KR100904631B1 - Preparation of functional hydrolysates from oyster using transglutaminase - Google Patents

Abstract

The present invention relates to an oyster enzyme hydrolyzate using transglutaminase and a method for producing the same. More specifically, after crosslinking oyster protein using transglutaminase, the protein is hydrolyzed twice. Preparation and hydrolysis of functional oyster enzyme hydrolysates using transglutaminase to hydrolyze cross-linked proteins with protamex and Neutrase to produce new functional peptides It is a product supplemented with phosphorus and Schisandra chinensis extract by optimizing the degradation product, and the hydrolyzate containing oyster hydrolyzate and herbal medicine has antioxidant activity, ACE inhibitory ability, neutral lipid and total cholesterol lowering function, and reduced arteriosclerosis index. Effect, superxoide radical lowering effect and hepatocyte Damage repair function was confirmed, and cytotoxicity was not shown, and oyster enzyme hydrolyzate was used for antioxidative effect, lowering blood pressure using ACE inhibitory effect, lowering of neutral lipid and total cholesterol level, alleviating atherosclerosis and restoring liver function damage. It can be used as raw materials and materials for functional health supplements, cosmetics and medicines.

Transglutaminase, oyster hydrolyzate, peptide, functional dietary supplement, total cholesterol, ACE inhibition, liver function recovery

Description

Functional oyster enzyme hydrolyzate using transglutaminase and preparation method thereof {Preparation of functional hydrolysates from oyster using transglutaminase}

The present invention crosslinks oyster proteins with transglutaminase, and then prepares oyster enzyme hydrolysates using proteolytic enzymes, and adds phosphorus and Schizandrae through optimization to these hydrolysates. The present invention relates to an improved preparation, in which phosphorus and schisandra chinensis were added through an optimization process for the purpose of enhancing the hepatoprotective effect of the hydrolyzate, thereby preparing a hepatoprotective agent.

Enzymatic hydrolysates of fish meat proteins have the advantage of modifying or enhancing the functional properties of the protein, and the physicochemical properties that govern the protein's functionality include the size, amino acid composition and binding sequence of the peptide or protein, and interactions with other compounds. (Damodaran, 1996). Enzymatic hydrolysates of proteins produce peptides with low molecular weights with different secondary structures, and peptides produced from muscle hydrolysates have antioxidant properties (Hattori et al., 1998; Saiga et al., 2003), hypotension (Arihara et al. , 2001; Suetsuna, 2002; Katayama et al., 2004), stimulating peripheral blood lymphocytes (Kayaser and Meisel, 1996), immunomodulatory activity (Duarte et al., 2006), hypocholesterolemia (Zhong et al., 2006; 2007). It is becoming.

 There are 212 patents registered worldwide for the food application of transglutaminase producing strains and transglutaminase. Of these, eight patents related to oysters, health foods, pharmaceuticals and peptides were produced, which contained reduced proteolytic enzyme activity for use in pharmaceuticals, including the maintenance of transglutaminase in the raw material at a preset state. Preparation of transglutaminase comprising (WO2007026922-A1), a protein that improves affinity for metal ions useful in nutrients, beverages and health foods (JP11322790-A), the production of a protein for use as an anticancer agent -trans glutaminenase Comparison of the Response of Active Proteins with Ligand Ligands (WO9813381-A; EP950665-A), Physiologically Active Peptide Modification Process by Reaction with Amino Donor and Microbial Transglutaminase (EP785276-A; WO9610089-A; JP8511619-X; US601071-A; CN1165539-A), Activation by Trans-glutaminase-Calcium Ions from Japanese Oysters and Gelling Reagents for Food (EP693556-A; WO9520662-A; JP7519983-X), Preparation of protein improved nutrition by introducing amino acids and / or peptides into protein using calcium independent transglutaminase (JP6007091-A), comprising transglutaminase Partial hydrolysates are prepared from wound treatments (EP598133-A; WO09320837-A; JP5516879-X), proteolytic enzymes and / or acids, and then treated with transglutaminase or diluted acid to remove bitter taste. Patents (JP4126039-A; JP2958801-B2) relating to food functional peptides that can widely use proteins have been registered.

Accordingly, the present invention has a new structure of physiological functionality, transglutaminase to the oyster protein to produce a peptide of less than 500 daltons of molecular weight corresponding to less than 5 amino acid residues that can be absorbed as it is without further degradation in the small intestine To form a crosslinked protein, and the crosslinked protein was digested with protease under optimal conditions to prepare a functional peptide. In addition, the present invention has been completed to utilize the optimal amount of phosphorus and Schisandra chinensis through an optimization process in order to enhance the recovery and protection of liver damage, and to use it as a material of functional supplements for restoring liver function. The purpose is to 1) make a new type of protein by crosslinking oyster protein with transglutaminase, and 2) to prepare a functional peptide that can be absorbed without digestion in the small intestine by hydrolyzing a new type of protein with protein hydrolase, 3) The purpose of the present invention is to provide food supplements, cosmetics, and medicines through the manufacture of products containing phosphorus and schizandra to reinforce the recovery function of liver function.

Looking at the effect of the oyster enzyme hydrolyzate using the present invention transglutaminase (transglutaminase), it has a new structure of physiological functionality (antioxidative capacity, lowering blood pressure, neutral and total cholesterol, lowering superoxide radicals), more in the small intestine To prepare a peptide with a molecular weight less than 500 daltons corresponding to less than 5 amino acid residues that can be absorbed as it is without functional degradation, a glucoprotein is transglutaminase to form a cross-linked protein, and the cross-linked protein is optimal. Functional peptides were prepared by digesting with proteolytic enzymes below. In order to restore liver damage and reinforce the protective function, supplements for optimal recovery of liver function were prepared by adding optimal amounts of phosphorus and Schizandrae through an optimization process. This manufacturing process and product development has the effect that it can be used as a raw material of health supplement food, cosmetics and medicines based on oysters, and thus, oysters with low utilization rate, damaged oysters and non-commercial oysters due to long-term freezing storage. The aim of this study was to show the effect of increasing fisherman's income and fishery material industry by developing processes for effective utilization of fishery resources and production of high value-added products.

The present invention crosslinks oyster proteins with transglutaminase, and then prepares oyster enzyme hydrolysates using proteolytic enzymes, and adds phosphorus and Schizandrae through optimization to these hydrolysates. The present invention relates to an improved preparation, in which a human liver and a Schizandra chinensis are added through an optimization process for the purpose of enhancing the hepatoprotective effect of the hydrolyzate, and a functional hydrolyzate, an ACE inhibitory peptide, and a herbal medicine include a functional health product. In order to use as a raw material and materials of the supplements, cosmetics and pharmaceuticals, the present invention has been completed in order to use as a material of functional products that can be processed in the form of liquid products, solid products, powders, and granules. It will be described in detail through the implementation of the present invention. Each example was repeated to confirm the reproducibility of the results.

 Example 1 Preparation of Functional Peptides through Formation of Proteins and Protein Hydrolysis Using Transglutaminase

The method for preparing oyster enzyme hydrolyzate using transglutaminase (hereinafter referred to as “TGase”) was performed as in FIG. 1.

 In detail, in the preparation of the raw material (1), various raw materials such as shellfish and fish meat including oysters and mussels, which are protein sources, are selected and selected, and twice the amount of tap water is added to the prepared raw material. Homogenizer (2) at 3000 rpm for 2 minutes at pH 6.7-7.0 using a homogenizer. 1% TGase (TG-K, Ajinomoto, Japan) was added, reacted at 30 ° C for 1 hour (3), cross-linked and hydrolysis temperature, enzyme concentration and reaction for primary hydrolysis (4). Considering the time and DPPH radical scavenging ability, Protamex 1% was reacted at 40 ° C. for 1 hour. After 1 hour of inactivation at 100 ° C., the optimum conditions of the same conditions for the first hydrolysis were selected again for the second hydrolysis (5), and 1% of Neutrase was reacted at 50 ° C. for 1 hour. Inactivate. This was centrifuged at 8000 rpm for 25 minutes (6) to precipitate the supernatant with 60% ethanol and centrifuged at 8000 rpm for 25 minutes and filtered with filter paper (7). Ethanol was evaporated from the filtrate (8) and filtered with a 0.45 um filter, followed by ultrafiltration (Ultra filtration, 9) using a 5 kDa ultra membrane (cut off limit: 5 kDa). The filtrate is lyophilized (10) and stored at -20 ° C. The functional peptide thus prepared was referred to as PN as a sample which did not use TGPN or TGase.

Example 2 Recovery of Functional Peptides for Industrialization

In [Example 2], the same method as in [Example 1] is not able to mass-produce the required amount of peptide for industrial application. Therefore, the method of [Example 2] is used for mass preparative method for industrial application.

 Figure 2 shows the scale-up method for the industrialization of oyster enzyme hydrolyzate using TGase. To explain this in more detail, two methods were used for scale-up. The first is the separation method of the polar substance using adsorption chromatography (1) (activated carbon, shinyo Pure Chemicals CO LTD, Osaka, Japan) is poured deionized water into the activated carbon adsorbent, and washed twice (2). 0.4 g / mL of oyster hydrolyzate using TGase is injected (3) into a column (2.8 × 19 cm void vol 120 mL). In the elution step (4), deionized water corresponding to about 2 times the volume of the column (250 mL) was run to collect fractions (capture of non-adsorbed material), and 20% ethanol containing 1% acetic acid and 50% ethanol were flowed in order. Fraction. This is evaporated in a rotary vacuum concentrator, and then lyophilized to powder (5).

 In the second method, ion-exchange chromatography (6) (industrial resin) is used to separate the ion level, and the ion-exchange resin is washed with 2 mM citrate buffer (pH 3.0) for resin equilibrium. Dissolve 0.4 g of oyster enzyme hydrolyzate using TGase as a sample in 10 mL of each buffer, and inject the sample solution into a column (2.8 x 19 cm void vol 90 mL) (3). In the elution step (4), non-adsorbed material was collected with 1 void volume (90 mL) of 5 mM buffer solution, and the adsorbents were eluted with each 5 mM buffer solution (pH 3.0) containing 0.5 M KCl to evaporate the buffer solution using a rotary evaporator. Lyophilization to powder (5).

Example 3 Optimization Conditions for Manufacturing a Mixed Product

Table 1 relates to the optimization conditions for the preparation of the product of the blended product, with the limit of each component in the range of 0.5-3% for oyster hydrolyzate, 3-6% for phosphorus extract, and 6-10% for Schizandra chinensis extract. As a result of the mixture preparation applied to the modified distance design, the phosphorus and oyster hydrolysates showed higher antioxidant activity than the Schisandra chinensis extract, and the ratio of phosphorus extract 3.8, Schisandra chinensis extract 8.2 and oyster hydrolyzate 3.0 was appropriate for the mixture preparation. The predicted antioxidant activity was 90.677% and the preference was 4.522 (Table 1).

Example 4 Cytotoxicity of Functional Hydrolysates

Figure 3 is a diagram showing the cytotoxicity of the functional hydrolyzate, hydrolyzate (TGPN) obtained by proteolytic treatment with transglutaminase, the product of adding phosphorus and Schizandra to enzyme hydrolyzate (TGPN + injin) + omija), the enzyme hydrolyzate (3PN) obtained by extracting protein with 3% NaCl solution and then treated with Protamex and Neutrase to improve yield is significantly toxic to cells up to 200 ug / mL. It is not shown.

Example 5 DPPH and ACE Inhibitory Effects of Functional Peptides

Table 2 shows the DPPH antioxidant activity and the ACE inhibitory activity of the purified substance. A functional peptide prepared with protamex and Neutrase, which are proteolytic enzymes using transglutaminase according to the present invention. (TGPN) does not use transglutaminase and has higher antioxidant activity and ACE inhibitory activity (hypertension) than peptide (PN) prepared by hydrolysis with Protamex and Neutrase. I had.

Example 6 Total Cholesterol Reduction Effect of SD Rats

Table 3 shows the total cholesterol reduction effect of SD rats, and the total cholesterol content in serum of each diet group tended to decrease in all oyster hydrolysis diet groups compared to the control group. In particular, 79.04 ± 12.50 mg / dL in the TGPN-200 (TGPN 200 mg) diet and 84.38 ± 16.20 mg / dL in the TGPNH-200 (TGPN, 200 mg optimal combination of human and Schizandra chinensis), 29.9% There was a significant reduction of 24.1%. In addition, in the case of the TGPN-100 (TGPN 100 mg), PN-100 (PN 100 mg), PN-200 (PN 200 mg) and TGPNH-100 (TGPN, the optimal combination of Injin and Schisandra chinensis) There was a decrease of 1.5-10.0%, but no significance was found. It was found that TGPN hydrolyzate had the effect of lowering serum cholesterol content, and the reduction effect was the least in PN hydrolyzate.

Example 7 LDL-Cholesterol Reduction Effect of SD Rats

Table 4 LDL-cholesterol-reducing effects of SD rats include cholesterol (VLDL) -cholesterol, low density (LDL) -cholesterol and high density (HDL) -cholesterol. LDL-cholesterol, which carries cholesterol in lipoprotein, is known to be a risk factor for cardiovascular diseases because of its high cholesterol content.

 As a result of measuring the effect of diet containing oyster hydrolyzate on LDL-cholesterol content, optimum phosphorus and Schisandra chinensis extract were added to TGPN hydrolyzate and TGPN hydrolyzate diet groups compared to the control group (55.41 ± 20.81 mg / dl). In one herb diet, blood levels of LDL-cholesterol decreased. In addition, when the amount of hydrolyzate added is larger, the reduction effect was shown. In particular, the TGPN diet decreased the most, with 44.87 ± 21.55 mg / dV in the TGPN-100 group and 33.67 ± 18.06 mg / dV in the TGPN-200 group, down 19% and 39.2%, respectively.

Example 8 Triglyceride Reducing Effect of SD Rats

Table 5 shows the triglyceride reduction effect of SD rats, and the content of triglycerides in blood or tissues increased when high fat diet or energy intake was ingested. The high triglyceride content in the blood has also been shown to be a risk factor for cardiovascular disease, and the triglyceride content in the blood tended to decrease in all oyster hydrolyzed diets. Among them, the herb mixture diet with optimal phosphorus and Schisandra chinensis extract added oyster hydrolyzate to 87.78 ± 9.32 mg / ㎗ in the Herb M-100 group, with a 7.8% reduction, and 77.44 in the Herb M-200 group. A significant reduction of 18.6% was recognized at ± 10.86 mg / dL. The neutral lipid (TG) content of TGPN oyster hydrolyzate and PN oyster hydrolyzate tended to decrease from 2.4% to 5.4% for TGPN hydrolyzate and 3.3% to 16.9% for PN oyster hydrolyzate. Was not. Neutral lipid content was decreased in both serum and liver tissues in the TGPN and Herb M diets. However, PN hydrolyzed diet group showed a slight decrease in serum, but liver tissue showed a tendency to increase the content of triglyceride rather than control group.

Example 9 Superoxide Radical Reduction Effect of SD Rats

Superoxide radicals (Superoxide radical; O ₂ ^-) is the triplet oxygen (triplet oxygen) accepts electrons ^{_{(3O 2 + e - → O}} 2 -) is generated, hydroperoxide radical (OOH), hydrogen peroxide, hydroxyl radical (OH It is converted into singlet oxygen or singlet oxygen, so it is generated in the early stage of free radical damage. Active oxygen generation mechanism of by varying the concentration of the oyster hydrolyzate to which is initially generated to measure the oxidative damage caused by superoxide radicals six weeks breeding then superoxide radicals in the serum (Superoxide radical; O ₂ ^-) content The results of the measurement are shown in Table 6.

 In the control group, the superoxide radical content was 266.23 ± 60.96 nmol / g protein, and in the case of TGPN-100 and TGPN-200, 246.49 ± 125.20 nmol / g protein and 173.82 ± 75.05 nmol / g protein were 7.4% and There was a reduction of 34.7%. In the PN oyster hydrolyzate diet group, the PN-NaCl-100 group increased 28% compared to the control group, and the PN-NaCl-200 group showed almost similar results. Herb M group showed significant reduction effect, especially Herb-M-200 group showed significant decrease effect of 32.6% with 179.34 ± 46.20 nmol / g protein. In addition, the TGPN group and Herb M group showed that the superoxide radical content decreased with concentration, indicating that TGPN intake greatly contributed to the generation of superoxide radical content.

Example 10 Hepatocyte damage repair effect of a preparation containing a functional hydrolyzate

Figure 4 is a diagram showing the effect of hepatocellular damage recovery of the preparation containing the functional hydrolyzate, after the damage of the liver cells with THA, the results of experiments of the recovery of hepatocellular damage of the preparation containing the functional oyster hydrolyzate, phosphorus and Schizandra In comparison with silimarine, positive hepatocytes were found to have higher hepatocellular damage repair effects, and these effects increased with increasing concentrations.

According to the present invention, the optimized preparation of the hydrolyzate prepared by using the enzyme after crosslinking the protein using TGase, the present hydrolyzate, and the phosphorus and Schizandra soluble extracts has antioxidant activity, ACE inhibitory activity, neutral lipid and total cholesterol lowering function, The effect of reducing arteriosclerosis index, reducing superoxide radical and restoring hepatocellular damage was confirmed, and cytotoxicity was not shown. Oyster hydrolyzate had antioxidative effect, blood pressure drop and neutrality using ACE inhibitory effect. It can be used as a raw material and material of functional health supplements, cosmetics, and medicines for lowering lipid and total cholesterol, relieving arteriosclerosis, and restoring liver function damage.

1 is a flow chart of a functional peptide manufacturing process through protein formation and protein hydrolysis using Transglutaminase

Figure 2 is a manufacturing process for improving the yield of the functional peptide

3 shows the cytotoxicity of functional hydrolysates

4 is a diagram showing the effect of restoring hepatocyte damage of a preparation containing a functional hydrolyzate

Claims (5)

(1) process of preparing the raw material for selecting and selecting the raw material of oyster which is a protein source; After adding twice the amount of tap water to the prepared raw material, the grinding (2) process of grinding for 2 minutes at 3000 rpm, pH 6.7 using a homogenizer; After the grinding (2) process, by adding 1% transglutaminase, reacting for 1 hour at 30 ° C. for cross-linking (3); Primary hydrolysis in which 1% of Protamex was reacted at 40 ° C. for 1 hour for hydrolysis to a raw material that had undergone the reaction (3), and then inactivated at 100 ° C. for 1 hour. 4) process; After the first hydrolysis (4), the second hydrolysis (5) process of reacting 1% Neutrase at 50 ℃ for 1 hour for secondary hydrolysis, inactivated at 100 ℃; , After the second hydrolysis (5), centrifugation at 8000 rpm for 25 minutes to precipitate the supernatant with 60% ethanol, followed by centrifugation (6) for 25 minutes at 8000 rpm; After centrifugation (6), filtration (7) of filtration with filter paper; An evaporation (8) step of evaporating ethanol from the filtrate filtered in the filtering (7) step; After the process of evaporation (8), after filtration with a 0.45 um filter, and the ultrafiltration (9) process of ultrafiltration using a 5 kDa ultrafiltration membrane, the filtered filtrate was lyophilized (10). Method for producing oyster enzyme hydrolyzate using transglutaminase, characterized in that the functional peptide is produced by protein hydrolysis Oyster enzyme hydrolyzate using transglutaminase, characterized in that the oyster enzyme hydrolyzate is produced by the method of claim 1 delete The oyster enzyme hydrolyzate according to claim 2, wherein the oyster enzyme hydrolyzate is used as a health supplement food material or a food material. The method according to claim 2, wherein the phosphorus and Schizandra chinensis extract are added to the oyster enzyme hydrolyzate to be used as one of the dietary supplements, food materials, and pharmaceutical materials for the recovery and enhancement of liver function and damaged cells. Functional Oyster Enzyme Hydrolysates Using Transglutaminase

Images