WO2019045492A2

WO2019045492A2 - Soy protein concentrate hydrolyzed under low moisture condition and preparation method thereof

Info

Publication number: WO2019045492A2
Application number: PCT/KR2018/010072
Authority: WO
Inventors: Hyojeong SEO; Sungwook Han; Youngho Hong; Seong Bo Kim; Seung Won Park
Original assignee: Cj Cheiljedang Corporation
Priority date: 2017-08-31
Filing date: 2018-08-30
Publication date: 2019-03-07
Also published as: WO2019045492A3

Abstract

The present disclosure relates to a method for preparing a soy protein concentrate hydrolyzed under a low moisture condition, a soy protein concentrate prepared by the method, and a feed composition comprising the same.

Description

SOY PROTEIN CONCENTRATE HYDROLYZED UNDER LOW MOISTURE CONDITION AND PREPARATION METHOD THEREOF

The present disclosure relates to a soy protein concentrate which is hydrolyzed under a low moisture condition and thus has a high content of low molecular weight peptides, and a preparation method thereof.

In general, animal feed contains fishmeal as a protein source, but the cost of fishmeal has been rising worldwide as the production thereof in fishmeal-producing countries has recently decreased, or the supply and demand of fishmeal have become unstable. Accordingly, there is an increasing demand for vegetable protein sources which can be used as a substitute for fishmeal, and efforts to develop the same are continuously underway.

Defatted soybean meal using soybean, which is a vegetable protein, is a by-product remaining after extracting the fat from soybeans having high protein and fat contents, and soy protein is extracted from the defatted soybean meal using water or an organic solvent (see Korean Patent No.　10-1612600). These soy proteins can be classified into processed soy protein products of defatted soy flour, soy protein concentrates, structured soy proteins, hydrolyzed soy proteins, or soy protein isolates depending on the extent to which non-protein ingredients such as water-soluble and non-water-soluble carbohydrates are removed from the defatted soybean meal. Because soy proteins have a high protein content, they can be used for livestock feed, in addition to processed meat products, processed milk products, and foods such as bread or snacks.

However, soy proteins contain soybean-derived saponins and thus may cause an inflammatory reaction in the bodies of fish, including salmon, thereby adversely affecting the growth thereof. In addition, soy proteins contain high molecular weight proteins having a molecular weight of 1,000　kDa or more consisting of protein subunits, and also contain a variety of anti-nutritional factors (ANFs), which can impair digestion. These anti-nutritional factors are mainly present in vegetable proteins and act as a factor for inhibiting the digestive ability of animals (Li et al., J Anim. Sci., 68:1790,1990).

Accordingly, in order to use soy proteins in animal feed, there is a need for research focusing on reducing anti-nutritional factors to increase the digestibility of feed and converting high molecular weight proteins into low molecular weight peptides.

Meanwhile, processed soy protein products which are currently produced, such as soy protein concentrates, soy protein isolates, or hydrolyzed soy proteins, are produced by chemical or enzymatic treatment. However, if a chemical is added for the production of such processed protein products, or a process related to the condition of the enzyme reaction is added, problems may arise in that the quality of the processed product may deteriorate and the time and cost required for producing soy protein hydrolysates may increase. In addition, since a conventional method for preparing soy protein hydrolysates includes a step of adding an enzyme in an aqueous solution phase, it has a disadvantage in that a step of drying the product must be additionally included therein. Further, a method for preparing a fermented soy protein using a novel Bacillus strain is disclosed in, for example, Korean Patent No.　10-1139027 and Korean Patent Laid-Open Publication No.　10-2015-0129238. However, both enzymatic hydrolysis and microbial fermentation methods have a problem in that a long and expensive drying process is additionally required in order to obtain a desired low molecular weight soy protein.

In addition, the related prior art includes US Patent Laid-Open Publication No.　12/811,188, but this discloses a means for performing hydrolysis of soybean using pepsin, pancreatin, or the like, which are proteases, after heat treatment. Further, US Patent No.　06/227602 discloses a method for treatment using bromelin- or papain-containing enzymes for improving the yield of edible proteins in legumes, and US Patent No.　06/541208 discloses a method of degrading trypsin inhibitor by treatment with starfish trypsin (DIT1) and carboxypeptidase B. Furthermore, Japanese Patent Nos.　1994-170414 and 2004-252174 merely disclose a method for hydrolyzing soy protein in aqueous solution and a method for using the same as a plant growth promoter which inhibits soybean trypsin using alkali protease through hydrolysis, respectively. In addition, although a method of enzymatic hydrolysis of soy protein using lactic acid bacteria is disclosed, hydrolysis of soy protein concentrate under a low moisture condition of the present disclosure is neither disclosed nor implied (Aguirre et al., Enzymatic hydrolysis of soy protein using lactic acid bacteria, Food Chemistry 111(2008) 976-982). Therefore, a method for preparing a soy protein concentrate having low molecular weight peptides which are hydrolyzed under a low moisture condition and a soy protein concentrate prepared therefrom have not been disclosed or suggested in any of the prior art.

The citation of a number of patent documents is shown and described in the specification of the present disclosure. The technical contents disclosed in these cited documents can be easily understood by those of ordinary skill in the art to which the present disclosure belongs with reference to the present specification as a whole.

[Prior Art Documents]

[Patent Documents]

(Patent Document 1) Korean Patent No.　10-0612600

(Patent Document 2) Korean Patent No.　10-1139027

(Patent Document 3) Korean Patent Laid-Open Publication No.　10-2015-0129238

(Patent Document 4) US Patent Laid-Open Publication No.　US　12/811518

(Patent Document 5) US Patent No.　US　06/227602

(Patent Document 6) US Patent No.　US　06/541208

(Patent Document 7) Japanese Patent No.　1994-170414

(Patent Document 8) Japanese Patent No.　2004-252174

[Non-Patent Documents]

(Non-Patent Document 1) Li et al., J. Anim Sci., 68:1790, 1990

(Non-Patent Document 2) Aguirre et al., Enzymatic hydrolysis of soy protein using lactic acid bacteria, Food Chemistry 111(2008) 976-982

Under these circumstances, the present inventors have made extensive efforts to develop a method for preparing a soy protein concentrate hydrolyzed under a low moisture condition. As a result, they have prepared a soy protein concentrate, which can enhance the digestibility for an animal since it has a high content of low molecular weight peptides and a low content of anti-nutritional factors, and thus can be applied as a feed material, thereby completing the present disclosure.

One object of the present disclosure is to provide a method for preparing a soy protein concentrate hydrolyzed under a low moisture condition.

Another object of the present disclosure is to provide a soy protein concentrate hydrolyzed under a low moisture condition which is prepared according to the preparation method above.

Still another object of the present disclosure is to provide a feed composition comprising the soy protein concentrate hydrolyzed under a low moisture condition.

Other objects and advantages of the present disclosure will become apparent from the detailed description together with the appended claims and drawings. The contents not described in this specification can be sufficiently recognized and inferred by those skilled in the technical field or similar technical field of the present disclosure, and thus the description thereof will be omitted.

The method for preparing the soy protein concentrate under a low moisture condition according to the present disclosure can increase hydrolysis efficiency while minimizing processing steps. In addition, the soy protein concentrate prepared by the method according to the present disclosure can enhance the digestibility for an animal since it has a high content of low molecular weight peptides and low content of anti-nutritional factors, and thus exhibits an excellent effect in the application as a feed material.

FIG.　1 is a diagram showing SDS-PAGE results of the soy protein concentrate.

FIG.　2 is a diagram showing TLC results of the soy protein concentrate.

FIG.　3 is a diagram showing SDS-PAGE results of the soy protein concentrate hydrolysate according to the moisture content using the protease derived from Bacillus licheniformis.

FIG.　4 is a diagram showing SDS-PAGE results of the soy protein concentrate hydrolysate according to the concentration and time of the protease, when the soy protein concentrate containing a moisture content of 25% by weight is hydrolyzed.

FIG.　5 is a diagram showing SDS-PAGE results of the soy protein concentrate hydrolysate according to the concentration and time of the protease, when the soy protein concentrate containing a moisture content of 15% by weight is hydrolyzed.

FIG.　6 is a diagram showing SDS-PAGE results of the soy protein concentrate hydrolysate according to the moisture content and the enzyme.

FIG.　7 is a diagram showing SDS-PAGE results of the soy protein concentrate hydrolysate according to the moisture content using the protease derived from Bacillus subtilis.

FIG.　8 is a diagram showing SDS-PAGE results of the soy protein concentrate hydrolysate at high temperature conditions.

Each description and embodiment disclosed in the present disclosure can be applied to other descriptions and embodiments, respectively. That is, all combinations of various elements disclosed herein fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not intended to be limited by the specific description described below.

The conventional method for preparing soy protein hydrolysates has a problem in that a long and expensive drying process is required as it includes steps of adding an enzyme in an aqueous solution state and drying the product. Therefore, it is necessary to develop a hydrolysate of a soy protein concentrate close to a solid state, which can save cost and time.

The present disclosure relates to a soy protein concentrate hydrolyzed under a low moisture condition and a preparation method thereof. More specifically, the present disclosure provides a method for preparing a soy protein concentrate hydrolyzed under a low moisture condition, comprising: preparing a soy protein concentrate; measuring the moisture content of the soy protein concentrate obtained in the above step; controlling the moisture content of the soy protein concentrate based on the measured moisture content in the above step; hydrolyzing the soy protein, in which the moisture content is controlled, by adding a protease thereto; and obtaining the soy protein concentrate hydrolyzed in the above step. The method can increase hydrolysis efficiency while minimizing processing steps. Accordingly, the hydrolyzed soy protein concentrate with enhanced digestibility can be prepared very efficiently, without adding a process related to the reaction conditions according to the type of enzymes.

In addition, the method according to the present disclosure includes a step of performing a hydrolysis reaction by adding an enzyme to the solid soy protein concentrate, and thus can enhance the hydrolysis efficiency of the soy protein concentrate without additionally including a drying step.

The soy protein concentrate hydrolyzed by the method according to the present disclosure has a feature in that it is suitable as a protein material for feed since the content of the anti-nutritional factors is low and the content of the low molecular weight peptides is increased so as to improve its digestibility for livestock.

As used herein, the term "soy protein concentrate" refers to a concentrate of protein extracted from soybean. The protein extracted from soybean is a protein produced by extracting soybean oil from soybean using hexane, which is an organic solvent, and then removing non-protein ingredients such as water-soluble and non-water-soluble carbohydrates from defatted soybean.

As used herein, the term "hydrolysis" refers to a reaction in which one large molecule reacts with water during a chemical reaction and decomposes into several ions or molecules and is applied to degradation of starch alpha bonds, fatty ester bonds, and protein peptide bonds.

As used herein, the term "protease" refers to a protein hydrolytic enzyme and is an enzyme that produces an amino acid or a peptide mixture. The types of enzymes used include pepsin, peptidase, and trypsin.

As used herein, the term "protein solubility" refers to the degree to which a protein is dissolved in water, and as more hydrophilic amino acids are present on the surface, the protein solubility increases by interacting with the ionic groups of the solvent.

According to one embodiment, the step of preparing the soy protein concentrate of the present disclosure can be carried out by various methods. It is preferred that the soy proteins are continuously supplied from the same type of soybean in the same region for use so as to maintain the same quality. In particular, the difference in protein content according to the types of soybean indicates the difference in protein content of the final product, and the quality of the hydrolyzed soy protein concentrate increases as the protein content of soybean increases. The soy protein concentrate may be prepared by first washing soy protein with alcohol and extracting soybean oil using an organic solvent such as hexane or the like, followed by further adding alcohol to the residual defatted soybean meal, thereby removing water-soluble and non-water-soluble carbohydrates other than the protein.

According to one embodiment of the present disclosure, the protein content of the soy protein concentrate may be controlled at the preparation step according to the type, quantity, acceptability, and solubility of a desired final low molecular weight peptide. The protein content may be measured by using a nitrogen titration method or the Kjeldahl instrument. The protein content of the soy protein concentrate may be in the range of, for example, 50% by weight to 70% by weight, specifically 55% by weight to 65% by weight, and 58% by weight to 62% by weight.

In addition, according to one embodiment of the present disclosure, the moisture content of the soy protein concentrate may be measured by a moisture meter using the volume titration method or coulometric titration method. In the step prior to controlling the moisture content, the moisture content of the soy protein concentrate may be in the range of 5% to 10%, specifically 6% to 8%.

According to one embodiment, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and GPC (gel permeation chromatography) may be used to measure the protein degradation and molecular weight distribution of the soy protein concentrate. The soy protein concentrate before hydrolysis may contain 70% or more of high molecular weight peptides having a molecular weight of 30　kDa or more and 30% or less of low molecular weight peptides having a molecular weight of 30　kDa or less. In the present disclosure, the peptides form a polymer of various combinations of amino acids via a peptide bond and generally consist of 2 to 50 amino acids bound together. The low molecular weight peptide is a small peptide having a low molecular weight and has the advantage of facilitating absorption during digestion, thereby increasing its digestibility when applied as animal feed. Depending on the type and composition ratio of the low molecular weight peptide included in the desired hydrolysate to be finally prepared, the conditions of the moisture content, protein content, or the like of the soy protein concentrate, which is the raw material, can be appropriately selected and used.

In addition, before the hydrolysis of the present disclosure, the soy protein concentrate may further contain a water-soluble sugar component such as glucose, sucrose, stachyose, raffinose, or the like.

The method for preparing the hydrolyzed soy protein concentrate of the present disclosure comprises a step of controlling the moisture content of the soy protein concentrate. Specifically, the moisture content may be controlled to 10% by weight to 40% by weight based on the total weight of the soy protein concentrate. More specifically, the moisture content may be controlled to 10% by weight to 30% by weight, 10% by weight to 25% by weight, 10% by weight to 20% by weight, and 10% by weight to 15% by weight based on the total weight of the soy protein concentrate.

When the condition of low moisture content is applied during the preparation of the soy protein hydrolysate, it has an advantage in that unlike the general protein hydrolysis, which occurs in a liquid phase, it does not require the additional drying step of the soy protein concentrate in the production process, thereby reducing production time and cost.

According to one embodiment, the soy protein concentrate hydrolyzed under a low moisture condition using the method of the present disclosure may contain 30% to 90% or more of the peptides having a molecular weight of 30　kDa or less. The content of the peptides having a molecular weight of 30　kDa or less may be more specifically 40% to 90%, 50% to 90%, 50% to 85%, 60% to 90%, 60% to 85%, 70% to 90%, 70% to 85%, 80% to 90%, or 80% to 85%, but is not limited thereto, and may vary depending on the amount of enzyme used, reaction time, or the like. Said % may refer to a percentage ratio of the partial area that represents a particular molecular weight range over the total area in the protein molecular weight distribution by GPC. The digestibility for an animal may be enhanced as content of the low molecular weight peptides increases, and accordingly, the soy protein concentrate having enhanced digestibility and the feed composition comprising the same can be prepared using the method of the present disclosure.

The method for preparing the hydrolyzed soy protein concentrate of the present disclosure comprises a step of hydrolyzing the soy protein, in which the moisture content is controlled, by adding a protease thereto. The amount of the protease to be added may be 0.05% by weight to 0.5% by weight based on the total weight of the soy protein concentrate. More specifically, the amount of the protease to be added may be 0.05% by weight to 0.35% by weight, 0.1% by weight to 0.35% by weight, or 0.2% by weight to 0.35% by weight.

According to one embodiment, the protease may be chemical enzymes or biological enzymes including those derived from microorganisms, specifically, may be at least one enzyme derived from microorganism selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis. The protease may be used by appropriately selecting the type and concentration of an appropriate enzyme and the reaction time according to the peptide composition or content of the desired hydrolyzed soy protein concentrate. According to one embodiment, the hydrolysis of the present disclosure may be performed at a temperature of 30°C to 90°C, more specifically at 50°C to 85°C, or 60°C to 80°C, for 30 minutes to 300 minutes, more specifically for 100 minutes to 280 minutes, 150 minutes to 250 minutes, or 180 minutes to 240 minutes to carry out an enzyme reaction.

According to one embodiment, the present disclosure provides a feed composition comprising the soy protein hydrolysate prepared by the above method. The content of the soy protein concentrate in the feed composition according to the present disclosure may be properly controlled depending on the kind and age of livestock to be applied, application form, desired effects, or the like. For example, it may be used in an amount of 1% by weight to 99% by weight, more specifically 10% by weight to 90% by weight, and 20% by weight to 80% by weight, but is not limited thereto.

For administration, the feed composition of the present disclosure may further include a mixture of at least one of an organic acid such as citric acid, fumaric acid, adipic acid, lactic acid, or the like; phosphate such as potassium phosphate, sodium phosphate, polyphosphate, or the like; a natural antioxidant such as polyphenol, catechin, tocopherol, vitamin C, green tea extract, chitosan, tannic acid, or the like; in addition to the hydrolyzed soy protein concentrate. If necessary, other typical additive such as an anti-influenza agent, a buffer, a bacteriostatic agent, or the like may be added. In addition, a diluent, a dispersing agent, a surfactant, a binder or a lubricant may be additionally added to formulate the composition into an injectable preparation such as an aqueous solution, a suspension, an emulsion, or the like, a capsule, a granule, or a tablet.

Moreover, the feed composition of the present disclosure may be used together with various auxiliary components such as amino acids, inorganic salts, vitamins, antioxidants, antifungal agents, antimicrobial agents, or the like, and a nutrient supplement, a growth accelerator, a digestion-absorption accelerator, and a prophylactic agent, in addition to the main ingredients including a vegetable protein feed such as pulverized or fragmented wheat, barley, corn, or the like, an animal protein feed such as blood meal, meat meal, fish meal, or the like, animal fat, and vegetable oil.

When the feed composition of the present disclosure is used as a feed additive, the feed composition may be added as it is or used together with other components, and may be appropriately used according to the typical method. The feed composition may be prepared in the administration form of an immediate-release formulation or a sustained-release formulation, in combination with non-toxic pharmaceutically acceptable carriers. The edible carriers may be corn starch, lactose, sucrose, or propylene glycol. The solid carrier may be in the administration form of tablets, powders, troches, or the like, and the liquid carrier may be in the administration form of syrups, liquid suspensions, emulsions, solutions, or the like. In addition, the administration agent may include a preservative, a lubricant, a solution accelerator, or a stabilizer and may also include other agents for improving inflammatory diseases and a substance useful for the prevention against viruses.

The feed composition of the present disclosure may be applied to an animal's diet, that is, a feed for many animals including mammals, poultry, fish, and crustaceans. It may be used in commercially important mammals such as pigs, cattle, goats, or the like, zoo animals such as elephants, camels, or the like, or livestock such as dogs, cats, etc. Commercially important poultry may include chickens, ducks, geese, or the like, and commercially grown fish and crustaceans such as trout and shrimp may also be included.

The feed composition according to the present disclosure may be mixed in an amount of approximately 10　g to 500　g, preferably 10　g to 100　g per 1　kg, based on the dry weight of the livestock feed. After being completely mixed, the feed composition may preferably be provided as mash or further subjected to a pelletizing, extensification, or extrusion process.

Hereinafter, the soy protein concentrate hydrolyzed under a low moisture condition and the preparation thereof according to the present disclosure will be described in more detail by way of Examples. However, these Examples are given for illustrative purposes only, and should not be construed as limiting the scope of the present disclosure. Hereinafter, the present disclosure will be described in detail by way of Examples.

[Example 1] Preparation of soy protein concentrate

The soy protein concentrate was prepared by washing the soy protein with alcohol to remove components other than soy protein, followed by concentrating the protein.

[Example 2] Analysis of soy protein concentrate

The moisture content, protein content, protein degradation and molecular weight distribution, and water-soluble sugar components of the soy protein concentrate prepared according to Example 1 were analyzed as follows.

1) Measurement of moisture and protein contents of soy protein concentrate

The moisture content of the soy protein concentrate prepared according to Example 1 was measured using a moisture meter, and the protein content was measured using the Kjeldahl instrument.

Table 1 shows the results of four measurements of the crude protein and moisture content (%) of the soy protein concentrate. As a result of the measurements, the soy protein concentrate of the present disclosure contained 6% to 8% moisture and about 60% protein.

No.	Crude protein (%)	Moisture (%)
1	59.33	8.27
2	58.59	7.92
3	61.13	7.12
4	59.5	6.18

2) Measurement of protein degradation and molecular weight distribution of soy protein concentrate through SDS-PAGE and GPC

In order to measure the molecular weight distribution of protein using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), 100　mg of the soy protein concentrate was suspended in 5　mL of 8　M urea solvent, sonicated, and then centrifuged at 8000　rpm for 10 minutes to isolate the supernatant. The supernatant was quantified as bicinchoninic acid and loaded onto SDS-PAGE gel.

FIG.　1 is a diagram showing SDS-PAGE results of the soy protein concentrate. With reference to FIG.　1, the soy protein concentrate of lane 2 contained proteins consisting of subunits having various molecular weights ranging from 20　kDa to 75　kDa (FIG.　1, lane 1: biomarker; lane 2: soy protein concentrate). The proteins contained in the soy protein concentrate include, for example, glycinin and β-conglycinin.

GPC (gel permeation chromatography) is a method to derive the protein molecular weight distribution in a sample to be measured, by analyzing standard proteins having different molecular weights to determine the retention time of each protein, and then by calculating a standard curve for the relationship between the molecular weight and the retention time. More specifically, after the retention time of the protein having a specific molecular weight is calculated, the chromatogram is divided into parts according to the time, and the ratio of partial area of each molecular weight range in the entire chromatogram area is calculated to derive the protein molecular weight distribution. In order to measure the protein molecular weight distribution of the soy protein concentrate using such GPC method, 100　mg of the soy protein concentrate was suspended in 5　mL of 8　M urea solvent, sonicated, and then centrifuged at 8000　rpm for 10 minutes to isolate the supernatant. Subsequently, the supernatant was filtered through a 0.45　μm syringe filter, and the filtrate was analyzed by GPC.

Molecular weight (kDa)	Protein distribution (%)
> 75	36.4
30 ~ 75	34.4
10 ~ 30	15.7
5~10	4.9
<5	8.6
Total	100.0

Table 2 shows the measurement results of the protein molecular weight distribution of the soy protein concentrate using GPC. According to Table 2, it was confirmed that the soy protein concentrate contained 70% or more of proteins having a molecular weight of 30　kDa or more and was mostly composed of polymer peptides.

3) Measurement of water-soluble sugar components of soy protein concentrate through TLC

In order to perform a qualitative analysis of water-soluble sugars in the soy protein concentrate by TLC (thin layer chromatography), 1　g of soy protein concentrate was mixed with 25　mL of distilled water, and the mixture was extracted at the boiling point thereof for 20 minutes. After the extraction process, the water-soluble sugar was sufficiently extracted through stirring at 180　rpm for 2 hours at 37°C. The resulting extract was centrifuged, and the water-soluble sugar components were identified by TLC from the supernatant.

FIG.　2 is a diagram showing TLC results of the soy protein concentrate. With reference to FIG.　2, the soy protein concentrate contained water-soluble sugar components such as glucose, sucrose, stachyose, and raffinose (FIG.　2, lane 1: control group; lanes 2 to 6: production batch for each of 5 species).

Water-soluble components	Production batch
Water-soluble components	1	2	3	4	5
Glucose	Small amount detected	Small amount detected	Small amount detected	Small amount detected	Small amount detected
Sucrose	Detected	Detected	Detected	Detected	Detected
Stachyose	Detected	Detected	Detected	Detected	Detected
Raffinose	Detected	Detected	Detected	Detected	Detected

Table 3 shows the water-soluble sugar components identified in the soy protein concentrate (5 production batches, 1 to 5). The soy protein concentrate included glucose, sucrose, stachyose, and raffinose, and in particular, glucose was included in a very small amount.

[Example 3] Measurement of protein hydrolysis of soy protein concentrate according to moisture content control and addition of protease

Seven experimental groups of the soy protein concentrate obtained in Example 1 were prepared.

The moisture content in the soy protein concentrate of each experimental group was measured, and then water was added thereto to control the moisture content to10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight, 35% by weight, and 40% by weight based on the total weight, respectively. In each experimental group, the protease derived from Bacillus licheniformis (Prozyme AK) was added in an amount of 0.35% by weight based on the weight of the soy protein concentrate. The enzyme reaction was induced at 60℃ for 4 hours in the soy protein concentrate experimental groups, to which the water and protease were added. In order to measure the protein degradation in the experimental groups in which the enzyme reaction was completed, they were subjected to heat treatment at 100°C for 20 minutes to terminate the enzyme reaction. The soy protein concentrate of each experimental group in which the enzyme reaction was terminated was obtained by drying and pulverization. The protein degradation and molecular weight distribution in each experimental group were measured by SDS-PAGE and GPC described in Example 2, 2).

FIG.　3 is a diagram showing SDS-PAGE results of the soy protein concentrate hydrolysate according to the moisture content using the protease derived from Bacillus licheniformis. With reference to FIG.　3, it was confirmed that as the moisture content decreased during the enzyme reaction, the amount of low molecular weight peptides increased, thereby increasing the degree of protein degradation. (FIG.　3, lane M: biomarker; lane 1: raw material of soy protein concentrate; lane 2: moisture content of 40% by weight; lane 3: moisture content of 35% by weight; lane 4: moisture content of 30% by weight; lane 5: moisture content of 25% by weight; lane 6: moisture content of 20% by weight; lane 7: moisture content of 15% by weight; lane 8: moisture content of 10% by weight).

Specifically, in the hydrolysates having high moisture contents of 40% by weight, 35% by weight, and 30% by weight, the existing polymer protein subunits (mainly 30　kDa or more) constituting the soy protein concentrate were clearly indicated relatively. However, at the moisture content of 25% by weight, some polymer bands of the protein subunits disappeared, and the bands of the low molecular weight peptides began to strongly appear. Further, it was confirmed that the hydrolysates under moisture conditions of 15% by weight and 10% by weight were mostly composed of the proteins having a molecular weight of 25　kDa or less.

MW (kDa)	Moisture content
MW (kDa)	40% by weight	35% by weight	30% by weight	25% by weight	20% by weight	15% by weight	10% by weight
>75	25.3	19.7	10.8	4.7	3.8	5.6	5.1
30-75	22.4	25.5	25.5	15.6	13.2	11.6	11.0
10-30	13.4	16.4	23.8	30.6	26.6	18.9	18.8
5-10	11.2	11.6	14.2	21.0	25.1	27.0	27.3
<5	27.8	26.7	25.7	28.2	31.4	36.9	37.8
Total	100.0	100.0	100.0	100.0	100.0	100.0	100.0

Table 4 shows the GPC measurement results of the soy protein concentrate hydrolysates according to the moisture content before hydrolysis. With reference to Table 4, as the moisture content decreased, the percentage of the proteins having a molecular weight of 75　kDa or more decreased, and the percentage of the low molecular weight peptides gradually increased. Specifically, the soy protein hydrolysates were composed of about 80% of the low molecular weight peptides having a molecular weight of 30　kDa or less under the moisture condition of 25% by weight, and the content of the low molecular weight peptide of 30　kDa or less increased as the moisture content further decreased.

That is, it was confirmed that the content of the low molecular weight peptides of 30　kDa or less was greatly increased when the hydrolysis was performed using the soy protein concentrate in which the moisture content was controlled to 25% or less. When the hydrolysis was performed at the low moisture condition, unlike the protein hydrolysis reaction which is generally carried out in a liquid phase, a process of drying the final product could be omitted as the final product of the reaction was solid, and ultimately, the time and cost required for the production of hydrolysates could be reduced.

[Example 4] Protein degradation and molecular weight distribution of soy protein concentrate hydrolysate according to concentrations of enzyme

The protein degradation and molecular weight distribution according to the concentrations of enzyme for treating the soy protein concentrate having the same moisture content (including the moisture contents of 25% by weight or 15% by weight based on the total weight) were measured.

The protein degradation and molecular weight distribution were measured in the same manner as in Example 3, except that moisture content was controlled to 25% by weight or 15% by weight based on the total weight, and that the protease was treated at each concentration of 0.05% by weight, 0.1% by weight, 0.2% by weight, and 0.35% by weight (relative to the weight of soy protein concentrate) and then reacted at 60°C. The enzyme reaction was performed at an interval of 1 hour to 4 hours. The samples for each time period and concentration were inactivated by heating at 100°C for 20 minutes after the enzyme reaction. Then, samples for analysis were prepared through drying and pulverization, and the protein degradation and molecular weight distribution thereof were measured by SDS-PAGE and GPC described in Example 2, 2).

FIG.　4 is a diagram showing SDS-PAGE results of the soy protein concentrate hydrolysates according to the concentration of the protease and time when the soy protein concentrate containing a moisture content of 25% by weight was hydrolyzed. With reference to FIG.　4, when the soy protein concentrate containing a moisture content of 25% by weight was hydrolyzed, the protein degradation was increased as the concentration of the protease and the reaction time increased (FIG.　4, lane M: biomarker; lane 1: raw material of soy protein concentrate; lane 2: 0.05% by weight, 1 hour; lane 3: 0.05% by weight, 2 hours; lane 4: 0.05% by weight, 3 hours; lane 5: 0.05% by weight, 4 hours; lane 6: 0.1% by weight, 1 hour; lane 7: 0.1% by weight, 2 hours; lane 8: 0.1% by weight, 3 hours; lane 9: 0.1% by weight, 4 hours; lane 10: 0.2% by weight, 1 hour; lane 11: 0.2% by weight, 2 hours; lane 12: 0.2% by weight, 3 hours; lane 13: 0.2% by weight, 4 hours; lane 14: 0.35% by weight, 1 hours; lane 15: 0.35% by weight, 2 hours; lane 16: 0.35% by weight, 3 hours; lane 17: 0.35% by weight, 4 hours).

Table 5 shows the results of GPC measurement of the hydrolysates of the soy protein concentrate according to the concentrations of the protease and time when the soy protein concentrate containing a moisture content of 25% by weight was hydrolyzed.

MW (kDa)	Enzyme concentration at 0.05% by weight				Enzyme concentration at 0.1% by weight
MW (kDa)	1 hour	2 hours	3 hours	4 hours	1 hour	2 hours	3 hours	4 hours
>75	8.5	8.5	9.0	5.9	8.7	6.2	8.0	4.7
30-75	47.4	42.8	43.0	34.8	44.4	36.6	40.1	29.6
10-30	24.4	27.5	27.0	33.2	26.2	32.6	29.6	36.2
5-10	8.1	9.4	9.4	12.4	8.9	11.9	10.3	14.7
<5	11.6	11.8	11.7	13.7	11.7	12.7	11.9	14.9
Total	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0
MW (kDa)	Enzyme concentration at 0.2% by weight				Enzyme concentration at 0.35% by weight
MW (kDa)	1 hour	2 hours	3 hours	4 hours	1 hour	2 hours	3 hours	4 hours
>75	5.7	3.4	2.9	3.0	2.2	2.4	2.2	2.3
30-75	37.2	25.3	22.7	21.3	18.3	17.3	16.3	14.7
10-30	32.7	38.2	38.3	37.5	35.6	35.4	33.8	29.3
5-10	11.8	17.0	18.4	19.2	23.4	22.8	23.8	26.0
<5	12.7	16.1	17.7	19.0	20.5	22.1	23.9	27.8
Total	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0

With reference to Table 5, it was confirmed that as the concentration of the enzyme or the reaction time increased, the high molecular weight protein was decomposed into low molecular weight peptides. More specifically, when the enzyme at a concentration of 0.1% by weight was used, the content of the peptides having a molecular weight of 30　kDa or less was about 50% or more on average. When the enzyme at a concentration of 0.2% by weight was used, the content of the peptides having a molecular weight of 30　kDa or less was about 70% or more on average. In addition, when the enzyme at a concentration of 0.35% by weight was used, the content of the peptides having a molecular weight of 30　kDa or less was about 80% or more on average.

FIG.　5 is a diagram showing SDS-PAGE results of the soy protein concentrate hydrolysates according to the concentration of the protease and time when the soy protein concentrate containing a moisture content of 15% by weight was hydrolyzed.

With reference to FIG.　5, when the soy protein concentrate containing a moisture content of 15% by weight was hydrolyzed, the protein degradation increased as the concentration of the protease and the reaction time increased (FIG.　5, lane M: biomarker; lane 1: 0.05% by weight, 1 hour; lane 2: 0.05% by weight, 2 hours; lane 3: 0.05% by weight, 3 hours; lane 4: 0.05% by weight, 4 hours; lane 5: 0.1% by weight, 1 hour; lane 6: 0.1% by weight, 2 hours; lane 7: 0.1% by weight, 3 hours; lane 8: 0.1% by weight, 4 hours; lane 9: 0.2% by weight, 1 hour; lane 10: 0.2% by weight, 2 hours; lane 11: 0.2% by weight, 3 hours; lane 12: 0.2% by weight, 4 hours; lane 13: 0.35% by weight, 1 hour; lane 14: 0.35% by weight, 2 hours; lane 15: 0.35% by weight, 3 hours; lane 16: 0.35% by weight, 4 hours).

Table 6 shows the results of GPC measurement of the hydrolysates of the soy protein concentrate according to the concentrations of the protease and time when the soy protein concentrate containing a moisture content of 15% by weight was hydrolyzed.

MW (kDa)	Enzyme concentration at 0.05% by weight				Enzyme concentration at 0.1% by weight
MW (kDa)	1 hour	2 hours	3 hours	4 hours	1 hour	2 hours	3 hours	4 hours
>75	2.5	2.2	2.2	2.6	2.4	2.5	2.3	2.9
30-75	14.9	16.1	15.0	14.9	14.8	14.2	13.7	12.4
10-30	26.4	31.5	27.7	26.2	27.3	25.0	23.0	20.5
5-10	27.9	25.9	27.7	27.8	27.8	28.7	29.3	30.1
<5	28.4	24.3	27.4	28.5	27.7	29.6	31.7	34.1
Total	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0
MW (kDa)	Enzyme concentration at 0.2% by weight				Enzyme concentration at 0.35% by weight
MW (kDa)	1 hour	2 hours	3 hours	4 hours	1 hour	2 hours	3 hours	4 hours
>75	2.6	2.7	2.2	2.6	2.4	2.4	2.2	2.2
30-75	13.4	12.8	11.9	12.3	11.2	10.8	10.6	8.9
10-30	23.3	20.7	18.3	18.0	18.0	16.3	15.1	14.0
5-10	29.2	29.8	30.3	30.1	30.2	30.0	29.6	29.3
<5	31.6	34.1	37.3	36.9	38.2	40.5	42.5	45.6
Total	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0

With reference to FIG.　6, it was confirmed that as the concentration of the enzyme or the reaction time increased, the high molecular weight protein was decomposed into low molecular weight peptides. More specifically, when the enzyme at a concentration of 0.1% by weight was used, the content of the peptides having a molecular weight of 10　kDa or less was about 60% or more on average, when the enzyme at a concentration of 0.35% by weight was used, the content of the peptides having a molecular weight of 10　kDa or less was about 70% or more on average. However, the content of the peptides having a molecular weight of 30　kDa or less did not significantly increase even when the concentration of the enzyme increased from 0.05% by weight to 0.35% by weight, and the content thereof was about 82% to 87% or more on average.

That is, it was confirmed that as the concentration of the enzyme and the reaction time increased, the hydrolysis rate was increased, and also that as the moisture content of the soy protein concentrate decreased, the rate of hydrolysis increased.

[Example 5] Measurement of protein degradation using protease derived from various species

(1) Protease derived from Bacillus amyloliquefaciens and Bacillus licheniformis

After measuring the moisture content in the soy protein concentrate obtained in Example 1, water was added to control the moisture content to 15% by weight and 25% by weight based on the total weight. Each experimental group was supplemented with 0.35% by weight of protease (Bision Biocam, Alphalase NP) derived from Bacillus amyloliquefaciens or protease (Bision Biocam, FoodPro Alkaline Protease) derived from Bacillus licheniformis based on the weight of the soy protein concentrate. The enzyme reaction was induced in the soy protein concentrate, to which the water and enzyme were added, at 60°C for 4 hours. In order to measure the degree of protein degradation in the experimental groups in which the enzyme reaction was completed, they were subjected to heat treatment at 100°C for 20 minutes to terminate the enzyme reaction. The soy protein concentrate in each experimental group was dried and pulverized. The degradation and the molecular weight distribution of protein in each experimental group were measured by SDS-PAGE and GPC described in Example 2, 2).

It was confirmed in both experiment results of SDS-PAGE (FIG.　6) and GPC (Table 7) on the soy protein concentrate treated with the enzyme that that the amount of the low molecular weight peptides was higher when the hydrolysis was performed at a moisture content of 15% by weight, as compared to when the hydrolysis was performed at a moisture content of 25% by weight (FIG.　6, lane M: biomarker; lane 1: raw material of soy protein concentrate; lane 2: protease derived from Bacillus amyloliquefaciens, moisture content of 25% by weight; lane 3: protease derived from Bacillus amyloliquefaciens, moisture content of 15% by weight; lane 4: protease derived from Bacillus licheniformis, moisture content of 25% by weight; lane 5: protease derived from Bacillus licheniformis, moisture content of 15% by weight). Specifically, according to FIG.　6, in

lanes

3 and 5 having a moisture content of 15% by weight, the existing high molecular weight peptide bands appeared blurry, while the low molecular weight peptide bands strongly appeared. Further, according to Table 7, showing the results of GPC experiment of the soy protein concentrate hydrolysates treated with the two enzymes, the composition ratios of the low molecular weight peptides having a molecular weight of 30　kDa or less were shown to be 33.9% and 68.1% at the moisture contents of 25% by weight and 15% by weight, respectively, when the protease derived from Bacillus amyloliquefaciens was used, and were shown to be 69.1% and 83.6% at the moisture contents of 25% by weight and 15% by weight, respectively, when the protease derived from Bacillus licheniformis was used. That is, when the proteases derived from Bacillus amyloliquefaciens and Bacillus licheniformis were used for the hydrolysis of the soy protein concentrate having a low moisture content, it was confirmed that the degradation of the soy protein concentrate was significantly increased.

MW (kDa)	Bacillus amyloliquefaciens-derived protease		Bacillus licheniformis-derived protease
MW (kDa)	Moisture content of 25% by weight	Moisture content of 15% by weight	Moisture content of 25% by weight	Moisture content of 15% by weight
>75	22.9	8.6	6.0	4.2
30-75	43.2	23.3	24.8	12.2
10-30	15.6	31.7	32.9	16.4
5-10	5.6	16.7	15.8	26.9
<5	12.7	19.7	20.4	40.3
Total	100.0	100.0	100.0	100.0

(2) Bacillus subtilis-derived protease

After measuring the moisture content in the soy protein concentrate obtained in Example 1, water was added to control the moisture content to 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight, 35% by weight, and 40% by weight based on the total weight. Each experimental group was supplemented with 0.35% by weight of protease derived from Bacillus subtilis (Benesol, Alkaline protease) based on the weight of the soy protein concentrate. The enzyme reaction was induced in the soy protein concentrate, to which water and the enzyme were added, at 45°C for 4 hours. Subsequently, in order to measure the degree of protein degradation in the experimental groups in which the enzyme reaction was completed, they were subjected to heat treatment at 100°C for 20 minutes to terminate the enzyme reaction. The soy protein concentrate in each experimental group was dried and pulverized. The molecular weight distribution of protein in each experimental group was measured by SDS-PAGE and GPC described in Example 2, 2).

As a result of the measurement, when the hydrolysis was performed using the enzyme, the amount of the low molecular weight peptides increased as the moisture content in the soy protein concentrate decreased. In particular, when the moisture content was at 20% by weight or less, specifically 15% by weight or less, it was confirmed that the amount of the low molecular weight peptides was significantly high (FIG.　7, lane M: biomarker; lane 1: moisture content of 40% by weight; lane 2: moisture content of 35% by weight; lane 3: moisture content of 30% by weight; lane 4: moisture content of 25% by weight; lane 5: moisture content of 20% by weight; lane 6: moisture content of 15% by weight; lane 7: moisture content of 10% by weight).

Table 8 shows the results of GPC measurement of the soy protein concentrate hydrolysates using the protease derived from Bacillus subtilis.

MW (kDa)	Moisture content of 40% by weight	Moisture content of 35% by weight	Moisture content of 30% by weight	Moisture content of 25% by weight	Moisture content of 20% by weight	Moisture content of 15% by weight	Moisture content of 10% by weight
>75	18.1	22.7	18.6	18.2	6.7	5.2	5.1
30-75	36.4	39.7	42.8	44.3	28.9	17.0	15.6
10-30	19.6	16.5	18.9	19.3	35.7	32.0	29.3
5-10	9.8	7.3	7.2	6.8	14.1	22.0	23.4
<5	16.1	13.8	12.4	11.4	14.6	23.9	26.6
Total	100.0	100.0	100.0	100.0	100.0	100.0	100.0

With reference to Table 8, when the soy protein concentrate having a moisture content of 10% by weight to 20% by weight was hydrolyzed, the content of the low molecular weight peptides having a molecular weight of 30　kDa or less was about 64% to about 80%. That is, it was confirmed that the protein degradation was greatly increased even when the enzyme derived from Bacillus subtilis was used in the hydrolysis of the soy protein concentrate having a low moisture content.

[Example 6] Preparation of soy protein concentrate hydrolysate at high temperature

The level of hydrolysis of the soy protein concentrate was confirmed when the enzyme reaction temperature was at 80°C. The soy protein concentrate containing a moisture content of 10% by weight was treated with 0.2% (raw material weight ratio) of the enzyme derived from Bacillus licheniformis (Prozyme AK) to carry out the enzyme reaction at 80°C for 4 hours. The degradation and molecular weight distribution of protein of the samples, in which the enzyme reaction was completed, were confirmed by SDS-PAGE and GPC described in Example 2, 2).

FIG.　8 is a diagram showing SDS-PAGE results of the soy protein concentrate hydrolysate at a high temperature.

With reference to FIG.　8, it was confirmed that the soy protein concentrate was hydrolyzed even at a high temperature of 80°C, thereby increasing the content of the low molecular weight peptides (FIG.　8, lane M: biomarker; lane 1: raw material of soy protein concentrate; lane 2: enzyme reactant).

Table 9 shows the GPC result of the soy protein concentrate hydrolysate at a high temperature.

With reference to Table 9, it was confirmed that the content of the low molecular weight peptides having a molecular weight of 30　kDa or less was about 84.2%, which was at a high level. Therefore, it was confirmed that the hydrolysates of the soy protein concentrate having a high content of low molecular weight peptide could be prepared even when the enzyme reaction was carried out at a high temperature of about 80°C in the hydrolysis of the soy protein concentrate having a low moisture content.

MW (kDa)	Moisture content of 10% by weight
>75	5.1
30-75	10.8
10-30	18.6
5-10	30.7
<5	34.9
Total	100.0

Claims

A method for preparing a soy protein concentrate hydrolyzed under a low moisture condition, comprising:

preparing a soy protein concentrate;

measuring the moisture content of the soy protein concentrate obtained in the above step;

controlling the moisture content of the soy protein concentrate based on the measured moisture content in the above step;

hydrolyzing the soy protein, in which the moisture content is controlled, by adding a protease thereto; and

obtaining the soy protein concentrate hydrolyzed in the above step.
The method of claim 1, wherein the moisture content in the step of controlling the moisture content of the soy protein concentrate is controlled to 10% by weight to 40% by weight based on the total weight of the soy protein concentrate.
The method of claim 1 or claim 2, wherein the hydrolyzed soy protein concentrate comprises 30% to 90% of peptides having a molecular weight of 30　kDa or less.
The method according to any one of claim 1 to claim 3, wherein the protease is added in an amount of 0.05% to 0.5% based on the total weight of the soy protein concentrate.
The method according to any one of claim 1 to claim 4, wherein the protease is at least one enzyme derived from microorganisms selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis.
The method according to any one of claim 1 to claim 5, wherein the step of hydrolyzing the soy protein concentrate is performed by an enzyme reaction at 30℃ to 90℃ for 30 minutes to 300 minutes.
The method according to any one of claim 1 to claim 6, wherein the step of preparing the soy protein concentrate is a step of preparing a soy protein concentrate which has a protein content of 50% by weight to 70% by weight and comprises at least one sugar selected from the group consisting of glucose, sucrose, stachyose, and raffinose.
A soy protein concentrate hydrolyzed under a low moisture condition which is prepared according to any one method of claim 1 to claim 7.
The soy protein concentrate of claim 8, wherein the hydrolyzed soy protein concentrate comprises 30% to 90% of peptides having a molecular weight of 30　kDa or less.
A feed composition comprising the soy protein concentrate of claim 8 or claim 9.