimental animals were fasted for 12 hours and then anesthetized with ethyl ether. After sacrifice, the thoracic cavity was opened and blood was collected from the aorta. Part of the blood was taken in EDTA-treated blood vessels to prevent blood clotting We measured total protein (T.Protein), albumin, alkaline phosphatase (ALP), aspartate transaminase (ALT), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), total cholesterol Lt; tb >

When compared to the control group, the rice peptide-treated group (SRP70) of the present invention showed no significant difference. In the case of ALP, the rice peptide-treated group had a higher value than the control group, but the value decreased . Especially, when AST, ALT, and T. Chol were closely related to liver damage, there was no significant difference between the two groups.

2. Histopathological examination

The control group and SRP70 were inoculated for 5 days. The animals were sacrificed for 12 hours and then anesthetized with ethyl ether. The thoracic cavity was opened, and blood was collected and liver and kidney were extracted and weighed. After the measurement, the liver and kidney were fixed in formaldehyde solution, fixed with new formaldehyde for the next day, and fixed with formalin, followed by paraffin embedding. Hematoxylin & eosin (H & E) staining was performed for the liver, and periodic acid staining (PAS) for the kidney was performed.

When necrosis occurs, mitochondria expand, nucleus collapse, destruction of cell membrane by cell organelle and expansion, and fusion of ribosome and lysosome occur. In addition, when the contents of the cells were extracellularly released to cause inflammation and H & E staining was performed, the cells became pinkish and the boundary between cells was not clearly observed. In the control and SRP70-treated groups, liver (Fig. 9) and kidney ), Both cytoplasmic and circular nuclei were distinct, and hepatic cell necrosis and infiltration of inflammatory cells showed normal liver tissue which was not found.

Experimental Example 14: Confirmation of liver protection effect of rice peptides in an animal experiment

1. Blood biochemical test

The rats were fed in the same manner as in Experimental Example 11. In the control group, 0.5 g / kg of rice peptide (SRP70) and 1.0 g of rice peptide (SRP70) of the Example after administration of physiological saline were orally administered to give the same stress as the experimental group / kg and 2.0 g / kg, respectively. Oral administration was repeated orally for 5 days once a day.

After 5 hours, t-BHP was intraperitoneally administered to the control and SRP70-treated experimental animals at a concentration of 1.5 mM / kg. After 18 hours, the animals were sacrificed by anesthetizing with ethyl ether, the thoracic cavity was opened and blood was collected from the aorta The serum was separated from the supernatant and analyzed for AST, ALT and LDH by FUJI DRI-CHEM 3500i (Fuji Photo Film Co., Osaka, Japan).

Significant differences were found in ALT, AST and LDH, which are indicators of liver failure. ALT lowered the concentration of SRP70 from 1.0 g / kg to the normal level without treatment with t- BHP. AST was decreased to normal level at 0.5, 1.0 and 2.0 g / kg, but there was no significant difference according to concentration. LDH levels were decreased to 1096.0, 996.5 and 822.7 (U / L) as the concentrations of the samples increased to 0.5, 1.0, and 2.0 g / kg, respectively.

2. Histopathological examination

The animals were sacrificed by anesthetizing with ethyl ether for 18 hours, and the control and SRP70 were administered for 5 days. The animals were sacrificed by the administration of t-BHP at a concentration of 1.5 mM / The animals were fasted for 12 hours, the thoracic cavity was opened, and blood was collected and the liver was weighed and weighed. After the measurement, hematoxylin and eosin (H & E) staining was carried out in the same manner as in Experimental Example 13 to observe the tissue.

Necrosis or lesions were not observed in the liver of the control group administered with saline solution alone or in the group administered with SRP70 at the concentration of 2.0 g / kg. However, in the liver tissues administered with t-BHP alone, lipid peroxidation and necrosis were observed around the blood vessels When compared with the surrounding area, it was brightly colored and the cell boundary was not clear. In the group administered with the t- BHP, the condition was improved as the concentration of the sample was increased, which was not different from that of the control group (FIG. 11).

3. Catalase, glutathione peroxidase activity and glutathione concentration determination

Hepatic homogenate was prepared by grinding the extracted liver from the histopathological examination with 5 mL of 50 mM potassium phosphate (pH 7.0, containig 1 mM EDTA) solution per g of liver. The liver homogenate was centrifuged (4 ° C, 10,000 g, 15 min) to prepare an enzyme source with the supernatant, and the catalase, glutathione peroxidase activity and glutathione concentration were measured and are shown in Table 19.

Catalase activity was expressed as 1 unit of 1 uM conversion of hydrogen peroxide as a substrate per minute under optimum conditions. In comparison with t - BHP alone group (31.7 U / mg protein) after administration of SRP70, t - BHP group showed 77.8, 113.9, 118.4 U / mg protein catalase activity.

Glutathione peroxidase is dehydratase (GSH-Px) activity of ^{molar extinction coefficient 43.6 M -1 cm -} 1 (6.22M -1 cm -1) by using the unit / mg protein (1 unit = 1 uM of H 2 O 2 degraded for a minute. When SRP70 was administered, the t- BHP-treated group had a control level of 0.5, 1.0, and 2.0 g / kg at a dose of 0.5 mg / kg, compared with the group administered t- BHP alone (5.4 U / Enzyme activity was shown, but there was no significant difference with increasing concentration.

Glutathione (GSH) measurement, following administration of SRP70 treated group, a t- BHP t- BHP only administered group (3.0 mM) in both the sample administration concentration 0.5, 1.0, 2.0 g / kg as compared to the normal And the activity was increased to a level similar to or higher than that of the liver.

Hereinafter, formulation examples of compositions containing the rice peptides of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

20 mg of the rice peptide powder of the examples

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

20 mg of the rice peptide powder of the examples

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

20 mg of the rice peptide powder of the examples

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

20 mg of the rice peptide powder of the examples

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O 26 mg

It is prepared by the above-mentioned component content per ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

20 mg of the rice peptide powder of the examples

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the conventional liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water and adjusted to 100 as a whole. To prepare a liquid agent.

Formulation example  6. Manufacture of health functional foods

20 mg of the rice peptide powder of the examples

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 [mu] g vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components may be mixed , Granules may be prepared and used in the manufacture of a health functional food composition according to a conventional method.

Formulation example  7. Preparation of Protein-Enriched Beverages

The rice peptide powder of Example 3,000 mg

Citric acid 1,000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to the flask to obtain a total of 900 mL

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 L container, It is used in the production of the functional beverage composition of the invention.

Formulation example  8. Rice Milk  Produce

The rice peptide powder of Example 12.58 g

Rice glycation solution 979.91 g

1 g of coconut powder

6.25 g of calcium carbonate

Vanilla and rice flavor 0.5 g

Xanthan gum 0.3 g

Rice peptides, liquid calcium carbonate and coconut powder were added to the saccharified rice flour, and the mixture was heated to completely dissolve the xanthan gum in hot water. Then, the mixture containing vanilla and rice flavor was mixed and heated and homogenized, sterilized and cooled to prepare rice milk do.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (10)

Hepatotoxicity is produced by hydrolyzing the rice protein, the peptide having a molecular weight of 300 to 700 Da is at least 80% by weight of the total peptide, glutamic acid is 15 to 25% by weight of the total amino acids, branched amino acid 12 to 20% by weight Rice peptides having hepatoprotective activity from chemicals or toxic chemicals indicated.
[Claim 2] The rice peptide according to claim 1, wherein the rice peptide has liver protection activity from a chemical or toxic substance showing hepatotoxicity, characterized in that the content of amino nitrogen is 10 to 1000 mg%.
[Claim 2] The rice peptide according to claim 1, wherein the rice peptide has hepatoprotective activity against chemicals or toxic substances exhibiting hepatotoxicity, characterized in that the water solubility at pH 4.6 to pH 12 is 90% or more.
According to claim 1, wherein the rice peptide is aspartic acid 9 to 12 parts by weight, threonine 3 to 5 parts by weight, serine 5 to 7 parts by weight, glutamic acid 19 to 22 parts by weight, proline 3 to 5 parts by weight, glycine 4 to 6 Parts by weight, alanine 5 to 7 parts by weight, valine 5 to 7 parts by weight, methionine 1 to 3 parts by weight, isoleucine 3 to 5 parts by weight, leucine 7 to 9 parts by weight, tyrosine 4 to 6 parts by weight, phenylalanine 5 to 7 Rice peptide having liver protection activity from a chemical or toxic substance showing hepatotoxicity, characterized by having an amino acid composition consisting of parts by weight, lysine 2 to 4 parts by weight, histidine 1 to 3 parts by weight and arginine 7 to 10 parts by weight.
[Claim 2] The rice peptide according to claim 1, wherein the peptide has hepatoprotective activity against a chemical or toxic substance exhibiting hepatotoxicity, characterized by fatigue recovery activity after exercise.
[Claim 2] The rice peptide according to claim 1, wherein the rice protein is derived from rice syrup cake and has a hepatoprotective activity against a chemical or toxic substance exhibiting hepatotoxicity.
An alpha-amylase treatment step of reacting rice syrup gourd with alpha-amylase; Removing the water-soluble substance for washing the rice syrup cake treated with the alpha-amylase; And a protease treatment step of reacting the washed rice syrup gourd with a protease. The method of claim 1, wherein the rice peptide is selected from claim 1.
Claim 1 to 6 liver protective food composition containing the rice peptide of any one selected from the active ingredient.
9. The liver protective food composition according to claim 8, which is a health functional food in a beverage form.
Claims 1 to 6, wherein hepatotoxicity prevention and treatment pharmaceutical composition containing the rice peptide of any one selected from the active ingredient.

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