KR20090095902A - Whey protein hydrolysates having anti-oxidative activity, process for preparing the same and use for food of the same - Google Patents

Abstract

A whey protein hydrolysate having antioxidative effect is provided to have antioxidation, DNA damage suppression, and anti-tumor effect and develop various functional health beverage. A whey protein hydrolysate having antioxidative effect is obtained by hydrolyzing whey protein concentrate with protease. The protease is neutrase, trypsin, flavorzyme, alcalase, and protamax. A method for producing the whey protein hyrolysate comprises: a step of treating the protease in whey protein; and a step of separating the reacted supernatant by spraying. A functional food comprises whey protein hydrolysate as an active ingredient.

Description

Whey protein hydrolysates having anti-oxidative activity, process for preparing the same and use for food of the same}

The present invention relates to a whey protein hydrolyzate having an antioxidant effect, a method for preparing the same, and a use thereof in a food thereof, and more particularly, to a high-quality protein obtained by hydrolyzing whey protein, a by-product formed in the manufacture of cheese from milk. Containing whey protein hydrolysate, (1) treating whey protein concentrate with protease enzyme; And (2) spray drying only the reaction supernatant; The present invention relates to a method for preparing the whey protein hydrolyzate and an antioxidant health food containing the whey protein hydrolyzate as an active ingredient.

Whey refers to the liquid that leaves and harvests the curd formed in the manufacture of cheese from milk. If not properly processed, these whey can result in loss of food resources and environmental and economic burdens. In addition, the consumption of cheese is increasing along with the current well-being trend, and the movement to manufacture farm-type cheese is also active. As the cheese production increases, whey may act as a pollution factor, but when it is used to develop foods, it can be used as a valuable material in various industries.

Such whey is a mixture of several proteins with various chemical, physical and functional properties. Therefore, in addition to playing an important role in human nutrition, in many cases also plays a special physiological action in vivo. Specifically, different types of whey proteins provide immunity to newborns, and in particular, small digested proteins may act as part of a bioactive peptide that confers physiological activity.

Whey proteins also provide a wide range of functionality, including solubility, viscosity, water retention, foam generation, emulsification, and gel formation. Whey protein products are stable to pH changes and can retain solubility over a wide range of pH. Due to these characteristics, whey protein can be widely developed as a functional material applied to a wide range of foods. In addition, hydrolysis of whey protein has the effect of improving nutritional properties and tissues, inhibiting degradation and improving tissues. If the protein in the natural state is limited in its functionality, the biologically active peptide obtained by hydrolysis by protease and the like possesses additional nutritional and functional properties, so that it can be usefully used for the manufacture of high value-added health foods and medicines. .

Indeed, peptides hydrolyzed from whey proteins have been shown to be involved in inhibiting the proliferation of cancer cells through various cytochemical studies. In addition, hydrolyzed whey protein containing probiotic lactic acid bacteria has been reported to play an important role in consumers and children, including children and the elderly by adding to various foods to promote calcium absorption and induce growth. When enzymes and probiotic lactic acid bacteria are added to the whey protein and reacted, alpha-lactophine (α-lactorphin) and beta-lactophine (β-lactorphin), which show ACE (Angiotensin Converting Enzyme) inhibitory effects, are bioactive peptides. Active peptides are also produced which are hydrolysates derived from alpha-lactalbumin. In addition, hydrolyzed peptides derived from beta-lactoglobulin (β-lactoglobulin) are also known to exhibit an ACE inhibitory effect. In particular, alpha-lactopine derived from alpha-lactoalbumin has been studied to increase the immune power by the proliferation of human peripheral blood lymphocytes (PBL). At this time, in order to generate the hydrolyzed active peptide, the reaction conditions must be optimized and have appropriate functionality.

Therefore, the present inventors continued efforts to develop functional health foods using whey protein. As a result, the whey protein was prepared by heat treating cheese whey, hydrolyzed by adding various proteases, and then spray drying only the reaction supernatant. As a result, hydrolyzates of whey protein having various protein contents were prepared. In addition, it was confirmed that the whey protein hydrolyzate has antioxidant, DNA damage inhibition and anti-tumor efficacy, and successfully completed the present invention by providing a functional health beverage to which the hydrolyzate was added.

It is an object of the present invention to provide whey protein, whey protein hydrolyzate, and a method of preparing them, and their use in addition to food having excellent antioxidant efficacy.

In order to achieve the above object, the present invention provides a whey protein hydrolyzate having an antioxidant effect.

In addition, the present invention (1) Whey protein treatment with the protease enzyme; And (2) separating and spray drying only the reaction supernatant; It provides a process for producing the whey protein hydrolyzate made of a process.

The present invention also provides a functional food comprising the whey protein hydrolyzate as an active ingredient.

In addition, the present invention provides a functional beverage comprising the whey protein hydrolyzate as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a whey protein and whey protein hydrolyzate having excellent antioxidant effects. Whey protein and whey protein hydrolyzate of the present invention, in addition to the antioxidant effect, has a DNA damage suppression and antitumor effect.

Specifically, the present invention provides a whey protein hydrolyzate obtained by hydrolyzing whey protein concentrate with a protease enzyme. The whey protein concentrate is (1) heat treated with cheese whey; And (2) filtered to concentrate; Preference is given to using those produced by the process. Also in addition to the above procedure, (3) at least one diafiltration (diafiltration); It is also preferable to use those prepared by adding the procedure.

In addition, the whey protein concentrate is preferably used in the range of 65 to 85% protein content, more preferably in the 70 to 80% protein content. It is also preferable to use a protein content in the range of 30 to 40%, and more preferably use a protein content of about 35%. In addition, the whey protein concentrate is hydrolyzed by treatment of at least one protease selected from neutrase, trypsin, flavozyme, alcalase and protamax. desirable.

The present invention (1) the cheese whey is heat-treated; And (2) filtered to concentrate; It provides a process for producing whey protein consisting of a process.

In addition, the present invention is in addition to the above process, (3) semi-filtered one or more times; It provides a process for producing whey protein by adding a process.

In the step (1), the cheese whey is preferably heat-treated by adjusting to pH neutral, and more preferably heat-treated for about 5 minutes at about 70 ° C. In addition, in the process (2), the whey is preferably cooled to about 50 ° C. and then ultrafiltered, and the ultrafiltration may be performed using a conventional method. In addition, it is preferable to semi-filter at least once according to the concentration of whey in the above (3) process, it is preferable to spray-dry for later storage, and the semi-filtration process and the spray-drying process may also use a conventional method. Do.

In addition, the present invention (1) Whey protein treatment with the protease enzyme; And (2) separating and spray drying only the reaction supernatant; It provides a process for producing the whey protein hydrolyzate made of a process.

In the step (1), the whey protein (a) removes 80 to 85% permeate from the whey; And (b) spray drying the concentrate; It is preferable to use those produced by the process. In addition, the whey protein preferably has a protein content of 30 to 40%, more preferably about 35% protein content.

In addition, in the step (1), the whey protein (a) removes 95 to 97.5% permeate from the whey; (b) adding the concentrate and the same amount of water; And (c) diafiltration to remove the same permeate; It is also preferable to use what is produced by the process. Further, in addition to the above process, (d) again adding the same amount of concentrated solution and the same amount of water and then semifiltered to remove the same amount of permeate; It is also preferable to use those produced by the process.

In addition, the whey protein preferably has a protein content in the range of 65 to 75%, and more preferably has a protein content in the range of about 70%. In addition, the whey protein preferably has a protein content of 75 to 85%, more preferably a protein content of about 80%.

In addition, the protease in the process (1) using at least one selected from neutrase, trypsin, flavozyme, alcalase and protamax It is preferable. In addition, the protease is preferably treated at a temperature of 35 to 45 ° C. for at least 3 hours, and after the hydrolysis reaction, it is preferable to inactivate the reaction by heating at a high temperature.

The present invention also provides a functional food comprising the whey protein and / or whey protein hydrolysate as an active ingredient. The functional food has antioxidant, DNA damage inhibition and / or antitumor efficacy.

The present invention also provides a functional beverage comprising the whey protein and / or whey protein hydrolysate as an active ingredient. The functional beverage has antioxidant, DNA damage inhibition and / or antitumor efficacy.

The functional food and the functional beverage are preferably formulated by adding probiotic lactic acid bacteria and sugars, citric acid, flavoring and / or pigments in addition to the whey protein and / or its hydrolyzate. In this case, the whey protein and its hydrolyzate are preferably those having a final protein content of 1.0% or less, and the whey protein hydrolyzate is preferably hydrolyzed to 5% or less, 3 to 4% It is more preferable to use those hydrolyzed in the range.

As described above, whey protein hydrolysate containing high quality protein hydrolyzed whey protein, which is a by-product formed in the manufacture of cheese from milk, (1) treating the whey protein concentrate with a protease enzyme; And (2) spray drying only the reaction supernatant; The present invention relates to a method for preparing the whey protein hydrolyzate and an antioxidant health food containing the whey protein hydrolyzate as an active ingredient.

The whey protein and / or hydrolyzate thereof prepared in the present invention exhibits excellent antioxidant, DNA damage suppression and antitumor efficacy and furthermore, probiotic lactic acid bacteria can be added to effectively produce various bioactive peptides as well as easy to discard milk. It also has the economic advantage of recycling by-products. Therefore, it can be widely applied in foods for children, sportsmen, weight adjusters and the elderly.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 Preparation of Whey Protein

Fresh mozzarella cheese whey supplied from Seoul Milk Singal Plant was adjusted to pH 7.0 and heat-treated at 70 ° C. for 5 minutes and then cooled to 50 ° C. The heat-treated whey was made of whey protein (WPC) by ultrafiltration. At this time, a spiral wound type ultrafilter equipped with a membrane having a molecular weight range of 20,000 Daltons of polysulfone was used. The whey was processed into a balance tank of the ultrafilter to reduce the volume in various ways. At this time, the pressure was applied to the inlet pressure 1.5 bar and the outlet pressure 5 bar, the temperature was 50 ℃ to inhibit the growth of bacteria. The filtration process removes the permeate and concentrates the whey to 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 and 97.5%, respectively, and then stops the process at the required amount of permeate. (Diafiltraltion). The whey protein (WPC) obtained above was spray dried using a spray dryer (Buechi, Switzerland) under the conditions of inlet temperature of 175 ° C and outlet temperature of 70 ° C after performing the ultrafiltration process in one or two stages according to its type. .

(1) Preparation of Whey Protein-35 (WPC-35)

Whey protein (WPC-35) with 35% protein content was ultrafiltration of 100 L whey at 50 ° C. to remove 80-85 L permeate, and then 15-20 L of retentate was separated and spray dried. . This ultrafiltration process results in the release of most of the lactose and water-soluble salts and water from the milk, leaving proteins and milk fats, including whey protein, in the concentrate. The whole process depends on the type of final product, which took a total of six hours to produce the WPC-35.

(2) Preparation of Whey Protein-70 (WPC-70)

Whey protein (WPC-70) with 70% protein content was ultrafiltration of 100 L whey at 50 ° C. to remove permeate from 95 to 97.5%, and then 5 to 2.5 L of the concentrate obtained therefrom and the same volume of water was added again. It was. Ultrafiltration process (diafiltration) was added to remove the water from the concentrate to remove the same amount of filtrate, and as a result, WPC-70 containing 70% protein was prepared.

(3) Preparation of Whey Protein-80 (WPC-80)

Whey protein (WPC-80) having a protein content of 80% was obtained in the same manner as in (2) above, and then added to the distilled water by adding 50 ° C. of distilled water as much as the concentrate. A second ultrafiltration process was performed to remove the permeate. As a result, WPC-80 containing 80% protein was prepared.

Example 2. Preparation of Whey Protein Hydrolyzate

Whey protein hydrolysates were prepared using the whey protein (WPC) prepared in Example 1 and commercially available whey protein powder as follows. First, we use neutrase, trypsin, flavozyme, alcalase and protamax as proteases, and the hydrolysis reaction is performed by Shoba's method (Shobba, KP). : Diss. Univ. Agric. Sci. Bangalore, India, 2002). 1 ml of each protease was added at a rate of 25 g of whey protein, and the reaction was performed at a temperature of 40 ° C. for 3 hours. Then, the hydrolysis reaction was terminated, the hydrolyzate was centrifuged and only the supernatant was separated. The WPC hydrolyzate obtained therefrom was spray dried in the same manner as in Example 1, and the spray dried hydrolyzate was immediately packaged and stored.

Example 3. Investigation of Physicochemical Properties of Whey Protein

The physicochemical properties of the whey protein prepared in Example 1 were investigated as follows. Total solids, protein, fat, lactose and ash of whey and spray-dried WPC were measured according to AOAC method (1980), lactose method by Lawrence method, NPN (nonprotein nitrogen compound) by Lowland method (Lawrence, AJ, The determination of lactose in milk products, J. Dairy Technol., 23: 103, 1963; Rowland, SJ, The determination of nitrogen distribution in milk, J. Dairy Res., 9: 42 , 1938). In addition, the pH of whey and spray-dried WPC was measured using a pH meter (Orion 420A, USA), the bulk density was investigated according to standard methods, and 5-hydroxy-methyl-2-perfural ( HMF; 5-hydroxy-methyl-2-furfural content was determined by Keeney and Bassette, J. Dairy Sci., 42: 945-960, 1959. .

In addition, the whey protein nitrogen index (WPNI) of spray-dried products was determined by the Mahmoud method (Mahmoud, R., Brown, BJ, Ernstrom, CA, J. Dairy Sci., 73: 1694-1699, 1990), and the solubility of the spray dried WPC was analyzed according to Mor and Foegeding, Food Technol., 44: 110-112, 1990. In addition, the viscosity was measured by using a viscometer (Brookfield, Mode LVDV-E) after reducing the powder to have a total solid content of 10%, foaming capacity (Phillips et al., J. Food Sci., 52: 1074-1078, 1987), with minor modifications, expressed as% overrun. In addition, the emulsifying ability was investigated according to the turbidometric method of Pierce and Kinsella, J. Agric. Food Chem., 26: 716-720, 1987 and the emulsifying activity index (EAI). ). The results are explained in detail for each in the following examples.

Example 4 Compositional Concentration of Whey Protein Concentrate and Permeate

In the present invention, in order to optimize the manufacturing process of whey protein, the composition of the concentrate and the permeate was investigated as follows. The results of chemical analysis of the concentrate obtained by reducing the capacity in several steps are shown in Table 1, and the composition of the permeate is shown in Tables 2 and 3. NPN content indicates that protein content is important because the retention of NPN during ultrafiltration is very low. Table 4 shows the physical properties of the whey investigated during the various stages of ultrafiltration, which were determined to be due to the increase in pH, specific gravity and viscosity as the concentration progressed, and the increase in pH due to the release of some of the minerals into the permeate.

Composition of Concentrates in Whey Ultrafiltration Volume reduction (%) Constituents (%) Total solids Protein NPN Fat Lactose Ash 0 6.28 0.78 0.05 0.05 4.90 0.55 10 6.75 1.01 0.05 0.10 5.06 0.58 20 7.26 1.24 0.05 0.20 5.20 0.62 30 7.80 1.47 0.06 0.30 5.32 0.66 40 8.25 1.79 0.06 0.40 5.44 0.71 50 8.74 1.96 0.06 0.50 5.54 0.76 60 9.20 2.16 0.07 0.60 5.63 0.81 70 9.62 2.27 0.07 0.70 5.69 0.86 80 10.62 3.20 0.08 0.80 5.74 0.88 90 14.36 6.78 0.08 0.90 5.78 0.90 95 21.30 12.58 0.08 1.00 5.80 0.92 97.5 22.29 14.30 0.08 1.20 5.85 0.94

Composition of Permeate in Whey Ultrafiltration Volume Reduction (%) Total solids protein lactose Protein Lactose Instantaneous Permeate Pooled permeate Instantaneous permeate Pooled permeate Instantaneous permeate Pooled permeate Whey - - - - - - 0 4.55 - 0.17 - 3.98 3.98 50 4.60 4.50 0.18 0.16 4.12 3.96 60 4.61 4.51 0.18 0.16 4.14 3.97 70 4.77 4.53 0.19 0.16 4.23 3.98 80 4.90 4.54 0.19 0.16 4.32 3.99 90 5.05 4.60 0.24 0.17 4.39 4.04 95 5.28 4.65 0.29 0.17 4.55 4.09 97.5 5.41 4.73 0.33 0.17 4.62 4.15

Composition of Permeate in Whey Ultrafiltration Ash NPN Instantaneous Permeate Pooled permeate Instantaneous Permeate Pooled permeate Whey - - - - 0 0.40 0.40 0.02 - 50 0.30 0.28 0.02 0.02 60 0.29 0.28 0.02 0.02 70 0.36 0.30 0.03 0.02 80 0.40 0.34 0.03 0.02 90 0.42 0.36 0.03 0.02 95 0.43 0.38 0.03 0.02 97.5 0.46 0.41 0.03 0.01

Physical Properties of Concentrates in Whey Ultrafiltration Volume reduction (%) Physical properties pH Specific gravity Viscosity (cP) 0 6.40 1.03 1.16 20 6.45 1.04 1.38 30 6.45 1.04 1.63 40 6.46 1.04 1.90 50 6.46 1.05 2.53 60 6.47 1.05 2.80 70 6.47 1.06 3.96 80 6.52 1.06 4.25 95 6.58 1.07 10.98 97.5 6.74 1.07 12.20

Example 5 Investigation of Yield in Preparation of Whey Protein

In the present invention, in order to investigate the production yield during the production of whey protein, the composition of the whey is concentrated in the ultrafiltration process and the composition is analyzed in the process of manufacturing the WPC and compared with the components of the first whey as follows: Was calculated: y (yield of component) = C1 / f C ₀ ; Where the initial concentration of the C ₀ specific component, the final concentration of the C ₁ specific component, and the f concentration factor. As a result, the spray-dried WPC yield was affected by the initial protein, and if the protein content of the raw whey was low, it was confirmed that the consumption of whey was high in producing WPC. In particular, WPC-35 showed higher yields compared to WPC 50, 70, and 80 high protein products. These results showed that lactose and ash were highly concentrated due to the low permeate removal rate and the low level of lactose removal.

Composition of Spray Dried Whey Protein Concentrate Component (%) Ultrafiltration and Ultrafiltration Steps UF concentrate (80% volume reduction) UF concentrate (97.5% reduction) Stage 1 Ultrafiltration (97.5% decrease) Stage 2 Ultrafiltration (97.5% decrease) Moisture 3.80 3.75 3.40 4.10 Protein 33.50 62.20 70.50 80.20 Lactose 53.80 24.25 16.30 6.30 Fat 1.45 4.40 5.20 5.80 Ash (minerals) 7.58 5.40 4.60 3.60

Chemical Composition of Spray Dried Whey Protein Concentrate Products Components (%) Moisture Protein Lactose Fat Ash WPC-35 3.80 33.50 53.67 1.45 7.58 WPC-70 3.40 70.50 16.30 5.20 4.60 WPC-80 4.10 80.30 6.30 5.80 3.60

Protein Quality of Spray Dried Whey Protein Concentrate product Components Total nitrogen (%) Nonprotein Nitrogen (% Total Nitrogen) Protease peptone (%) Whey Protein Nitrogen (%) Lysine (g / 100g Protein) Denaturation degree (%) WPC-35 5.40 8.90 11.40 79.70 8.40 23.0 WPC-70 11.15  7.10 9.95 82.95 9.33 27.0 WPC-80 12.63  6.25 9.20 84.55 10.75 32.0

Example 6 Investigation of Physicochemical Properties of Whey Protein Hydrolysates

In the present invention, in order to investigate the physicochemical properties of whey protein hydrolyzate, total solids, protein, fat, ash, lactose content, viscosity, pH, specific gravity, HMF, whey protein nitrogen index (WPNI) ), Solubility, foaming ability, emulsifying ability was analyzed by the same method described in Example 3. In addition, the degree of hydrolysis was determined by Alder-Nissen, J. Agri. 24: 24-26, 1979) was examined by measuring the Trinitro benzene sulfonic acid (TNBS).

As a result, as shown in Table 9 and Table 10, enzymatic hydrolysis had no effect on the composition of WPC-70H, WPC-80H. Protein, lactose, fat and ash contents were not significantly changed in hydrolyzed samples and non-hydrolyzed WPC-70 and WPC-80 (p <0.05). It was confirmed that only the form of was changed and the total nitrogen content was not changed.

In addition, the pH, bulk density, and insolubility index of whey protein (WPC) and its hydrolysates were measured and the results are shown in Table 8.

Physical Properties of Spray Dried Whey Protein Concentrate Properties Product WPC-35 WPC-70 WPC-80 pH 6.58 6.74 6.78 Bulk density (g / ml) 0.52 0.45 0.40 Dispersability (%) 95.40 97.00 98.30 Wettability (sec) 35 65 80 Insolubility index (ml) 0.45 0.55 0.60 Sinkability (% transmission) 48 58 62

Chemical Composition of Spray Dried Whey Protein and Hydrolysates thereof Products Components (%) Moisture Protein Lactose Fat Ash WPC-70 4.41 70.40 14.70 6.40 4.05 WPC-70H 4.50 70.25 14.55 6.38 4.08 WPC-80 4.55 80.60 4.90 7.20 2.80 WPC-80H 4.62 80.40 4.88 7.22 2.82

Physicochemical Properties of Spray Dried Whey Protein and Hydrolysates thereof Products Attributes pH Bulk density (g / ml) Insolubility index (ml) HMF (nmol / 100g) WPNI (mg N / g) WPC-70 6.68 0.35 0.85 1.30 59.45 WPC-70H 6.55 0.42 0.62 3.20 71.75 WPC-80 6.72 0.37 0.75 0.60 74.59 WPC-80H 6.60 0.44 0.53 1.80 87.61

(1) pH Characteristics of Whey Protein Hydrolyzate

Hydrolysates of Whey Proteins WPC-70 and WPC-80 WPC-70H and WPC-80H were found to have lower pH than unhydrolyzed Whey Protein WPC-70 and WPC-80 samples. The reason was determined that the hydrolyzed sample is exposed to a lot of -COOH groups on the peptide by hydrolysis. This decrease in pH during hydrolysis has already been reported similarly in enzyme treatment experiments with various proteases (Adler-Nissen 1981, Mahmoud 1984). However, it was judged that such a decrease in pH was not so low that it cannot be used for food.

(2) density of whey protein hydrolyzate

Hydrolysates of Whey Proteins WPC-70 and WPC-80 WPC-70H and WPC-80H were measured to have a bulk density of 0.42 and 0.44 g / ml, resulting in unhydrolyzed whey proteins WPC-70 and WPC-80 The samples were significantly higher compared to the density of 0.35 and 0.37 g / mL. Therefore, it was determined that hydrolysis produced free amino acids with reduced particle size and peptides of small molecular weight, resulting in high density (Adler-Nissen 1981, Mahmoud 1984). In particular, the samples hydrolyzed with neutrase have a higher density and thus have a greater advantage in the packaging of dry products than samples without hydrolysis.

(3) Insoluble Index of Whey Protein Hydrolysates

The insoluble indices of whey proteins WPC-70 and WPC-80 showed 0.85 and 0.75, while the whey protein hydrolysates WPC-70H and WPC-80H were measured to decrease insoluble indices of 0.62 and 0.53 (see Table 11). ). This phenomenon is thought to be due to protein hydrolysis increasing the thermal stability of the protein, because the improved thermal stability reduced the degeneration of whey protein. Thus, the degree of heat treatment before and after spray drying was similar in both hydrolyzed and non-hydrolyzed WPC concentrates, while hydrolyzed WPC-70H and WPC-80H were not hydrolyzed WPC-70 and WPC. WPNI was measured higher than -80. These results indicate that the hydrolysis process reduces the degree of denaturation of whey protein.

Solubility of Spray Dried Whey Protein and Hydrolysates thereof pH Solubility WPC-70 WPC-70H WPC-80 WPC-80H 2 92.4 97.5 90.2 96.0 3 89.0 94.5 87.0 93.0 4 83.0 90.0 81.0 88.0 5 80.0 89.0 78.0 86.0 6 92.0 96.0 90.0 94.5 7 94.5 98.0 92.0 96.5 8 94.0 98.5 92.5 96.0

Example 7 Investigation of Maillard Reactions of Whey Protein Hydrolysates

Maillard reactions occurring in spray dried WPC products are expressed as HMF content. HMF content was much higher in WPC-70 and WPC-70H compared to WPC-80 and WPC-80H (see Table 11). These results were thought to be due to the high lactose content of WPC-70. However, the spray-dried WPC hydrolysates of the two samples showed high HMF content compared to their WPC, so hydrolysis affected the HMF content.

The Maillard reaction occurs as a result of the reaction between lysine and lactose, and since the hydrolyzed product has a high content of free lysine, the reaction was promoted to determine that the HMF content was high. The high HMF content and the high degree of browning change in the hydrolyzed powder were attributed to the high reaction propensity due to the high ratio of free amino acids and peptides. The degree of denaturation measured as an undenatured protein shows the effect of enzymatic hydrolysis, and the hydrolyzed WPC samples showed higher undenatured protein (WPNI) content than unhydrolyzed WPC. The positive effects of this hydrolysis were also observed for insoluble index, WPNI and bulk density.

Example 8 Viscosity Investigation of Whey Protein Hydrolyzate

The viscosity of the recombinant sample of spray dried WPC (WPC-70 and WPC-80) and its hydrolyzate was measured between pH 2-8. The hydrolyzed samples WPC-70H and WPC-80H showed a significant decrease in viscosity compared to the non-hydrolyzed WPC, and the viscosity of the non-enzymatic WPC samples was very high in the pH range of 4-5. Hydrolysis caused a significant decrease in viscosity, but WPC was found to have a very high viscosity at various pH ranges. On the other hand, in the case of hydrolyzed samples, the degree of viscosity change with pH was not so great compared to their non-hydrolyzed samples. The degree of viscosity reduction at the time of hydrolysis showed 1.54-1.65 units. This viscosity reduction is important for the use of protein hydrolysates in beverages. The change in viscosity may be due to the breakdown of some peptide bonds to produce protein hydrolysates (Adler-Nissen, J. Enzymatic hydrolysis of food proteins, Elsevier Applied Sci. Publ., New York, 1986).

 That is, during hydrolysis, long chains and large molecular weight proteins are broken down into shorter chains and lower molecular weight peptides, thereby reducing the viscosity.

Example 9 Investigation of Foam Formation of Whey Protein Hydrolyzate

The effect of WPC enzymatic hydrolysis on foaming ability in relation to the action of pH is shown in Table 12 below. As can be seen from the fact that the foaming ability expressed by overrun was markedly increased at all pH in the WPC hydrolyzate, the hydrolysis positively affected the foaming performance. The degree of improvement was particularly evident at the isoelectric point pH. Improvements in the foaming performance of enzymatic hydrolysis on WPC have been observed in other studies (Vkjayakumar et al. 2000). The phenomenon of improved foaming can be explained by the release of small molecules, peptides and amino acids, due to hydrolysis. These small peptides and amino acids tend to dissolve well in water and form bubbles well, which quickly diffuses proteins at the water interface, reducing the surface tension. This facilitates the encapsulation of bubbles. This foaming ability plays an important role in certain beverages.

Example 10 Investigation of Emulsification Capacity of Whey Protein Hydrolysate

The emulsification capacity of the spray dried WPC and WPC hydrolyzate was measured by Emulsifying activity index (EAI) from pH range 2 to 8. Higher EAI was observed at all pHs compared to the non-hydrolyzed samples in the hydrolyzed samples WPC-70H and WPC-80H. Hydrolyzed WPC showed higher EAI unit difference from 5.7 to 6.3 m 2 / g compared to unhydrolyzed protein in emulsification capacity (see Table 14). An improvement in emulsifying capacity has been observed in many hydrolysis experiments with different proteins and enzymes (Turgeon et al., J. Food Sci., 57: 601-604, 1992). These phenomena are thought to cause changes in the emulsification capacity by exposing the hidden hydrophobic interior to the water environment due to the alteration of protein molecular structure by hydrolysis (Phillips and Beuchat, 1981). Oil and hydrophobic moieties may explain the improvement in emulsification in the smooth interaction with water to produce hydrophobic moieties and cohesive surface membranes (Turgeon et al., J. Food Sci., 57: 601-604). , 1992).

Viscosity of Spray Dried Whey Protein and Hydrolysates thereof pH WPC-70 WPC-70H WPC-80 WPC-80H 2 7.5 6.4 8.2 6.9 3 8.0 6.5 8.7 7.1 4 8.8 7.0 9.5 7.5 5 9.1 7.2 9.9 7.9 6 8.4 6.8 9.1 7.4 7 8.0 6.4 8.7 7.1 8 7.6 6.3 8.3 6.9

Foaming Performance of Spray Dried Whey Protein and Its Hydrolysates pH Foaming (%) WPC-70 WPC-70H WPC-80 WPC-80H 2 420 450 460 495 3 440 475 485 525 4 480 530 520 570 5 530 595 570 630 6 470 520 505 550 7 400 440 445 480 8 340 370 390 420

Emulsification degree of spray dried whey protein and its hydrolyzate pH Emulsion activity index (EAI ㎡ / g) WPC-70 WPC-70H WPC-80 WPC-80H 2 34.2 39.4 36.4 42.0 3 33.0 38.0 35.4 41.0 4 25.2 31.7 27.8 34.5 5 22.4 29.4 24.2 31.7 6 36.1 40.5 38.2 43.7 7 38.2 43.7 40.5 46.6 8 41.0 47.5 43.5 50.5

Example 11 Investigation of DNA Damage Inhibition of Whey Protein Hydrolysate

The inhibition of DNA damage of the whey protein hydrolysate of the present invention was investigated using lymphocytes as follows. First, blood was collected from two healthy adult males, and only 5 ml of fresh whole blood was isolated using histopark (Histopaque 1077). Whey proteins hydrolyzed with four types of enzymes (alcalase, flavozyme, neutrase, protamex) were treated with lymphocytes at a concentration of 50 μg / ml in total 16 samples at different reaction times (1 to 4 hours). In addition, the lymphocytes were pre-reacted at 37 ° C. for 30 minutes and then treated with 200 μM hydrogen peroxide at 4 ° C. for 5 minutes to cause oxidative damage to the lymphocytes.

The DNA damage inhibition activity was then subjected to a Comet assay, where lymphocytes were mixed with 75 μl of 0.5% agarose gel (LMA) and 1.0% normal agarose (NMA) precoated. The suspension of cell and LMA were evenly distributed over the slide. The cover glass was covered there and stored in a 4 ° C. refrigerator. When the gel was hardened, the cover glass was removed, and then 75 μl of a 0.7% LMA solution was covered thereon. 1% Triton X-100 was added immediately prior to use in cold alkali lysis buffer (2.5 M NaCl, 100 mM Na ₂ EDTA, 10 mM Tris), and then the slides were immersed in a cold dark for 1 hour. Cell crushed slides were placed in a slide electrophoresis tank and filled with electrophoresis buffer (300 mM NaOH, 10 mM Na ₂ EDTA, pH> 13) at 4 ° C. to release the DNA for 40 minutes and treated to reveal alkali-vulnerable sites. Electrophoresis was performed for 20 minutes under a voltage of 25V / 300 ± 3 mA, and additional damage to DNA by light was prevented by covering the electrophoretic tank with a dark cloth. After the electrophoresis, the slides were dried three times by washing in a 0.4 M Tris buffer solution (pH 7.4) for 10 minutes. The nuclei were stained with ethidium bromide at a concentration of 20 μl / ml, covered with a cover slide, and observed on a fluorescence microscope (Leica, Germany). Each nucleus image on a CCD camera (Nikon, Japan) was analyzed on a computer equipped with a Komet 4.0 comet image analyzing system (Kinetic Imaging, UK).

Specifically, the degree of inhibition of DNA damage caused by hydrogen peroxide and damage by whey protein hydrolyzate in lymphocytes is shown in the% tail DNA in the tail portion separated from the nucleus to the tail portion, the DNA content in the nucleus head, and nucleus. The tail moment (TM) value of the distance (tail length, TL) or tail length of DNA fragments from the product multiplied by the percentage of DNA contained in the tail is measured and expressed. Two slides were made in each treatment group, and the DNA damage of each 100 cells was measured, and each treatment group was tested at least three times. All data were processed using the SPSS-PC + statistical package, the percentage and mean ± standard error (SE) were calculated according to each item, and ANOVA analysis was performed to compare the degree of DNA damage inhibition. F value was calculated and the difference of significance was verified by Duncan's multiple range test. All statistical significance was evaluated at 5% level.

The results are as shown in Fig. 3 and Table 15. When oxidative stress induced hydrogen peroxide in lymphocytes at a concentration of 200 μM, the% tail DNA, which represents the relative amount of damaged DNA released from the nucleus, was 43.% compared to 8.2% of the negative control treated with PBS buffer. Significantly higher, confirming DNA damage. Whey protein hydrolyzate hydrolyzed by various enzymes at 50 ㎍ / ㎖ concentration significantly reduced the DNA damage was similar to the negative control treated with PBS buffer only. In addition, it was found that whey protein hydrolyzate was excellent in inhibiting DNA damage due to oxidative stress regardless of the type of hydrolase or hydrolysis reaction time.

Effect of Whey Protein Hydrolyzate on DNA Damage Head DNA (%) Tail moment Tail length (㎛) Negative control 91.8 ± 0.5 3.1 ± 0.7 33.3 ± 6.7 Positive control 57.0 ± 2.8 137.7 ± 13.1 250.9 ± 13.6 Alcalse 1 hr 89.5 ± 2.5 7.4 ± 3.2 48.1 ± 13.4 2 hr 93.2 ± 0.6 3.3 ± 0.5 37.3 ± 6.3 3 hr 91.0 ± 0.5 3.6 ± 0.8 36.8 ± 4.2 4 hr 89.3 ± 0.3 5.4 ± 0.4 39.5 ± 3.7 Flavozyme 1 hr 91.9 ± 1.3 4.9 ± 2.1 32.9 ± 0.5 2 hr 91.5 ± 0.8 3.8 ± 0.3 33.1 ± 3.2 3 hr 91.6 ± 0.1 2.8 ± 0.4 26.5 ± 0.8 4 hr 91.7 ± 0.5 3.3 ± 0.7 32.3 ± 3.3 Neutrase 1 hr 90.0 ± 0.1 3.9 ± 0.8 26.0 ± 3.2 2 hr 91.3 ± 0.1 3.8 ± 0.4 31.8 ± 1.4 3 hr 91.0 ± 0.8 4.3 ± 0.8 33.9 ± 6.5 4 hr 91.2 ± 0.0 3.0 ± 0.2 24.3 ± 0.6 Protamex 1 hr 91.7 ± 0.1 3.7 ± 0.3 37.4 ± 2.6 2 hr 90.8 ± 0.2 6.0 ± 1.0 49.1 ± 3.4 3 hr 89.2 ± 0.7 5.6 ± 1.0 37.0 ± 0.8 4 hr 90.2 ± 0.6 4.1 ± 0.2 29.2 ± 2.1

Example 12. Antioxidant Activity of Whey Protein Hydrolyzate

In the present invention, in order to investigate the antioxidant activity of whey protein hydrolyzate, it was used that the excess iron causes an oxidative stress state that increases hydrogen peroxide (OH ·) production (see FIG. 5). Excessive iron was administered to experimental animals to induce oxidative stress An experimental diet containing cheese whey protein (WPC), cheese whey protein hydrolysate (WPH) and probiotics was prepared and fed for 6 weeks. Antioxidant activity of each sample was evaluated at in vivo levels, and the degree of DNA damage in leukocytes and colon tissue, total antioxidant activity in plasma (TRAP), conjugated dienes, and antioxidant vitamins (retinol, α-, and γ-tocopherols) Antioxidant enzymes (SOD, GSH-Px, Catalase) in erythrocytes were measured, and the data were analyzed and statistically analyzed for validity.

(1) Effects on weight gain, dietary intake and dietary efficiency

The mean dietary intake was significantly higher in the WPC and WPH + Pro groups than in the NFe and HFe groups, but the weight gain was significantly higher in the Pro and WPH + Pro groups than in the NFe and HFe groups. Low levels of WPH and probiotics were found to be beneficial in weight loss.

Food intake (g / d) Weight gain (g / d) FER NFe ^{1 )} 18.4 ± 0.4 ^{2) a4 )} 3.7 ± 0.1 ^c 19.4 ± 0.5 ^b HFe 18.6 ± 0.4 ^a 3.5 ± 0.2 ^bc 18.5 ± 1.2 ^b WPC 18.8 ± 0.3 ^ab 3.4 ± 0.1 ^abc 17.7 ± 0.8 ^b WPH 19.7 ± 0.2 ^b 3.0 ± 0.2 ^ab 14.8 ± 0.9 ^a Pro 19.4 ± 0.3 ^ab 2.9 ± 0.2 ^a 14.7 ± 1.0 ^a WPH + Pro 19.6 ± 0.2 ^b 3.0 ± 0.2 ^a 14.8 ± 1.2 ^{a 1)} NFe: Normal control group, HFe: High Fe supplemented group, WPC: High Fe + whey protein concentrates, WPH: High Fe + whey protein hydrolysates. Pro: B. polyfermenticus SCD (3 × 10 ⁸ ) and L. lactics (3 × 10 ⁸ ), WPH + Pro: High Fe + whey protein hydrolysates + B. polyfermenticus SCD (3 × 10 ⁸ ) and Lactobacillus (3 × 10 ⁸ ) ²⁾ Mean ± SE. ³⁾ FER: Food efficiency rate. ⁴⁾ Values with different superscript within a column are significantly different at p <0.05.

(2) Effect on organ weight

The effect of the experimental diet on organ weight per 100 g of rats was examined as follows. Liver weight was significantly lower in the WPH, Pro and WPH + Pro groups than in the NFe group. Heart weight was significantly higher in the HFe group than in the other groups. In the spleen, the HFe group was significantly higher than the WPH, Pro and WPH + Pro groups, and the weight of the kidneys was not significantly different between the groups.

(g / 100 g B.W) Liver Heart Kidney Spleen NFe ^{1 )} 4.18 ± 0.11 ^{2) c3 )} 0.54 ± 0.02 ^a 0.96 ± 0.02 ^{ns4 )} 0.30 ± 0.01 ^bc HFe 4.09 ± 0.08 ^bc 0.59 ± 0.01 ^b 0.97 ± 0.02 0.33 ± 0.02 ^c WPC 3.96 ± 0.11 ^abc 0.53 ± 0.02 ^a 0.94 ± 0.01 0.30 ± 0.01 ^abc WPH 3.83 ± 0.13 ^ab 0.52 ± 0.01 ^a 0.95 ± 0.02 0.28 ± 0.01 ^ab Pro 3.68 ± 0.09 ^a 0.50 ± 0.01 ^a 0.93 ± 0.02 0.26 ± 0.01 ^a WPH + Pro 3.88 ± 0.12 ^ab 0.51 ± 0.01 ^a 0.95 ± 0.01 0.29 ± 0.01 ^{ab 1)} NFe: Normal control group, HFe: High Fe supplemented group, WPC: High Fe + whey protein concentrates, WPH: High Fe + whey protein hydrolysates. Pro: B. polyfermenticus SCD (3 × 10 ⁸ ) and L. lactics (3 × 10 ⁸ ), WPH + Pro: High Fe + whey protein hydrolysates + B. polyfermenticus SCD (3 × 10 ⁸ ) and Lactobacillus (3 × 10 ⁸⁾ ) ²⁾ Mean ± SE. ³⁾ Values with different superscript within a column are significantly different at p <0.05. ⁴⁾ ns: not significant

(3) Effects on Plasma Antioxidant Vitamins

The effects on the plasma antioxidant vitamin concentration of each diet were investigated as follows. Plasma retinol was significantly higher in the WPH + Pro group than in the iron-administered group (HFe). In the γ-tocopherol group, the WPH + Pro group was significantly higher than the NFe group, and the α-tocopherol group was significantly higher in the WPC, Pro and WPH + Pro groups than the HFe group.

Retinol ^* γ-Tocopherol ^* α-Tocopherol ^* NFe ^{1 )} 0.43 ± 0.04 ^{2) ab3 )} 0.15 ± 0.02 ^a 14.08 ± 1.74 ^ab HFe 0.38 ± 0.02 ^a 0.16 ± 0.01 ^ab 12.80 ± 0.59 ^a WPC 0.44 ± 0.02 ^ab 0.19 ± 0.02 ^ab 17.12 ± 1.64 ^b WPH 0.41 ± 0.03 ^ab 0.17 ± 0.02 ^ab 15.45 ± 1.68 ^ab Pro 0.44 ± 0.04 ^ab 0.20 ± 0.02 ^ab 21.37 ± 1.35 ^a WPH + Pro 0.50 ± 0.02 ^b 0.33 ± 0.02 ^b 25.49 ± 0.91 ^{a 1)} NFe: Normal control group, HFe: High Fe supplemented group, WPC: High Fe + whey protein concentrates, WPH: High Fe + whey protein hydrolysates. Pro: B. polyfermenticus SCD (3 × 10 ⁸ ) and Lactobacillus (3 × 10 ⁸ ), WPH + Pro: High Fe + whey protein hydrolysates + B. polyfermenticus SCD (3 × 10 ⁸ ) and Lactobacillus (3 × 10 ⁸ ) ^{2 )} Mean ± SE. ³⁾ Values with different superscript within a column are significantly different at p <0.05. ^* Corrected by plasma triglycerides

(4) effect on erythrocyte antioxidant enzyme activity

The effect on the erythroid antioxidant enzyme activity of each diet is shown as follows. Erythrocyte catalase activity was significantly higher in the WPH + Pro group than in the other groups except WPC. GSH-Px was significantly increased in the WPH group compared to the Pro group.

Catalase (K / gHb) GSH-Px (U / gHb) NFe ^{1 )} 170.6 ± 24.6 ^{2) a3 )} 286.1 ± 21.8 ^ab HFe 182.3 ± 16.7 ^a 268.0 ± 19.3 ^ab WPC 212.9 ± 26.3 ^ab 282.3 ± 33.0 ^ab WPH 163.7 ± 21.4 ^a 318.1 ± 23.0 ^b Pro 166.7 ± 14.8 ^a 238.5 ± 29.2 ^a WPH + Pro 247.0 ± 21.3 ^b 280.0 ± 12.5 ^{ab 1)} NFe: Normal control group, HFe: High Fe supplemented group, WPC: High Fe + whey protein concentrates, WPH: High Fe + whey protein hydrolysates. Pro: B. polyfermenticus SCD (3 × 10 ⁸ ) and Lactobacillus (3 × 10 ⁸ ), WPH + Pro: High Fe + whey protein hydrolysates + B. polyfermenticus SCD (3 × 10 ⁸ ) and Lactobacillus (3 × 10 ⁸ ) ^{2 )} Mean ± SE. ³⁾ Values with different superscript within a column are significantly different at p <0.05.

(5) Effect on DNA damage of white blood cells and colon cells

To investigate the effect of oxidative stress induced by excessive iron administration on the DNA damage of leukocytes and colon tissues, we analyzed the comit using leukocytes and colon cells. As a result, it was observed that the DNA damage of leukocytes and colon cells was significantly increased compared to the normal control group when excessive iron was administered. Leukocytes showed a significant decrease in DNA damage in all experimental diets compared to HFe, especially in WPH + Pro group, similar to normal control (NFe). In the case of colon cells, DNA damage was significantly decreased in all experimental diet groups compared to HFe group, and DNA damage was significantly decreased in CS and MeCS groups compared to HFe group, and in WPC, WPH and WPH + Pro groups. It was also significantly decreased compared to the normal control group.

Example  13. Whey  Enzymatic Hydrolysis of Proteins

In the present invention, in order to optimize the enzymatic hydrolysis process of WPC, spray-dried WPC recombined with various protein contents was hydrolyzed using neutrase, trypsin and chymotrypsin enzymes. The most suitable hydrolysis conditions were investigated as follows. Results related to the effect of the type of enzyme, the ratio of enzyme and substrate on the degree of hydrolysis are shown in Table 20 and Table 21.

Enzymatic Hydrolysis Using Various Enzymes Time (min) enzyme Neutraze Trypsin Chamotrypsin Degree of hydrolysis (%) 20 4.17 2.79 3.43 40 4.58 3.22 4.21 60 5.03 3.87 4.59 90 5.82 4.29 4.97 120 6.02 4.68 5.33 150 6.19 4.84 5.59 180 6.31 5.01 5.75 CD 0.14

E: S Ratio of Neutrase Enzyme in WPC Hydrolysis Time (min) E: S ratio CD CD 1: 25 1: 50 1: 100 20 4.14 3.87 3.39 0.231 40 4.55 4.29 2.81 60 5.01 4.53 4.07 90 5.83 4.80 4.39 120 6.08 5.04 4.66 150 6.34 5.29 4.90 180 6.57 5.50 5.12 240 6.69 5.59 5.18 CD 0.243

In addition, in order to compare the performance of the degree of hydrolysis of the enzyme, the performance of various enzymes obtained as a result of measuring the degree of hydrolysis is shown in Table 22. When hydrolysis was performed using neutrase, trypsin, and chymotrypsin, respectively, the degree of hydrolysis after incubation for 3 hours was 6.31, 5.01, and 5.75, respectively. Neutrase showed a high degree of hydrolysis of 6.31% compared to the performance of chymotrypsin (5.75%) and trypsin (5.01%). Statistical analysis showed that the type of enzyme had a significant effect on the degree of hydrolysis. Neutrase was the most effective for hydrolysis of WPC, followed by chymotrypsin and trypsin. As shown in Table 22, the availability, efficiency, and price of the enzyme when using the Neutrase enzyme was examined.

Enzymatic Hydrolysis Using Proteolytic Enzymes Time (min) enzyme Neutraze Trypsin Chymotrypsin Degree of hydrolysis (%) 20 4.17 2.79 3.43 40 4.58 3.22 4.21 60 5.03 3.87 4.59 90 5.82 4.29 4.97 120 6.02 4.68 5.33 150 6.19 4.84 5.59 180 6.31 5.01 5.75 CD 0.14

Example 14. Preparation of Functional Beverages

In order to prepare a functional health beverage to which the whey protein hydrolyzate of the present invention was added, a mixed beverage was prepared by adding sugar, citric acid, flavoring, and coloring matter to the whey protein. Placebo was recombined with 20.8, 77.50, 1.15, 0.28, and 0.28, respectively, in the combination ratio (%). Recombinant 77.50, 1.15, 0.28 and 0.28, and sample 2 with probiotics added to the hydrolyzed WPC and sugar, citric acid, flavoring, and pigment were 20.8, 77.50, 1.15, 0.28, 0.28 and Recombinant to 0.1.

Compounding ratio of drink (%) Placebo Sample 1 Sample 2 WPC 20.80 0.00 0.00 H-WPC 0.00 20.80 20.80 Sugar 77.50 77.50 77.40 Citric acid 1.15 1.15 1.15 Spices 0.28 0.28 0.28 Pigment 0.28 0.28 0.28 Probiotic 0.00 0.00 0.10 Sum 100.00 100.00 100.00

Figure 1 schematically shows the manufacturing process of the whey protein WPC-35 of the present invention.

Figure 2 schematically shows the manufacturing process of whey protein WPC-70.

Figure 3 shows the effect of whey protein hydrolyzate of the present invention on the inhibition of DNA damage in human lymphocytes.

Figure 4 shows the image of the comit analysis using the whey protein hydrolyzate of the present invention (NC is negative control, PC is positive control).

5 shows a pattern in which oxidative stress is caused by excess iron.

Claims (16)

Whey protein hydrolyzate with antioxidant effect. The method of claim 1, The whey protein hydrolyzate is whey protein hydrolyzate, characterized in that obtained by hydrolyzing the whey protein concentrate with a protease enzyme. The method of claim 2, The whey protein hydrolyzate is characterized in that the protein content of 65 to 85% range that is used. The method of claim 2, The whey protein concentrate is whey protein hydrolyzate, characterized in that using a protein content of 30 to 40% range. The method of claim 2, The protease is whey protein hydrolyzate, characterized in that at least one selected from neutrase, trypsin, flavozyme, alkalase and protamax. (1) treating the whey protein with a protease enzyme; And (2) separating and spray drying only the reaction supernatant; Process for producing whey protein hydrolyzate of claim 1 consisting of a process. The method of claim 6, In the step (1), the whey protein (a) removes 80 to 85% permeate from the whey; And (b) spray drying the concentrate; Manufacturing method characterized by using the one produced by the process. The method of claim 7, wherein The whey protein is characterized in that the protein content of 30 to 40% range. The method of claim 6, In the step (1), the whey protein (a) removes 95 to 97.5% permeate from the whey; (b) adding the concentrate and the same amount of water; And (c) diafiltration to remove the same permeate; Manufacturing method characterized by using the one produced by the process. The method of claim 9, In addition to the above process, (d) again adding the same amount of concentrate and water and then semi-filtering to remove the same amount of permeate; Manufacturing method characterized in that the production by the process. The method of claim 9, The whey protein is characterized in that the protein content of 65 to 75% range. The method according to claim 9 or 10, The whey protein is a manufacturing method, characterized in that the protein content of 75 to 85% range. The method of claim 6, In the process (1), the protease is at least one selected from neutrase, trypsin, flavozyme, flavorase, alcalase, and protamax. . The method of claim 13, The protease is characterized in that the treatment for at least 3 hours in the temperature range 35 to 45 ℃. A functional food comprising the whey protein hydrolyzate of claim 1 as an active ingredient. A functional beverage comprising the whey protein hydrolyzate of claim 1 as an active ingredient.

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