KR20060012436A - Method for producing selenium peptide by yeast aytolysis - Google Patents

Abstract

The present invention relates to a method for producing selenium peptides by yeast autoenzyme digestion, specifically, preparing selenium yeast by culturing yeast in a selenium-containing medium; Separating and crushing the selenium yeast, adding an autoenzyme promoter, and obtaining an autoenzyme-containing product containing selenium peptide by autoenzyme digestion; And it is excellent in antioxidant activity and comprises a step of separating selenium peptide of low molecular weight, it relates to a method for producing selenium peptides by yeast autoenzyme digestion. The production method of the present invention provides an inorganic selenium and autoenzymatic conditions that can be converted to organic selenium in cultivation of selenium yeast, from which a low toxicity and low molecular weight selenium peptide with high bioabsorption rate and high utilization and excellent antioxidant activity The selenium peptide produced by the method of the present invention can be usefully used as a pharmaceutical composition, dietary supplement, animal feed or cosmetic additive having antioxidant activity.

Selenium, Yeast, Autoenzyme Degradation

Description

Production method of selenium peptide by yeast autoenzyme digestion {METHOD FOR PRODUCING SELENIUM PEPTIDE BY YEAST AYTOLYSIS}             

1 is a result of measuring the antioxidant activity of selenium peptide obtained by acid hydrolysis of selenium protein with different concentrations of HCl,

2 is a result of measuring the antioxidant activity and the average molecular weight of selenium peptide obtained by enzymatic hydrolysis of selenium protein using a proteolytic enzyme,

YP: yeast protein,

YPE: yeast protein hydrolyzate by Pronase E treatment,

YPK: yeast protein hydrolyzate by protease K treatment,

SeP: Se-protein,

SePE: Se-protein hydrolyzate by Pronase E treatment,

SePK: Se-protein hydrolysates by protease K treatment

Figure 3a is a result of examining the effect of cell disruption on the antioxidant activity of selenium peptides produced from the autolysis before autolysis, Figure 3b is a view of sodium selenate and sodium selenite (sodium selenite) This is a result of comparing the production of selenium peptide according to the concentration,

4 is a result of investigating the effects of NaCl ( FIG. 4A ) and ethanol addition ( FIG. 4B ) on protein efflux, antioxidant activity and average molecular weight, respectively, as autoenzyme promoters during autoenzyme digestion .

5 is to change the concentration of NaCl without the addition of ethanol ( Fig. 5a ), or to fix the concentration of ethanol to 5% and to change the concentration of NaCl ( Fig. Investigation of the effects on protein efflux, antioxidant activity and mean molecular weight,

6 is a result of investigating the effect of selenium concentration on the antioxidant activity of the selenium peptide produced by autoenzyme digestion in selenium yeast culture,

Figure 7 is the result of measuring the antioxidant activity change of selenium peptide with the time of autoenzyme digestion,

8 is a result of measuring the antioxidant activity of each selenium peptide separated from the autoenzyme digest of selenium yeast by FPLC system.

The present invention relates to a method for producing selenium peptides by yeast autoenzyme digestion, and more particularly, to prepare a selenium yeast by culturing yeast in a selenium-containing medium; Crushing the selenium yeast culture medium, and then adding an autoenzyme promoter and obtaining an autoenzyme product including selenium peptide by autoenzyme digestion; And it is excellent in antioxidant activity and comprises a step of separating selenium peptide of low molecular weight, it relates to a method for producing selenium peptides by yeast autoenzyme digestion.

Selenium is a trace mineral that is essential to the human body and is an important component of antioxidant enzymes that protect cells from free radicals produced by oxygen-consuming metabolism, and also works well in the normal functioning of the immune system and the thyroid gland. Is an essential ingredient in. Selenium first became known as a nutrient for the first time in 1957, when liver cirrhosis symptoms observed in mice lacking vitamin E could be prevented by administration of trace amounts of selenium. Subsequently, it became important that selenium was present in the form of selenocysteine as a component of glutathione peroxidase (GPx) (Arteel, GE and Sies, H, Environmental toxicology and pharmacology 10 (4): 153-158, 2001; Anonymous, Nutr Rev. 45 (11): 344-5, 1987; Anonymous, Nutr Rev. 49 (2): 62-4, 1991).

Selenium, which is absorbed into the body through food, causes insufficient blood circulation, heart disease, joint diseases, vision disorders, decreased sexual function, and cell aging. In particular, deficiency of selenium is known not only to lower the anticancer ability but also to cause the heart disease, keshan disease or the joint disease, kashin-Beck disease (Tapiero, H., et al., Biomedicine & pharmacotherapy 57 (3): 134-144, 2003).

In humans, selenium is effective in improving immune function, preventing cancer and inhibiting cancer metastasis as well as antioxidant activity (Combs, GF and WP Gray, Pharmacol. Ther. 79: 179-192, 1998; Jiang, C, et al. , Mol.carcinog . 26: 213-225, 1999; McKenzie, RC, et al., Clinical and experimental dermatology 25 (8): 631-636, 2000), particularly when the skin is damaged from oxidative stress such as ultraviolet rays. (McKenzie, R, et al., Clinical and experimental dermatology 25 (8): 631-636, 2000; Emonet, N, et al., J. photochem.photobiol. B Biol. 40: 84-90, 1997).

So far, about 10 selenium-containing proteins have been isolated and identified in animal tissues. For example, glutathione peroxidase (GPx), thioredoxin reductase, and selenoprotein P. , Selenoprotein W, selenoprotein R and the like. It has been found that selenium-containing proteins are mostly related to antioxidant mechanisms, and their exact physiological and biochemical functions have not yet been fully identified.

GPx, a representative selenoprotein, is an antioxidant enzyme that removes hydrogen peroxide, lipid peroxides and phospholipids from metabolism, regulates inflammatory responses, and protects lipids, proteins and DNA from oxidative attack. The antioxidant activity of GPx is dependent on glutathione (GSH), which is reduced by the use of two molecules of glutathione when selenocysteine (R-SeH) in the active site of GPx reacts with peroxides (Arteel, GE and Sies, H, Environmental toxicology and pharmacology 10 (4): 153-158, 2001). However, GPx is a high molecular weight protein of 84 KDa, which is difficult to be used for therapeutic purposes because of its low stability. Therefore, there are many studies to develop GPx mimics (Liu, JQ, et al., Biochimica et Biophysica Acta. 1481 (2): 222-228, 2000; Ren, X, et al., The Biochemical Journal 359 (2): 369-374, 2001; Su, D, et al., Biochemical and Biophysical Research Communications 285 (3): 702-707, 2001; Su, D, et al., Archives of Biochemistry and Biophysics 395 (2): 177-184, 2001).

Selenium has different bioavailability depending on its form. Inorganic selenium, such as selenite and selenate, can act as an oxidation promoter during digestive absorption and is highly toxic, and has a very low bioavailability, which is contained in livestock excretion and is thus released into rivers and soils. May cause environmental problems that accumulate. In order to solve this problem of inorganic selenium, it has been proposed to use organic selenium having low toxicity and high bioavailability in the form of binding to amino acids such as selenomethionine. Organic selenium, which is synthesized through vegetables, fish, and yeast, is mostly selenomethionine, which is absorbed into the body as a constituent of the protein, and then stored in the protein and then gradually supplied to the selenium through methionine metabolism. Selenium is released through urine or breathing. Therefore, organic selenium is combined with amino acids such as selenomethionine is an ideal structure with the best bioabsorption rate.

Such organic selenium may be provided by using selenium yeast for converting inorganic selenium into organic form or by directly synthesizing organic selenium. Selenium yeast is an ideal source of selenium because it is a nutritional source that supplies minerals, vitamins, trace elements, and proteins as well as selenium. In yeast, selenium can be randomly introduced into the protein in the selenium-added medium because Met-tRNA does not distinguish between methionine and selenomethionine. In particular, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) does not have a UGA codon, so selenocysteine (21 amino acid introduced into the selenoprotein by recognizing the UGA codon) will be present only to the degree of conversion of selenomethionine (Stadtman, TC, Advances in Inorganic Biochemistry 10: 157-176, 1994). However, the production of organic selenium using selenium yeast has a good conversion rate from inorganic selenium, but since the molecular weight of organic selenium produced therefrom is very high, the actual bioavailability and absorption is very low, There is a problem in that a medium must be used and an additional cost for the treatment of inorganic selenium occurring after incubation is required.

On the other hand, organic selenium can be synthesized directly, as the organic selenium thus synthesized is known as ebselene (ebselene). However, the synthesized organic selenium shows an antioxidant activity, but has a problem that the selenium-containing antioxidant enzyme glutathione peroxidase-like activity is low and its solubility in water is very low. In addition, selenium-containing amino acids, such as selenomethionine, have lower toxicity than inorganic selenium, but when used alone, exhibit some toxicity and have a very high cost of use (Schrauzer, GN, The Journal of nutrition 130 (7)). : 1653-1656, 2000).

In order to overcome the problems of using the conventional selenium, the present inventors determined that supplying selenium in the form of a peptide can further reduce toxicity and facilitate intestinal absorption and transdermal absorption into the skin.

Accordingly, the present inventors have overcome the problems of inorganic selenium having high toxicity and low bioavailability, and researched to produce organic selenium having low toxicity, high absorption rate and high utilization rate through yeast, and crushing it after culturing selenium yeast The present invention has been completed by developing a method for producing low molecular weight selenium peptides having low toxicity and high antioxidant activity while being easy to use and absorb in vivo through the addition of an enzyme decomposition promoter.

It is an object of the present invention to provide a method for producing low molecular weight selenium peptides having low toxicity, high bioavailability, high absorption rate and excellent antioxidant activity using autoenzymatic digestion of yeast.

In order to achieve the above object, the present invention provides a method for producing selenium peptide by yeast autoenzyme digestion.

In addition, the present invention provides a low molecular weight selenium peptide having low toxicity, high bioavailability and high absorption, and excellent antioxidant activity produced by the method.

In addition, the present invention provides a pharmaceutical composition containing the selenium peptide as an active ingredient, dietary supplements, livestock feed and cosmetic composition for addition.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing selenium peptides using autoenzyme digestion of yeast in order to produce low molecular weight selenium peptides having low toxicity, high bioavailability, high absorption rate and excellent antioxidant activity.

Specifically, the production method of the present invention

1) culturing yeast in a selenium-containing medium to prepare selenium yeast;

2) crushing the selenium yeast culture;

3) adding an autoenzyme promoter to the cell lysate and obtaining an autoenzyme from the selenium peptide by autoenzyme digestion; And,

4) separating the selenium peptide having excellent antioxidant activity and low molecular weight from the autoenzyme digest.

In more detail, the step 1) is a step of preparing selenium yeast by culturing yeast in a selenium-containing medium in order to convert inorganic selenium to organic selenium. Yeast strains usable in the present invention include Saccharomyces cerevisiae , Saccharomyces uvarium , and Saccharomyces cerevisiae are preferred. Selenium yeast is a state in which selenium is organically bound in yeast cells. When yeast strains are cultured in a medium containing inorganic selenium, inorganic selenium is replaced with sulfur, a cell component of yeast, during fermentation to bind organic matter. . At this time, the medium to be used is preferably adjusted to include as little sulfur as possible for the substitution of inorganic selenium and sulfur, a cell component, YM, YPD, YPS or YEPD may be used as such a medium.

In general, when the yeast strain is cultured in a medium containing inorganic selenium, the growth of the yeast strain is inhibited than when cultured in a medium containing no selenium, the growth inhibition is shown to be stronger as the selenium concentration is increased Known. Accordingly, the present inventors have found that the most suitable inorganic selenium that can be converted into organic selenium by the selenium yeast culture, sodium selenite (sodium selenate) of the yeast strain than sodium selenite (sodium selenite) that was previously used for cultivation of selenium It is confirmed that the degree of growth inhibition is about 5 times lower and shows a similar level of conversion to organic selenium (see FIG. 3b ), and sodium selenate is provided as an appropriate inorganic selenium raw material in culturing selenium yeast.

Step 2) is to recover the cells by centrifuging the selenium yeast culture prepared in step 1) at 2000 to 3000 g for 10 to 20 minutes, washed twice with distilled water and the cells using an ultrasonic ultrasonicator Shredding. In the case of performing autoenzyme after crushing the cells, the protease is initially activated, and thus the protein effluent and antioxidant activity are increased as compared with the use of non-broken cells (see FIG. 3A ).

Step 3) is a step of obtaining an autoenzyme product of selenium peptide by adding an autoenzyme promoter to the cell lysate and autoenzymatically digesting it.

The present inventors first measured the antioxidant activity by hydrolyzing the protein produced from selenium yeast through acid hydrolysis and enzymatic hydrolysis. As a result, it was confirmed that as the acid concentration increased during acid hydrolysis, the antioxidant activity of selenium protein that had before hydrolysis was lost (see FIG. 1 ), whereas the antioxidant activity increased rather during enzyme hydrolysis ( FIG. 2 ) . Reference). Thus, the present inventors attempted to produce selenium peptides from selenium proteins produced from selenium yeast through autoenzymatic decomposition of proteins into small peptides using proteases possessed by yeast.

To this end, step 3) adds an autoenzyme promoter to the cell lysate and produces selenium peptides by autoenzyme digestion. The autoenzyme promoter is added to promote autoenzyme digestion in yeast autoenzyme and to prevent contamination from the outside (Boonraeng, S, et al., Nat. Sci. 34: 270). -278, 2000; Son, SM and Kim, JS, et al., Korean J. Food Sci.Technol . 35 (2): 201-205, 2003), NaCl, ethanol, dextrose, thiamin, ethyl acetate, chloroform or Mixtures of these can be used, with 3-7% NaCl being preferred.

In a preferred embodiment of the present invention, by using NaCl and ethanol in the concentration range of 1 to 10% as an autolytic enzyme promoter, the antioxidant activity of selenium peptides produced therefrom was investigated. First, each of them was added to the cell lysate about 1 to 7% and induces autoenzyme digestion. As a result, the ethanol was added as an autoenzyme promoter only in the group with only 5% ethanol. In one experimental group, there was no overall difference (see FIG. 4B ). In addition, the addition of NaCl showed similar protein efflux and GPx activity as the control group, but showed higher GPx activity compared to ethanol addition, and the average molecular weight of the protein after autoenzymatic degradation was clearly smaller (see FIG. 4A ). Based on the results, the present inventors investigated the effects by adding NaCl and ethanol together to produce selenium peptides that increase the outflow of protein and maintain the GPx activity while maintaining a low average molecular weight. As a result, when NaCl and ethanol were added together, there was no obvious increase in protein outflow or GPx activity.In addition, when ethanol was added, even though NaCl was added, a large molecular weight peptide was produced. It was found to be inadequate to produce selenium peptides (see FIGS. 5A and 5B ).

In addition, the present invention, in order to investigate the proper selenium concentration in the medium for the autoenzyme digestion of yeast, the selenium concentration was changed to 10, 20, 50 and 100 ppm with sodium selenate to incubate and autoenzymatically digest the yeast. As a result, it was confirmed that the GPx activity of the selenium protein produced from selenium yeast was the highest when 20 ppm selenium was added (see FIG. 6 ).

In addition, the present invention, in order to investigate the effect of autoenzyme time of yeast on GPx activity of autoenzyme, autoenzyme by each time period while enzymatically digesting selenium yeast cultured in 20 ppm selenium medium for 72 hours GPx activity and average molecular weight of were measured. As a result, it was confirmed that autoenzymatic degradation of yeast drastically reduced the molecular weight of the peptide during the initial 6 hours and increased GPx activity for 24 hours (see FIG. 7 ).

Based on the above results, the autoenzymatic digestion of yeast for producing the selenium peptide of the present invention is 10 to 50 ppm of sodium selenate, preferably 20 to 50 ppm of S. cerevisiae yeast strain of Saccharomyces After inoculation into the contained YM medium, incubated for 40 to 48 hours at 200 to 250 rpm, 30 ℃ conditions to prepare a selenium yeast, and 3 to 7% NaCl in the crushed solution obtained by crushing the selenium yeast culture with an ultrasonic mill It is preferred to add and autolyze at 50 ° C. for 24 to 48 hours.

Step 4) is a step of separating the selenium peptides having excellent antioxidant activity and low molecular weight by fractionating an autoenzyme product including the selenium peptides obtained from the above, superdex, superose, sephacryl ) Or selenium peptides produced by autoenzymatic digestion of yeast using chromatography using a sephadex column, and then the antioxidant activity and average molecular weight of each fraction were measured to determine low molecular weight selenium peptides, preferably Separates selenium peptides having a molecular weight of 1000 to 7000 Da. In a preferred embodiment of the present invention, the fraction of selenium peptides by fast protein liquid chromatography (FPLC) using a superdex column, and then measured the antioxidant activity of each fraction, the peptide having excellent molecular weight around 5,000 Da It was confirmed to show activity (see FIG. 8 ).

As described above, selenium peptides having excellent low molecular weight antioxidant activity produced by autoenzymatic decomposition of yeast have low toxicity, high bioavailability and high absorption rate, and have a high antioxidant activity. It can be usefully used.

The composition comprising the selenium peptide prepared according to the present invention exhibits antioxidant activity and can be used in a variety of drugs, foods and beverages for immune strengthening, prevention of albinism, or prevention and treatment of cancer diseases. Examples of the food to which the selenium peptide of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health supplements.

The pharmaceutical composition comprising the selenium peptide prepared according to the present invention as an active ingredient can be administered orally or parenterally during clinical administration, and can be used in the form of general pharmaceutical preparations.

In other words, the pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used Or using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, Sucrose (Lucose) or lactose (Lactose), gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The effective dose of selenium peptide of the present invention is 3 to 200 mg, preferably 5 to 20 mg, and may be administered 1 to 4 times a day.

Selenium peptide of the present invention is a drug that can be used with confidence even for long-term administration for prevention purposes because there is little toxicity and side effects.

The bioavailability and high absorption rate and low molecular weight selenium peptides prepared according to the present invention can be added to foods or beverages for the purpose of showing anti-oxidative activity, for strengthening immunity, preventing myopathy or preventing and treating cancer diseases. At this time, the amount of the selenium peptide in the food or beverage is generally added to the health food composition 0.01 to 0.1% by weight, preferably 0.02 to 0.1% by weight of the total food weight, the health beverage composition is 0.003 based on 100 ml To 0.02 g, preferably 0.005 to 0.01 g.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the selenium peptide as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. The above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The natural carbohydrate can be used in the range conventional in the art, and generally can be used at about 1 to 20 g, preferably about 5 to 12 g per 100 ml.

In addition to the above, the composition for food addition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the composition of the present invention may contain a natural fruit juice and a pulp for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the selenium peptide of the present invention can increase the selenium content in the livestock body by adding it to feed and ingesting the livestock, and the animal food produced in this way can be used as a good source of selenium that is likely to be insufficient through daily diet. . The feed composition for livestock according to the present invention may contain selenium peptide at 0.1 to 10% by weight, preferably 0.3 to 0.8% by weight relative to the feed composition. The feed composition may contain other conventional ingredients in addition to selenium peptides and may be ingested to livestock by conventional methods, which may differ depending on the type and purpose of livestock, which are well known in the art. It can be easily determined by those skilled in the art. According to the present invention, the type of livestock or livestock products in which selenium accumulates in muscle is not particularly limited, and it is possible to climb to the table of mankind in general, and includes all livestock products such as poultry, beef, pork, eggs, and milk. do.

In addition, the selenium peptide according to the present invention has excellent antioxidant activity, and thus exhibits the effects of skin aging and skin whitening, such as inhibiting the activity of collagen degrading enzymes and increasing collagen synthesis. It is included in a range.

Cosmetics containing the selenium peptide of the present invention may be in the form of a lotion or cream, containing all or part of abrasives, purified water, glycerin, buffers, flavorings, oils, alcohols and other ingredients used as conventional ingredients. To prepare a lotion by adding the selenium peptide of the present invention having antioxidant activity to the conventional composition as described above. In this case, the amount of selenium peptide to be added may be appropriately selected according to the absorbency, inactivation rate, excretion rate, age, gender and condition of the active ingredient in the skin, and 0.01 to 5.0% by weight of the total cosmetic composition components It is preferable.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Culture of Selenium Yeast and Isolation of Selenium Protein

Saccharomyces cerevisiae (ATCC 7752) was used as a yeast strain, and sodium seleide in YM medium (glucose 10g / l, peptone 5g / l, yeast extract 3g / l, malt extract 3g / l, Difco) Nate (sodium selenate, Sigma) was added at a concentration of 20 ppm and then incubated for 48 hours at 250 rpm, 30 ℃.

Cells were recovered by centrifuging the selenium yeast culture at 3,000 g for 20 minutes, then washed twice with distilled water and crushed by using an ultrasonic ultrasonicator. After cell debris was centrifuged at 3,000 g for 30 minutes to remove cell debris, the protein was precipitated for 30 minutes at 80% concentration of ammonium sulfate. Salts were removed by dialysis, and proteins isolated therefrom were stored by lyophilization.

Example 2 Acid Hydrolysis and Enzymatic Hydrolysis

Acid hydrolysis was added to 2 mg of selenium protein prepared in Example 1 at concentrations of 0, 1, 2 and 3 N, and hydrolyzed at 80 ° C. for 1 hour and neutralized with NaOH. Enzymatic hydrolysis was performed by adding protease K and pronase at a ratio of 1:50 to selenium protein, respectively, and hydrolyzing at 37 ° C. for 3 hours. After hydrolysis, the reaction solution was heated at 90 ° C. for 10 minutes to remove protease activity. The antioxidant activity of the selenium peptides produced by each degradation method was measured according to the following method.

Determination of antioxidant activity due to selenium is indicated primarily by glutathione peroxidase (GPx) activity. GPx activity was measured using the method of Paglia, DE and Valentine, WN, et al., J. Lab. Clin. Med. 70: 158-169, 1967.

Sample to be measured in 0.4 ml of a mixture of 1 mM EDTA, 1 mM NaN ₃ , 0.2 mM NADPH, 1 mM glutathione, and 1 U / ml glutathione reductase in 50 mM potassium phosphate buffer (pH 7.0) 0.05 ml was added. After the reaction solution was reacted for 10 minutes in a 37 ° C thermostat, the absorbance (340 nm) was measured for 5 minutes by adding 0.05 ml of 5 mM H ₂ O ₂ solution. The absorbance coefficient of NADPH was 6.22 mM −1 cm −1, and 1 μM NADPH was consumed for 1 minute. GPx activity was expressed as protein activity and protein quantification was performed using BCA protein assay kit (pierce).

As a result, during acid hydrolysis, as the acid concentration increased, the antioxidant activity of the original selenium protein was lost ( FIG. 1 ), since amino acids having a thiol group such as methionine or cysteine are decomposed by the acid. . Selenomethionine has a structure similar to methionine in the form of the sulfur position of methionine substituted with selenium, and thus, selenomethionine is also decomposed by acid and loses antioxidant activity.

The selenium content of the protein produced in the medium containing selenium was 2.3 [mu] g / mg protein, while the protein that was not was insignificant. Due to the difference in selenium content in these proteins it can be seen that the antioxidant activity is also different ( Fig. 2 ). In addition, the antioxidant activity was increased by enzymatic hydrolysis, which allows hydrolysis with proteolytic enzymes to selectively degrade the protein to produce peptides with specific activity through changes in protein structure and reduction in molecular weight ( Byun, HG and Kim, SK, Process Biochemistry 36 (12): 1155-1162, 2001; Kunio Suetsuna, Mar. Biotechnol. 2: 5-10, 2000) are in line with existing research reports on bioactive peptides. .

The selenium content in the protein was measured by the following method.

That is, the cells were hydrolyzed at 60 ° C. for 12 hours using 60% HNO ₃ and the protein was used diluted in 1% HNO ₃ . The standard curve was measured in the range of 0 to 100 ppb each time, and after diluting the sample pretreated to the above range using a graphite furnace atomic absorption spectrophotometer (AAS 5EA, Analytical Jena, Germany) 196 The amount of selenium was measured at nm. Selenium is stabilized by media deformation because it is subject to much interference from other materials (Cho, KH and Suh, JK, et al., Analytical Science & Technology 12 (2): 130-135, 1999; Hernandez-Caraballo, EA , et al., Atomic spectroscopy 57 (12): 2159-2165, 2002). To modify the medium, palladium is added to a final concentration of 10 ppm or 3 ppm of Mg (NO ₃ ) is added to the palladium. In the present invention, selenium was analyzed by adding 10 ppm of palladium.

Example 3 Establishment of Autoenzyme Degradation Conditions

<3-1> Effect of Cell Fracture on Autoenzyme Degradation

In the yeast autoenzyme, various enzymes work in combination. First, cell wall is decomposed by cell wall degrading enzyme, and then proteolysis is performed by three kinds of proteolytic enzymes (Boonraeng, S, et al., Nat . Sci . 34: 270-278, 2000). Thus, the present inventors incubated selenium yeast according to the method described in Example 1 in order to investigate how the crushed cells and then used for autoenzymatic degradation after culturing selenium yeast in an ultrasonic grinder Cell lysate obtained by crushing and centrifugation was autoenzymatically at 50 ° C. for 48 hours. At this time, the unbroken yeast suspension was used as a control.

As a result, in the case of autoenzymatic digestion after cell crushing, protease was initially activated, resulting in more protein outflow and increased GPx activity of selenium peptides compared to the control group without crushing the cell ( FIG. 3 ).

<3-2> Effect of Autoenzyme Additive

In order to determine how the promoter affects selenium peptide production during autoenzyme digestion, the present inventors have discovered that NaCl and ethanol (Boonraeng, which are known to promote autoenzyme digestion at a concentration range of 1 to 10% and prevent contamination from outside), respectively. S, et al., Nat. Sci. 34: 270-278, 2000; Son, SM and Kim, JS, et al., J. Food Sci. Technol. 35 (2): 201-205, 2003) The following experiment was performed using the enzyme decomposition promoter.

First, after culturing selenium yeast as in Example 1 , the cells were recovered in a sterile state and cells were crushed using an ultrasonic mill. The autoenzyme promoter was added to the cell lysate at different concentrations or added together and reacted at 50 ° C. and 250 rpm for 48 hours. After the reaction was completed, the reaction solution was centrifuged (3,000 g, 30 minutes) to remove cell residues and the antioxidant activity was measured in the same manner as in <Example 2> . The average molecular weight was measured as follows.

That is, the average molecular weight of the peptide was measured using gel permeation chromatography (GPS). Ultrahydrogel 250 (Ultrahydrogel 250, Waters) was used as a column. At this time, using eluate potassium phosphate buffer solution (0.01 M, pH 7.0) at a flow rate of 1 ml / min, the molecular weight standard curve is vitamin B12 (MW: 1,355 Da), RNase A (13,700 Da), BSA ( 66,000 Da). Average molecular weight was calculated according to the following equation (1).

Average Molecular Weight = ∑ (MW × Concentration) / ∑ Concentration

As a result of autoenzymatic digestion after adding 1 to 7% of ethanol, there was no significant difference in the amount of effluent protein and only slightly increased in the group to which 5% ethanol was added ( FIG. 4B ). However, even though the addition of NaCl showed a similar protein efflux and GPx activity as the control group, the average molecular weight of the protein after autoenzymatic degradation was markedly lower and the GPx activity was higher than that of ethanol addition ( FIG. 4A ). Based on the above results, the effect of the addition of NaCl and ethanol to increase the protein effluent and the average molecular weight of small peptides was examined. At the time of addition, even though NaCl was added, it was confirmed that a large molecular weight peptide was produced, indicating that the addition of ethanol was inadequate for producing a small molecular weight peptide ( FIGS. 5A and 5B ).

<3-3> Effect of Selenium Concentration on Autoenzyme Degradation

In order to investigate the effect of selenium concentration on the antioxidant activity of autoenzyme products in cultivation of selenium yeast, yeast was cultured and autoenzymatically changed with selenium concentration to 10, 20, 50 and 100 ppm with sodium selenate. . As a result, the highest protein per selenium content and GPx activity in the yeast autoenzyme cultured by adding 20 ppm selenium ( FIG. 6 ). Table 1 below compares the autoenzyme effect of yeast cultured in 20 ppm selenium-added medium with that without autoenzyme digestion.

Control Autoenzyme digestion Ratio (control: autoenzyme digestion) Protein Runoff (mg Protein / g Cells) 84.79 ± 0.45 87.73 ± 1.34 1: 1.03 Selenium Concentration (mg Se / mg Protein) 6.35 ± 0.15 14.15 ± 0.06 1: 2.22 Total selenium recovery (mg Se recovery / g cell) 538.30 1241.03 1: 2.31 GPx activity (unit / mg protein) 99.32 ± 5.26 326.97 ± 6.53 1: 3.29 Total GPx Activity (Units / g Cells) 8421..37 28686.95 1: 3.41

As shown in Table 1 , it can be seen that the autoenzyme digestion of yeast cultured in a medium containing 20 ppm of selenium produces selenium peptides that show significantly increased GPx activity compared to the control. The protein outflow by autoenzyme was similar to that without autoenzyme, but GPx activity per protein increased more than three times.

<3-4> Effect of Autoenzyme Decomposition Time

In order to investigate the effect of yeast autoenzyme time on the antioxidant activity of autoenzyme products, the enzyme was incubated in selenium yeast in 20 ppm selenium supplemented medium, and the enzyme was digested for 72 hours. GPx activity and mean molecular age of were measured.

As a result, the autoenzyme digestion of yeast cultured in a medium containing 20 ppm of selenium rapidly decreased the molecular weight during the initial 6 hours and GPx activity increased up to 24 hours ( FIG. 7 ).

Example 4 Production of Low Molecular Selenium Peptides by Autoenzyme Degradation

Based on the results of Example 3, Saccharomyces cerevisiae was inoculated in YM medium to which 20 ppm sodium selenate was added and incubated at 250 rpm and 30 ° C. for 48 hours. Cells were collected by centrifuging the selenium yeast culture at 3,000 g for 20 minutes, washed twice with distilled water, and crushed by using an ultrasonic ultrasonicator. After 5% NaCl was added to the cell lysate, autogenolysis was performed at 50 ° C. for 48 hours. After the end of the enzymatic digestion, cell residues were removed by centrifugation, and the autoenzymes obtained therefrom were fractionated by fast protein liquid chromatography (FPLC) to separate selenium peptides by size. At this time, Superdex 30 (Amersham Biosciences) was packed into a 16 mm / 60 cm column, distilled water was flowed at a rate of 0.8 ml / min, and the eluted peptide was separated into three fractions. Fraction A is the collection of fractions 6-9 with a dissolution time of 50-90 minutes. Fraction B is a collection of fractions 14 to 17 of 130 to 170 components and fraction C is a collection of fractions 18 to 21 of 170 to 210 components. GPx activity and average molecular weight of the fractions A, B and C were measured and compared with the control.

As a result, it was confirmed that the average molecular weight of the peptide around 5,000 Da (fraction B) showed GPx activity increased by about 2.5 times or more compared to the control ( Fig. 8 ).

Preparation Example 1 Preparation of Cream

Stearic acid, cetostearyl alcohol, caprylic capric triglyceride, mineral oil and butylene glycol are added to the beaker in the water at 75 ° C. Warmed to give an oil phase. Selenium peptide, water, glycerin, Tween 80 and potassium hydroxide were added to the oil phase prepared by mixing, stirred at 1200 to 1500 rpm and then cooled and left at room temperature for 1-2 days. The content of the ingredients in the cream is described below and the total weight is 100 g.

Selenium Peptide 100.0 mg

Stearic acid 3.0 mg

Cetostearyl alcohol 2.0 mg

Caprylic Capric Triglyceride 5.0 mg

Mineral oil 8.0 mg

Butylene Glycol 3.0 mg

Glycerin 6.0 mg

Tween 80 2.5mg

Potassium hydroxide 0.5 mg

Distilled water

Preparation Example 2 Preparation of Beverage

Selenium peptide, vitamin C, powdered vitamin E, ferric nitrate, zinc oxide, nicotinic acid amide, vitamin A, vitamin B1 and vitamin B2 of the present invention were prepared by mixing well.

The components of the beverage are as follows.

Selenium Peptide 0.1 g

15.0 g of vitamin C

7.5 g of powdered vitamin E

Ferric Iron 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B1

0.3 g of vitamin B2

Distilled water

As described above, the production method of the low molecular weight selenium peptide having antioxidant activity using the autoenzyme digestion of the yeast of the present invention provides an inorganic selenium and autoenzyme conditions that can be converted into organic selenium in the culture of selenium yeast, Since selenium peptides produced by the method of the present invention have low toxicity and low molecular weight while exhibiting high bioavailability and utilization rate and exhibiting excellent antioxidant activity, dietary supplements or additives for dietary supplements and livestock having antioxidant activity It can be usefully used.

Claims (15)

A method for producing low molecular weight selenium peptide by yeast autoenzyme digestion. The method of claim 1, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae and Saccharomyces uvarium . The method of claim 1, i) culturing the yeast in a selenium-containing medium to produce selenium yeast; ii) crushing the selenium yeast culture; iii) adding an autoenzyme promoter to the cell lysate and obtaining an autoenzyme comprising selenium peptide by autoenzyme digestion; And iv) a method of producing selenium peptides, comprising the step of separating selenium peptides having excellent antioxidant activity and low molecular weight from the autoenzyme digests. The method of claim 3, wherein the medium of step i) comprises selenium at a concentration of 20 to 50 ppm. The method of claim 3, wherein the selenium contained in the medium of step i) is sodium selenate. The method of claim 3, wherein the autoenzyme promoter of step iii) is 3 to 7% NaCl. The method of claim 3, wherein the autoenzyme of step iii) is carried out at 50 ° C. for 24 to 48 hours. The method of claim 3, wherein step iv) is to separate selenium peptides having a molecular weight of 1000 to 7000 Da using autoenzyme column chromatography. The method of claim 8, wherein said column is selected from the group consisting of superdex, superose, sephacryl and sephadex. 10. A low molecular weight selenium peptide having antioxidant activity produced by the method of claim 1. The selenium peptide according to claim 10, which has a molecular weight of 1000 to 7000 Da. A pharmaceutical composition having an antioxidant activity containing the selenium peptide of claim 10 as an active ingredient. Health supplements having antioxidant activity containing the selenium peptide of claim 10 as an active ingredient. Cosmetic composition having an antioxidant activity containing the selenium peptide of claim 10 as an active ingredient. Animal feed composition having antioxidant activity containing the selenium peptide of claim 10 as an active ingredient.

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