Method for producing hydrolyzed peptides of soy protein isolate with increased glutamine synthetase activation efficacy and tyrosine content

Abstract

The present invention relates to a method for producing a hydrolyzed peptide of a soy protein isolate having increased glutamine synthetase (GS) activation efficacy and tyrosine content, and more specifically, to a method for preparing a hydrolyzed peptide of a soy protein isolate having increased glutamine synthetase (GS) activation efficacy and tyrosine content, the method comprising the steps of: hydrolyzing a soy protein isolate into a low-molecular-weight mixed peptides using a hydrolase; and fractionating the hydrolyzed soy protein isolate by ion exchange chromatography. The hydrolyzed peptide of the isolated soy protein produced by the production method of the present invention is expected to be used as a health functional food or medicine for preventing, alleviating or treating diseases caused by a decrease in GS activity (diseases caused by depression, anxiety disorder or stress).

Description

Method for producing hydrolyzed peptides of soybean protein isolate with enhanced glutamine synthetase activation efficacy and tyrosine content

[0001] The present invention relates to a method for producing a hydrolyzed peptide of soybean protein isolate having enhanced glutamine synthetase activation efficacy and tyrosine content.

[0002] The present invention was carried out as an "Individual Basic Research Project" of the National Research Foundation of Korea under the Ministry of Science and ICT (or affiliated therewith) under the "Discovery of Innovative New Drug Candidates with Antidepressant Effects Based on the Activation Mechanism of Glutamine Synthetase" project (Project Unique Number: 2710013047, Project Number: 2022R1A2C1005296); as an "Group Research Support Project" of the National Research Foundation of Korea under the Ministry of Science and ICT (or affiliated therewith) under the "Anti-aging Biomaterial Cell Factory Regional Innovation Research Center" project (Project Unique Number: 2710012807, Project Number: 2021R1A5A8029490); This work was carried out with the support of the "Development of Neurofunctional Biopharmaceutical Materials" project (Project ID: 2710071353, Project ID: CP000116) carried out as a "Public Research Results Value Creation Technology Promotion Project" of the Science and Technology Commercialization Agency under (or affiliated with) the Ministry of Science and ICT.

[0003] Peptides, which are protein hydrolysates, are polymers of amino acids. When a small number of amino acids are linked, they are called peptides. When many amino acids are linked, they are called proteins. The linkages between amino acids in these peptide and protein structures are formed by amide or peptide bonds. In addition to their nutritional function as a nitrogen source, peptides have physiological activities such as taste, antioxidant, and antibacterial effects. They also have bioregulatory functions such as immune regulation, hormones, neurotransmitters, and antihypertensive effects, and are attracting attention in the pharmaceutical and food industries. Functional protein hydrolysates are hydrolysates derived from natural food ingredients produced by hydrolyzing natural protein food ingredients. Depending on the production conditions of the hydrolysate, they exhibit various physiological functions. Methods for producing functional hydrolysates include enzymatic hydrolysis using proteolytic enzymes. Enzymatic hydrolysis is economical and can be mass-produced using food ingredients.

[0004] Meanwhile, soybeans are native to Northeast Asia, centered around China, and have been cultivated in Korea for approximately 1,500 years. Soybeans are composed of the edible part and the embryo, with the cotyledons (cotyledons) of the edible part, which constitute approximately 90-92%, and contain approximately 40% protein and 20% fat, making them not only an inexpensive yet high-quality protein source, but also recognized for their nutritional value and importance as a physiologically active substance, as they lower blood cholesterol, which is the cause of various adult diseases, and are effective in preventing arteriosclerosis, myocardial infarction, stroke, and high blood pressure, as well as diabetes and liver disease. These soybeans are widely used in foods such as fermented products (soybean paste, cheonggukjang, red pepper paste, soy sauce), sprouted products (soy sprouts), and other processed foods (soy milk, tofu, cooking oil), and defatted soybeans, soy protein concentrate, soy protein isolate, and textured soy protein are used as food ingredients. Since each soy protein isolate has different functional characteristics, many studies are being conducted to prove the functionality and physiological activity effects of food ingredients. Among the methods for improving the functional characteristics of proteins, manufacturing modified soy protein through hydrolytic enzyme treatment has the highest practical applicability. It has been revealed that soy protein enzymatic hydrolysates have physiological functions that cannot be explained simply by amino acid aggregates, and soy peptides, which are mainly hydrolysates of soy protein, have been revealed to be involved in cholesterol-lowering effects, antihypertensive activity, anticancer activity, antioxidant activity, and anti-arteriosclerosis, and are being re-evaluated as new food materials with disease prevention and functional efficacy in addition to their basic purpose of satisfying nutritional supply and palatability.

[0005] As a technology related to the hydrolysis of soy protein isolate, the Angiotensin-Ⅰ Converting Enzyme inhibitory effect of soy protein isolate hydrolysate is disclosed in the Journal of the Korean Society of Food Science and Nutrition (J Korean Soc Food Sci Nutr 39(1), 8-13 (2010), and a method for producing a high protein hydrolysate is disclosed in Korean Patent No. 0555221. However, a method for producing a hydrolyzed peptide of soy protein isolate with enhanced glutamine synthetase activation efficacy and tyrosine content has not yet been disclosed.

[0006] The present invention was derived from the above-mentioned needs, and the present invention provides a method for producing a hydrolyzed peptide of soybean protein isolate having enhanced glutamine synthetase activating efficacy and tyrosine content, and has completed the present invention by confirming that the hydrolyzed peptide of soybean protein isolate produced by the method of the present invention has enhanced tyrosine content and enhanced glutamine synthetase activating efficacy.

[0007] In order to achieve the above purpose, the present invention comprises: (1) a step of hydrolyzing soy protein isolate into a low-molecular-weight mixed peptide using a hydrolytic enzyme;

[0008] (2) After the above step (1), a step of inactivating the hydrolytic enzyme, filtering and drying to obtain a hydrolyzate of soy protein isolate; and

[0009] (3) A method for producing a hydrolyzed peptide of soybean protein isolate with enhanced glutamine synthetase (GS) activation efficacy and tyrosine content, including a step of obtaining a fraction of the hydrolyzate obtained in step (2) using an ion exchange chromatography technique under conditions of pH 3.0 to 3.4 or pH 6.2 to 6.6, is provided.

[0010] In addition, the present invention provides a hydrolyzed peptide of soybean protein isolate having enhanced glutamine synthetase (GS) activation efficacy and tyrosine content, manufactured by the manufacturing method of the present invention.

[0011] The present invention relates to a method for producing a hydrolyzed peptide of soybean protein isolate having enhanced glutamine synthetase activation efficacy and tyrosine content, and more specifically, a method for producing a hydrolyzed peptide of soybean protein isolate having enhanced glutamine synthetase (GS) activation efficacy and tyrosine content, comprising the steps of: hydrolyzing soybean protein isolate into low-molecular-weight mixed peptides using a hydrolytic enzyme; and fractionating the peptides using an ion exchange chromatography technique. The peptides produced by the method have enhanced glutamine synthetase (GS) activation efficacy and tyrosine content.

[0012] Figure 1 shows the results of confirming the degree of hydrolysis of a single protein-decomposing enzyme according to reaction time.

[0013] Figure 2 shows the results of examining the degree of hydrolysis of complex proteolytic enzymes according to reaction time. A is Alcalase, P is Protamex, N is Neutrase, and F is Flavorzyme.

[0014] Figure 3 shows the results of confirming the tyrosine content of hydrolyzed product by a single proteolytic enzyme according to reaction time.

[0015] Figure 4 shows the results of confirming the tyrosine content of hydrolyzed product by complex proteolytic enzyme according to reaction time.

[0016] Figure 5 shows the results of confirming the optimal hydrolysis conditions for the protein content of alkaline hydrolysate.

[0017] Figure 6 shows the results of confirming the optimal hydrolysis conditions for the tyrosine content of alkaline hydrolysate.

[0018] Figure 7 shows the results of confirming the optimal hydrolysis conditions for the protein content of the complex protein decomposition enzyme.

[0019] Figure 8 shows the results of confirming the optimal hydrolysis conditions for the tyrosine content of the complex protein decomposition enzyme.

[0020] Figure 9 shows the results of examining changes in glutamine synthetase activity according to treatment with alkaline hydrolysate and its pH fractions (Alc3.2 and Alc6.4). PN stands for peroxynitrite.

[0021] The present invention comprises the steps of (1) hydrolyzing soy protein isolate into low-molecular-weight mixed peptides using a hydrolytic enzyme;

[0022] (2) After the above step (1), a step of inactivating the hydrolytic enzyme, filtering and drying to obtain a hydrolyzate of soy protein isolate; and

[0023] (3) A method for producing a hydrolyzed peptide of soybean protein isolate with enhanced glutamine synthetase (GS) activation efficacy and tyrosine content, comprising: obtaining a fraction of the hydrolyzate obtained in step (2) using an ion exchange chromatography technique under conditions of pH 3.0 to 3.4 or pH 6.2 to 6.6.

[0024] In the above step (1), the hydrolytic enzyme is preferably one or two complex enzymes selected from Alcalase, Protamex, Neutrase and Flavorzyme, more preferably Alcalase, but is not limited thereto.

[0025] The low-molecular-weight mixed peptide obtained in the above step (1) is preferably a low-molecular-weight mixed peptide of 400 kDa or less, but is not limited thereto.

[0026] In the above step (1), hydrolysis is preferably performed for 1 to 3 hours under conditions of a temperature of 40 to 70°C and a pH of 7 to 9, and more preferably for 2 hours under conditions of a temperature of 60°C and a pH of 8, but is not limited thereto.

[0027] In the above step (3), the ion exchange chromatography technique is cation exchange chromatography, but is not limited thereto.

[0028] A preferred example is (1) a step of adding Alcalase to soy protein isolate at a concentration of 1 to 2% (w / v) and hydrolyzing the resulting mixture into low-molecular-weight mixed peptides of 400 kDa or less under conditions of 40 to 70°C and pH 7 to 9 for 1 to 3 hours;

[0029] (2) After the above step (1), a step of inactivating the hydrolytic enzyme, filtering and drying to obtain a hydrolyzate of soy protein isolate; and

[0030] (3) A method for producing a hydrolyzed peptide of soybean protein isolate with enhanced glutamine synthetase (GS) activation efficacy and tyrosine content, including a step of obtaining a fraction by eluting the hydrolyzate obtained in step (2) with citric acid using a cation exchange chromatography technique under conditions of pH 3.0 to 3.4 or pH 6.2 to 6.6, but is not limited thereto.

[0031] In addition, the present invention relates to a hydrolyzed peptide of soybean protein isolate with enhanced glutamine synthetase (GS) activation efficacy and tyrosine content manufactured by the above manufacturing method.

[0032] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0033]

[0034] [Materials and Methods]

[0035] 1. Materials

[0036] The soy protein isolate used in the experiment was manufactured by Shandong Yuxn Bio-Tech Co., Ltd. (China) using 100% defatted soybeans as the raw material. 20 kg of soy protein isolate was purchased from Jeongwoo Trading Co., Ltd. (Cheongju, Korea) in October 2022 and used as a sample. 2.4 ℓ of Alcalase, 0.8 ℓ of Neutrase, 500 mg of Protamex, and Flavorzyme, which are proteolytic enzymes for hydrolysis, were purchased from Biosis (Busan, Korea). All reagents used in the examples of the present invention were for analytical use.

[0037]

[0038] 2. Preparation of hydrolyzate and optimization of hydrolysis conditions

[0039] To set the hydrolytic enzyme and hydrolysis time, 5 g of soybean protein isolate was suspended in 25 mM Tris-Cl (pH 7.5) and diluted to 100 ml. 5 ml of the soybean protein isolate suspension was added to a 15 ml conical tube, and 0.5 ml of the enzyme solution was added so that the enzyme-to-substrate ratio was 2% (w / v or v / v). The soybean protein isolate and enzyme mixture were reacted at a reaction temperature of 50°C for 120 minutes. 1 ml of the reaction solution was taken into each test tube at 15, 30, 45, 60, 90, and 120 minutes, and 20% TCA (Trichloroacetic acid) solution was added so that the final concentration was 4% to stop the enzyme reaction. The reaction mixture was centrifuged at 10,000 rpm for 10 minutes (MC-12, Benchmark Scientific Inc, NJ, USA), and the protein concentration of the supernatant was measured using the Biuret method (Spies, 1957). The degree of hydrolysis of the supernatant was calculated using the following equation (Equation 1).

[0040] (Formula 1) Degree of hydrolysis (DH, %) = (Protein content of supernatant / total protein content) × 100

[0041] Meanwhile, in order to confirm the degree of hydrolysis of the two types of enzyme mixtures, each enzyme was added to the soybean protein isolate suspension at 1% (w / v, v / v) so that the total enzyme-to-substrate ratio was 2%, and hydrolysis was performed at the same reaction temperature and time to measure the hydrolysis effect of the different types of enzyme mixtures.

[0042] To optimize the hydrolysis conditions, an experimental design was performed using the Box-Benken method in the range of enzyme to soy protein isolate concentration ratio (1-3%), reaction temperature (50-70℃), and pH (6-8) as dependent variables, and the protein concentration of the hydrolyzate and the tyrosine content in the hydrolyzate were measured as response values.

[0043]

[0044] 3. Scale-up of the tyrosine-rich fraction

[0045] The hydrolysate manufacturing process was scaled up to separate a large amount of tyrosine or a fraction containing tyrosine from the hydrolysate. 180 ℓ of purified water (1:12, w / v) was added to 15 kg of soybean protein isolate in an extraction mixer (custom-made, Myungbo Industrial, Korea), and the pH was adjusted to 8.0 with 1 N HCl solution. After mixing the suspension well, the temperature was adjusted to 60 ℃, and 225 g of the hydrolytic enzyme Alcalase was added to 1.5% of the weight of the soybean protein isolate. ® 2.4LFG (Novozyme) was added and hydrolyzed for 2 hours while stirring at 30 rpm at 60°C.

[0046] After hydrolysis, the enzyme was inactivated by heating at 90℃ for 30 minutes and cooled to room temperature. The filtrate filtered through a 60-mesh filter was frozen in a deep-temperature freezer and then freeze-dried (LP100, Ilshin Biobase, Korea). The freeze-dried product was used as a sample for fraction separation. For fraction separation using batch column chromatography, 550 g of the hydrolyzate was suspended in 4 ℓ of 0.01 N HCl solution and centrifuged at 4,000 rpm for 20 minutes (Supra R22, Hanil Scientific, Korea) to recover the supernatant. The protein content of the supernatant was measured by the Biuret method. A column (SAC-Bio-72-60G-20, LISUAE Science, China) was packed with 2 kg of SP SepFast 6HF resin (BioToolomics Ltd, England), and 25 mM sodium citrate (pH 3.2) was flowed to equilibrate the resin. Resin equilibrium was confirmed by the pH value measured using pH paper. 1.8 L (110 g, pH 2.0) of hydrolysate was loaded onto the paralleled column to bind amino acids or peptides to the resin, and then 5 L of sodium citrate (pH 3.2) was used as the eluent and eluted at a flow rate of 20 mL / min to obtain a pH 3.2 fraction. In addition, 5 L of 25 mM sodium citrate (pH 6.4) was used at the same flow rate to fractionate the column, and a pH 6.4 fraction was obtained.

[0047] After fractionation, 2.5 ℓ of 0.1 N NaOH solution was used to elute undissolved substances, the resin was washed with distilled water to pH 7.0, and then equilibrated with 25 mM sodium citrate (pH 3.2) and used to prepare fractions.

[0048]

[0049] 4. Amino acid composition of hydrolysates and fractions

[0050] 5 mg of freeze-dried hydrolysate and 2 ml of each fraction were placed in a test tube, and 5 ml of 6 N HCl solution was added to the hydrolysate and 2 ml of 12 N HCl solution was added to the fraction, and the tube was completely sealed while filling with nitrogen. The tube was hydrolyzed for 24 h in a heating block (HB-96D, Daihan Scientific Co., Ltd, Seoul, Korea) at 110°C. The hydrolyzed sample was filtered through a 3G-glass filter and completely concentrated using a rotary vacuum evaporator (N-1110, Eyela, Tokyo, Japan) at 50°C or lower. After diluting with 5 ml of loading buffer (pH 2.2) for amino acid automatic analysis and filtering through a 0.20 μm cylindrical filter, 40 μl was injected into an amino acid automatic analyzer (Biochrom 30, Biochrom, Cambridge, UK) and the amino acid composition was analyzed according to the analysis program.

[0051]

[0052] 5. Confirmation of the in vitro glutamine synthetase (GS) activation effect

[0053] Peroxynitrite (PN) and the reactants were added to the mouse mPFC lysate, mixed well, and left on ice for 5 min. For each reaction, the reactants were mixed as follows: 1 μl of lysate + 1 μl of PN (finally 10-fold concentrated) + 1 μl of hydrolysate (finally 10-fold concentrated) + 7 μl of GS digestion buffer (50 mM imidazole, pH 6.8). After reacting the mixture on ice for 5 min, 40 μl of GS digestion buffer and 50 μl of GS assay buffer (50 mM imidazole-HCl, pH 6.8, 25 mM L-glutamine, 12.5 mM hydroxylamine, 12.5 mM sodium arsenate, 1 mM MnCl2, and 0.08 mM ADP) were added and reacted at 37°C for 30–60 min. The reaction was terminated by adding 100 μl of stop buffer (90 mM FeCl3, 1.8 N HCl, and 1.45% TCA), and the absorbance was measured at 560 nm using a microplate reader. GS activity was expressed as the amount of the final product, γ-glutamylhydroxamate, and the unit is μM / min / μg protein.

[0054] [Statistical Analysis]

[0055] Statistical analysis of standard deviation, analysis of variance, and regression analysis, as well as experimental design, were performed using the statistical program JMP (ver. 12, SAS Institute, Cary, NC, USA).

[0056]

[0057] Example 1. Preparation of hydrolyzate and optimization of hydrolysis conditions.

[0058] To determine the hydrolytic enzyme, two-stage hydrolysis conditions were set to obtain the degree of hydrolysis and hydrolysate of soybean protein isolate at 50°C, pH 7.5, and 15 to 120 minutes. Thereafter, the content of tyrosine or peptides containing tyrosine residues in the hydrolysate of the obtained soybean protein isolate was measured using a fibrinolytic activity test using absorbance (Anson method).

[0059] As a result, when hydrolyzed using a single enzyme, the hydrolysis degrees of Alcalase, Protamex, and Neutrase, excluding Flavorzyme, did not show a large difference, but the initial hydrolysis degree was relatively high for Alcalase and decreased after 60 minutes (Fig. 1). The above results suggest that the optimal pH range of Alcalase is pH 7-9, and as the degree of hydrolysis increases, the pH rapidly decreases below the optimal pH range due to the increase in the hydrogen ion concentration of the hydrolyzate. Although Alcalase does not show a large difference in the hydrolysis degree of Protamex, it is the enzyme most frequently used industrially because the price of the enzyme is relatively low.

[0060] Meanwhile, when the degree of hydrolysis was measured by mixing two enzymes, the degree of hydrolysis of Alcalase + Protamax mixed enzyme (A + P) was relatively high, and the degree of hydrolysis of Nutrase + Flavorzyme or Nutrase + Alcalase was relatively low (Fig. 2). It was determined that the reason the degree of hydrolysis was lower than that of other mixed enzyme groups when Flavorzyme was mixed was because Flavorzyme is an endo and exo type. When Flavorzyme is included as a complex enzyme, it is used to remove the bitter taste derived from the hydrolyzate by cutting the terminal amino acid of the hydrolysis fragment.

[0061] Additionally, the tyrosine content was measured over reaction time using the Anson method using single enzymes and complex enzymes. When single enzymes were used, the amount of tyrosine produced was relatively high when Alcalase and Protamax were used, while the amount of tyrosine produced was relatively low when Flavorzyme was used (Fig. 3).

[0062] At a reaction time of 60 minutes, the amount of tyrosine produced according to the combination of complex enzymes treated was significantly higher when Alcalase + Protamax and Alcalase + Nutrase complex enzymes were treated, but at a reaction time of 120 minutes, there was no difference in the amount of tyrosine produced according to the type of complex enzyme (Fig. 4).

[0063] Meanwhile, nutrazyme is rarely used domestically, requiring a separate order. When using a complex enzyme, the optimal temperature and pH for each enzyme must be adjusted, necessitating a two-stage hydrolysis process, complicating the unit process. However, if flavor improvement through terminal cleavage of the hydrolyzed fragment is desired, the use of flavorzyme is preferable.

[0064] The ultimate goal of the present invention is to produce a low-molecular-weight peptide containing a high content of tyrosine (Tyr) for the activation of glutamine synthetase. Therefore, the hydrolysis conditions were optimized using the single enzyme "Alcalase," which produces a large amount of tyrosine and has a simple unit process, and the combined enzyme "Alcalase + Flavorzyme" to eliminate the bitter taste of the hydrolyzate.

[0065] Optimization of hydrolysis conditions was performed by performing surface reaction analysis using the Box-Benken method, setting the enzyme-to-substrate ratio as 1-3%, the temperature range of 50-70℃, the pH range of 6-8, and the reaction times of 60 and 90 minutes for Alcalase and Alcalase+Flavorzyme complex enzymes, respectively, as independent variables (Tables 1 and 2).

[0066] Experimental design for optimal hydrolysis of alkaline hydrolysate according to the Box-Benken method Experiment number Alcalase, % Temperature, ℃ pH Protein, g / 100g Tyr, g / 100g1150710.240.722160610.360.693160811.170.844170710.370.68525069.760.576250810.600.85726079.980.7482607 9.840.70926079.790.701027069.190.5811270811.471.011235078.890.621336068.980.5714360810.920.821537079.720.68

[0067] Experimental design for optimal hydrolysis of hydrolysates using complex enzymes according to the Box-Benken method Experiment number Alcalase + Flavorzyme, % Temperature, ℃ pH Protein, g / 100g Tyr, g / 100g1150711.230.692160611.320.573160811.760.874170711.250.735250611.400.666250811.280.957260711.230.688260711 .130.699260711.400.6910270611.150.3811270811.460.9112350710.500.6613360610.860.6114360811.220.9415370711.280.70

[0068] The protein and tyrosine contents of the hydrolysate prepared with Alcalase alone varied depending on the hydrolysis conditions, but the protein contents of the hydrolysate prepared with Alcalase and Flavorzyme complex enzymes did not differ significantly. The enzyme-to-substrate concentration, reaction temperature, and pH value all showed a significant correlation with the protein concentration of the hydrolysate, which is the reaction value (p<0.05). The protein concentration was 11.33 g / 100 g at an Alcalase concentration of 1%, 65°C, and pH 8 (Fig. 5). Meanwhile, the Alcalase concentration and pH showed a significant correlation with the tyrosine content, which is the reaction value (p<0.05). The optimal hydrolysis conditions were an Alcalase concentration of 1.5%, a reaction temperature of 60°C, and pH 8.0, at which time the tyrosine content was 0.88 g / 100 g (Fig. 6).

[0069] Hydrolyzate using Alcalase and Flavorzyme complex enzyme showed a significant correlation only with the complex enzyme concentration (p<0.05), and the protein concentration was 11.63 g / 100 g at an enzyme concentration of 1%, a reaction temperature of 65°C, and pH 8.0 (Fig. 7).

[0070] Meanwhile, tyrosine concentration showed a significant correlation only with pH (p<0.05), and there was a difference in reaction temperature with enzyme concentration of 1%, reaction temperature of 60℃, and pH 8.0 (Fig. 8).

[0071]

[0072] Example 2. Scale-up standard manufacturing process

[0073] The scale-up process extended the hydrolysis time from 1 to 2 hours under the identified optimal hydrolysis conditions (Table 3).

[0074] Conditions for hydrolysis and fractionation to obtain Tyr-rich fraction from soy protein isolateStep-by-step process (Unit process)Description(Description)Input raw material without hydrolysis processing into the reaction tank -Input soy protein isolate (SPI, 15 kg) -Input purified water (180 kg)Hydrolysis -Add 1.5% (v / v) alcalase -60℃, pH 8.0,mix reaction by stirring at 30 rpm for 2 hoursInactivation -Inactivate protease at 90℃ for 30 minutesCool to room temperatureFiltration -Filter through 60 mesh screenDry (freeze or spray)Tyrosine-rich fractionDried powder (hydrolysate)Loaded on column -SPIPreparation of hydrolysate: 110 g hydrolysate / 2 ℓ of 0.01 M HCl-Centrifugation at 4000 rpm / 20 minutes,take supernatant-SP SepFast Load the above supernatant onto a column packed with 6HF (pH 3.2) 1st elution - pH 3.2 fraction elution with 25 mM sodium citrate (pH 3.2) 2nd elution - pH 6.4 fraction elution with 25 mM sodium citrate (pH 6.4) Concentration - Concentrate the fraction at 80°C and dry (freeze or spray)

[0075] As mentioned above, the extension of the reaction time was considered in consideration of the heat transfer time in a large reactor, and when the reaction time was 2 hours (120 minutes), compared to 1 hour (60 minutes) in the production of alkaline hydrolysate, the protein content and tyrosine content were somewhat reduced, but it was judged to be appropriate in terms of simplification and economic feasibility of the industrialization process according to hydrolysis.

[0076]

[0077] Example 3. Analysis of the composition ratio of constituent amino acids and GS activation efficacy

[0078] (1) Analysis of the composition ratio of constituent amino acids

[0079] The amino acid composition of soybean protein isolate and each fraction was analyzed. The pH 3.2 fraction showed a slightly increased content of proline and methionine compared to soybean protein isolate hydrolysate, whereas glutamic acid, leucine, lysine, and arginine were slightly decreased, and tyrosine content was significantly increased.

[0080] Meanwhile, the total amino acid content of the pH 6.4 fraction was approximately 10 times that of the pH 3.2 fraction. Comparing the amino acid composition ratios, the composition ratio of basic amino acids increased compared to the pH 3.2 fraction.

[0081] Amino acid composition ratio according to pH fraction of hydrolysate of soy protein isolate Amino acid SPI hydrolysate pH 3.2 Fraction pH 6.4 Fraction Mean ± Standard Deviation (g / g) % (w / w) Mean ± Standard Deviation (g / g) % (w / w) Mean ± Standard Deviation (g / g) % (w / w) Asp 0.080 ± 0.000 10.53 0.080 ± 0.000 10.66 0.627 ± 0.000 8.09 Thr 0.032 ± 0.000 4.23 0.037 ± 0.000 5.000 319 ± 0.001 4.12 Ser 0.043 ± 0.000 5.61 0.036 ± 0.000 4.82 0.368 ± 0.001 4.75 Glu 0.143 ± 0.000 18.84 0.117 ± 0.001 1 5.701.111±0.00314.34Pro0.050±0.0006.600.076±0.00110.120.686±0.0088.86Gly0.033±0.0004.410.037±0.0005.020.327±0.0004.22 Ala0.033±0.0004.360.036±0.0004.760.337±0.0004.351 / 2Cys0.004±0.0000.490.007±0.0000.960.031±0.0000.40Val0.031±0.0004.090 .024±0.0013.240.281±0.0003.36Met0.008±0.0001.060.014±0.0011.930.098±0.0001.27Ile0.033±0.0004.370.036±0.0014.850.346±0 .0014.47Leu0.054±0.0007.060.045±0.0006.040.550±0.0007.10Tyr0.023±0.0003.080.039±0.0005.200.363±0.0024.69Phe0.042±0.00 05.570.046±0.0006.110.504±0.0116.51His0.023±0.0003.070.025 ±0.0003.290.363±0.0024.68Lys0.051±0.0006.690.031±0.0004.200 .541±0.0106.99NH30.011±0.0001.450.006±0.0000.840.088±0.0001.14Arg0.065±0.0008.490.055±0.0007.310.806±0.00110.40Total0.760±0.002100.000.746±0.001100.037.746±0.011100.00.

[0082] (2) Confirmation of activation efficacy of glutamine synthetase (GS) GS activity was confirmed by treating two types of oyster hydrolysates (PNY, TGPN) and four types of soybean hydrolysates (Alc / Alc, Alc / Fla, Alc 3.2 fraction, Alc 6.4 fraction) (Fig. 9).

[0083] Alc / Alc and Alc / Fla, provided in powder form, were dissolved in 20 mM citrate buffer, and all hydrolysates were tested at a final concentration of 0.39 mg / ml. Oyster hydrolysates PNY, TGPN, Alcalase, and the combined enzyme Alcalase and Flavorzyme hydrolysates did not increase GS activity, but the pH 3.2 and 6.4 fractions of Alcalase hydrolysate significantly increased GS activity. In particular, the pH 6.4 fraction (Alc6.4) showed an activity equivalent to that of the peptide Tyr-Gln (YQ) in the 1.0 mM and 0.1 mM PN treatment groups.

Claims

1. (1) A step of hydrolyzing soy protein isolate into low molecular weight mixed peptides using a hydrolytic enzyme; (2) After the above step (1), a step of inactivating the hydrolytic enzyme, filtering and drying to obtain a hydrolyzate of soy protein isolate; and (3) A method for producing a hydrolyzed peptide of soybean protein isolate with enhanced glutamine synthetase (GS) activation efficacy and tyrosine content, comprising: obtaining a fraction of the hydrolyzate obtained in step (2) using an ion exchange chromatography technique under conditions of pH 3.0 to 3.4 or pH 6.2 to 6.

6.

2. A method for producing a hydrolyzed peptide of soybean protein isolate with enhanced activation efficacy of glutamine synthetase (GS) and increased tyrosine content, characterized in that in the step (1), the hydrolyzed enzyme is one or two complex enzymes selected from Alcalase, Protamex, Neutrase, and Flavorzyme.

3. A method for producing a hydrolyzed peptide of soybean protein isolate with enhanced glutamine synthetase (GS) activation efficacy and tyrosine content, characterized in that the low-molecular-weight mixed peptide obtained in step (1) is a low-molecular-weight mixed peptide of 400 kDa or less in the first paragraph.

4. In the first paragraph, a method for producing a hydrolyzed peptide of soybean protein isolate with enhanced glutamine synthetase (GS) activation efficacy and tyrosine content, characterized in that the hydrolysis in step (1) is performed for 1 to 3 hours under conditions of a temperature of 40 to 70°C and a pH of 7 to 9.

5. A method for producing a hydrolyzed peptide of soybean protein isolate with enhanced glutamine synthetase (GS) activation efficacy and tyrosine content, characterized in that in the first paragraph, the ion exchange chromatography technique in the step (3) is cation exchange chromatography.

6. In paragraph 1, (1) A step of adding Alcalase to soy protein isolate at a concentration of 1 to 2% (w / v) and hydrolyzing it into a low-molecular-weight mixed peptide of 400 kDa or less under conditions of 40 to 70°C and pH 7 to 9 for 1 to 3 hours; (2) After the above step (1), a step of inactivating the hydrolytic enzyme, filtering and drying to obtain a hydrolyzate of soy protein isolate; and (3) A method for producing a hydrolyzed peptide of soybean protein isolate with enhanced glutamine synthetase (GS) activation efficacy and tyrosine content, comprising: a step of obtaining a fraction by eluting the hydrolyzate obtained in step (2) with citric acid using a cation exchange chromatography technique under conditions of pH 3.0 to 3.4 or pH 6.2 to 6.

6.

7. A hydrolyzed peptide of soybean protein isolate with enhanced glutamine synthetase (GS) activation efficacy and tyrosine content, manufactured by any one of the manufacturing methods selected from items 1 to 6.

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