KR20090037085A - Method of preparing whey protein hydrolysates using protease and its usage - Google Patents

Abstract

A method for preparing whey protein hydrolysates by using protease is provided to obtain whey protein hydrolysates by hydrolyzing whey protein which is the by-products of cheese, and to prepare iron bond-reinforcing protein by using the hydrolysates. A method for preparing whey protein hydrolysates by using protease comprises the steps of: (i) preparing whey protein solution of pH 7-9 by using concentrated whey protein and (ii) adding an alcalase enzyme (Bacillus licheniformi) in the whey protein solution and hydrolyzing the solution at 45-55°C. The Alcalase enzyme is used in the amount of 0.1-5 parts by weight based on the whey protein solution 100 parts by weight.

Description

Method for preparing whey protein hydrolyzate using proteolytic enzyme and use of hydrolyzate prepared thereby {METHOD OF PREPARING WHEY PROTEIN HYDROLYSATES USING PROTEASE AND ITS USAGE}

The present invention relates to a method for producing whey protein hydrolyzate using proteolytic enzymes and to the use of the hydrolyzate prepared thereby, more specifically, various milks using enzyme hydrolyzate having excellent iron binding ability and low cost. The present invention relates to a method for preparing whey protein hydrolysates using proteolytic enzymes applicable to dairy products and functional drinks, and to the use of the hydrolyzates prepared thereby.

In Korea, the dairy industry and the dairy processing industry are very difficult due to the opening of livestock imports following the WTO and FTA, and milk consumption is emerging as the most efficient way to get rid of them. In addition, domestic consumption of dairy products continues to increase, especially cheese consumption is rapidly increasing [Source: Korea Dairy Association, cheese consumption: 6.7 thousand tons ('90) → 12.4 thousand tons ('95) → 44.9 thousand tons ('00) ) → 68.9 thousand tons ('04)].

Cheese can consume about ten times the amount of milk, making it the most suitable dairy product to promote milk consumption.

However, whey, a by-product of cheese production, is 5-6 times cheaper than casein, the same milk protein, and despite its excellent functional, nutritional and physiological activities, it is mostly due to economic problems in the separation and processing processes. The use of by-products is urgently needed for feed or disposal.

In addition, as consumers' interest in high quality and high functional foods has recently increased, the development of various high value added beverages and foods is urgently needed. In addition, the necessity of developing new high value-added enhanced milk and dairy products to cope with reduced milk consumption has emerged.

Domestic iron deficiency patients are reported to account for about 25% of the adult disease, and is increasing rapidly every year, it is necessary to develop preventive, fortified drinks and treatment of patients with iron deficiency anemia.

Iron is an essential nutrient, most of which is combined with protein and absorbed in the duodenum of the small intestine. Iron deficiency is one of the most common nutritional deficiencies. Iron deficiency anemia is caused by very low absorption rate from food to the body.

In order to facilitate the absorption of iron in the body, iron must be in a solubilized state. In particular, proteins and peptides have been reported to help solubilize iron. Enzyme hydrolysates and chelated iron show absorption rates of about 80-90%, while conventional iron powders show absorption rates of only 7-20%.

On the other hand, whey, a by-product produced during cheese production, contains useful ingredients such as proteins, lactose, vitamins, and minerals. Therefore, various studies have been conducted for its use. There is no research.

In addition, whey is mostly disposed of in the dairy industry, and environmental problems are contributing to the development of functional materials using them.

Recently, as consumers' interest in high-quality and high-functional foods has increased, the development of various high value-added dairy products is urgently needed, and as the necessity of developing new high value-added enhanced milk and dairy products to cope with reduced milk consumption has emerged, the present inventors have By using whey, a by-product produced during manufacturing, to isolate and develop natural iron-binding proteins, it was applied to milk, dairy products and other functional beverages to increase the productivity of domestic dairy and dairy industries and contribute to the health of the people.

The present invention has been made to solve the problems such as utilization of the excellent whey resources and lowering the absorption rate of conventional anemia, the object of the present invention is excellent iron binding ability from the protein of whey which is a by-product produced during cheese manufacturing In addition, it is thought that the iron-binding protein can be developed from an inexpensive enzyme hydrolyzate to be used in milk, dairy products and other functional beverages in the future, thereby completing the present invention.

It is an object of the present invention to provide a method for preparing hydrolyzate for applying whey protein, a by-product produced in cheese production, to milk, dairy products and beverages.

Another object of the present invention is to provide an iron binding protein having excellent iron binding ability and a method for producing the same using the hydrolyzate.

The present invention is to prepare a whey protein solution of pH 7-9 using concentrated whey protein,

Adding an alkalase enzyme ( Bacillus licheniformi ) to the whey protein solution to hydrolyze at 45-55 ° C.

Provided are methods for preparing whey protein hydrolysates using proteolytic enzymes.

In addition, the present invention is to adjust the whey protein hydrolyzate obtained by the above method to pH 4-6,

Reacting the iron compound with the whey protein hydrolyzate,

The product obtained after the reaction is subjected to ion chromatography to provide a method for producing an iron binding enhancing protein comprising the step of obtaining a fraction.

The present invention also provides an iron binding enhancing protein obtained by the above method.

The present invention also provides milk, dairy products or beverages comprising the iron binding fortification protein.

By using the whey protein discarded by the present invention, iron-binding enhanced protein is obtained from the hydrolyzate having high iron binding ability and low cost, and can be effectively applied to additives such as milk, dairy products and various beverages. .

Whey protein hydrolysates using proteolytic enzymes according to the invention

Using whey protein concentrate (WPC) to prepare a whey protein solution of pH 7-9,

Alcalase enzyme ( Bacillus licheniformi , Bacillus licheniformi ) is added to the whey protein solution and is prepared through a step of hydrolysis at 45-55 ° C.

First, whey protein concentrate is mixed with water to prepare a whey protein solution of pH 7-9.

The whey protein concentrate may be used to collect by-products that are commercially available or produced during cheese production, and are not particularly limited in the present invention.

The pH of the whey protein solution is then adjusted to optimize the activity of subsequent alcalase enzymes.

Next, the hydrolyzate is prepared by adding an alkalase enzyme to the whey protein solution to perform hydrolysis.

The alkalase enzyme is a hydrolytic enzyme of Bacillus licheniformi derived from microorganisms, and is used in an amount of 0.1 to 5 parts by weight of an alkalase enzyme based on 100 parts by weight of a whey protein solution.

Referring to the preferred Experimental Example 1 of the present invention, the hydrolysis rate as a result of using alkalase, esparase, prabozyme, neutrase, pancreatin, papain, pepsin, protase, trypsin, etc. of hydrolysis) was very good (13.60% alkalase) (see Figure 1).

In addition, the results of electrophoresis confirmed that the whey protein hydrolyzate obtained using the alkalase was most efficiently hydrolyzed (see FIG. 2).

In particular, the present invention uses a whey protein hydrolyzate obtained by the above method to produce an iron binding enhancing protein.

Specifically, the whey protein hydrolyzate obtained by the above method is adjusted to pH 4-6.

Next, the hydrolyzate and the iron compound are reacted at 30 to 45 ° C. for 30 minutes to 2 hours.

The iron compound is FeSO ₄ · 7H ₂ O, and reacts with the hydrolyzate.

Next, the product obtained after the reaction is subjected to ion chromatography to obtain a fraction containing an iron binding enhancing protein.

Referring to Experimental Example 2 of the present invention, the iron solubility of the whey protein hydrolyzate was examined and it was confirmed that the alkalase hydrolyzate exhibited high solubility for iron.

In addition, the iron content of the fraction obtained through ion chromatography through Experiment 3 confirmed the iron binding protein of 0.21 ppm in fraction 1.

The iron binding enhancer protein thus obtained contained various amino acids, contained high Lys, Ala, Phe, etc., and did not contain Pro, Gly, Tyr, etc. (Experimental Example 4).

Such iron binding fortification protein according to the present invention can be variously applied to the production of milk, dairy products or beverages comprising the same.

Whey protein, which is used as a raw material, is currently used as a part of feed, and the rest is discarded, causing environmental problems. As the present invention, an enzyme hydrolyzate using whey protein is added as an additive of milk and dairy products to promote iron absorption. As such, it is expected to contribute to strengthening the competitiveness of the dairy and dairy industries and to improving public health.

In particular, the iron-binding protein according to the present invention has excellent competition potential in terms of price and efficacy between lactoferrin and lactoferricin, which have been added to conventional milk and dairy products for the purpose of iron fortification.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Experimental Example  One: Whey  Protein singer Decomposition  Hydrolysis Characterization

A commercial whey protein concentrate (containing 80% protein) was adjusted to pH 8.0 to prepare a whey protein solution.

Trypsin and neutrases (manufactured by Novozyme), protase and papain (manufactured by Amanosa), alkalase, esparase, prabozyme, pancreatin and pepsin (manufactured by Sigma) , Pepsin concentrated whey protein solution adjusted to pH 2) was added to 100 parts by weight of protein to 1 part by weight and hydrolyzed at 50 ℃ for 4 hours to obtain a hydrolyzate as a supernatant after centrifugation.

A: trinitrobenzene sulfonic acid method

In order to determine the hydrolysis rate of the various hydrolyzates obtained above, trinitrobenzene sulfonic acid was measured using the method, and the results are shown in FIG. 1.

1 is a graph showing the change of hydrolysis rate with time of hydrolyzate produced by various hydrolytic enzymes.

Referring to FIG. 1, the final (240 minutes) hydrolysis rate was as high as 13.60%, 12.80%, and 13.91% of alkalase, prabozyme, and pancreatin, as shown in FIG. 1.

B: electrophoresis

Electrophoresis was performed to investigate the change in the hydrolysis pattern of the various hydrolyzates obtained above, and the results are shown in FIG. 2.

Figure 2 is a graph showing the hydrolysis pattern over time of the hydrolyzate produced by various hydrolytic enzymes.

As shown in Figure 2, the alkalase (LAN 3) hydrolyzate was found to hydrolyze most of the main components most efficiently.

From the above results, the alkalase hydrolyzate which had been most ideally hydrolyzed was obtained.

Experimental Example 2: Analysis of iron solubility of whey protein hydrolysate

Each hydrolyzate obtained in Experimental Example 1 was dissolved in 1% distilled water, adjusted to pH 5.0, and 0.1% (w / v) FeSO ₄ 7H ₂ O (Sigma) was reacted at 37 ° C for 1 hour.

Subsequently, iron binding ability was indirectly investigated by confirming iron solubility of the hydrolyzate reacted with iron, and is shown in FIG. 3 and Table 1 below. At this time, the iron dissolving power was investigated by expressing the ion content of the supernatant as a percentage of the total iron ion content, and the iron ion was measured using an inductively coupled plasma spectrometer (ICP).

line Protein or hydrolysate Iron melting ability One Whey Protein Concentrate 72.40% 2 Alcalase Hydrolyzate 97.56% 3 Esparase hydrolyzate 85.3% 4 Prabozyme hydrolyzate 84.12% 5 Neutrase Hydrolyzate 82.33% 6 Pancreatin Hydrolyzate 80.02% 7 Papain Hydrolysate 80.04% 8 Pepsin hydrolysate 83.30% 9 Protease hydrolyzate 82.20% 10 Trypsin hydrolyzate 88.30%

3 is a graph showing the iron dissolving ability of the hydrolyzate produced by various hydrolase enzymes.

Referring to Table 1 and FIG. 3, the highest iron binding ability was 97.56% in the hydrolyzate using an alcalase (line 2) enzyme. As a result, alkalase hydrolyzate exhibits the highest solubility in iron, which is the most suitable and inexpensive enzyme among hydrolysates derived from a variety of commonly used.

Experimental Example 3: Isolation of Iron Binding Enhancing Proteins

The iron-bonded protein in the alkalase hydrolyzate having excellent iron binding ability obtained in Experimental Example 2 was obtained by ion chromatography (Ion exchange chromatography), and the iron content of each fraction was measured.

4 is a spectrum obtained by performing ion chromatography to separate iron binding enhancing protein from alkalase hydrolyzate.

Referring to FIG. 4, it can be seen that two fractions are obtained as a result of ion chromatography.

5 is a graph showing the iron content of each fraction obtained after performing ion chromatography.

Referring to FIG. 5, the iron content of the fractions of each fraction was measured to obtain 0.21 ppm in fraction 1 (F-1) and 0.03 ppm in fraction 2 (F-2).

Experimental Example  4: Analysis of Amino Acid Composition in Iron Binding Proteins

The iron-binding protein of fraction 1 recovered in 3 in the experiment was analyzed for amanoic acid, and the results are shown in Table 2 below. At this time, the amino acid composition of the whey protein is shown for comparison.

amino acid Whey Protein Concentrate (%) Iron binding protein of alcalase fractions (%) Asp Thr Ser Glu Pro Gly Ala Val Cys Met Ile Leu Tyr Phe Lys His Arg  7.55 7.49 6.56 16.34 8.27 3.87 9.07 4.90 0.41 1.66 4.83 11.57 1.70 2.90 9.75 1.69 1.44  4.13 2.27 2.45 6.81 ND ND 17.24 3.19 7.02 2.56 3.23 8.20 ND 16.58 18.09 5.88 2.35 total 100.00 100.00

Referring to Table 2, the concentrated whey protein had the highest concentration of Glu of 16.34%, but in the alcalase fraction (iron binding protein), Lys (18.09%) and Ala (17.24), known as iron binding sites, are bivalent cations. %), Phe (16.58%) was relatively high, Pro, Gly, Tyr was not detected.

1 is a graph showing the change of hydrolysis rate with time of hydrolyzate produced by various hydrolytic enzymes.

Figure 2 is a graph showing the hydrolysis pattern over time of the hydrolyzate produced by various hydrolytic enzymes.

3 is a graph showing the iron dissolving ability of the hydrolyzate produced by various hydrolase enzymes.

4 is a spectrum obtained by performing ion chromatography to separate iron binding enhancing protein from alkalase hydrolyzate.

5 is a graph showing the iron content of each fraction obtained after performing ion chromatography.

Claims (7)

Using whey protein concentrate to prepare a whey protein solution of pH 7-9, Adding an alkalase enzyme ( Bacillus licheniformi ) to the whey protein solution to hydrolyze at 45-55 ° C. Method for preparing whey protein hydrolyzate using proteolytic enzyme. The method of claim 1, A method for producing whey protein hydrolyzate using proteolytic enzyme, which is used at 0.1 to 5 parts by weight of an alkalase enzyme based on 100 parts by weight of the whey protein solution. Whey protein hydrolysate obtained by the method of claim 1 is adjusted to pH 4-6, Reacting the iron compound with the whey protein hydrolyzate, Performing ion chromatography on the product obtained after the reaction to obtain a fraction. Method for preparing iron binding fortification protein. The method of claim 3, The iron compound is FeSO ₄ · 7H ₂ O It is a method for producing iron binding fortification protein. The method of claim 3, The reaction of the hydrolyzate with the iron compound is carried out for 30 minutes to 2 hours at 30 ~ 45 ℃ manufacturing method of iron binding enhancing protein. Iron binding fortification protein obtained by the method of claim 3. Milk, dairy products or beverages comprising the iron binding fortified protein of claim 6.

Images