KR100673433B1 - Method for preparing whole-soy oil having low molecular weight which is improved sensory evaluation and nutrition - Google Patents

Abstract

The method for producing low molecular weight soybean milk with improved sensory beauty and nutritional properties according to the present invention is pulverized by adding 5-9 times of water to soybeans soaked after washing with water, and then added with proteolytic enzyme and 30 minutes to 1 hour 30 at 60-60 ° C. After hydrolysis by shaking for 15 minutes and processing for 15 seconds to 10 minutes at 100 ~ 145 ° C, the whole soymilk has excellent sensory and nutritional properties such as sugar content, solid content, calcium binding ability, low molecular weight of soy hydrolysate and free amino acid content. There is an excellent effect that can be produced.

Soymilk, hydrolysis, protease, whole soymilk

Description

Method for preparing whole-soy oil having low molecular weight which is improved sensory evaluation and nutrition

1 is a graph showing the activity according to the pH of the protease B used in the present invention.

Figure 2 is a graph showing the pH stability of the protease B used in the present invention.

Figure 3 is a graph showing the activity according to the temperature of the protease B used in the present invention.

4 is a schematic diagram showing a process of preparing a hydrolyzate using soybean powder.

Figure 5 is a graph showing the yield of the hydrolyzate obtained by treating different protein hydrolases in soybean powder compared to the non-enzymatic control.

Figure 6 is a graph showing the solid content of the hydrolyzate obtained by treating different protein hydrolases in soybean powder compared to the non-enzymatic control.

7 is a graph showing the degree of hydrolysis of the hydrolyzate obtained by treating different protein hydrolases in soybean powder compared to the control without enzyme treatment.

Figure 8 is a graph showing the total phenolic content of the hydrolyzate obtained by treating different protein hydrolases in soybean powder compared to the non-enzymatic control.

Figure 9 is a graph showing the yield of the hydrolyzate obtained by treating different concentrations of proteolytic enzymes in soybean powder compared to the non-enzymatic control.

10 is a graph showing the solid content of the hydrolyzate obtained by treating different concentrations of proteolytic enzymes in soybean powder compared to the non-enzymatic control.

11 is a graph showing the degree of hydrolysis of the hydrolyzate obtained by treating different concentrations of proteolytic enzymes in soybean powder compared to the control without enzyme treatment.

Figure 12 shows the results of SDS-PAGE electrophoresis to determine the degree of hydrolysis of the protein by treating different concentrations of protease in soybean powder.

Figure 13 is a graph showing the total phenolic content of the hydrolyzate obtained by treating different concentrations of proteolytic enzymes in soybean powder compared to the control without enzyme treatment.

14 is a graph showing the yield of the hydrolyzate according to the different hydrolysis time when the hydrolyzate by hydrolyzing the protein hydrolase in soybean powder compared to the control without the enzyme treatment.

15 is a graph showing the solid content of the hydrolyzate according to the different hydrolysis time when the hydrolyzate is treated with protein hydrolase in soybean powder compared with the control without the enzyme treatment.

FIG. 16 is a graph showing the degree of hydrolysis of hydrolyzate according to different hydrolysis time when hydrolyzing by treating soybean powder with protein hydrolase, compared with the control without enzymatic treatment.

17 is a graph showing the results of SDS-PAGE electrophoresis according to different hydrolysis time when hydrolyzing soybean powder treated with protein hydrolase.

Figure 18 is a graph showing the total phenolic compound content of the hydrolyzate according to the different hydrolysis time when the hydrolyzate by hydrolyzing the protein hydrolase in soybean powder compared to the control group without the enzyme treatment.

19 shows the ridge analysis results for the effects of hydrolase concentration and hydrolysis time on yield and soluble solids of soybean powder hydrolysates.

20 shows ridge analysis results for the effect of hydrolase concentration and hydrolysis time on the degree of hydrolysis and calcium binding of soybean powder hydrolyzate.

Figure 21 shows the ridge analysis results for the effect of hydrolase concentration and hydrolysis time on the total phenolic content of soybean powder hydrolyzate.

Figure 22 shows the range of hydrolysis conditions for optimum yield of soybean powder hydrolysis, soluble solids, degree of hydrolysis, calcium binding capacity and total phenolic content.

Fig. 23 shows the results of DPPH free radical scavenging activity analysis of soybean powder hydrolysates treated with optimum conditions by reaction surface analysis.

Figure 24 shows the results of the superoxide radical scavenging activity analysis of soybean powder hydrolyzate treated with optimum conditions by reaction surface analysis.

FIG. 25 shows the results of angiotensin converting enzyme inhibitory activity analysis of soybean powder hydrolysates treated with optimum conditions by reaction surface analysis. FIG.

Figure 26 shows the results of SDS-PAGE electrophoresis to determine the molecular weight distribution of the soybean powder hydrolyzate treated with the hydrolysis enzyme under the optimum conditions by reaction surface analysis.

Figure 27 shows the results of SDS-PAGE electrophoresis to determine the degree of hydrolysis of the protein according to the amount of hydrolysis during hydrolysis using soybean.

Figure 28 shows the electrophoresis pattern of soy hydrolyzate according to the concentration of hydrolase during hydrolysis using soybean.

Figure 29 shows the electrophoresis pattern of soy hydrolyzate with hydrolysis time during hydrolysis using soybean.

Figure 30 shows the electrophoresis pattern of soybean hydrolyzate according to sterilization temperature during hydrolysis using soybean.

Figure 31 compares the electrophoresis pattern of the optimized soy hydrolyzate and the control of the present invention.

The present invention relates to a method for producing low molecular weight soybean milk with improved sensory beauty and nutritional properties, and more specifically, 5 to 9 times the amount of soybeans soaked in water, followed by grinding and hydrolysis by the addition of protein hydrolase. The present invention relates to a method for producing an excellent soybean milk in terms of sensory and nutritional properties such as sugar content, solid content, calcium binding ability, low molecular weight of soy hydrolysate and free amino acid content.

Soybean ( Glycine max L. Merrill ) is a legume and is native to Manchuria and is known to have been cultivated in Korea since the 4th-5th century BC. Soybeans contain not only large amounts of protein (40%) and lipids (20%), but also carbohydrates and vitamin groups, and the processed products are rich in calcium, phosphorus, magnesium, and other inorganic components such as iron, zinc and copper. It is contained. Traditionally, Asian countries, including Korea, have traditionally used soy protein in the form of doenjang, soy sauce, and red pepper paste using decomposing enzymes by microbial groups and used it as a corrosion in diet. In addition, many components of soybean have been known to have various physiological activities such as antihypertensive effect, anti-mutation, anticancer, and thrombolytic function (Kim, CS et al. Korean J. Food Sci. Technol . 2000 , 32 , 25-304 ).

In recent years, by ingesting a large amount of animal protein accumulates a large amount of cholesterol in the body, causing atherosclerosis diseases, interest in the use of plant protein is increasing. Soybeans are receiving new attention because they are re-evaluating that soybeans contain high quality protein and high content of essential fatty acids, making them a nutritionally superior food ingredient (Park, YW J. Korean Soc. Nutr . 1993 , 22 , 643-649). Soy protein is one of the most widely used vegetable protein. It is relatively inexpensive and has a wide range of uses in order to enhance the functional and nutritional properties of processed foods. It lowers serum cholesterol content and has anti-cancer effects, prevention of osteoporosis and improvement of renal dysfunction (Kim, JS Korea Soybean Digest . 1996 , 13 , 17-24; Sung, MK Korea Soybean Digest . 1996 , 13 , 19-31).

In general, in order to use a protein widely in food can be modified the functional properties, the method is mainly used chemical modification and enzymatic modification. Hydrolysis of proteins with acids or alkalis increases the concentration of salts or creates harmful substances. Therefore, many modifications by enzymes have been used, and thus the functional and nutritional value of soybeans, peanuts, cottonseed and rapeseed proteins. Improvements have been reported (Kim, SY et al. J. Agric. Food Chem . 1990 , 38 , 651; Kang, YJ Korean J. Food Sci. Tech . 1984 , 16 , 211; Sekul, AA et al. J. Agric.Food Chem . 1978 , 26 , 855; Lacroix, M. et al. J. Food.Sci. 1983 , 48 , 1644; Ponnampalan, R. et al. J. Food Sci . 1987 , 52 , 1552); Peptide production by enzymes is a method used to develop peptide-like new material. The protein is used to break down a large protein into small peptides and amino acids using proteolytic enzymes, and among them, the desired peptide. Or hydrolysis from peptides to amino acids Is to develop a new peptide (peptide) using the reverse reaction of the process. Proteolytic products include free amino acids, oligopeptides, and low molecular weight proteins, which have different physiological activities from the original proteins after their degradation, and are being developed as new biomaterials. It has been suggested that the well-adjusted can improve the functionality, these hydrolysates are very effective in inhibiting aging and carcinogenesis, and the antioxidant and physiological activities have been revealed (Yeum, DM et al. J. Korean) . Soc.Food Nutr . 1993 , 22 (2) , 226-233; Susumu, M. et al. Agric. Biol. Chem . 1985 , 49 , 1405; Toshiro, M. et al. Biosci. Biotech. Biochem . 1993 , 57 , 922; Kim, SB et al. Korean J. Food Sci. Technol . 1989 , 21 (4) , 492-497; 山口Nippon Shokuhin Kogyo Gakkkaishi . 1979 , 26 , 14; Adler-Nissen, J. J. Agric Food Chem . 1976 , 26 , 14).

The hydrolyzate of soy protein is attracting attention as a new functional food material. The function of soy protein is changed by processing conditions such as pH, salt concentration, temperature, etc. and is restricted in its use. Attempts have been made to modulate functionality (Yamaguchi, N. et al. Nippon Shokuhin Kogyo Gakkaishi , 1975 , 22 , 431-435). Thus, partial hydrolysis by enzymes has been suggested to increase the water solubility of the hydrolyzate, to change the functionality, as a new functional food. For example, it is widely used as a nutrient enhancer for soft drinks and juice drinks, and its value as a soluble protein food that is easily digestible and absorbed for patients with dysfunction of nitrogen nutrient intake is greatly appreciated. Some studies have been made on the improvement of new nutritional and processing functionalities by partial hydrolysis by treating enzymes with soy protein (Kim, DS et al. Kyungsung University Journal. 1998 , 19 (1) , 667-676). Changes in functional properties are known to be due mainly to changes in protein molecules such as changes in protein structure, molecular weight reduction, etc., due to hydrolysis by enzymes (Kinsella, JF In Food Protein, Applied Sci. Publishers, London. 1982 , 51; Choi, TR et al. J. Food Sci . 1982 , 47 , 1713).

Soybean milk is a representative soybean processed product that has increased digestibility and protein utilization of soybean. It is well absorbed, rich in essential amino acids, rich in essential fatty acids, rich in minerals such as iron, phosphorus, potassium, arteriosclerosis and lipid metabolism. Improvement, nerve function, anti-aging and isoflavones are effective in preventing breast cancer, and demand is increasing day by day due to nutritional excellence and low cholesterol content. On the other hand, the lack of calcium, which is essential for growing children and the elderly, is one of the big problems of soymilk, which is recognized as a high-protein milk substitute. When calcium is added, the soy protein and calcium of soy milk combine and precipitate. Therefore, some studies have been reported in which some calcium is added (Weingartner, KE et al. J. Food Sci . 1983 , 48, 256; Hirotsuka, M. et al. J. Food Sci . 1984 , 49 , 1111), the treatment is complicated and the practical use is poor due to the precipitation of calcium salt. However, the treatment of protease, which was used to improve the functional properties of proteins, may increase calcium intolerence due to a decrease in protein molecular weight and a change in protein structure, which may solve the calcium deficiency problem of soy milk. (Pyun, JW et al. Korean J. Food. Sci. Technol . 1996 , 28 (6) , 995-1000).

Therefore, the development of biological materials through functional control suitable for processing purposes and conditions using soy protein is required.

In view of the above, the present inventors compared and investigated the functionalities of soybean hydrolysates according to enzymatic selection and optimization of hydrolysis conditions according to hydrolysis conditions of soybeans and soybean proteins, and improved functionality and no bitter taste. We have developed a method for producing differentiated low-molecular frontal milk, which can be used as whole head and powder.

Accordingly, it is an object of the present invention to provide a method for producing low molecular weight soybean milk with improved sensuality and nutritional properties.

The above object of the present invention is crushed by adding 5-9 times the amount of water to the soybean soaked after washing, and then hydrolyzed by adding protein hydrolase and shaking at 30-60 ° C. for 30 minutes to 1 hour 30 minutes. Achieved by treatment at 145 ° C. for 15 seconds to 10 minutes.

The present invention provides a method for producing low molecular frontal milk.

In the method for producing a low molecular soybean milk of the present invention, after washing with water, it is pulverized by adding 5-9 times of water to soybeans soaked, and then hydrolyzed by adding protein hydrolase and shaking for 30 minutes to 1 hour and 30 minutes at 30 to 60 ° C. After that it includes the treatment for 15 seconds to 10 minutes at 100 ~ 145 ℃.

Most preferably, the method for preparing low molecular soybean milk according to the present invention is crushed by adding 7 times the amount of water to soybean soaked after washing with water, followed by adding 0.2% protein hydrolase and shaking at 100 rpm for 1 hour at 50 ° C. for hydrolysis. After 15 seconds at 130 ℃ is good in terms of sensory and nutritional aspects such as sugar content, solid content, calcium binding capacity, low molecular weight and free amino acid content of soy hydrolysates.

Proteinase Protin KM-70,000Pu / g used in the present invention is Aspergillus duck material ( Aspergillus) Oryzae ) is an enzyme derived from an enzyme that converts an amino acid using a protein as a substrate and mainly contains amylase. Endo-type proteinases and exo-types are included together, making it the most useful enzyme for seasoning or soybean peptide production of protein sources. In addition, the protease is in the form of fine dry powder (fine dry powder) is pale yellow, has a peculiar fermentation odor and has a physical property easily soluble in water. In addition, the optimum pH is 7.0, the pH stability range is 5.0 ~ 8.0, the optimum temperature is 50 ℃, has a temperature stability at 35 ℃ or more and has a chemical characteristic that is completely inactivated when treated at 60 ℃ 30 minutes (Fig. 1 to 3).

The analysis method used in the present invention is as follows.

1) yield

The yield of soybean is expressed as a percentage of the raw material minus the dry weight of the residue after hydrolysis.

2) solid content

The solids content of the hydrolyzate was determined by measuring the moisture content of the supernatant after hydrolysis using an infrared moisture determination balance (Kett, FD-240, Japan) and subtracting the water content to obtain the solid content. .

3) Hydrolysis degree measurement

Determination of the degree of hydrolysis of soy protein using protease was carried out in a test tube containing 2 mL of 20% (w / v) trichloroacetic acid (TCA), taking 2 mL of each sample hydrolyzate. After mixing, centrifugation (3,000 × g, 10min), a certain amount of the centrifuged supernatant was taken to measure the amount of 10% TCA soluble protein, and the degree of hydrolysis was calculated from the following equation.

4) calcium binding capacity

The amount of protein at 280 nm was measured by absorbance of the supernatant obtained by adding 30 mM CaCl ₂ to the hydrolyzate and centrifuging at 8000 rpm for 10 minutes (Pyun, JW et al. Korean J. Food. Sci. Technol . 1996 , 28 (6) , 995-1000) were used to compare the degree of coagulation and to determine the binding capacity with calcium.

5) SDS-PAGE Electrophoresis

To investigate the effect of protease treatment of the hydrolyzate on soy protein, 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Standard proteins and protein markers were purchased from Sigma, USA, and sample buffer (60 mM Tris-HCl, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, 0.1% bromophenol blue, 10 mL distilled water). After hydrolysis, the same amount of sample buffer was added to the centrifuged supernatant of the hydrolyzate which stopped the enzymatic reaction, and then heated in 100 ° C. water for 5 minutes to induce complete denaturation of the protein, followed by electrophoresis (Mini-Protean, BIO-RAD Laboratories, Inc. USA.). The electrophoretic buffer solution was a 0.025 M-Tris base-0.192 M glycine (pH8.3) solution containing 0.1% (w / v) SDS. Waving was performed at room temperature for 2 hours using a Blue Al-250 solution (Coomasie blue R-250; 1.0 g Coomassie blue R-250, 450 mL methanol, 450 mL H ₂ O, 100 mL glacial acetic acid). Decolorization was carried out by changing the reagents twice over 30 minutes by pouring the decolorizing reagent (100mL methanol, 100mL glacial acetic acid, 800mL H2O) slightly so that the gel was submerged.Additionally, the gel was completely submerged and decolorized for 18 hours. . The band stained on the gel obtained at this time was compared with the protein markers and observed the degree of hydrolysis according to the hydrolase treatment.

6) Total Phenolic Compound

The total phenolic compound content of the hydrolyzate was colorimetrically determined by the Pauline-Dennis method (Amerine, MA et al. Wiley & Sons, New York , 1980 , 176-180). In other words, 2 mL of a hydrolyzate was added with 2 mL of a phenol reagent (Folin-Ciocalteu's reagent), mixed, and after 3 minutes, 2 mL of 10% Na ₂ CO ₃ was added thereto, stirred, and left at room temperature for 1 hour, followed by UV-visible spectroscopy. Absorbance was measured at 700 nm using a photometer (UV-visible spectrophotometer; UV-1601, shimadzu, Japan). At this time, using tannic acid (tannic acid) as a standard material was converted from the standard curve prepared by the above method.

7) Determination of DPPH free radical scavenging activity

α, α-diphenyl-β-picrylhydrazyl (α, α-diphenyl-β-picrylhydrazyl; DPPH) radical scavenging activity was measured as follows. That is, 12 mg of DPPH was dissolved in 100 mL of absolute ethanol, and the absorbance of the DPPH solution was diluted to about 1.0 at 517 nm using a 50% ethanol solution as a control. After mixing 4 mL of DPPH solution with 1 mL of sample solution for 30 seconds, measure the change in absorbance at 517 nm using a UV-vis spectrophotometer (UV-1601, Shimadzu, Japan). DPPH free radical scavenging activity was calculated.

As: absorbance at sample addition port

Ac: absorbance of sample-free

8) Determination of Superoxide Radical Scavenging Activity

Superoxide radical (O ₂ ^{· -1} ) scavenging activity was determined by the xanthine-xanthine oxidase cytochrome C reduction method (Iio, M. et al. Agric. Biol. Chem ., 1985 , 49 , 2173). 0.2 mL of hydrolyzate, 1.2 mL of 50 mM phosphate buffer (pH 7.8), 0.2 mL of 1 mM xanthine, 0.2 mL of 0.05 mM cytochrome C, and 0.2 mL of xanthine oxidase diluted at 550 nm with an absorbance change of 0.02 per minute. After mixing and reacting for exactly 3 minutes, the absorbance at 550 nm was measured using a UV-vis spectrophotometer (UV-1601, Shimadzu, Japan) and expressed as superoxide radical scavenging activity from the following equation.

·O₂ ^- 소거활성(%) =

O ₂ ^- scavenging activity (%) =

As: absorbance at sample addition port

Ac: absorbance of sample-free

9) Angiotensin Converting Enzyme (ACE) Inhibitory Activity

(1) Preparation of coenzyme liquid and substrate

The crude enzyme solution was suspended in rabbit lung acetone powder (Sigma Chemical Co.) with 50 mM sodium borate buffer (pH 8.3), extracted with enzyme at 4 ° C, and then centrifuged at 3,000 rpm for 40 minutes. The supernatant containing about 1.2 unit / mL ACE activity was used as the crude enzyme solution. The activity of the enzyme solution was defined as 1 unit of the amount of enzyme that liberates 1 μM hippuric acid from the substrate at 37 ° C. for 1 minute.

The substrate was used to prepare a 12.5 mM hyperyl-histidyl-leucine solution using 50 mM sodium borate buffer containing 300 mM NaCl.

(2) ACE inhibitory activity

Angiotensin converting enzyme (ACE) inhibitory activity of the sample is only kuswi method (Cushman) and chyung (Cheung); a (Chshman, DW Cheung, HS Biochem Pharmacol 1971, 20, 1637-1648..) Modified by the following method Measured. 50 μL of the sample solution was added 50 μL of ACE coenzyme solution and 100 μL of 50 mM sodium borate buffer (pH 8.3), followed by preincubation at 37 ° C. for 5 minutes. 50 μl of substrate solution was added thereto, followed by further reaction at 37 ° C. for 30 minutes, and then 250 μL of 1N HCl was added to stop the reaction. (In the blank test, 50 μL of distilled water was added instead of the sample, and the control group included both the sample and the substrate, but the reaction was stopped by adding 250 μL of 1N HCl, and then 50 μL of the sample was added thereto.) 1 mL of ethyl acetate was added thereto for 15 seconds. After stirring, the mixture was centrifuged at 3,000 rpm for 10 minutes to take 1 mL of the supernatant. The supernatant was completely dried at 120 ° C. for 30 minutes and then dissolved by stirring for 15 seconds by adding 3 mL of 1 M NaCl and measuring absorbance at 228 nm. The ACE activity inhibition rate was shown by the following equation.

A: absorbance of the reaction solution in which distilled water was added instead of the sample

B: absorbance of sample added reaction solution

C: absorbance of the reaction solution at the beginning of the reaction by adding 1 M HCl to stop the reaction

10) free amino acids

To determine the free amino acid content of the soy hydrolyzate, 80 mL of 75% ethanol (v / v) solution was added to the protein hydrolyzate, and free amino acid was extracted with reflux cooling for 1 hour in a water bath maintained at 80 ° C. The extract was cooled, filtered (Watman No. 1), concentrated under reduced pressure to 100 mL with distilled water, 50 mL of this was added, and 25% TCA solution was added thereto in the same amount, refrigerated for 1 hour to precipitate protein, followed by centrifugation. (3,000 rpm, 20 min). The supernatant was taken and 100 mL of diethyl ether was added thereto, followed by extraction three times to remove lipids, pigments and fat-soluble substances. The aqueous layer was concentrated under reduced pressure and dried at 40 ° C., dissolved in 10 mL of loading buffer solution (0.2 N lithium citrate, pH 2.2), and filtered through a 0.22 μm membrane filter using an amino acid autoanalyzer. The conditions were analyzed as shown in Table 1.

Operating conditions of amino acid autoanalyzers for the analysis of free amino acids division Condition Instrument Biochrom 30 (U.K.) Buffer solution Lithium citrate buffer Buffer flow rate 0.33 mL / min Ninhydrin flow rate 0.33 mL / min Column temp. 37 ℃ Injection volume 40 μl

11) Calcium endogenous comparison

Optimized conditions In the treated soybean powder hydrolyzate and non-hydrolase-free treatment, powdered seaweed calcium and liquid ionized calcium were added equally to the calcium content of 110 mg / 100 mL. Each calcium was added and stirred for 1 hour. In order to confirm the binding of calcium and protein, centrifugation at 8000 rpm for 10 minutes to precipitate protein-bound calcium, and the supernatant was incubated at 550 ° C in a sinter furnace, dissolved in 5 mL of 6 N-HCl, and left at room temperature for 12 hours. It was analyzed by filtration with 0.45 ㎛ membrane filter and calcium content was measured using an inductively coupled plasma spectrometer (JY-38S, Jobin Yvon, France).

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited thereto.

Experimental Example 1: Preliminary Investigation of Preparation Conditions of Whole Soymilk

Since the soybean pulverized by steaming easily deteriorates, the preparation conditions of the soybean milk preliminarily using soybean powder were investigated as follows.

Step 1: Preparation of Soy Hydrolyzate

In the present invention, the soybean grown in Gyeongbuk province in 2004 was used to use a powder pulverized more than 100 mesh (mesh). General components of the samples were analyzed by the AOAC method (AOAC Official Methods of Analysis . 15 th ed. Association of Official Analytical Chemists, Wsahington. DC. 1990 , 148) and are shown in Table 2 below. For soybean milk, golden soybeans purchased from Sangju (Gaya agricultural) were used.

General ingredient content(%) Soybean Powder moisture 8.5 Crude protein 40.8 Crude fat 20.5 View minutes 4.2 Carbohydrate 26.0

Protease is A (Protin KM-90,000Pu / g), B (Protin KM-70,000Pu / g), C, which is suitable for the preparation of low molecular whole soymilk according to the present invention through preliminary experiments of the currently available enzyme. (Protin KM-100,000Pu / g) and D (Protin KM-200,000Pu / g) four {purchased: Keimyung FedEx, Korea} were selected and used.

Samples were prepared by the method of Cha and Yoon (Cha, MH; Yoon, S. Modification of functional properties of soy protein isolate by proteolytic enzymes.Korean J. Food.Sci.Technol . 1993 , 25 (1) , 39-4553). In other words, 10 g of soybean powder was suspended in 100 mL of distilled water to make a 10% (w / v) substrate solution, and 0.2% of each protease was added so that the ratio of the enzyme to the substrate solution was 100: 2. v / w), and hydrolyzed by shaking at 100 rpm for 2 hours at 50 ° C, inactivating the enzyme at 80 ° C for 10 minutes to stop the reaction, and using the supernatant centrifuged at 8,000 rpm for 20 minutes as a hydrolyzate. (FIG. 4).

Second Step: Screening of Protease

A (Protin KM-90,000Pu / g), B (Protin KM-70,000Pu / g), C (Protin KM-100,000Pu / g) and D (Protin KM-200,000Pu / g) used to prepare the low molecular frontal milk ) The characteristics of the proteolytic enzymes of four species {purchased from: Keimyung Fudex, Korea} were compared.

Protease A (Protin KM-90,000Pu / g) has been shown to have a Protease activity of 68,900 U / g. B (Protin KM-70,000Pu / g) has a protease activity of 54,200 U / g, Viable bacteria count is 10 CFU / g, heavy metals such as lead are less than 40 µg / g, and Arsenic Was 3 µg / g or less. C (Protin KM-100,000Pu / g) had protease activity of 99,300 U / g with 10 CFU / g viable bacteria, 40 μg / g or less for heavy metals such as lead, and 3 μg / g or less for arsenic. D (Protin KM-200,000Pu / g) had a polyphenol oxidase activity of 127,000 POU / g with a viable bacterial count of 10 CFU / g and an arsenic of 3 μg / g or less.

In order to select protease suitable for hydrolysis of soybean powder and SPI by protease, 10% (w / v) soybean powder solution was prepared, respectively, for protease A, B, C and Add 0.2% (v / w) of D each, shake at 100 rpm for 2 hours at 50 ° C, stop enzymatic reaction at 80 ° C for 10 minutes, centrifuge at 8,000 rpm for 20 minutes, and then use supernatant to yield, Solid contents, hydrolysis degree, calcium binding ability and total phenolic compound content were selected.

The results of comparing the yield, solid content, degree of hydrolysis, calcium binding ability, SDS-PAGE electrophoresis and total phenolic compounds of the soybean powder, which were added to four kinds of protease for 2 hours, were as follows.

The yield according to protease treatment was slightly higher than that of the control as shown in FIG. 5, and there was no significant difference between treatments, but it was relatively high at 43.2% in protease B treatment. Yields by protease type showed a big difference.

As a result of analyzing the solid content according to the protease treatment, the soybean powder hydrolyzate was generally lower in FIG. 6 than in the control.

As a result of measuring the degree of hydrolysis according to protease treatment, as shown in FIG. 7, the degree of hydrolysis was higher in the protease B and C treatment groups than in the untreated group. Protease B treatment showed the highest value of 23.3%. In the study report of Ahn (65), the isolated soy protein prepared from genuine soybean was treated with α-chymotrypsin, which was 4.09% in the untreated group and 15.35% in the untreated group. It was similar to, but slightly lower in the enzyme treatment group.

Calcium binding capacity of protease treatment was determined by adding 30 mM CaCl ₂ by the method of stool and the like (Pyun, JW et al. Korean J. Food. Sci. Technol . 1996 , 28 (6) , 995-1000). After measuring the turbidity of the supernatant obtained by centrifugation at 280nm it is shown in Table 3 to determine the binding degree with calcium. Compared to 0.107 of the untreated group, the protease B treated group was 0.421, which is very high. However, the protease B treated group is thought to have less protein and calcium binding.

As a result of the total phenol content according to the protease treatment, there was no significant difference between the untreated or protease treatment sections, as shown in Figure 8, the increase in the total phenol content according to the hydrolysis of soy protein is Pratt (Yu, RN et al. J. Korean Soc. Food Sci. Nutr. 1996 , 25 , 1031-1036) hydrolyzes soy protein and genistein, an isoflavone that is a phenolic antioxidant The total phenolic content of the isoflavones was increased as reported by extracts of daidzein and glycidin.

Comparison of Calcium Binding Ability of Soybean Powder According to Kinds of Hydrolase Hydrolase treatment Soybean Powder Control 0.107 ± 0.002 ¹⁾ Enzyme A Treatment 0.116 ± 0.000 Enzyme B Treatment 0.421 ± 0.014 Enzyme C Treatment 0.188 ± 0.003 Enzyme D Treatment 0.105 ± 0.007 [Note] 1) Turbidity

Step 3: Investigate the Effect of Protease Treatment

Protease treatment is an important determinant of protein degradation, using protease B, which was shown to be relatively good in all assays as a result of protease selection to investigate the effects of protease treatment. After making 10% (w / v) soybean powder solution, the ratio of protease B to substrate solution is 0.1, 0.2, 0.3, 0.4, 0.5% (v / w) for 2 hours at 50 ℃. Treatment was hydrolyzed and hydrolyzate was prepared and compared.

As a result, the yield was not significantly different according to the protease concentration as shown in Figure 9, but slightly increased from 42.0% to 46.3% as the concentration increased.

Solid content according to protease concentration is shown in FIG. 10. Compared with 1% treatment, it increased little by little but showed similar tendency without big difference.

As a result of examining the degree of hydrolysis of soybean powder according to the protease concentration, it was shown in FIG. 11. As the protease concentration increased, the difference between the protease concentrations in the set range (1-5%) was not significant.

The results of SDS-PAGE electrophoresis are shown in FIG. 12 to determine the degree of hydrolysis of the protein according to protease treatment. In all sections, it was below 14,300 Da and clear band around 14,300 Da and 3,500 Da, and faint band of wide distribution around 6,500 Da, but there was no significant change according to the concentration.

The results of comparing the calcium binding capacity according to the protease concentration are shown in Table 4 below. Protease concentration increased up to 4% but decreased slightly at 5% concentration.

The total phenolic compound content according to protease concentration was shown in FIG. 13. A slight increase in protease concentration up to 5% was shown.

Comparison of Calcium Binding Ability of Soybean Powder According to Hydrolase Content Hydrolase Content (%) Soybean Powder One 0.291 ± 0.004 ¹⁾ 2 0.328 ± 0.004 3 0.342 ± 0.018 4 0.408 ± 0.003 5 0.394 ± 0.003 [Note] 1) Turbidity

Step 4: Investigate the Effect of Protein Hydrolysis Time

To investigate the effects of protease treatment time, a 10% (w / v) soybean powder solution was prepared so that the ratio of protease B to substrate solution was 0.2% (v / w) at 30 ° C. The components of the hydrolyzate obtained by hydrolysis for, 60, 90, 120, 150, 180, 240 and 300 minutes were investigated.

Yield according to protease treatment time was not significantly different as the soybean powder hydrolyzate increased as shown in FIG. 14, but increased slightly from 40.1% to 46.2% at 300 min treatment as time passed.

As a result of the solid content with the protease treatment time, as shown in FIG. 15, it was found to increase with time, and it was found to increase from 4.05% to 4.89%.

The result of measuring the degree of hydrolysis according to protease treatment time is shown in FIG. 16. There was no significant difference from 23.8% to 24.5%.

SDS-PAGE electrophoresis according to protease treatment time was shown in FIG. 17. It was distributed below 14,300 Da, and two clear bands around 6,500 Da and one band around 3,500 Da appeared, but there was no significant change with the hydrolysis time. Appeared.

As a result of the calcium binding capacity according to the protease treatment time, as shown in Table 5, 0.190 and 0.390 at 0.3 minutes in the 30-minute treatment group were continuously increased to 0.381 at 300 minutes. Increasing the value seems to reduce the binding of soy protein to calcium.

The total phenolic compound content according to protease treatment time is shown in FIG. 18. It was 64.2mg% in 30 minutes treatment and increased to 91.4mg% in 300 minutes treatment.

Comparison of Calcium Binding Ability of Soybean Powder with Hydrolysis Time Hydrolysis Time (min) Soybean Powder 30 0.117 ± 0.002 ¹⁾ 60 0.246 ± 0.016 90 0.220 ± 0.063 120 0.445 ± 0.193 150 0.237 ± 0.083 180 0.340 ± 0.013 240 0.390 ± 0.005 300 0.381 ± 0.002 [Note] 1) Turbidity

Step 5: Experimental Design for Optimizing Hydrolysis Conditions

Hydrolysis of proteins can be made differently by different variables, such as the type of protease, the concentration of enzyme on the substrate, the hydrolysis time.

In the present invention, the response surface methodology (RSM) 54 was used to monitor the hydrolysis characteristics and optimize the hydrolysis conditions. The experimental plan 55 of the factor (independent) variable (X _i ) by the central composite design (Table 6) is the concentration of the factor, ie protease, considered as an important variable in proteolytic conditions as shown in Table 6 below. (0, 0.2, 0.4, 0.6, 0.8%, X ₁ ) and the hydrolysis time (1, 3, 5, 7, 9 hrs, X ₂ ) are encoded in 5 steps of -2, -1, 0, 1, 2 According to the central synthesis plan was set to 10 sections as shown in Table 7 to perform a hydrolysis experiment.

In addition, the dependent variables (Y _n ) affected by these factor variables, that is, the quality factors of the hydrolysis conditions, yield (Y ₁ ), solid content (Y ₂ ), degree of hydrolysis (Y ₃ ), calcium binding capacity (Y ₄ ), The total phenolic compound (Y ₅ ) was used. Three replicates were used and the average value was used for the regression analysis. Prediction by regression analysis was performed using a statistical analysis system (SAS) program (56). As a result of the regression analysis, the critical point is not the maximum or the minimum but the saddle point. In this case, the optimum point was obtained by ride anaysis.

Hydrolysis Levels of Soybean Powder in Factor Variable Planning X _i Hydrolysis conditions Level -2 -One 0 One 2 X ₁ Hydrolase Content (%) 0 0.2 0.4 0.6 0.8 X ₂ Hydrolysis time (hrs) One 3 5 7 9

Central Synthesis Scheme for Optimizing Hydrolysis of Soybean Powder Experiment number ¹⁾ Hydrolase Content (%) Hydrolysis time (hrs) One 0.2 (-1) 3 (-1) 2 0.2 (-1) 7 (1) 3 0.6 (1) 3 (-1) 4 0.6 (1) 7 (1) 5 0.4 (0) 5 (0) 6 0.4 (0) 5 (0) 7 0.4 (0) 1 (-2) 8 0.4 (0) 9 (2) 9 0 (-2) 5 (0) 10 0.8 (2) 5 (0) 1) Number of experimental conditions by factor variable plan

The yield, solids content, degree of hydrolysis, calcium binding capacity and total phenolic compound content of the supernatant of soybean powder hydrolyzed by various conditions by the central synthesis scheme are shown in Table 8 below. Response surface regression was performed using each result, and regression equations for yield, solid content, degree of hydrolysis, calcium binding capacity, and total phenolic compound content were obtained.

Yield, Solids Content, Hydrolysis Degree, Calcium Binding Capacity and Total Phenolic Compound Content of Supernatant of Soybean Powder Hydrolyzed by Various Conditions by Central Synthetic Scheme Experiment number ¹⁾ yield(%) Solid content (%) Degree of hydrolysis (%) Calcium binding capacity (O.D.) Total Phenolic Compound (mg%) One 43.60 ± 0.56 5.17 ± 0.50 ²⁾ 20.62 ± 1.71 0.18 ± 0.01 53.90 ± 3.96 2 46.10 ± 0.75 5.75 ± 0.49 22.08 ± 0.48 0.19 ± 0.01 71.50 ± 1.98 3 52.10 ± 0.85 6.07 ± 0.76 25.42 ± 0.16 0.20 ± 0.01 90.05 ± 2.76 4 53.93 ± 1.10 6.40 ± 0.14 25.14 ± 0.40 0.22 ± 0.01 123.35 ± 3.18 5 49.57 ± 0.60 5.83 ± 0.25 24.81 ± 1.45 0.21 ± 0.01 100.00 ± 6.22 6 48.40 ± 1.35 5.73 ± 0.64 23.60 ± 0.38 0.21 ± 0.01 104.75 ± 2.33 7 47.00 ± 1.10 4.87 ± 0.40 21.79 ± 2.07 0.17 ± 0.01 57.05 ± 0.21 8 52.07 ± 0.70 6.60 ± 0.10 24.50 ± 0.81 0.22 ± 0.02 115.04 ± 5.90 9 33.90 ± 0.96 5.20 ± 0.14 7.19 ± 0.86 0.10 ± 0.01 67.43 ± 8.90 10 56.17 ± 0.21 6.45 ± 1.20 25.28 ± 0.72 0.23 ± 0.01 123.00 ± 3.50 NOTE 1) Number of test conditions by factor variable plan 2) Mean ± standard deviation value (n = 3)

As can be seen from the results of Table 8, the yield of the hydrolyzate of soybean powder was found to range from 33.90 to 56.17%, and the regression expression was affected by the protease concentration rather than the hydrolysis time. In addition, the soluble solids of soybean powder did not show a big difference in the range of 4.87 to 6.60%, and the regression equation was not significantly affected by the protease concentration and the hydrolysis time.

Y _Yield

_{= 29.9015 + 6.6797X 1 + 0.7922X 2} - 0.5959X 1 2 _{- 0.0418X 1 X 2 - 0.002187X 2} 2

Y _{Soluble solids}

= 3.8459 + 0.2340X ₁ + 0.2847X ₂ + 0.0016X ₁ ² _{- 0.0156X 1 X 2 - 0.0040X 2} 2

The solubility and calcium binding capacity of soybean powder were found to have a greater effect on protease concentration than hydrolysis time, and the total phenol content was shown to have a greater effect on hydrolysis time than protease concentration. The results are shown.

Y _{Degree of hydrolysis}

_{= 3.4689 + 6.5591X 1 + 1.6166X 2} - 0.5225X 1 2 - 0.1087X 1 X 2 - 0.0906X 2 2

Y _{Calcium binding capacity}

_{= 0.0850 + 0.0327X 1 + 0.0091X 2} - 0.0030X 1 2 _{+ 0.0010X 1 X 2 - 0.0007X 2} 2

Y _{Total phenol}

_{= 23.2645 + 5.2851X 1 + 11.1269X 2} - 0.2367X 1 2 + 0.9812X 1 X 2 - 0.8098X 2 2

The reaction surface for the yield of soybean powder hydrolyzate showed the form of the maximum point, and R ² was 0.9951, which was recognized as significant within 1%. As a result of ridge analysis (FIG. 19), the maximum yield was obtained. The value was predicted to be 102.6%. Optimum hydrolysis conditions at this time were 1.29% protease concentration and 46.72 hours hydrolysis time (Table 9). The reaction surface for soluble solids of soybean powder hydrolyzate showed the form of saddle point and R ² was 0.9544, which was significant within 1%. As a result of ridge analysis (FIG. 19), the optimum hydrolysis conditions were predicted to be 7.10% of protease concentration and 7.52 hours of hydrolysis time (Table 9).

R ² of the soybean powder was 0.9155, and significance was recognized within 5%. As a result of ridge analysis (FIG. 20), the maximum value of the degree of hydrolysis was 26.62%.

The response surface of calcium soybean powder hydrolyzate showed the form of the maximum point, and R ² was 0.9315, which was recognized as significant within 5% (FIG. 20). The reaction surface for the total phenolic compound content of soybean powder hydrolyzate showed the form of saddle point, and R ² was 0.9196, which was significant within 5%, and the result of ridge analysis (FIG. 21), the maximum was predicted to be 139.35 mg%.

Predicted Levels of Hydrolysis Conditions for Maximum Response by Ridge Analysis reaction R ² Pro> F X ₁ ¹⁾ X ₂ ²⁾ Maximum Morphology yield 0.978 0.0022 7.21 7.39 56.12 Saddle Solid content 0.954 0.0087 7.10 7.52 6.67 Saddle Degree of hydrolysis 0.916 0.0286 5.70 5.49 26.62 Maximum Calcium binding capacity 0.932 0.0191 7.14 11.15 0.25 Maximum Total Phenolic Compound Content 0.920 0.0261 7.26 7.32 139.35 Saddle ¹⁾ X ₁ : hydrolase content (%) ²⁾ X ₂ : hydrolysis time (hrs)

Step 6: prediction and empirical test of optimal hydrolysis conditions

Optimum hydrolysis conditions were predicted as the range of overlapping parts when superimposing a contour map of reaction variables such as yield, solid content, degree of hydrolysis, calcium binding capacity, and total phenolic compounds. In addition, the center point was set in the predicted range and substituted into the regression equation, and the predicted optimal value was verified.

For optimum hydrolysis conditions of soybean powder, the central synthesis program is used to control the dependence of protease concentration and hydrolysis time on yield, solids content, degree of hydrolysis, calcium binding capacity and total phenolic compound content. Superimposing the map (contour map) showed a range of hydrolysis conditions satisfying all the properties of the hydrolyzate (FIG. 19). As a result of making a superimposed map using a contour map of the change of the hydrolyzate, the predicted condition range is a hatched portion of FIG. 22, and a protease as shown in Table 10 below. The concentration was 5.05 ~ 6.60% and the hydrolysis time was 6.5 ~ 9.0 hours (hrs). In order to verify the above prediction model equation, the hydrolysis conditions were set to any optimal point within the optimum condition range, that is, 6.0% of protease concentration and 7.7 hours of hydrolysis time, and the actual hydrolysis conditions were repeated and the hydrolyzate of the hydrolyzate The results of the measurement were shown in Table 11. As a result of prediction at the hydrolysis optimum point, yield was 52.66%, solid content 6.72%, degree of hydrolysis 30.04%, calcium binding capacity 0.27 (O.D.) and total phenolic compound content 164.99 mg%.

Table 11 shows the actual values obtained by actual experiments under the same conditions as the predicted values of the components related to the quality of soybean powder hydrolyzate, wherein the actual values under any conditions are the yield of hydrolyzate, solid content, degree of hydrolysis, calcium The quality characteristics such as binding capacity and total phenolic compound content showed similar tendency compared with the predicted values. Such optimum hydrolysis conditions may vary somewhat depending on the properties considered for the hydrolyzate.

Optimum Range of Reaction Parameters for Soybean Powder Hydrolysis by Superimposing Contour Maps reaction Optimal range Hydrolase Content (%) 5.05 ～ 6.60 Hydrolysis time (hrs) 6.50 ～ 9.00

Comparison of predicted and observed values on soy flour hydrolyzate reaction Predicted value Observed value Yield (%) 52.66 50.43 ± 0.54 ¹⁾ Solid content (%) 6.72 6.68 ± 0.19 Degree of hydrolysis (%) 30.04 28.59 ± 0.46 Calcium binding capacity (O.D.) 0.276 0.29 ± 0.02 Total Phenolic Compound Content (mg%) 164.99 170.09 ± 2.84 NOTE 1 Values are mean ± standard deviation (n = 3).

Step 7: search for functionality of the optimal hydrolyzate

The hydrolyzate treated under optimized conditions according to the central synthesis program was used to compare with the untreated plot. At this time, the optimized condition was used for analysis after hydrolysis by setting the center point of X ₁ and X ₂ among the overlapped ranges when superimposing each contour map. DPPH free radical scavenging activity, superoxide radical scavenging activity, angiotensin converting enzyme inhibitory activity, free amino acid, SDS-PAGE electrophoresis and calcium endurance of soybean powder optimum hydrolysates were analyzed.

DPPH-free radical scavenging activity, superoxide radical scavenging activity, and angiotensin converting enzyme inhibitory activity for the preparation of hydrolysates using optimal protease concentration and hydrolysis time obtained by reaction surface analysis , Free amino acid, SDS-PAGE electrophoresis was analyzed.

1) DPPH free radical scavenging activity

DPPH free radical scavenging activity of soybean powder hydrolysates treated with optimum conditions by reaction surface analysis was analyzed. As shown in FIG. 23, the optimal treatment was 35.31% higher than that of 12.85% of the no treatment.

2) superoxide radical scavenging activity

Superoxide radical scavenging activity of soybean powder hydrolysates treated with optimum conditions by reaction surface analysis was analyzed. As shown in FIG. 24, the optimum treatment was increased from 21.54% to 27.04%.

3) Angiotensin converting enzyme inhibitory activity

Angiotensin converting enzyme inhibitory activity of soybean powder hydrolysates treated with optimal conditions by reaction surface analysis was analyzed. As shown in FIG. 25, the soybean powder hydrolyzate was increased by about 10% from 53.68% of the non-treated group to 62.16% of the optimal treated group. Yu et al. (Yu, RN et al. J. Korean Soc. Food Sci. Nutr. 1996 , 25 , 1031-1036) were used to determine the spontaneous hypertension of ultrafiltration peptides isolated from soybean hydrolysates (SHR). Blood pressure lowering effect), which is estimated to be expressed through lipid-improving actions such as total cholesterol and triglyceride in blood and inhibition of angiotensin I converting enzyme (ACE) activity in thoracic artery. In addition, Shin et al. (Shin, JK et al. Korea J. Food Sci. Technol ., 1995 , 27 , 230-234) also reported that blood pressures in which several peptides derived from soy sauce, miso and soybean hydrolysates act as ACE inhibitors. As confirmed the drop activity, the results showed similar results to the ACE inhibitory activity of the hydrolyzate.

4) free amino acids

The free amino acids of soybean powder and hydrolyzate of SPI treated with optimum conditions by reaction surface analysis were analyzed. As shown in Table 12, the results of the hydrolyzate of soybean powder showed that the total free amino acids and essential amino acids increased very high in the optimum conditions. Total amino acids increased from 77.57 mg% to 730.16 mg% and essential amino acids increased from 8.66 mg% to 361.99 mg%. It is believed that the soy protein is broken down into amino acids by protease, resulting in a sharp increase in hydrolysis.

Comparison of Free Amino Acids of Hydrolysates of Soybean Powder Treated with Optimum Conditions by Response Surface Methodology Free amino acids Soybean Powder Hydrolase untreated Hydrolase treatment Urea 0.91 ND ¹⁾ L-Aspartic Acid ND 0.15 L-Threonine 1.02 45.34 L-Serine 1.18 25.70 L-Glutamic Acid 4.95 64.28 L-Sarcosine ND 3.23 L-α-Aminoadipic Acid 0.56 1.26 L-Proline 0.48 17.31 Glycine 0.76 9.58 L-Alanine 3.33 33.93 L-Citrulline 0.28 2.38 L-α-Amino-n-butyric acid ND 1.02 L-Valine 4.04 54.35 L-Cystine ND 0.75 L-Methionine ND 1.41 L-Cystathionine ND 9.49 L-Isoleucine 1.11 43.36 L-Leucine 1.42 90.53 L-Tyrosine ND 33.95 β-Alanine 0.78 5.84 L-Phenylalanine 1.07 55.98 L-Homocystine ND 0.43 γ-amino-n-butyric acid 2.75 2.78 Ethanolamine 0.94 0.86 Ammonium Chloride 13.02 14.25 δ-hydroxylysine 0.26 0.62 L-Ornithine ND 0.31 L-Lysine 1.68 56.90 1-Methyl-L-histidine 0.00 2.77 L-Histidine 1.91 24.29 L-Tryptophan ND 14.13 L-Anserine ND 6.33 L-Arginine 35.11 106.68 TA ²⁾ 77.57 730.16 EA ³⁾ 8.66 361.99 ¹⁾ ND: Not detected ²⁾ TA: Total amino acid ³⁾ EA: Essential amino acid; Thr + Val + Met + Ile + Leu + Phe + Lys + Trp )

5) SDS-PAGE Electrophoresis

As a result of SDS-PAGE electrophoresis as shown in FIG. 26 to determine the molecular weight distribution of the protease-treated soybean powder hydrolysate by the reaction surface analysis method, the soybean powder had five coarse grains in the non-protease treated group. The band appeared. In the protease treatment, soybean powder did not appear in the top three bands of 14,300 Da, two thick bands of 6,500 Da or less appeared, and SPI showed two bands of 6,500 Da or less. Lee et al. (Lee, CH et al. Korea J. Food Sci. Technol ., 1984 , 16 , 228-234) reported that electrophoresis of soybeans may result in 7-18 bands depending on the variety. (Cha, MH et al. Korean J. Food. Sci. Technol . 1993 , 25 (1) , 39-45) reported that native SPI (native SPI) showed about nine distinct bands. In the present invention, there are five bands, so that protease B induced more hydrolysis to proteins than enzymes used by Lee and Cha, or is considered to be a difference depending on the varieties of soybean.

6) Calcium endogenous comparison

Calcium endogenous laboratory algae calcium (34.08% calcium) was purchased from Bio Delta Korea Co., Ltd. was used to receive liquid calcium (4.00% calcium) from Keimyung Fudex Co., Ltd.

Soybean powder hydrolyzate treated with protease under optimal conditions and processed without protease in soybean powder and optimized protease After the addition of powdered seaweed calcium and liquid liquid calcium in one sphere, centrifugation of precipitated calcium precipitated and centrifuged to precipitate the calcium content of the hydrolyzate as shown in Table 13 The amount of liquid calcium was lowered by 15mg% after centrifugation compared to the amount added, and the liquid calcium was reduced to 75.60mg% in the non-protease treated group after centrifugation compared to 130mg%. In one group, 116.74 mg% showed no decrease in calcium content compared to the control group. This suggests that protease treatment of soy protein will resolve the problem of protein precipitation and calcium binding due to reduced molecular weight and changes in protein structure, thereby solving the problem of precipitation of protein and calcium (Pyun, JW et. Al., Korean J). Food, Sci. Technol , 1996 , 28 (6) , 995-1000). Therefore, the protease treatment of soybean is thought to be possible to produce high calcium soymilk with calcium.

Comparison of calcium binding ability by adding powdered algae calcium and liquid liquid calcium to the protease-treated and optimum protease-treated spheres (unit: mg%) Aqua calcium Liquid calcium Control Filtration Control Filtration Control Soybean Powder 130.58 14.39 113.41 75.60 optimization Soybean Powder 137.33 15.11 112.45 116.74

Example 1 Preparation of Whole Soymilk

After washing 100g of dried soybeans three times, it is immersed in tap water for 7-8 hours (summer season) and is called 2.3 times the weight of soybean. Heat at 100 ° C. in boiling water for 20 minutes and strip. Peeled soybeans were ground in 15,000 rpm for 10 minutes using a homogenizer, but the amount of water added was 7 times the weight of dry soybeans. Crushed whole soybean oil was hydrolyzed by adding proteolytic enzyme and shaking at 100 rpm for 1 hour at 50 ° C, and then inactivated for 10 minutes at 100 ° C to stop the reaction to remove soybean hydrolysate. Used.

Experimental Example 2: Investigation of the effect of the amount of water in the preparation of the frontal milk

In order to obtain an optimized soybean hydrolyzate, the effect of hydrolysis was investigated. Soybean oil is prepared by making the amount of hydrolyzate 5, 7, 9 times (w / v) of dry soybean weight, and then add 0.2% of protease B, the best soy protein hydrolase during soybean powder hydrolysis. After hydrolysis at 50 ° C. for 1 hour, the soybean hydrolyzate obtained by inactivating the enzyme at 100 ° C. for 10 minutes was investigated.

Soy milk and tofu, a by-product from the process of making soybeans, have been soluble in soybeans, but they contain valuable amounts of protein and carbohydrates. Therefore, protease (protease B) was added to whole soybean milk using whole soybeans, which retained the nutrients in the sebum, and hydrolyzed for 1 hour, soybean hydrolyzate pH, sugar, solid content, calcium binding capacity, SDS-PAGE electrophoresis and free The result of comparing amino acids was as follows.

As shown in Table 14, the pH and the sugar content according to the amount of the hydrolyzate were 5, 7, 9 times the amount of the hydrolyzate to 6.340, 6.367, 6.447. However, the sugar content of soy hydrolyzate decreased as the amount of hydrolysis increased.

  As a result of analyzing the solid content of soy hydrolyzate, Table 15 showed the highest amount of 11.543% at 5 times the amount of hydrolyzate. Current standards and standards for foods refer to soy milk fluids of more than 7% of the soybean solids extracted from soybeans. Therefore, it is considered that soybean hydrolyzate in all sections can be used as a raw material of soymilk.

The calcium binding capacity of soy hydrolyzate according to the amount of hydrolysis was determined by the method of stool (Pyun, JW et al. Korean J. Food. Sci. Technol . 1996 , 28 (6) , 995-1000). The degree of binding to and is shown in Table 16. As the amount of hydrolyzate increased, the calcium binding capacity of soy hydrolyzate tended to be lower from 0.664 to 0.349.

To determine the degree of hydrolysis of the protein according to the amount of hydrolysis, the results of SDS-PAGE electrophoresis are shown in FIG. 27. In the 5 times hydrolysis treatment, the band appeared at 55kDa, 33kDa, 24kDa or less, 17kDa, 11kDa, but in the 7 times and 9 times hydrolysis treatment, the top 1 band did not appear, and the distribution was less than 33kDa.

Free amino acids of soy hydrolysates were analyzed. As shown in Table 17, 120.61mg% of total free amino acids and 42.69mg% of essential amino acids were found to be very high. Total free amino acids in the 7- and 9-fold hydrolysates increased from 81.91 mg% to 85.86 mg%, while essential amino acids decreased from 31.39 mg% to 27.98 mg%.

As a result, the low molecular weight of soy hydrolyzate according to the amount of hydrolysis was 7 and 9 times higher. When used as a stock solution of soymilk, 7 times the amount of solids, but the high amount of essential amino acids was found to be most suitable.

PH and sugar content of soy hydrolyzate according to the amount of hydrolysis Hydrogen amount (times) pH Sugar content 5 6.340 ± 0.026 4.100 ± 0.100 7 6.367 ± 0.015 2.767 ± 0.058 9 6.447 ± 0.006 2.433 ± 0.058

Solids Content of Soy Hydrolysates According to Hydrolysis Amount Hydrogen amount (times) Solid content (%) 5 11.543 ± 0.035 7 9.010 ± 0.265 9 7.713 ± 0.657

Calcium Binding Ability of Soy Hydrolysates According to Hydrolysis Amount Hydrogen amount (times) Calcium binding capacity 5 0.664 ± 0.005 ¹⁾ 7 0.455 ± 0.001 9 0.349 ± 0.001 ¹⁾ Diluted 25 times of the supernatant with 30mM CaCl ₂ added to the sample.

Changes in Free Amino Acids of Soy Hydrolysates According to Hydrolysis Amount Free amino acids Hydrogen amount (times) 5 7 9 Urea ND ¹⁾ ND 6.73 Hydroxy-L-poline 18.29 11.95 11.56 L-Threonine 0.65 0.46 0.27 L-Serine ND ND 2.54 L-Glutamic Acid 2.49 1.70 1.67 L-Sarcosine 3.54 1.57 3.53 L-α-Aminoadipic Acid 1.21 0.12 ND L-Proline 0.58 ND ND Glycine 1.30 1.19 1.12 L-Alanine 3.52 2.32 2.41 L-Citrulline 0.26 0.29 0.25 L-α-Amino-n-butyric acid 0.36 0.21 0.24 L-Valine 1.87 1.63 1.38 L-Cystine 0.81 ND ND L-Methionine 0.47 0.37 0.29 L-Cystathionine 4.40 5.16 5.11 L-Isoleucine 1.50 1.41 1.02 L-Leucine 11.91 9.15 7.75 L-Tyrosine 0.30 ND ND β-Alanine 8.54 5.30 5.50 L-Phenylalanine 8.95 6.67 5.46 D, L-β-Aminoisobutyric Acid 1.44 1.07 0.97 L-Homocystine 1.44 1.27 0.97 γ-amino-n-butyric acid 1.87 1.26 1.30 Ethanolamine 0.46 1.39 ND Ammonium Chloride ND ND ND δ-hydroxylysine 3.13 0.35 ND L-Ornithine 0.84 0.50 0.26 L-Lysine 13.15 8.54 9.42 1-Methyl-L-histidine 0.85 0.58 0.63 L-Tryptophan 4.20 3.18 2.39 L-Anserine 3.27 2.00 2.39 L-Carnosine 1.96 ND ND L-Arginine 17.05 12.29 10.26 TA ²⁾ 120.61 81.91 85.86 EA ³⁾ 42.69 31.39 27.98 ¹⁾ ND: Not detected ²⁾ TA: Total amino acid ³⁾ EA: Essential amino acid; Thr + Val + Met + Ile + Leu + Phe + Lys + Trp )

Experimental Example 3: Investigation of the effect of hydrolytic enzyme treatment concentration on the preparation of soymilk

To investigate the effects of protease treatment, the ratio of protease B to 0.05, 0.2, and 0.35% of soybean milk was hydrolyzed at 50 ° C for 1 hour, and then the enzyme was digested at 100 ° C for 10 minutes. The components of the soybean hydrolyzate obtained by inactivation were investigated.

Important factors in the degradation of soy protein are the type and concentration of protease and hydrolysis time. After setting the hydrolyzate of the soybean hydrolyzate to optimize, in order to determine the effect of protease concentration, each protease B was added 0.05, 0.20, 0.35%, and hydrolyzed at 50 ° C. for 1 hour. Soy hydrolysates obtained by inactivating enzymes were prepared and compared.

As a result, pH and sugar content are shown in Table 18. There was no significant difference in pH according to protease concentration, but sugar content increased from 2.433 to 3.167 brix with increasing protease concentration.

The solid content according to the protease concentration was 8.762%, 9.010%, 9.217% when the protease concentration was 0.05%, 0.20%, 0.35%, as shown in Table 19. As the protease concentration increased, the solid content increased little by little, but there was no big difference.

Table 20 shows the results of comparing the calcium binding capacity according to the protease B concentration. Protease concentration of 0.05% was the lowest at 0.281, but protease concentration of 0.20% was high as 0.455, and protease concentration of 0.35% was high at 0.478. This suggests that as the concentration of protease B increases, the binding ability of calcium and protein decreases.

SDS-PAGE electrophoresis results are shown in FIG. 28 to determine the degree of hydrolysis of the protein according to the protease concentration treatment. Hydrolysis was carried out at a molecular weight of less than 33kDa in all sections, and as the concentration of protease increased, it was confirmed that it had a low molecular weight of less than 17kDa.

Changes in free amino acids of soy hydrolyzate according to protease concentration are shown in Table 21. Total amino acids were 27.28 mg%, 81.91 mg% and 129.54 mg%, while essential amino acids were increased to 6.52 mg, 31.39 mg% and 43.21 mg%. It is thought that the soy protein is broken down into amino acids by protease, resulting in a sharp increase in hydrolysis.

Therefore, as a result of analyzing the properties of soy hydrolyzate according to protease concentration, it was found to be excellent at 0.20% overall and was set to 0.20% of protease B.

PH and Sugar Content of Soy Hydrolysates According to Hydrolase Concentration Protease Concentration (%) pH Sugar content 0.05 6.563 ± 0.012 2.433 ± 0.115 0.20 6.367 ± 0.015 2.767 ± 0.058 0.35 6.497 ± 0.020 3.167 ± 0.040

Solid Contents of Soy Hydrolysates According to Hydrolase Concentration Protease density(%) Solid content (%) 0.05 8.762 ± 0.136 0.20 9.010 ± 0.265 0.35 9.054 ± 0.102

Calcium Binding Ability of Soy Hydrolysates According to Hydrolase Concentration Protease concentration (%) Calcium binding capacity 0.05 0.281 ± 0.002 ¹⁾ 0.20 0.455 ± 0.001 0.35 0.478 ± 0.015 ¹⁾ Diluted 25 times of the supernatant with 30mM CaCl ₂ added to the sample.

Changes in Free Amino Acids of Soy Hydrolysates According to Hydrolase Concentration Free amino acids Protease Concentration (%) 0.05 0.20 0.35 Urea ND ¹⁾ ND ND Hydroxy-L-poline 8.11 11.95 20.16 L-Threonine ND 0.46 0.75 L-Serine ND ND ND L-Glutamic Acid 1.29 1.70 3.10 L-Sarcosine 1.29 1.57 2.96 L-α-Aminoadipic Acid ND 0.12 1.08 L-Proline ND ND 0.87 Glycine 0.70 1.19 1.36 L-Alanine 0.96 2.32 3.81 L-Citrulline 0.76 0.29 0.29 L-α-Amino-n-butyric acid ND 0.21 0.35 L-Valine 0.51 1.63 2.04 L-Cystine ND ND 0.70 L-Methionine 0.32 0.37 0.51 L-Cystathionine ND 5.16 7.41 L-Isoleucine ND 1.41 1.84 L-Leucine 1.50 9.15 13.41 L-Tyrosine ND ND 0.44 β-Alanine 1.28 5.30 8.46 L-Phenylalanine 1.75 6.67 8.14 D, L-β-Aminoisobutyric Acid ND 1.07 1.66 L-Homocystine ND 1.27 1.44 γ-amino-n-butyric acid 0.94 1.26 1.85 Ethanolamine ND 1.39 1.43 Ammonium Chloride ND ND ND δ-hydroxylysine 0.34 0.35 1.25 L-Ornithine 0.19 0.50 0.23 L-Lysine 2.45 8.54 15.83 1-Methyl-L-histidine 0.55 0.58 0.83 L-Tryptophan ND 3.18 0.69 L-Anserine ND 2.00 1.69 L-Carnosine ND ND 4.22 L-Arginine 4.36 12.29 20.72 TA ²⁾ 27.28 81.91 129.54 EA ³⁾ 6.52 31.39 43.24 ¹⁾ ND: Not detected ²⁾ TA: Total amino acid ³⁾ EA: Essential amino acid; Thr + Val + Met + Ile + Leu + Phe + Lys + Trp )

Experimental Example 4: Investigation of the effects of hydrolysis time on the preparation of soymilk

In order to investigate the effects of protease treatment time, the ratio of protease B to soybean milk was 0.2% and hydrolyzed at 50 ° C. for 30, 60, and 90 minutes, respectively, and then at 100 ° C. for 10 minutes. The components of soy hydrolyzate obtained by inactivating the enzyme were investigated.

Soybean obtained by hydrolysis at 50 ° C for 30 minutes, 60 minutes, and 90 minutes to determine the effect of hydrolysis time after optimization of soybean hydrolysate and protease concentration Hydrolysates were prepared to investigate their effects.

As a result, pH and sugar content are shown in Table 22. PH did not show a significant difference from 6.367 to 6.640. The sugar content increased significantly with the hydrolysis time to 2.167, 2.767, and 3.333 brix.

The solid content tended to increase from 8.510% to 9.317% with the hydrolysis time as shown in Table 23.

As a result of comparing the calcium binding capacity according to the hydrolysis time is shown in Table 24. Soybean hydrolyzate was 0.349, 0.455 at 60 minutes, 0.548 at 90 minutes, and calcium endurance increased as the hydrolysis time increased.

SDS-PAGE electrophoresis results are shown in Figure 29 to determine the degree of hydrolysis of soy protein. In all treatment periods, hydrolysis was performed at 33kDa, 24kDa, 17kDa, and 11kDa or less, and it was confirmed that the soy protein of macromolecules had a low molecular weight of less than 17kDa as the concentration increased.

The change in the free amino acid of soy hydrolyzate with hydrolysis time is shown in Table 25. Total amino acids increased from 47.14 mg% to 177.80 mg% over time, while essential amino acids increased from 15.33 mg%, 31.39 mg%, and 58.95 mg%. From the above results, it was found to be excellent at 90 minutes, but because of slight odor, the hydrolysis time was set to 60 minutes.

PH and Sugars of Soy Hydrolysates with Hydrolysis Time Hydrolysis time (min) pH Sugar content 30 6.440 ± 0.020 2.167 ± 0.058 60 6.367 ± 0.015 2.767 ± 0.058 90 6.437 ± 0.015 3.333 ± 0.058

Solids Content of Soy Hydrolyzate with Hydrolysis Time Hydrolysis time (min) Solid content (%) 30 8.510 ± 0.064 60 9.010 ± 0.265 90 9.317 ± 0.093

Calcium Binding Ability of Soy Hydrolysates with Hydrolysis Time Hydrolysis time (min) Solid content (%) 30 0.349 ± 0.002 ¹⁾ 60 0.455 ± 0.001 90 0.548 ± 0.003 ¹⁾ Diluted 25 times of the supernatant with 30mM CaCl ₂ added to the sample.

Free Amino Acid Changes of Soy Hydrolyzate with Hydrolysis Time Free amino acids Hydrolysis time (min) 30 60 90 Urea 2.06 ND ¹⁾ 10.07 Hydroxy-L-polline 7.94 11.95 24.98 L-Threonine 0.21 0.46 1.47 L-serine ND ND 5.24 L-glutamic acid 1.14 1.70 3.74 L-sarcosine 1.49 1.57 4.94 L-α-aminoadipic acid ND 0.12 1.29 L-proline ND ND 2.17 Glycine 0.64 1.19 1.70 L-alanine 1.35 2.32 5.16 L-Citrulline 0.17 0.29 0.31 L-α-amino-n-butyric acid ND 0.21 0.53 L-valine 0.82 1.63 2.59 L-cystine ND ND 0.68 L-methionine ND 0.37 0.91 L-cistathioneine 2.00 5.16 4.79 L-isorucin 0.46 1.41 2.51 L-leucine 3.36 9.15 17.37 L-tyrosine ND ND 0.45 β-alanine 3.65 5.30 9.96 L-phenylalanine 2.85 6.67 14.19 D, L-β-aminoisobutyric acid 0.87 1.07 ND L-homocystine 0.91 1.27 2.15 γ-amino-n-butyric acid 0.92 1.26 1.60 Ethanolamine 0.85 1.39 0.35 Ammonium chloride ND ND ND δ-hydroxylysine ND 0.35 5.80 L-ornithine ND 0.50 0.91 L-lysine 5.48 8.54 18.92 1-methyl-L-histidine 0.40 0.58 0.96 L-Tryptophan 1.87 3.18 0.98 L-anserine ND 2.00 2.54 L-carnosine ND ND 5.57 L-arginine 7.43 12.29 22.93 TA ²⁾ 47.14 81.91 177.80 EA ³⁾ 15.33 31.39 58.95 ¹⁾ ND: Not detected ²⁾ TA: Total amino acid ³⁾ EA: Essential amino acid; Thr + Val + Met + Ile + Leu + Phe + Lys + Trp )

Experimental Example 5: Investigation of the effects of sterilization temperature on the preparation of soymilk

In order to investigate the effect of sterilization temperature, the ratio of protease B in soybean milk was 0.2% and hydrolyzed at 50 ° C for 1 hour, followed by high temperature short time sterilization (HTST) at 115, 130 and 145 ° C. The components of the soybean hydrolyzate obtained by inactivating the enzyme by ultra high temperature sterilization for 15 seconds were investigated.

The soybean hydrolysates were optimized and sterilized at 115, 130 and 145 ° C. for 15 seconds at high temperature to investigate their effects.

Effect of Sterilization Temperature on Soy Hydrolysate pH and Sugars are shown in Table 26. The pH was 6.220 to 6.240, and there was almost no change in pH according to the sterilization temperature. The sugar content was 2.950 brix at sterilization temperature 115 ℃ and increased to 3.200 and 3.250 brix at 130 ℃ and 145 ℃, but there was no significant difference.

As a result of analyzing the solid content of soy hydrolyzate, Table 27 showed that the tendency to increase from 9.360% to 10.043% with increasing sterilization temperature.

As a result of comparing the calcium binding capacity according to the sterilization temperature is shown in Table 28. As the sterilization temperature was increased to 0.426 and 0.474 at 115 ° C and 0.493 at 145 ° C, the calcium endurance continued to increase.

SDS-PAGE electrophoresis results are shown in FIG. 30 in order to see the difference in molecular weight of soy protein according to sterilization conditions. As a result of the decomposition pattern, all the treatment sections on the electrophoresis showed similar patterns with molecular weights of 33kDa, 24kDa, 17kDa and 11kDa.

Changes in free amino acids of soy hydrolysates are shown in Table 29. Total amino acid increased from 101.40mg% to 148.47mg% according to the sterilization temperature, and essential amino acid increased rapidly from 35.71mg% to 41.15mg% at 115 ℃ and 130 ℃ sterilization but increased to 42.45mg% at 145 ℃ However, no significant difference was found. pH, sugar content, solid content, and electrophoresis were not significantly affected by sterilization temperature, but in terms of protein nutrition, it was found to be appropriate when sterilized at 130 ° C.

PH and Sugar Content of Soy Hydrolysates According to Sterilization Temperature Sterilization Temperature (℃) pH Sugar content 115 6.220 ± 0.000 2.950 ± 0.071 130 6.220 ± 0.007 3.200 ± 0.000 145 6.240 ± 0.007 3.250 ± 0.071

Solid Contents of Soy Hydrolysates According to Sterilization Temperature Sterilization Temperature (℃) Solid content (%) 115 9.360 ± 0.431 130 9.804 ± 0.264 145 10.043 ± 0.019

Calcium Binding Capability of Soy Hydrolysates with Sterilization Temperature Sterilization Temperature (?) Calcium binding capacity 115 0.426 ± 0.001 ¹⁾ 130 0.474 ± 0.002 145 0.493 ± 0.004 ¹⁾ Diluted 25 times of the supernatant with 30mM CaCl ₂ added to the sample.

Free Amino Acid Changes of Soy Hydrolyzate with Sterilization Temperature Free amino acids Sterilization Temperature (℃) 115 130 145 Urea ND ND 29.97 L-aspartic acid ND ND ND Hydroxy-L-polline 11.49 10.27 11.83 L-Threonine 0.49 0.51 0.55 L-serine ND ND 3.08 L-glutamic acid 1.95 1.81 1.81 L-sarcosine 3.87 4.21 3.62 L-α-aminoadipic acid 1.08 1.21 1.45 L-proline 0.71 0.66 0.52 Glycine 1.47 1.40 1.36 L-alanine 3.03 2.90 3.06 L-Citrulline 0.35 0.44 0.37 L-α-amino-n-butyric acid 0.40 0.43 0.46 L-valine 1.51 1.47 1.57 L-cystine 0.88 0.88 1.01 L-methionine 1.48 1.35 1.81 L-cistathioneine 5.59 7.50 7.47 L-isorucin 1.23 1.29 1.29 L-leucine 9.84 10.21 10.48 L-tyrosine 0.72 0.33 0.72 β-alanine 7.52 7.88 8.08 L-phenylalanine 8.39 8.92 8.70 D, L-β-aminoisobutyric acid 1.24 1.65 1.44 L-homocystine 1.34 1.27 1.24 γ-amino-n-butyric acid 1.25 1.26 1.28 Ethanolamine 0.02 0.53 0.46 Ammonium chloride ND ND ND δ-hydroxylysine 1.97 3.02 2.77 L-ornithine 0.78 0.85 0.83 L-lysine 12.78 13.18 13.57 1-methyl-L-histidine 0.56 0.70 0.76 L-Tryptophan ND 4.23 4.48 L-anserine 3.86 3.84 3.48 L-carnosine ND 2.06 1.98 L-arginine 15.62 16.84 16.96 TA ²⁾ 101.40 113.09 148.47 EA ³⁾ 35.71 41.15 42.45 ¹⁾ ND: Not detected ²⁾ TA: Total amino acid ³⁾ EA: Essential amino acid; Thr + Val + Met + Ile + Leu + Phe + Lys + Trp )

Experimental Example 6: Comparison of Optimal Frontal Fluid and Conventional Frontal Fluid of the Present Invention

The effects of soy hydrolyzate and protease B-treated controls prepared under optimal conditions were investigated.

The pH and sugar content of the optimized soy hydrolyzate and the control are shown in Table 30. pH was 6.220 and 6.550, respectively, and the control was high. These results indicate that when the hydrolysis of the protein occurs by the enzyme, the carboxyl group of the amino acid is exposed to decrease the pH of the solution. The sugar content was 3.200 and 2.750 brix, soybean hydrolyzate was high. It is believed that the sugar content is high due to the hydrolysis of the components of the sugar as it is hydrolyzed by the enzyme.

As a result of analyzing the solid content, in Table 31, the optimized soy hydrolyzate was lower than the control. This is the same as the tendency to appear earlier when comparing the soybean powder hydrolyzate with the control.

Table 32 shows the results of comparing the calcium binding capacity of the soybean hydrolyzate treated with the optimum conditions and the control group. Compared to 0.106 of the control group, the optimized soybean hydrolyzate was 0.474. The treatment of protease, which is used to improve the functional properties of soymilk protein, is reported to reduce calcium molecular weight and change the protein structure, thereby increasing calcium intolerance, which can resolve the calcium deficiency of soymilk. There is. These results show that the optimized soybean hydrolyzate has higher calcium endurance than the control.

SDS-PAGE electrophoresis results are shown in FIG. 31 to show the difference in protein molecular weight between the optimized soy hydrolyzate and the control. In the electrophoresis, the optimum conditions were treated with molecular weight bands less than 33kDa, 24kDa, 17kDa, 11kDa, but the control group appeared in the molecular weight band 17kDa or more. This study did not reveal the electrophoretic pattern for low molecular weight fractions with molecular weights of less than 10,000, but if the results of the study and the constituent amino acid sequence are complemented in the future, it may provide important information on the functional activity of each fraction. I think there will be.

Protein nutritional quality assessment is shown in Table 33 by analyzing the changes in the free soybean hydrolyzate and the free amino acid of the control group, which can be seen through the amino acid composition. Some of the amino acids are not made in our bodies are made but not enough. Essential amino acids in adults include threonin, valine, methionine, isoleucine, leucine, phenylalanine, lysine, and tryptophan (tryptophan)

The total amino acid of the optimized soybean hydrolyzate was 113.09mg% and the essential amino acid was 41.15mg%, but the control group was 7.88mg% and 0.45mg%, respectively, and the protein component was decomposed into amino acids and essential amino acids during protease B treatment. Could know.

Proteolytic products include free amino acids, oligopeptides, and low molecular weight proteins, each of which has a different biological activity from the first after being degraded from the original protein, and is being developed as a new biomaterial using the same. Therefore, it is proposed that the function can be improved by adjusting hydrolysis degree well. In addition, these hydrolysates are very effective ingredients for anti-aging or carcinogenesis and have been reported to have antioxidant and physiological activity.

PH and sugar content of optimized soy hydrolyzate and control pH Sugar content Optimized Soy Hydrolysate 6.220 ± 0.007 3.200 ± 0.000 Control 6.550 ± 0.021 2.750 ± 0.071

Solid content of optimized soybean hydrolyzate and control Solid content (%) Optimized Soy Hydrolysate 9.804 ± 0.264 Control 10.437 ± 0.028

Calcium Binding Capacity of Optimized Soy Hydrolysates and Controls Calcium binding capacity Optimized Soy Hydrolysate 0.474 ± 0.002 ¹⁾ Control 0.106 ± 0.001 ¹⁾ Diluted 25 times of the supernatant with 30mM CaCl ₂ added to the sample.

Free Amino Acid Changes of Optimized Soy Hydrolysates and Controls Free amino acids Soy Hydrolysate Optimized Soybeans Control Urea ND ¹⁾ 0 L-aspartic acid ND ND Hydroxy-L-polline 10.27 2.33 L-Threonine 0.51 ND L-serine ND 0.27 L-glutamic acid 1.81 ND L-sarcosine 4.21 ND L-α-aminoadipic acid 1.21 ND L-proline 0.66 ND Glycine 1.40 0.45 L-alanine 2.90 0.27 L-Citrulline 0.44 0.28 L-α-amino-n-butyric acid 0.43 0.21 L-valine 1.47 ND L-cystine 0.88 ND L-methionine 1.35 ND L-cistathioneine 7.50 ND L-isorucin 1.29 ND L-leucine 10.21 ND L-tyrosine 0.33 ND β-alanine 7.88 ND L-phenylalanine 8.92 ND D, L-β-aminoisobutyric acid 1.65 ND L-homocystine 1.27 ND γ-amino-n-butyric acid 1.26 0.49 Ethanolamine 0.53 ND Ammonium chloride ND ND δ-hydroxylysine 3.02 ND L-ornithine 0.85 ND L-lysine 13.18 0.45 1-methyl-L-histidine 0.70 ND L-Tryptophan 4.23 ND L-anserine 3.84 ND L-carnosine 2.06 ND L-arginine 16.84 3.12 TA ²⁾ 113.09 7.88 EA ³⁾ 41.15 0.45 ¹⁾ ND: Not detected ²⁾ TA: Total amino acid ³⁾ EA: Essential amino acid; Thr + Val + Met + Ile + Leu + Phe + Lys + Trp )

As described above, the method for producing the low molecular soybean milk according to the present invention is pulverized by adding 5-9 times of water to the soybean soaked after washing with water, and then adding protein hydrolase and 30 to 1 hour 30 minutes at 30 to 60 ° C. After hydrolysis and hydrolysis for 15 seconds to 10 minutes at 100 ~ 145 ℃ to prepare excellent soybean milk in terms of sensory and nutritional properties such as sugar content, solid content, calcium binding capacity, low molecular weight and free amino acid content of soy hydrolysate It is a useful invention in the food industry because it has an excellent effect.

Claims (3)

After washing with water, it is crushed by adding 7 ~ 9 times of water to soybeans soaked. It is an enzyme derived from Aspergillus Oryzae . The optimum pH is 7.0 and the pH stability range is 5.0 ~ 8.0, and the optimum temperature is 50 ℃ and 35 ℃. In the above-described process, it is completely deactivated when treated at 60 ° C for 30 minutes, and then hydrolyzed by adding 0.05 ~ 0.35% of protein hydrolase having chemical properties that inhibit enzyme, and shaking at 35-50 ° C for 30 minutes to 1 hour. Method of producing a whole soymilk having a low molecular weight of 11kDa ~ 33kDa without removing the busy paper, characterized in that after treatment for 15 seconds to 10 minutes at 100 ~ 145 ℃. delete The whole soymilk prepared by the method of claim 1 having a low molecular weight of 11 kDa to 33 kDa and has no bitter taste.

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