KR20180049679A - A method of manufacturing bioactive collagen peptides by using subcritical water and enzymes, and a liposome comprising bioactive prepared therefrom - Google Patents

Abstract

The present invention relates to a method for pretreating pig skin and hydrolyzing the same using subcritical water, and hydrolyzing a hydrolysate having hydrolyzed using the subcritical water with a proteolytic enzyme, which is an eco-friendly method without using an organic solvent, wherein the obtained bioactive collagen peptide contains a large amount of beneficial amino acids in the pig skin, and thus can be used as a main or supplementary ingredient for various medicines, cosmetics, and foods. The stability and absorption in the human body of the bioactive collagen peptide are increased through a liposome containing the bioactive collagen peptide prepared by the method to increase the biological availability, and thus can be useful in various industry fields such as medicine, beauty, or food. In addition, by using a pig skin which is not a major meat material of pigs but a supplementary accessory, it is expected to increase profits of pig farms, enhance the health of people, and satisfy the desire of consumers who prefer high value-added functional products.

Description

The present invention relates to a method for producing a bioactive collagen peptide by using an asymmetry factor and an enzyme, and a liposome comprising a bioactive collagen peptide produced by the method, and a liposome comprising bioactive prepared therefrom }

The present invention relates to a method for producing a bioactive collagen peptide using subcritical water and an enzyme from a pork pulp, and a liposome comprising the bioactive collagen peptide thus prepared.

More specifically, the present invention relates to a method for producing a bioactive bioactive collagen peptide having a high bioavailability while containing a large amount of free amino acid useful for human body by an environmentally friendly method using an asymmetry factor and a protease, and a method for producing a bioactive collagen peptide To increase the bioavailability of bioactive collagen peptides and maximize bioavailability and stability of bioactive collagen peptides.

Collagen is a fibrous protein that is one of the proteins that make up the skin, bones, and tendons. In particular, we have about 20% protein in our body, which accounts for about 30% of collagen. The molecular structure of such collagen has a structure in which two polypeptide chains are wound and bonded to each other by hydrogen bonding. Collagen having such a structure is a main component of connective tissue in the human body and is distributed and distributed in skin, bones, joints, blood vessels, membranes of organs and organs to support and maintain the human body. Cellular adhesion, support of body and organs, Cell proliferation, and hemostasis. It is a protein that is essential for the body to form the body and impart organic functions. However, collagen is more destroyed and released than collagen synthesized with age, and collagen, which is larger than normal protein, is not easily digested and absorbed in the body, and it is known that it is released by about 90%.

Collagen is widely used in medicine (capsule tablets, tourniquet, artificial skin, etc.), cosmetics (creams, ointments, etc.) and foods (binders, extender, beauty drinks etc.) Anti-aging, growth promotion, prevention of osteoporosis and inhibition of blood pressure increase. In particular, when collagen metabolism is activated by stimulating the synthesis of collagen in the skin, it is believed that the components of the dermatrix are increased, thereby improving wrinkles, improving elasticity, strengthening the skin, and healing wounds. In recent years, The important function of collagen in the skin has been revealed.

Collagen can be divided into polymer collagen and low-molecular collagen depending on the molecular weight. Since polymer collagen is a typical insoluble protein and its physical properties and digestion absorption ability are greatly changed according to the processing method, various hydrolysis processes The process of low molecular weight is indispensable.

Biologically active peptides are those in which a specific part of a protein contains an amino acid sequence (peptide) capable of exerting any physiological action. Bioactive peptides are known to be highly active and safe food additives and are known to be easily absorbed into the body. In this respect, bioactive peptides can be used in a wide variety of industries such as functional medicines, cosmetics or foods.

The two-step process, which is mainly used for producing bioactive collagen peptide from polymer collagen, is a process in which a collagen with a molecular weight of about 300 kDa is heat-treated with an acid or an alkali at a temperature of 45 ° C or higher and extracted with a soluble collagen of 100 kDa or less will be. Generally, the acid treatment method is used for the pork, and the various protein hydrolytic enzymes are added to the pork collagen to lower the molecular weight to 5 kDa or less. Afterwards, the product is finally produced powdery bioactive collagen peptide product by washing, neutralization, extraction, filtration, sterilization cooling, drying and pulverization. However, the process of low molecular weight conversion of pork collagen by an acid treatment method has problems such as equipment corrosion by acid, wastewater treatment, low collagen yield, and odor generation peculiar to a material.

Therefore, the inventors of the present invention have studied a method of obtaining a horseradish hydrolyzate by an environmentally friendly method without using an organic solvent, unlike a conventional preparation method, and by hydrolyzing the horseradish using a sub- (Korean Patent Laid-open Publication No. 10-2016- 0096354) has completed the invention which can obtain a pork pulp hydrolyzate having a large amount of pork but having a pork specific odor removed.

In addition, proteolytic enzymes can be used together to optimize the hydrolysis of collagen using the asymptotic coefficient. The relationship between process pressure and enzyme activity has recently been introduced through a number of documents including chymotrypsin, pepsin, trypsin, flavorzyme, protease E, Proteases such as alcalase are known to maintain or improve activity under high pressure. Research on ultra high pressure has been actively carried out at home and abroad, but in most studies, studies have been carried out mainly on microbial killing or inhibition of enzyme activity using relatively high pressure of 300 MPa or more. Therefore, there is no study on the effect of the subcycle environment, which is a relatively low pressure level, on collagen hydrolase.

On the other hand, it is important to develop collagen formulations in order to promote absorption of the body and direct penetration into the skin as well as low molecular weight processes of collagen. This is because absorption of polymer collagen is not easy to be absorbed in the human body, and it is also difficult to apply collagen to various industrial fields because of its low permeability to skin when applied to skin. In order to increase the penetration of the skin, a carrier capable of transferring useful components from the epidermis layer through the stratum corneum to the dermis is required. The main mechanism requires a delivery system consisting of a hydrophobic membrane for passing the relatively hydrophobic stratum corneum, and the delivery system must be nanoparticulated to allow the hydrophilic high purity collagen peptide to pass through the vesicular pathway do. Therefore, in order to improve the utilization of collagen, a process for increasing the penetration into the skin as well as the body after the hydrolysis with bioactive collagen peptide is urgent.

In this regard, in the field of pharmaceuticals, much research is being conducted to apply nanobiotechnology to drug delivery systems (DDS). The drug delivery system is a system that can efficiently deliver the required amount of drug to the organ and tissue cell by minimizing the side effects of the drug and maximizing the efficacy and effectiveness. Specifically, the useful substance is destroyed by a lipid physiologically active substance which is good in functionality but is limited in its use due to odor or already existing substances, or decomposed by temperature, oxidation, light, enzymes in the digestive tract, pH and other nutrients In order to overcome the limitations of the use of materials with reduced activity, it is possible to use solid dispersions, micro-emulsions, nano-emulsions, liposomes, pellets, Studies have been conducted on nano-sized carriers that enable direct ingestion of functional materials, such as improving the preservation of substances, controlling the rate of dissolution, blocking odors and taste, which are problematic for feeding, by using matrix tablets and the like have.

Among them, liposome refers to a kind of artificial cell that self-assemble to form a double or multiple membrane so that phospholipids having a hydrophilic and hydrophobic part are dispersed in an aqueous solution state so that the phospholipid becomes thermodynamically stable. The greatest benefit of this is in the bilayer structure. Liposomes Because liposomes are hydrophobic between bilayer membranes and hydrophilic inside cells, liposomes can capture both hydrophilic and hydrophobic materials. The bioactive collagen peptide can also be captured inside the liposome as a hydrophilic substance. However, liposomes have the best absorption ability and penetration ability of the human body, but they are easily destroyed by the external environment or weakened in stability. In particular, the functional materials in the cell may diffuse or move outward due to an increase in water vapor pressure or a difference in osmotic pressure, which may result in breakage of the liposome membrane and deterioration of stability. Therefore, there is a problem in that the effect corresponding to the original purpose becomes very small as the storage time passes.

Accordingly, the present inventors have studied an optimal process for producing a povidone collagen as an environmentally-friendly and energy-efficient bioactive collagen peptide, and have also found that a biologically active collagen peptide containing bioactive collagen peptide The research focused on the importance of liposomes. As a result, we found an optimal condition for the hydrolysis process for low - molecular - weight biodegradation of insoluble polymer collagen contained in Porphyra into bioactive collagen peptide using environmentally friendly asymmetry factors and enzymes. At this time, the produced bioactive collagen peptide After continuing the experiment to increase the bioavailability (human body absorbency, skin penetration ability and stability), the present invention was completed by studying a liposome which is a stable and skin-friendly protective film.

It is an object of the present invention to provide an optimum method for producing a bioactive collagen peptide from a hull using an asymptotic factor and an enzyme in an environmentally friendly manner.

It is another object of the present invention to provide a liposome comprising the bioactive collagen peptide in order to enhance the bioavailability and bioavailability of the bioactive collagen peptide produced according to the above method.

In order to solve the above-mentioned technical problems, the present invention provides a method for producing a bioactive collagen peptide from pork using an asymptotic modulus and an enzyme of 190 to 230 ° C and 1,100 to 2,600 kPa.

Preferably, in one specific embodiment of the present invention, there is provided a method of producing a bioactive collagen peptide from a pork pulp comprising the steps of:

(S1) cutting the pork slices and immersing them in water at 90 to 110 DEG C for 50 to 90 seconds to remove fat and impurities of pork slices;

(S2) homogenizing the immersed poultice of step (S1) in 0.5 to 1.5 weight percent water of the pomegranate weight;

(S3) hydrolyzing the homogenized pans of the step (S2) using an asymptotic factor of 190 to 230 DEG C and 1,100 to 2,600 kPa; And

(S4) hydrolyzing the hydrolyzate of step (S3) using a protease.

According to the present invention, a method for hydrolyzing a pork pulp followed by hydrolysis using an asymptotic ratio and hydrolyzing the hydrolyzate obtained therefrom with a proteolytic enzyme is an eco-friendly method which does not use an organic solvent, Collagen peptides contain a large amount of useful amino acids in pork, and can be used as main ingredients or auxiliary materials for various medicines, cosmetics, foods, and the like. For example, it can be used as a capsule refining, a tourniquet, an artificial skin, a suture, a functional cream, an ointment, a mask pack, collagen containing pork, a functional food, a functional beverage, a nutritional supplement or a protein-

The poultice used in the present invention is a conventional pig husk by-product, and there is no particular limitation on its use.

The step (S1) of the present invention is a step of immersing the poultice so as to remove the fat and residues remaining on the poultice, and after finishing the poultry, the poultice is cut and then heated at 90 to 110 ° C in water, preferably at 100 ° C for 50 to 90 seconds , Preferably 50 seconds to 70 seconds, more preferably 60 seconds.

At this time, when the temperature of the water is lower than 90 ° C, the fat and residues of the poultry can not be removed cleanly, and when the temperature of the water is higher than 110 ° C, the active ingredient in the poultry may leak out.

If the immersion time is less than 50 seconds, the fat and residues of the poultry can not be removed cleanly, and if the immersion time is more than 90 seconds, the effective ingredient in the poultry may be leaked.

The step (S2) of the present invention is a step of homogenizing the immersed pork of the step (S1) in water at a weight ratio of 0.5 to 1.5, preferably 0.9 to 1.1, more preferably equal to (1-fold) to be.

As a specific example, water can be pretreated by adding water in an amount equal to the weight of the pomegranate, and then homogenizing the pomegranate at 20,000 to 25,000 rpm for 2 to 8 minutes using a high-speed homogenizer.

The step (S3) of the present invention is a step of hydrolyzing the homogenized poultice of the step (S2) by using a limulus coefficient.

In the present invention, subcritical water means high-temperature and high-pressure water which maintains a liquid state in a state where a pressure of 0.4 to 40 MPa is applied between a boiling point (100 ° C) of water and a critical temperature of water (374.2 ° C) do. The subcritical modulus used in the present invention is an asymptotic modulus of 190 to 230 DEG C and an asymptotic modulus of 1,100 to 2,600 kPa, preferably 200 to 220 DEG C and 1,850 to 2,350 kPa, more preferably an amorphous modulus of 210 DEG C and 2,100 kPa To thereby produce a biologically active collagen peptide by hydrolyzing the poultry. For example, in the present invention, the submersion coefficient is obtained by using the submersion counting apparatus shown in FIG.

The step (S4) of the present invention is a step of hydrolyzing the hydrolyzate of the step (S3) into a protease, further hydrolyzing the hydrolyzate of the step (S3) and converting the hydrolyzate into a bioactive collagen peptide to be.

The protease of step (S4) of the present invention is a protease which hydrolyzes the hydrolyzate of step (S3) into a bioactive collagen peptide, preferably an alkaline protease.

When an alkaline protease is used as the protease in step (S4) of the present invention, hydrolysis is preferably performed at 55 to 65 ° C and pH 7.5 to 8.5.

In the present invention, the bioactive collagen peptide means collagen having a molecular weight of 10 kDa or less.

Also, the present invention provides a liposome comprising a biologically active collagen peptide prepared from pork slices using a limulus coefficient.

In one specific embodiment of the present invention, the liposome comprising the bioactive collagen peptide is a bilayer liposome, more specifically a thin film hydration method using bioactive collagen peptide and anion or cationic lecithin to form a single layer Liposomes can be produced. Thereafter, the bilayer liposome is produced by mixing a solution of the biopolymer and the monolayer liposome, which are primarily produced, by a layer-by-layer electrostatic deposition principle, or by mixing the monolayer liposome with an anion Bilayer liposomes can be produced by thin membrane hydration using cationic lecithin.

The anionic lecithin used in one specific embodiment of the present invention is Lipoid S75, a product of Lippo Ltd. (Germany), and the anionic lecithin Lipoid S75 contains phosphatidylcholine (69.3%), phosphatidylethanolamine (9.8%) and Risophosphatidylcholine (2.1% ), And their fatty acids are composed of palmitic acid (1720%), stearic acid (25%), oleic acid (812%), linoleic acid (5865%) and linolenic acid (46%).

The cationic lecithin used in one specific embodiment of the present invention is 1,2-dioloyo-3-trimethylammonium propane (DOTAP).

In the present invention, the lecithin is an anion or a cation, and is not limited to a substance that forms a circular or elliptical closed membrane structure including the prepared bioactive collagen peptide.

The lecithin-containing solution is an aqueous solvent, preferably water, phosphate buffered saline, tris buffered saline or acetic acid buffer solution.

In the present invention, the single-layer liposome is not limited thereto as long as it can trap the bioactive collagen peptide in lecithin.

In one specific embodiment of the present invention, the biopolymer used to form the bilayer liposome by coating the single-layer liposome formed primarily is chitosan or pectin.

In one specific embodiment of the present invention, the biopolymer can produce a bilayer liposome by the layered electrostatic deposition principle using the single layer liposome, but the production method of the bilayer liposome is not limited thereto.

The liposome capturing the bioactive collagen peptide obtained according to the present invention can be used as a main material or a sub ingredient of various medicines, cosmetics, foods, and the like. For example, it can be used as a capsule refining, a tourniquet, an artificial skin, a suture, a functional cream, an ointment, a mask pack, collagen containing pork, a functional food, a functional beverage, a nutritional supplement or a protein-

As described above, according to the present invention, a method of hydrolyzing a hyssop with a protease by pretreating the hyssop and then hydrolyzing the hydrolyzed hydrolyzate using the asymptotic modulus and using the hemicycle factor is an eco-friendly method that does not use an organic solvent , And thus the bioactive collagen peptide thus obtained contains a large amount of useful amino acid in the pork pulp and can be used as a main ingredient or a sub ingredient in various medicines, cosmetics, foods, and the like. The liposome containing the bioactive collagen peptide prepared according to the above method increases the stability of the bioactive collagen peptide and the absorbability in the body to increase the bioavailability and is useful for various industrial fields such as medicine, . In addition, it is expected that the use of pork, which is not a main meat material of pigs, can be used to increase the incomes of pig farmers as well as to meet the desire of consumers who prefer functional products of national health and high added value.

Fig. 1 shows an example of a sub-coefficient processing apparatus used in the present invention.
FIG. 2 is a graph comparing the temperature (left) and pressure (right) change curves of Samples I to VI subjected to hydrolysis using subclaims according to the present invention.
FIG. 3 is a graph comparing the chromaticities of Samples I to VI subjected to hydrolysis using subclaims according to the present invention.
Fig. 4 is a photograph of the appearance of Samples I to VI hydrolyzed using pigs pretreated according to the present invention and limulus coefficients.
FIG. 5 is a graph comparing the measured pH values of Samples I to VI hydrolyzed using subclaims according to the present invention.
Fig. 6 is a graph comparing the protein contents of Samples I to VI hydrolyzed using subclaims according to the present invention.
Fig. 7 is a graph comparing free amino acid contents of Samples I to VI hydrolyzed using subclaims according to the present invention.
FIG. 8 is a 3D model graph showing changes in free amino acid contents of Samples I to VI hydrolyzed using subclaims according to the present invention.
FIG. 9 is a graph showing the molecular weights of samples I to VI hydrolyzed using subclaims according to the present invention by using an electrophoresis method. FIG.
10 is a graph showing changes in molecular weight (Molecular weight, Da) analyzed by gel permeation chromatography of Samples I to VI hydrolyzed using subclaims according to the present invention.
11A and 11B show the results of measurement of protein content according to the type of each enzyme and the reaction time.
12A and 12B show the results of measurement of free amino acid content according to enzyme type and reaction time, respectively.
13 compares free amino acid contents according to enzyme types and mixing ratios.
FIG. 14 is a photograph of the sample hydrolyzed for 24 hours under the optimum conditions after mixing 5% Pigmented preprocessed sample and 5% enzyme solution at a ratio of 500: 1.
FIG. 15 shows changes in the chromaticity of a sample hydrolyzed for 24 hours under the optimum conditions after mixing 5% Pigmented preprocessed sample and 5% enzyme solution at a ratio of 500: 1.
16 shows the degree of hydrolysis according to the type of enzyme in terms of protein and free amino acid content.
17 shows the pH change of the hydrolyzate by the enzyme.
18A to 18F show the molecular weights of the hydrolyzate using alkalase, flavorzyme, nutraceutical, proteamic, bromelin, and papain depending on the hydrolysis reaction time, respectively, with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
FIG. 19 is a graph showing the molecular weights of hydrolyzates using alkaline, flavor, nutraceutical, proteomic, bromelin, and papain when the hydrolysis reaction time has elapsed after 24 hours was measured using sodium dodecylsulfate-polyacrylamide Gel electrophoresis (SDS-PAGE).
Fig. 20 is a graph comparing the contents of free amino acids and proteins of 5% and 50% pork samples used according to the present invention.
FIGS. 21A and 21B show protein and free amino acid contents of a sample treated according to the present invention, respectively, showing hydrolysis and fractionation treatment using an asymptotic factor and hydrolysis and fractionation treatment using an enzyme, respectively.
FIG. 22 shows a comparison of the molecular weights of Porphyra hydrolyzate using the subcycle according to the present invention, wherein a is the control (raw sample, blue), b is the asymptotic count (black) and the subcount / fraction (1-3 kDa) (Red), c is the enzyme treatment (black) and the enzyme / fraction (1-3 kDa) treatment (red), respectively.
FIG. 23 is a graph comparing the amino acid compositions of Porphyra hydrolyzate using enzyme and enzyme according to the present invention.
24A and 24B are graphs showing the measurement of the radical-trapping activity and the reducing power of ABTS at different concentrations of Sample IV and CP-II prepared according to the present invention.
25A and 25B are graphs showing the particle characteristics of the bilayer liposome according to the concentration of chitosan and pectin of the present invention, respectively.
26A and 26B are graphs respectively showing the collection efficiency of the bilayer liposome according to the chitosan and the concentration of the pectin of the present invention.
27a to 27d are a field emission-transmission electron microscope (FE-TEM) photographs of liposomes according to the present invention, wherein FIG. 27a shows a blank anion monolayer liposome with a scale bar of 50 nm, FIG. 27 (b) shows a blank anion single-layer liposome with a reference of 20 nm, FIG. 27 (c) shows an anion single-layer liposome containing a bioactive collagen peptide as a reference 50 nm, (Inner layer) / cation (outer layer)) are shown in the FE-TEM photographs with the liposome as the reference 50 nm.
FIG. 28 is a graph comparing the stability of the liposome according to the present invention with the storage period and the storage temperature, wherein A, B and C are the particle sizes, and D, E and F are the zeta potentials, respectively.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

< Manufacturing example  1> Donkey's  Pretreatment

Dungpyu was purchased from Jaein, Chungbuk Sangdong, Majang-dong, Seongdong-gu, Seoul and stored frozen until pretreatment. After thawing, the pork slices were cut into 1 cm x 1 cm (width x length) and immersed in boiling water for about 60 seconds to remove fat and impurities. The immersion process was carried out four times to remove foreign substances and fat. After the immersion process, the raw pork was mixed with distilled water in a ratio of 1: 1, homogenized at 22,000 rpm for about 3 minutes, and pretreated to have a solid content of about 50%.

< Example  1> Weighing factor  Hydrolysis using

The pretreated pigs were mixed with distilled water 1: 9 and re-homogenized. (5%) was packed into a commercially available sealed glass container (125 mL), packed in a metal lid with a heat resistant polyamide film, injected into a processing apparatus, and processed according to the conditions shown in Table 1 below. Pressure transducers and temperature sensors (K-type) were installed to observe the pressure and temperature changes during the process. The pressure and temperature data were collected by the data acquisition system (34970 A, Agilent, USA). Temperature and pressure data were used for further process intensity calculations.

sample pressure ( kPa ) Temperature (℃) Time to reach (min) Processing time (min) Duration (min) Sample I 350 150 3.5 10 8.7 Sample II 660 170 5.1 10 9.4 Sample III 1100 190 5.5 10 9.9 Sample IV 2100 210 6.5 10 10.4 Sample V 2600 230 6.8 10 10.5 Sample VI 3900 250 7.7 10 11.2

< Experimental Example  1> Subcounter  Analysis of temperature and pressure measurements of treated samples I to VI

FIG. 2 shows change curves of temperature (left) and pressure (right) of Samples I to VI subjected to hydrolysis using the subclaim coefficient according to Example 1. Referring to FIG. 2, since the subcritical state was induced by increasing the volume of the pressure vessel used in Example 1, the temperature and pressure were increased in proportion to each other. After reaching the target pressure and temperature, the same holding time of 10 minutes was maintained under all conditions, so that the plateau region was observed on the graph.

(P _d , kPa · min) by integrating time-pressure and temperature curves to integrate the degree of exposure of the sample to high pressure and high temperature conditions, And the temperature dose (T _d , ° C · s) were calculated. The amount of pressure and the amount of temperature mean the amount of pressure and heat applied to the sample from the start time ( t _i ) to the end time ( t _f ) of the process for calculating the asymptotic count, respectively. . The results are shown in Table 2 below.

[Equation 1]

[Formula 2]

sample Processing time (min) Temperature ( ℃ · min ) The amount of pressure ( kPa · min ) Sample I 22.2 3,012 5,513 Sample II 24.5 3,618 10,181 Sample III 25.4 4,031 16,477 Sample IV 26.9 4,539 29,564 Sample V 27.3 4,825 39,227 Sample VI 28.9 5,579 56,819

As shown in Table 2, the amount of pressure and the amount of temperature were 3012 占 폚 min and 5513 kPa 占 min at the sample I (150 占 폚 and 350 kPa), 5579 占 폚 min and 56819 占 폚 at the sample VI (250 占 폚 and 3900 kPa) kPa · min. Since the process intensity should be considered in terms of energy efficiency as well as the purity hydrolysis efficiency, it was used to set optimal pressure and temperature conditions by comparing with hydrolysis efficiency. The optimal temperature and pressure were found to be 210 ℃ and 2100 kPa, respectively. The optimum temperature and pressure were obtained by free amino acid analysis.

< Experimental Example  2> Subcounter  process Hydrolyzate  Chromaticity analysis

Samples I to VI obtained by treating with the sublimation coefficient in Example 1 were subjected to centrifugation (4,000 rpm, 20 DEG C, 20 minutes) to remove solids, followed by chromaticity analysis by taking only the supernatant.

The colorimetry was performed using a colorimeter (CR-400, Konica Minolta, Inc., Tokyo, Japan) and CIE L ^* indicating lightness, CIE a ^* indicating yellowness and redness CIE b ^{* &} lt; / RTI &gt; At this time, a calibration plate (CIE L ^* 94.49, CIE a ^* 0.66, CIE b ^* 3.32) was used as a standard color. The total color difference (ΔE) of the control after each asymmetry treatment was calculated using the following equation (3).

[Formula 3]

L ₁ ^* , a ₁ ^* , b ₁ ^* are values indicating the chromaticity of the control, and L ₂ ^* , a ₂ ^* , b ₂ ^* are chromaticities after the submixing process.

The change in the chromaticity (L *) of the hydrolyzate subjected to the asymmetry, which is the result of substituting the respective numerical values into the above equation 1, is shown graphically in FIG. 3. From this, the chromaticity difference increases as the temperature of the sub- .

FIG. 4 is a photograph of the hydrolyzate treated with the asymmetric hydrolyzate of the control and Sample I to Sample VI. As the temperature increased, the transparency tended to increase as compared with the control, which was the sample before treatment. Respectively. It was analyzed that the lightness of the collagen solution was increased by the low molecular weight through the hydrolysis using the asymptotic coefficient, and the lightness value was increased.

< Experimental Example 3>  process Hydrolyzate  pH analysis

The pH was analyzed with the supernatants of Samples I to VI taken in Experimental Example 2 above. The pH was measured three times using a pH meter (S220 SevenCompact ™ pH / Ion, Mettler Toledo, Greifensee, Switzerland).

FIG. 5 is a graph showing changes in pH depending on the treatment temperature of the ash process, which is the measurement result. As shown in FIG. 5, the pH tends to increase significantly with increasing the temperature of the subcritical water treatment, which is caused by an increase in the [OH] value due to self-ionization during hydrolysis I guess.

< Experimental Example  4> Subcounter  process Hydrolyzate  Protein content change analysis

Protein content was analyzed by Bicinchronic acid assay. This method is a colorimetric assay in which copper ions are reacted with tryptophan, tyrosine, and cysteine in an alkaline condition to produce a purple color. The protein is colored at a wavelength of 562 nm and the difference in the amount of protein can be compared according to the degree of purple color. In this experiment, protein content was indicated using bovine serum albumin (Sigma) as a standard.

As a result, as shown in FIG. 6, the protein content of the re-homogenized sample (control) mixed with 1: 9 of the pretreated rice hull of Example 1 was about 27.41% And 170 [deg.] C (sample II), the protein content was higher than that of the control. It was estimated that this was due to the coagulation effect of pork collagen caused by temperature increase. However, the protein content decreased significantly with the increase of the ash treatment temperature, and the protein content decreased to about 8.2% at the temperature condition of 250 ℃ (sample VI).

< Experimental Example  5> Subcounter  process Hydrolyzate  Free amino acid content change analysis

Free amino acid content was determined by free amino acid quantification method using 2,3,6-trinitrobenzenesulfonic acid (TNBSA, Thermo Scientific). The quantification method is a method in which TNBSA and primary amine react with each other to form a coloring product to confirm the degree of hydrolysis of the protein. The control and samples I to VI are measured using an ultraviolet / visible ray spectrophotometer at a wavelength of 335 nm Respectively. In this experiment, the content of free amino acid was indicated by measuring the content of L-leucine in the control and Samples I to VI.

As shown in FIG. 7, the content of free amino acid showed a tendency to increase with increasing the subconditioning treatment temperature, unlike the content of the protein in Experimental Example 4, and increased sharply from 230 ° C (Sample V). It is considered that the asymmetric hydrolysis of the protein of pork collagen is effective for the low molecular weight.

In order to evaluate the effect of increased temperature (T) and pressure (P) on the free amino acid content (A _f ) for the optimization of the energy efficiency during the asymptotic counting process, The results are shown in Table 3 below. In this experiment, A _f was defined as indicating the degree of hydrolysis of the protein.

 Coefficients Pr> | t | R ²  values Estimated standard error  (竜) β ₀ 6.145133 0.0001 0.99 0.42 β ₁ (T) 0.000808 0.9806 β ₂ (P) -0.040416 0.0001 β ₃ (T ² ) 0.000095 0.6375 β ₄ (P ² ) -0.000004 0.0001 beta ₅ (TxP) 0.000231 0.0001

[Formula 4]

As shown in Equation 4 and Table 3, the temperature (β ₁ ) in the polynomial regression equation showed a linear positive coefficient, but it was not statistically significant (P> 0.05). In the case of pressure (β ₂ ), negative coefficients were shown in linear regression and statistically significant. The regression equation showed positive coefficients for temperature (β ₃ ) in nonlinear quadratic terms, but it was not statistically significant (P> 0.05). For pressure (β ₄ ), statistically significant negative coefficients were obtained for the nonlinear quadratic term. From the results of the polynomial regression equation, it was found that the increase of the pressure alone can not hydrolyze the protein. Compared with other factors such as temperature and treatment time, the pressure had little effect on the hydrolysis of proteins during the asymptotic counting. From Table 3 and FIG. 8, it was found that it is preferable to simultaneously treat (β ₅ ) with the appropriate pressure and temperature (P <0.05) in order to promote the hydrolysis of proteins using the subcodes.

< Experimental Example  6> electrophoresis Hydrolyzate  Molecular weight analysis

Electrophoresis was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% acrylamide gel.

As shown in FIG. 9, when the molecular weight was confirmed by SDS-PAGE, a large number of bands were clearly observed in the polymer domain in the control, At a temperature of 190 ° C (sample III) or more, only a band having a molecular weight of 10 kDa or less was present. It is considered that this is due to a sufficient hydrolysis effect. Particularly, in the case where the subconditioning treatment temperature is higher than 210 DEG C (sample IV), no band is observed, and as a result, the bioactive collagen peptide It is considered to be hydrolyzed.

< Experimental Example  7> gel permeation chromatography ( GPC ) Hydrolyzate  Molecular weight measurement

Molecular weight determination of the hydrolyzate was measured using gel permeation chromatography (high performance liquid chromatography (HPLC) system). In the control, no peaks appeared in the molecular weight range of 67 kDa or less, so it is difficult to produce low-molecular-weight peptides by simple pretreatment. In order to produce low molecular weight peptides, there was. As shown in FIG. 10, when the molecular weight ranges from 70 kDa to 4 kDa are examined, the peak shifts from 70 kDa to 4 kDa as the temperature increases. In particular, in order to produce a low-molecular-weight peptide having a molecular weight of 400 Da or less, it has been confirmed that it is very important to treat the peptide with an amylase having a temperature of 190 ° C (sample III) or more. The maximum peak has a tendency to increase with increasing temperature treatment.

Finally, as shown in Experimental Example 1 and Experimental Example 5, when using an excessively high temperature and pressure to produce a low molecular weight peptide, it is not preferable from the viewpoint of excessive free amino acid production and energy efficiency. It turned out to be the optimum condition to treat.

< Example  2> Hydrolysis method using enzyme

In Example 2, a pork rib hydrolyzate was prepared using commercially available enzyme for food. Using the above enzyme, Alcalase, Flavourzyme, and Nutrase of Novozymes and Protamex, Bromelain, and Papain of Daejongsang are used. The hydrolysis was carried out at the optimal temperature and pH condition of each enzyme. Table 4 shows the optimum temperature and pH of each enzyme.

Enzyme name Optimum temperature ( ^o C ) Optimum pH Enzyme name Optimum temperature ( ^o C ) Optimum pH Al-Karadze 60 8 ProTamex 60 7 Nukraje 45 8 Bromelin 60 7 Flavor 60 6.5 Papain 60 7

The optimum pH values shown in Table 4 were adjusted to 0.1 N hydrochloric acid (HCl) and 5% enzyme solution by mixing 5% pre-treatment samples prepared in Example 1 and a 5% enzyme solution at a ratio of 100: 1, 500: Sodium hydroxide (NaOH). The samples were hydrolyzed for 0, 1, 3, 6, 12, and 24 hours in an incubator at the optimum temperature, and samples were taken at each hour and then adjusted to optimum pH again. The sample was heated to 95 DEG C for about 10 minutes to inactivate the enzyme in the sample, and then cooled in an ice box. After the sample was centrifuged (4,000 rpm, 20 ° C, 20 minutes), the supernatant was taken and the following physicochemical analysis was carried out.

< Experimental Example  8> Analysis of protein content by enzyme type and reaction time

In Example 1, the protein content of the hydrolyzed sample was measured according to the enzyme type and the reaction time by mixing the 5% povidone pretreated sample and the 5% enzyme solution at a weight ratio of 100: 1. The results are shown in Figs. 11A and 11B. Protein content was mainly affected by the enzyme type. In particular, the changes in protein content after hydrolysis of alkarase, nutraceutical, and protamex showed similar tendencies, and the protein content rapidly decreased after 1 hour.

< Experimental Example  9> Analysis of free amino acid content according to enzyme type and reaction time

As shown in Figs. 12A to 12B, the free amino acid content of the control (0 hour) contained a low concentration of free amino acid of about 3 mg / mL. On the other hand, as the reaction proceeded, the free amino acids formed according to the type of enzyme showed a significant difference. Especially, the content of free amino acid after 24 hours was the highest with alkarase of 25 mg / mL or more, It was confirmed that the order of decrease was in the order of tamax, nutraceutical, bromelin, flavor, papain. In addition, the effect of reaction time was observed, and it was observed that the content of free amino acid in all enzymatic hydrolysis reaction was rapidly increased within 1 hour.

< Example  3> Effect according to enzyme type and mixing ratio

In Example 3, hydrolysis was carried out by mixing the pretreated 5% solids of Example 1 with a 5% enzyme solution at a ratio of 100: 1, 500: 1, and 1000: 1. FIG. 13 shows the comparison of the free amino acid contents according to the types and mixing ratios of the enzymes. The protein content showed a large difference depending on the mixing ratio, and the higher the enzyme concentration, the more effective the hydrolysis reaction. The hydrolytic capacity of the enzyme was highest in the same manner as in Experimental Example 9, and even when the reaction was carried out at different mixing ratios, free amino acid content was the same.

< Example  4> Enzyme Hydrolyzate  Physicochemical characterization

In order to analyze the physicochemical properties of the hydrolyzate by enzyme, 5% povidone pretreated samples and 5% enzyme solution were mixed at a ratio of 500: 1, and hydrolysis was carried out for 24 hours at each optimum condition shown in Table 4, The degradation product was produced. Thereafter, the hydrolyzate was centrifuged (4,000 rpm, 20, 20 minutes), and then the supernatant was removed after solids were removed, and the physicochemical analysis was carried out. Fig. 14 shows an external view of the supernatant. The measurement of the change in chromaticity of the supernatant is shown graphically in Fig. The chromaticity change showed the same pattern as the appearance photograph. The highest brightness and chromaticity difference values were observed in the hydrolyzed samples of the flavor, and the samples hydrolyzed with other enzymes showed similar values.

16 shows the degree of hydrolysis according to the type of enzyme in terms of protein and free amino acid content. Compared with the protein content, the sample with the highest protein content was hydrolyzed into flavor, and the sample with the lowest protein content was hydrolyzed with nutraceutical. In contrast, the free amino acid content showed a tendency opposite to that of the protein content. As the protein was hydrolyzed by the enzyme, the free amino acid content was relatively increased. This is considered to be a very useful index for selecting suitable industrial enzymes for producing bioactive collagen peptide from pork.

17 shows the pH change of the hydrolyzate by the enzyme. It can be seen that most enzymes tend to increase in pH during hydrolysis.

< Experimental Example  10> Enzyme utilization by electrophoresis Hydrolyzate  Molecular weight analysis

Electrophoresis was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% acrylamide gel.

The molecular weight pattern confirmation by SDS-PAGE was performed according to the type of enzymes and the reaction time, and hydrolyzed with alkarase, flavorzyme, nutraceutical, proteomax, bromelin, and papain Were confirmed. The results are shown in Figs. 18A to 18F. As shown in FIGS. 18A to 18F, hydrolysis was carried out for each enzyme for 36 hours, and the molecular weight was determined by taking samples at each time. As the reaction time progressed, it was observed that the band corresponding to each molecular weight was shifted from the polymer domain to the low molecular domain, and it was observed that the degree of hydrolysis was significantly different depending on the type of the enzyme. In this experiment, the enzyme with the best hydrolytic activity in 8 was only able to identify low molecular bands of less than 10 kDa after 6 hours of hydrolysis reaction time as an alkarase. The next most highly hydrolyzable enzymes were nutraceuticals and bromelaine. The enzymes with the lowest hydrolysis were identified as flavozyme and papain. Comparing the molecular weight patterns after one hour of hydrolysis reaction time, it was possible to identify low-molecular-weight bands in nutraceuticals. However, the molecular weight concentrations below 10 kDa declined with time, while those with molecular weights of 10-20 kDa The band of the range was maintained. In addition, when the hydrolysis time was shorter than 36 hours, it was confirmed that there were various bands ranging from high molecular weight to low molecular weight, and these enzymes increased the hydrolysis reaction time or enzyme concentration And that there is a need to increase it.

19 shows the molecular weight of the hydrolyzate using alkarase, flavorzyme, nutraceutical, pro-thamex, bromelin, and papain when the hydrolysis reaction time elapsed after 24 hours was confirmed by SDS-PAGE will be. It was confirmed that the hydrolysis effect of alkarase was the best. As a result, in the subsequent powdering experiment, an attempt was made to produce bioactive collagen peptide by alkarase.

< Example  5> Nongpie Hydrolyzate Powdered

Based on the above experimental results, optimum conditions for hydrolysis of asymmetric and enzymatic hydrolysis were calculated. The enzymatic hydrolysis was carried out under the conditions of 60 ° C and pH 8 with an alkaline enzyme (Alkalase enzyme solution: pork sample = 1: 500) at 210 ° C and 1100 kPa. The reaction was allowed to proceed for 24 hours.

In Example 5, a 50% povidone pretreated sample was hydrolyzed in place of the 5% povidone pretreatment sample of Example 1 to increase the production yield of the povidone hydrolyzate. Fig. 20 shows the results of comparing the protein and free amino acid contents of the 5% pork pretreatment sample and the 50% pork pretreatment sample. Protein content was increased to 17.81 mg / mL for 5% Porphyra hydrolyzate and 195.60 mg / mL for 50% hydrolyzate, indicating that the protein content increased proportionally. Similarly, the free amino acid content showed a similar pattern to the protein content. Therefore, in the subsequent powdering experiment, the hydrolysis reaction was carried out using the ash factor and the enzyme as a pretreatment sample of 50% pork.

In order to produce only bioactive collagen peptide having a molecular weight of 3 kDa or less, the produced hydrolyzate was separated and purified by ultrafiltration. Table 5 shows the production yield of Porphyra hydrolyzate according to the optimal hydrolysis conditions.

The yield of raw pork protein samples was about 27.27%, and the yield tended to decrease during the hydrolysis process. The yield of the enzyme hydrolysis was significantly lower than that of the hydrolysis process. The yield of 3 kDa low molecular weight samples was about 6.55% for hydrolysis of submersible hydrolysis and 12.42% for hydrolysis of enzyme.

process Powdering yield (%) process Powdering yield (%) Donpy biotype sample (Control) 27.27 Donpy biotype sample (Control) 27.27 Hydrolysis
(210 degrees / 2100 kPa) 9.27 Enzyme hydrolysis
(Alcalase: Donkey = 1: 500, 24 hours) 21.76 Hydrolysis / fractionation
(1-3 kDa) 6.55 Enzymatic hydrolysis / fractionation
(1-3 kDa) 12.42

< Experimental Example  11> Hydrolyzate  Powder and powder Fraction  Protein and free amino acid content analysis

FIG. 21A shows the protein and free amino acid contents of a sample obtained by hydrolyzing and fractionating 50% of the pre-treatment sample of Example 5 by submixing. Figure 21 (b) shows the protein and free amino acid contents of the samples subjected to enzyme hydrolysis and fractionation thereof.

The pork collagen control contained about 12% protein, which was hydrolyzed with 31% protein and hydrolyzed with 20% protein. Further, when the bioactive collagen peptide was produced by fractionation (3 kDa) of the hydrolyzate, 26% (subcritical / fraction) and 21% (enzyme / fraction) proteins were contained, respectively.

On the contrary, when the free amino acid content was compared, the control value of the pork pretreatment sample contained about 0.76% free amino acid, and the free amino acid content of the pork pretreatment sample was increased as the fraction of the hydrolyzate was fractionated . In addition, it was confirmed that the free amino acid content was more produced by enzymatic hydrolysis than by the hydrolysis of the asymmetric hydrolysis. That is, when the bioactive collagen peptide was produced by fractionation (3 kDa) of the hydrolyzate produced by the hydrolysis of the hydrolyzate, the free amino acid contained 6.02% (subcritical / fraction) and 14.16% (enzyme / fraction) there was.

< Experimental Example  12> Measurement of Molecular Weight by Gel Permeation Chromatography

22 shows the result of molecular weight measurement by gel permeation chromatography. a showed a peak at 20 kDa or more in the 50% pretreatment sample of Example 2 (control), indicating that the ponytail collagen was very high molecular weight. However, the hydrolysis using the asymmetry factor and the enzyme tended to decrease the molecular weight. The hydrolysis of the hydrolyzate showed a molecular weight of about 12.6 kDa and a molecular weight of 6.9 kDa at the hydrolysis of the enzyme Respectively. It was found that bioactive collagen peptide was produced in a range of about 4 kDa when fractionated through 3 kDa membrane after hydrolysis using an asymmetry factor and an enzyme.

 In the present invention, in order to produce a bioactive collagen peptide, hydrolysis was carried out by using an asymptotic coefficient (210 DEG C and 2100 kPa) and an enzyme (alkarazine), and it was found that a highly effective collagen peptide capable of producing a bioactive collagen peptide Method.

< Experimental Example  13> Amino acid composition analysis

FIG. 23 shows the amino acid composition of the hydrolyzate obtained by fractionating the hydrolyzate using enzyme and enzyme and the hydrolyzate fractioned to 3 kDa by ultrafiltration. The amino acid concentration of the 50% pretreatment sample (control) of Example 2 was measured to be 35 / / mL or less in most cases, and the glycine and L-glutamine contents were high in the control.

L-proline showed the highest content and the content of glycine, L-histidine, L-alanine and L-cystine was lowered in the order of amino acid composition. When the hydrolyzate was fractionated, the amino acid content of the fraction tended to be lower than that of the fraction before the fractionation, and the content of glycine in the amino acid was the highest.

When hydrolyzed with an enzyme, amino acid composition changes were observed. When the enzyme treatment was carried out, L-isoleucine showed the highest content. In addition, when the hydrolyzate using the enzyme was fractionated, the amino acid composition of the fraction was found to be larger than that before the fractionation, and glycine and L-cystine were found to be high values.

As a result of analysis of amino acid composition, the amino acid composition of the hydrolyzate using the asymptotic factors is predominantly L-proline and glycine, while the amino acid composition of the hydrolyzate using the enzyme is predominantly glycine and L-cystine . These results show that the hydrolysis method combining the asymmetry factor and the enzyme can produce a bioactive collagen peptide containing different amino acid compositions.

< Experimental Example  14> Subcounter  According to fraction Nongpie Bioactivity  Collagen Peptides  Comparison of Physicochemical Properties of Powder

As the biologically active collagen peptide samples, hydrolyzed 50% pork pretreated samples of Example 2 at 210 ° C and 2100 kPa, which is the optimum process for hydrolysis of hydrolyzate, was used. The fraction of the biologically active collagen peptide was CP-I in the range of 1 kDa or less, CP-II in the 1 kDa-3 kDa, CP-III in the 3 kDa-5 kDa, CP-IV in the 5 kDa-10 kDa, And more than 10 kDa was named CP-V. The protein content, free amino acid content and yield of the fractionated bioactive collagen peptide CP-I to CP-V are shown in Table 6 below. The protein and free amino acid contents of the subcondition optimum condition sample IV (210 ° C and 2100 kPa) of Example 1 were 28.07% and 9.80%, respectively. The protein content of CP-I to CP-V of Experimental Example 14 was increased to 17.55 to 39.06% in order of CP-I <CP-II <CP-III <CP-IV <CP-V, -I The highest measured sample (10.93%). However, there was no significant difference according to the fraction size in the other samples (P> 0.05). The yield of fractionated biologically active collagen peptide decreased with decreasing cut molecular weight and was 1.75% in CP-I sample (1.75%).

Peptides  sample   Protein content ( % )   Free amino acid content ( % ) yield( % )   CP-I  17.55 ± 0.39 10.93 ± 0.01 1.75   CP-II  27.43 ± 1.44 3.16 ± 0.14 12.15   CP-III  28.71 ± 1.60 1.24 ± 0.57 20.84   CP-IV  32.23 ± 1.49 1.54 ± 1.57 22.65   CP-V  39.06 ± 1.77 1.14 ± 1.57 27.95

The composition of the constituent amino acids according to the fraction is shown in Table 7 below. CP-II (530,841 mg / kg)> CP-IV (520,018 mg / kg) CP> II (573,206 mg / kg) -V (434,878 mg / kg) > CP-I (409,257 mg / kg). Particularly, the CP-II samples contained glycine (248,077 mg / kg), proline (148,126 mg / kg), glutamine (141,003 mg / kg), alanine (103,555 mg / kg) and arginine .

And the absorbance at a wavelength of 734 nm was measured. Ascorbic acid (6.25 mg / mL) was used as a positive control for comparison. The ABTS radical-scavenging activity of the samples was calculated by Equation 5 below.

[Formula 5]

Where A control is the absorbance in the absence of the sample and A sample is the absorbance of the sample.

In order to test the ABTS radical scavenging activity, pork was hydrolyzed under subcritical conditions of 210 ° C and 2,100 kPa (sample IV) and then fractionated to 1 to 3 kDa (CP-II). In general, the antioxidant activity test system is known to be more useful in analyzing low molecular peptides than polymer peptides and proteins. One method for measuring antioxidant activity is the ABTS radical-scavenging assay. The assay analyzes the ability of antioxidants based on their ability to inhibit the formation of stained ABTS +, a blue-green chromophore that absorbs the 734 nm wavelength. The radical-trapping activity and the reducing power of ABTS of sample IV and CP-II at different concentrations are shown in FIGS. 24A and 24B. Vitamin C was used as a control for ABTS radical - capture activity and reducing power.

In the ABTS radical scavenging activity experiment of Experimental Example 15, the lowest ABTS radical-scavenging activity was observed in the pigs control after the pretreatment of Example 1. On the other hand, as shown in FIG. 24A, it was observed that the activity of ABTS radical scavenging increased significantly in the sample IV and the CP-II. The ABTS radical-capture efficiency of sample IV and CP-II exceeded 80% when the sample concentration was above 5 mg / mL. Sample IV and CP-II at the 5 mg / mL concentration also showed ABTS radical-collection efficiency equivalent to vitamin C of 62.5 mg / mL. However, there was no significant difference between sample IV and CP-II.

The reducing power was measured by modifying the Oyaizu method. 1 mL of 0.2 M phosphate buffer solution (pH 6.6) and 1 mL of 1% potassium ferricyanide were mixed and cultured at 50 ° C for 20 minutes, followed by addition of 10% trichloroacetic acid. 2 mL of the cultured mixed solution was mixed with 2 mL of distilled water and 0.4 mL of 0.1% ferric chloride. After 10 minutes from mixing, the samples prepared in the above procedure were measured using a spectrophotometer at a wavelength of 700 nm. 0.12 mg / mL ascorbic acid was used as a positive control for comparison. High absorbance at a wavelength of 700 nm means that the sample has a high reducing power.

The reductive power analysis showed a tendency similar to the result of the ABTS radical-trapping activity as a whole. Measuring the reducing power, which reflects the ability of the antioxidant to electron-donate, is considered to be a direct way to determine the overall antioxidant capacity. Compounds with reducing power are capable of reducing oxidized intermediates of the peroxidation reaction (Ambigaipalan, Al-Khalifa, & Shahidi, 2015).

The Fe ^{3 +} -Fe ^{2 +} transformation was strongly blue, and could be observed by measuring the change in absorbance at a wavelength of 700 nm. FIG. 24B is a graph showing the reduction power of Sample IV and CP-II at different concentrations, and the reducing power of the samples was 0.081 to 0.269. The reducing power of Sample IV and CP-II increased as the concentration increased. Compared with the control, the reducing power of Sample IV and CP-II was much higher than that of the control, but no significant difference was observed between Sample IV and CP-II.

From the results of Experimental Example 15, it is possible to produce a protein hydrolyzate exhibiting a high ABTS radical-scavenging activity and a reducing power, which means that even if the submerged filtration treatment is not carried out, Could know.

<Experimental Example 16> Measurement of anti-aging effect

The tyrosinase inhibitory activity was measured as follows. A premixture solution containing 70 μL of 0.1 M phosphate buffer (pH 6.8), 30 μL of mushroom tyrosinase (167 U / mL; Sigma-Aldrich, USA) and 20 μL of sample was incubated at 30 ° C. And cultured for 5 minutes. Then 100 μL of 3,4-dihydroxyphenyl-1-alanine (L-DOPA; Sigma-Aldrich, USA) was added to the preliminary solution to cause an enzyme reaction. The absorbance was measured at a wavelength of 492 nm for 20 minutes to observe the formation of L-DOPA. The activity inhibition rate was calculated by the following equation (6).

[Formula 6]

Wherein A is a mixed solution of tyrosinase to which no sample is added, B is a mixed solution in which a sample and tyrosinase are not added, C is a mixed solution in which a sample and tyrosinase are added, , But tyrosinase means a mixed solution not added.

The measurement of inhibitory activity of collagenase activity was measured as follows. A 50 mM tricine buffer solution (pH 7.5) containing 10 mM calcium chloride and 400 mM sodium chloride was prepared and then 1.0 mM of N- [3- (2-furyl) acryloyl] -leucine glycine proline alanine (Sigma-Aldrich, USA) and 0.2 mg / mL of collagenase. The solution was prepared with and without the sample for the reaction, respectively. The reaction was terminated by the addition of citric acid (6%). Thereafter, the reaction mixture solution was separated by adding ethyl acetate. The absorbance of the supernatant of the separated solution was measured at a wavelength of 345 nm. The ratio of the inhibitory activity was calculated by the following equation (7).

[Equation 7]

Wherein A is a mixed solution of collagenase to which no sample is added, B is a mixed solution in which a sample and collagenase are not added, C is a mixed solution in which a sample and collagenase are added, D is a sample in which collagenase Agar means a mixed solution not added.

Measurement of the inhibitory activity of elastase activity was measured as follows. N-succinyl alanine alanine alanine p-nitroanilide (SucAlaAlaAlPNA; Sigma-Aldrich, USA) was used as a substrate and the release of p-nitroalanine was observed at 25 ° C for 20 minutes. 1 [mu] g of type IV prokinetic creatase elastase (PPE) was dissolved in 1 mL of 0.2 M Tris-hydrochloric acid buffer solution (pH 8.0). The reaction mixture contained 0.2 M Tris-HCl buffer (pH 8.0), 1 ppm PPE, 0.8 mM succinyl alanine alanine alanine p-nitroanilide, a sample, and a substrate. The absorbance at 214 nm was measured and the inhibitory activity of the elastase activity was calculated by the following equation (8).

[Equation 8]

Wherein A is a mixed solution of elastase to which no sample is added, B is a mixed solution in which a sample and elastase are not added, C is a mixed solution in which a sample and elastase are added, and D is a sample Elastase means a mixed solution not added.

The inhibitory activity of tyrosinase, collagenase and elastase was used to confirm the anti-aging effect, and the results are shown in Table 8 below.

process Tyrosinase inhibition rate (%) Collagenase inhibition (%) Elastase inhibition rate (%) Vitamin C 96.64 + 0.010 50.09 ± 0.009 23.00 ± 0.000 Control 32.21 + 2.325 1.16 ± 2.039 Not Measured Sample IV 33.56 ± 1.162 48.98 占 .000 22.22 ± 2.238 CP-II 26.85 + - 3.487 44.80 7.727 Not Measured

As for the inhibition rate of tyrosinase, vitamin C (1 mg / mL, positive control) showed the highest inhibitory activity of tyrosinase activity. However, no significant difference was observed between the pretreated lard control, sample IV, and CP-II of Example 1. In general, melanin is involved in the formation of dots and freckles in the skin. And tyrosinase is the main enzyme for synthesizing melanin. This mechanism of tyrosinase activity inhibiting ability can play an important role in skin whitening effect for cosmetic purposes.

Collagen and elastin are important components that provide structural stability to connective tissue and skin, accounting for 70-80% of skin weight. However, collagen is rapidly damaged by aging or by the production of collagenase. This reduces the moisture and elasticity of the skin and wrinkles. As shown in Table 8, the inhibition rates of the collagenase activity of the control, the sample IV and the CP-II were 1.18%, 48.98%, and 44.80%, respectively. There was no significant difference between samples IV and CP-II with respect to inhibition of collagenase activity.

Elastin accounts for 2 to 4% of the dermal matrix and is known to have significant anti-wrinkle properties with collagen. Skin exposed to ultraviolet rays increases the production of elastase and reduces skin elasticity. As a result, skin wrinkles and pigmentation occur and the skin becomes thick. As shown in Table 8, the inhibitory rate of Elastase activity of Sample IV was 22.22%, which was equivalent to the inhibition rate of vitamin C, which is a positive control. However, the inhibitory effect of elastase inhibition was not determined in the pituitary control and CP-II.

Overall, vitamin C showed activity inhibition in the following order: tyrosinase (96.64%)> collagenase (50.09%)> elastase (23.00%). On the other hand, Sample IV showed activity inhibition in the following order: collagenase (49.98%)> tyrosinase (33.56%)> elastase (22.22%). CP-II showed active inhibition in the following order: collagenase (44.80%)> tyrosinase (26.85%). As a result of Experimental Example 16, it was found that Sample IV had a stronger anti-aging effect than CP-II, and also had high collagenase activity inhibitory ability. Therefore, it has been found that the bioactive collagen peptide having various functionalities can be produced without hydrolysis of pork collagen by using the subclaim coefficient without using an additional purification method such as ultrafiltration.

Therefore, bioactive collagen peptide having excellent functionalities can be produced without additional fractionation process by using the sublimation factor of 190 ° C and 2,100 kPa (sample-IV), which is an optimal condition for polymer collagen hydrolysis, and the bioactive collagen peptide prepared by this method It can be used as a health functional food or cosmetic material in the future.

Example 6 Production of Bi-active Liposome Containing Biologically Active Collagen Peptide

The anionic lecithin used in the manufacture of bi-active collagen peptide-containing liposomes was Lipoid S75 (Lipoid S75), which is a phosphatidylcholine (69.3%), phosphatidylethanolamine (9,8%) and Risophosphatidylcholine (2.1% ), And their fatty acids consist of palmitic acid (1720%), stearic acid (25%), oleic acid (812%), linoleic acid (5865%) and linolenic acid (46%).

The cationic lecithin used in the preparation of the bilayer liposomes containing the bioactive collagen peptide was Avanti Polar Lipids (USA) product as 1,2-dioloyo-3-trimethylammonium propane (DOTAP).

The conditions for preparing the single layer liposomes are shown in Table 9 below. (1% or 3%), the output of the ultrasonic processor (20% or 40%) and the time (2 minutes, 3 minutes, 5 minutes) in order to produce various monolayer liposomes .

Formulation
Lecithin type
Lecithin charge Concentration (%, w / v) Ultrasonic Processor Condition lecithin Collet
Sterol Bioactivity  Collagen peptide (CP) Print(%) Time (minutes) NL 1 Lipoid S75 Negative




One




0.2 0 20 5 NL 2 Lipoid S75 Negative 0 40 2 NL-CP 1 Lipoid S75 Negative One 20 3 NL-CP 2 Lipoid S75 Negative 3 20 2 NL-CP 3 Lipoid S75 Negative 3 20 3 NL-CP 4 Lipoid S75 Negative 3 20 5 NL-CP 5 Lipoid S75 Negative 3 40 2 PL 1 DOTAP Positive 0 20 5 PL 2 DOTAP Positive 0 40 2 PL-CP 1 DOTAP Positive One 20 2 PL-CP 2 DOTAP Positive 3 20 3 PL-CP 3 DOTAP Positive 3 20 5

In Table 9, CP is a bioactive collagen peptide, NL is an anionic liposome, NL-CP is an anionic liposome including a bioactive collagen peptide, PL is a cation liposome, and PL-CP is a cation liposome including a bioactive collagen peptide it means. In addition, the number of each formulation is intended to distinguish the concentration of the bioactive collagen peptide, the output of the ultrasonic processor, and the time condition.

The conditions of the bilayer liposome formulation are shown in Table 10 below. First, biopolymers, chitosan and pectin, were prepared at different concentrations for the production of bilayer liposomes, and a single layer liposome and a biopolymer solution were mixed. This method is based on the layered electrostatic deposition principle. Chitosan is mixed with liposomes made of anionic lecithin because of its positive charge, and pectin phosphorus is mixed with liposomes made of cationic lecithin because it has a negative charge. The concentration (w / v) of chitosan was determined to be 0, 0.0015%, 0.003%, 0.006%, 0.012%, 0.025%, 0.05%, 0.1%, 0.2% Was set to 0, 0.0078%, 0.015%, 0.031%, 0.062%, 0.125%, 0.25%, 0.5%, 1.0%, or 2.0%. Another method for the production of bilayer liposomes is to recapture the monolayer liposomes back to anion and cationic lecithin. In this method, a thin film hydration method, which is a method of producing the above-mentioned single layer liposome formed primarily, was used, and liposomes prepared from anion and cationic lecithin were respectively coated with anion and cationic lecithin.

The Inner layer Outer layer NL-CP / CHI Lipoid S75 Chitosan (CHI) PL-CP / LMP DOTAP Low methoxyl pectin (LMP) NL-CP / NL Lipoid S75 Lipoid S75 NL-CP / PL Lipoid S75 DOTAP PL-CP / PL DOTAP DOTAP PL-CP / NL DOTAP Lipoid S75

In Table 10, NL-CP / CHI is a liposome coated with an anionic liposome including bioactive collagen peptide coated with chitosan, PL-CP / LMP is a liposome coated with cationic liposome containing bioactive collagen peptide, NL-CP / NL is a liposome prepared by coating anion liposome containing bioactive collagen peptide with anion lecithin, NL-CP / PL is a liposome coated with cationic lecithin, anion liposome containing bioactive collagen peptide, PL- CP / NL refers to a liposome obtained by coating a cationic liposome containing a bioactive collagen peptide with anion lecithin, and PL-CP / PL refers to a liposome coated with cationic lecithin by cationic liposome containing a bioactive collagen peptide.

< Experimental Example  17> Bioactivity  Collagen Peptides  Included Liposomal  characteristic

The particle characteristics of the single-layer liposomes produced in the various compositions of Example 6 were compared as shown in Table 11 below. The particle size and particle dispersity index (PDI) of vacant anions and cationic liposomes (NL and PL) were 100 nm (PDI 0.402) and 88.6 nm (PDI 0.290), respectively. Their zeta potentials were 42.1 mV, and 63.1 mV, respectively. The zeta potential refers to the charge of a particle and is used as a reference measure to determine the stability of the particle. It can be judged that the particles are stably distributed at ± 30 mV. It is known that the higher the zeta potential, the better the repulsive force of the particles, so that the coagulation does not occur.

The effect of bioactive collagen peptide concentration (0, 1, 3%) on the particle size of NL-CP 1 or NL-CP 2 (negative charge liposome), PL-CP 1 or PL-CP 2 (positive charge liposome) The particle dispersity showed no significant difference after addition of the bioactive collagen peptide, but their zeta potential absolute values showed a tendency to decrease significantly as the bioactive collagen peptide concentration increased. The decrease in zeta potential is thought to be due to the biocompatibility of the bioactive collagen peptide with both positive and negative charges. Therefore, the charge is reduced when the bioactive collagen peptide is captured by the liposome.

The collection efficiency of NL-CP 1 (1% bioactive collagen peptide) sample was 90.8%, which was higher than that of NL-CP 3 (3% bioactive collagen peptide) sample 71.9%. On the other hand, the collection efficiencies of PL-CP 1 and PL-CP 2 samples were 60.8% and 68.0%, respectively.

&Lt; Example 7 > Effect according to operation time and output of ultrasonic processor

As shown in Table 11, when the operation time (2, 3, 5 minutes) and the output (20%, 40%) of the ultrasonic wave processor were examined, the effect of the operation time of the ultrasonic wave processor Size, and PDI values of the samples showed a tendency to decrease significantly, and the collection efficiency also tended to decrease. However, neither negatively charged or positively charged liposomes showed any effect on the output of the ultrasonic processor. Therefore, the optimal sonication conditions were set to 5 min in operation time and 20% in output (NL-CP 4, PL-CP 3).

<Experimental Example 18> Particle characteristics of bi-layer liposome using biopolymer

To prepare the bilayer liposome, the single-layer liposome prepared in Example 6 was used. In the case of the anionic single-layer liposome, chitosan, which is a cationic biopolymer, and pectin, which is an anionic biopolymer in the case of a cationic single liposome, Lt; / RTI &gt; By the layered electrostatic deposition principle, the concentration of chitosan or pectin was set at an appropriate concentration, so that the biopolymer having different charge could be prepared.

At lower concentrations of chitosan and pectin, agglutination of liposome particles (2-4 ㎛ particles) occurred. The concentration of chitosan or pectin with optimal particle size was 0.05% (NL-CP / CHI) and 0.5% (PL-CP / LMP) nm (PL-CP / LMP). The reason for the increase in the particle size of the biopolymer-based bilayer liposome relative to that of the single-layer liposome is that the biopolymer chitosan or pectin exhibits a thickness property around the liposome membrane.

As shown in FIG. 25A, the zeta potential of NL-CP / CHI shifted from a negative value to a positive value as the concentration of chitosan increased, and the optimum chitosan concentration was 0.025%. Addition of 0.025% or less of chitosan causes surface charge neutralization, resulting in intergranular aggregation. When the chitosan concentration is 0.025% or more, bridging flocculation between liposome particles occurs due to the presence of free chitosan. Therefore, it can be said that the liposome at the optimum concentration is coated with chitosan particles as a whole.

The change in zeta potential of the PL-CP / LMP formulation is similar to the NL-CP / CHI formulation. As shown in Fig. 25B, the zeta potential of PL-CP / LMP shifted from a positive value to a negative value as the concentration of pectin increased, and the optimum pectin concentration was measured to be 0.25%.

FIG. 26A shows the results of measurement of the collection efficiency by varying the concentrations of chitosan in the NL-CP / CHI formulations at 0.003%, 0.025%, and 0.2%. FIG. 26B shows the results of measurement of the collection efficiency by varying the concentration of pectin in the PL-CP / LMP formulation at 0.062%, 0.25%, and 2.0%. The collection efficiency of NL-CP / CHI formulation decreased with increasing chitosan concentration, but the collection efficiency of PL-CP / LMP formulation increased as the concentration of pectin increased The tendency to increase was shown.

In order to investigate the particle characteristics of the bilayer liposomes coated with lecithin having different electric charges, particle characteristics according to the ultrasonic treatment effect were further analyzed. Table 12 below shows particle characteristics of the double-layer liposomes without additional sonication.

The particle size, zeta potential and collection efficiency of NL-CP were about 75.7 nm (PDI = 0.325), -14.4 mV, and 67.4%, respectively. The particle size, zeta potential, and collection efficiency of PL-CP were 76.2 nm (PDI = 0.298), 20.6 mV, and 66.9%, respectively. The particle size of the bilayer liposomes (LL-CP = NL-CP / NL, NL-CP / PL, PL-CP / PL or PL-CP / NL) after coating the outer layer (NL or PL) (PDI> 0.6), respectively. PDI is a method of measuring the particle size distribution of a dispersion system, ranging from 0 to 1.0. A high PDI value (> 0.6) means that the particle dispersion is large, meaning that there is large droplet or intergranular aggregation. As can be seen in Table 12 below, the PDI values in the bilayer liposomes exceeded 0.624 in all cases. And this means that the liposomes are not uniform in particle size. The zeta potential values of NL-CP / NL and NL-CP / PL changed from 14.4 mV to 17.7 mV (NL-CP / NL) and 24.6 mV (NL-CP / PL) respectively. The zeta potential values of PL-CP / PL and PL-CP / NL changed from 20.6 mV to 32.1 mV (PL-CP / PL) and 18.8 mV (PL-CP / NL), respectively. In particular, the addition of PL (cationic liposome) to the NL-CP formulation changed the potential of NL-CP / PL from -14.4 mV to a positive value of 24.6 mV. On the other hand, the potential of PL-CP / NL did not change from positive to negative. This is because the concentration of NL (anionic liposome) in the outer layer is not sufficiently high to completely surround the PL-CP. The collection efficiency of NL-CP / NL and NL-CP / PL increased from 64.7% to 74.9% (NL-CP / NL) and 78.8% (NL-CP / PL) ). The bilayer liposomes have a somewhat higher collection efficiency due to their relatively large size compared to single-layer liposomes. In general, hydrophilic encapsulation is proportional to the concentration of lecithin and the internal size of the liposome. The difference in collection efficiency indicates that the liposome has a higher level of NL or PL coating.

As shown in Table 13 below, after further sonication, the size of the bilayer liposomes is smaller than without ultrasound treatment. The zeta potential values of the bilayer liposomes treated with the ultrasonic treatment were similar to those of the bilayer liposomes without the ultrasonic treatment. After ultrasonic treatment, the collection efficiency was 61.3% to 73.0%.

< Experimental Example  19> Field  Emission-transmission electron microscope (FE- TEM ) Image analysis

FIG. 27 (a) shows a blank anion single-layer liposome as a reference 50 nm, FIG. 27 (b) shows an anion single-layer liposome as a reference 20 nm and FIG. 27c shows an anion single-layer liposome containing a bioactive collagen peptide as a reference 50 nm, and an anion (inner layer) / cation (outer layer) liposome containing a bioactive collagen peptide as a reference 50 nm, respectively. The size of the droplets of the sample was 60-70 nm in the FE-TEM image, consistent with the results obtained using a size analyzer. As shown in Figs. 27A and 27B, the empty liposome showed a clean follicle. On the other hand, as shown in Fig. 27C, visible aggregation occurred in the NL-CP4 sample. From this, it was possible to predict the negatively charged residues of the liposome binding to the positively charged residues of the bioactive collagen peptide. In addition, the addition of a coating layer having an opposite charge to the NL-CP4 formulation results in the agglomeration of a stronger vesicle as shown in Figure 27d, which results in the adherence of the double strand as compared to NL-CP4 This is because the repulsive force that hinders it is lowered. That is, the bioactive collagen peptide appears to interact with the particles to form a more stable layer of liposomes.

The stability of the liposome was evaluated by varying the particle size and zeta potential at 25 캜 for 30 days, 37 캜 for 24 hours, and 60 캜 for 4 hours, and the results are shown in Fig. To measure the physical stability, additional NL-CP, PL-CP, NL-CP / PL and PL-CP / NL formulations with ultrasonic treatment were selected. No significant changes in particle size were observed during the measurement of the stability of single-layer liposome (L-CP) formulations containing bioactive collagen peptides at 25 ° C for 30 days. On the other hand, when the temperature was increased, the particle size and zeta potential absolute value of single layer liposomes increased. Particularly, the particle size of NL-CP increased from 74.2 to 88.5 nm and the zeta potential changed from -14.4 to -18.6 mV. All monolayer liposome formulations containing bioactive collagen peptides had a particle size of 90 nm or less. This means that in the case of all single-layer liposome formulations comprising the bioactive collagen peptide, the stable properties are obtained, with relatively small changes occurring during storage.

Liposomes (L-CP) containing bioactive collagen peptides were prepared by combining thin membrane hydration and ultrasonication emulsification. The formulations were prepared under various conditions and were prepared with different concentrations of lecithin charge (negative or positive charge lecithin), bioactive collagen peptide (CP) and sonication time and power. Bilayer liposomes containing biologically active collagen peptides were prepared using biopolymers (chitosan: CHI and low methoxyl pectin: LMP) and charged liposomes (negative charge liposomes: NL, positively charged liposomes: PL). NL-CP and PL-CP were found to have a smaller particle size below 70 nm and a collection efficiency of over 60% when sonicated at 20% power for 5 minutes. In addition, bilayer liposomes (NL-CP / NL, NL-CP / PL, PL-CP / PL, PL-CP / NL) comprising biologically active collagen peptides composed of liposomes And exhibits a narrower size distribution compared to NL-CP / CHI or PL-CP / LMP. In terms of storage stability, both monolayer liposomes coated with NL or PL and monolayered liposomes coated were physically stable.

Claims (5)

A method for producing a bioactive collagen peptide having a molecular weight of 10 kDa or less, comprising the steps of:
(S1) cutting the pork slices and immersing them in water at 90 to 110 DEG C for 50 to 90 seconds to remove fat and impurities of pork slices;
(S2) homogenizing the immersed poultice of step (S1) in 0.5 to 1.5 weight percent water of the pomegranate weight;
(S3) hydrolyzing the homogenized pans of the step (S2) using an asymptotic factor of 190 to 230 DEG C and 1,100 to 2,600 kPa; And
(S4) hydrolyzing the hydrolyzate of step (S3) using a protease. The method for producing a bioactive collagen peptide according to claim 1, wherein the protease is Alcalase. A bioactive collagen peptide produced by the method of claim 1 or 2. A liposome comprising the bioactive collagen peptide of claim 3. 5. The liposome of claim 4, wherein the liposome comprises a bilayer of an inner layer and an outer layer.

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