KR100339836B1 - Manufacturing Method for High-concentration Protein Hydroly-sis by Using Hydrochloric Acid and Enzymatic Treatments - Google Patents

Abstract

The present invention relates to a method for preparing protein hydrolyzate of high concentration by hydrolyzing degreased powdered soybean protein (DSF: Defatted Soybean Flour) with hydrochloric acid, and more specifically, the protein concentration when soybean protein is enzymatically digested. In order to prevent gelation during heat sterilization, the hydrolyzed soy protein is partially hydrolyzed with low concentration of hydrochloric acid and enzymatically decomposed with protease and peptidase to increase the protein content. It relates to a method for producing the soy protein hydrolyzate described above.

In particular, the hydrolyzate of the present invention does not contain chlorohydrins, which are harmful to the human body, have high nitrogen solubility and hydrolysis, and have uniform amino nitrogen and molecular weight distribution. In addition, there is no unpleasant smell peculiar to soybean odor, fluidity is good, and the color is light yellow, so the appearance is good, so it is easy to apply to the production of new soy sauce or nutritional food.

Description

Manufacturing Method for High-concentration Protein Hydroly-sis by Using Hydrochloric Acid and Enzymatic Treatments

The present invention relates to a method for preparing protein hydrolyzate of high concentration by hydrolyzing degreasing powdered soy protein (DSF: Defatted Soybean Flour) with hydrochloric acid. More specifically, soy protein has a high protein content, and in order to prevent gelation during heat sterilization, the soy protein is partially hydrolyzed to low concentrations of hydrochloric acid and deendoprotease (endo-protease). The present invention relates to a method for producing a high protein hydrolyzate having a protein content of 12% or more by hydrolysis with an enzyme of) and an exo-protease line.

The DSF hydrolyzate of the present invention is a monochloropropanediol (MCPD: 3-chloro-1,2-propanediol) or dichloropropanol (DCP: 1,3-dichloro-2-propanol) known to be harmful to the human body It is a high concentration of soy protein that does not contain chlorohydrins and can be used for the production of new soy sauce containing no harmful substances or special foods for nutrition.

In the prior art related to the present invention, Korean Patent Publication No. 85-717 relates to a method for recovering amino acids by acid, alkali or enzymatic digestion of soy protein. Korean Patent Publication No. 88-884 relates to a method for producing rapid soy sauce using a strain having good enzymatic activity. EP 0 223 560 (1986) relates to the flavor control of protein hydrolysates. On the other hand, Collier, PD, Cromie, DD0. And Davis, AP, JAOCS, 68 (10), 785, 1991) published a mechanism for the formation of chloropropanol in protein hydrolysates, and also Clegg, KM and McMillan AD, J. Food.Tech. 9, 21, 1974) discloses an enzyme digest of a dietary protein with a bitter taste but is not related to the present invention.

As a technique for conventional protein hydrolysis, when hydrolyzed with high concentration of hydrochloric acid in soybean during the production of chemical soy, monochloropropanediol (MCPD: 3-chloro-1,2-propanediol), known as a carcinogen, dichloropropanol ( DCP: 1,3-dichloro-2-propanol) is produced and contained in soy sauce, which has been a social problem and is being replaced with brewed soy sauce to eliminate such fertilization. In other words, the decomposition of soy protein with hydrochloric acid is difficult to prevent the production of chlorohydrin, so we are trying enzymatic digestion method, but in high concentration, it is difficult to decompose soy protein into amino acid and economical method because it takes long time to decompose. This has not been done.

The present invention provides a method for preparing high protein hydrolysates by combining two methods of treating DSF with hydrochloric acid and hydrolyzing with enzyme, so that the maximum protein concentration of soy protein does not gel and does not cause high viscosity during enzymatic degradation. By establishing an optimal hydrochloric acid concentration that does not produce carcinogens due to the acid decomposition and to provide a light, clean and physically stable hydrolyzate containing a high concentration of protein.

1 is a comparison of the degree of hydrolysis (DH) of soy protein with and without acid treatment

2 is a comparison of nitrogen solubility (NSI) of protein hydrolysates according to enzyme combination

3 is nitrogen solubility of the hydrolyzate of protein according to hydrochloric acid concentration.

Figure 4 shows the changes in nitrogen solubility of protein hydrolysates according to hydrochloric acid concentration treatment and two combination enzyme treatment

5 is a degree of hydrolysis during enzymatic degradation of soy protein pretreated by hydrochloric acid concentration.

6 is a change of α -amino nitrogen ( α -Amino-Nitrogen) according to the hydrochloric acid concentration treatment and two combination enzyme treatment

Figure 7 changes in the peptide chain length of the enzyme digestion of soy protein pretreated by hydrochloric acid concentration

Using a DSF (protein content of 57.0%, carbohydrate 20.9%, fat 1.1%, commercially available products) into a four-necked round flask, it was dispersed in distilled water at a protein concentration of 8-14w / w% and heated in a mantle heater to obtain a temperature of 50 When the temperature is 0 ° C., 0.01-1.ON hydrochloric acid is added to heat the solution at 95 ° C. for 40 minutes, and the hydrochloric acid is cooled to 50 ° C. and neutralized to pH 6.0-6.2 using soda ash.

Alcalase enzyme used in the present invention is an endo-protease family of enzymes from Novo, Denmark, and is a serine protease obtained from Bacillus licheniformis and is active at pH 6-8. This has a good characteristic. Flavozyme is also an exo-protease family, and peptidase and fungal protease from Aspergillus oryzae have good activity at pH 5-7. Has characteristics. In the present invention, these alcalase and flavozyme were used for hydrolysis.

Alkalase (20A.U./kg protein) was added to 100 g of the neutralized hydrolysis solution, and the enzyme was digested at 50 ° C. for 3 hours. To increase hydrolysis, endo-protease (alkalase (20A.U./kg protein) and exo-protease (flavozyme 25 LAPU / g protein) was added for enzymatic degradation for 21 hours. Alcalase 0.002% (v / w) and flavozyme 0.005% (w / w) was added respectively.

100 g of the first enzymatically treated sample was placed in a 250 ml flask and sterilized for 15 minutes at 105 ° C., and the enzyme treatment solution cooled to 50 ° C. was filtered through a membrane filter (d = 0.45 μm) to remove cells and the hydrolyzate at 4,000 rpm. Enzymatic filtrate was obtained by centrifugation for 20 minutes, and amino nitrogen (FIG. 6) and molecular weight distribution (FIG. 7) were measured.

Viscosity of the present invention was measured by the viscosity of the acid-treated solution (Brookfield cylindrical viscometer Model LVT) by dispersing the DSF at 50 ℃, the degree of hydrolysis is the degree of peptide chain cleavage (Adler-Nissen method) in the case of acid hydrolysis In the case of enzymatic degradation, it was measured by the ninhydrin method, and the average peptide chain length was estimated by the degree of hydrolysis. Nitrogen Solubility Index (NSI) was measured by the Kjeldahl method for enzymatic filtrate, amino nitrogen was measured by formol titration method with 0.02N-NaOH solution, and the molecular weight distribution of hydrolyzate was gel permeation chromatography. (Superdex Peptide HR 10/30 column, Pharmacia, Sweden) and the absorbance was measured by a UV detector at 214nm.

Hereinafter, the present invention will be described by the following examples. However, the present invention is not limited by these.

Example 1 Physical Properties After Acid Treatment of DSF

As described in detail above, the protein content of 8%, 10%, 12%, and 14% of the protein dispersion was measured, and the color and gelation phenomena of the hydrochloric acid concentration were observed based on 12% of the protein content. The results are shown in Table 1. The concentration of hydrochloric acid was increased from 0.O1N to 1.ON, and the color was changed from light yellow to dark brown. Gelation was severe at hydrochloric acid concentrations of 0.02N, 0.5N and 0.75N. Gelling hardly occurred at hydrochloric acid concentrations of 0.55 N, 0.1 N, and 0.2 N, and light yellow to brown color showed good fluidity.

After hydrochloric acid treatment at 95 ° C. for 1 hour, the degree of gelation at 50 ° C. is expressed as follows.

+++: very strong, ++: strong, ±: almost none,-: none

The viscosity and pH change according to hydrochloric acid concentration and the change in viscosity after heat treatment at 95 ° C. were measured as shown in Table 2 below.

* PH and viscosity measured at 50 ° C after hydrochloric acid treatment without heating, heat treated at 95 ° C for 1 hour, and neutralized at pH 6.0-6.2, 50 ° C with soda ash (Na ₂ C0 ₃ ) to measure viscosity.

Example 2 Effect of Enzyme Activity on Acid Treatment and Neutralization of DSF

When neutralization with hydrochloric acid treatment and soda cycle, salt is produced, thus affecting the enzyme activity. Alkalase is reacted with the substrate at 50 ° C. for 3 hours, and the results are shown in FIG. 1.

The hydrolysis degree of the acid-treated samples was significantly higher than that of the no addition group, which was influenced by hydrochloric acid treatment on the structure of DSF particles.

Hydrolysis of the protein and carbohydrates of DSF is minimized in low concentration hydrochloric acid, but by heat treatment at 95 ° C, the structure of the particles can be easily opened by enzymatic reaction. Salts formed by alkali neutralization of 0.O5N-0.2N hydrochloric acid did not have a reverse reaction.

Example 3 Effect of Enzyme Mixture on Protein Hydrolysis

The hydrolyzate treated in Example 2 was treated with alkalase, an endo-peptidase, for 3 hours, and then DSF was digested with an enzyme mixture of exo-peptidase, flabozyme, peptidase NP-2, and promod-194 for 21 hours, The enzymatic digestion yielded a water-soluble protein, and the results are shown in FIG. 2 as Nitrogen Solubility Index (NSI).

When treated with flavozyme alone, it showed 41.9% nitrogen solubility, and the enzymes in which flavozyme and alcalase were mixed showed 65.2% and 65.7% nitrogen solubility. In order to increase nitrogen solubility, the concentration of flavozyme was increased from 25 LAPU / g protein to 50 LAPU / g protein, but the nitrogen solubility was slightly increased.

Example 4 Effect on Combination of Hydrochloric Acid Treatment and Enzyme Treatment

DSF was treated with 0.O5N-0.2N hydrochloric acid and alkalase for 3 hours, and then mixed with flavozyme and alkalase for 21 hours, followed by enzymatic digestion for 21 hours, as shown in FIGS. 3 and 4. However, it was found that NSI increased relatively as the concentration of hydrochloric acid increased as 85.8% in 0.15 hydrochloric acid, 92.1% in 0.1N hydrochloric acid, and 95.8% in 0.2N hydrochloric acid.

DSF was treated according to hydrochloric acid concentration and enzymatically decomposed with alkalase and flavozyme for 24 hours, resulting in degree of hydrolysis (DH) of 20.7% in 0.5N hydrochloric acid, 23.0% in 0.1N hydrochloric acid and 0.2N hydrochloric acid. The result is shown in FIG. 5 as 29.5%. Treatment of DSF according to hydrochloric acid concentration and enzymatic digestion with the above combination enzyme resulted in the measurement of amino nitrogen ( α -amino nitrogen content). Similar results were obtained with NSI, 1.58mg / ml in 0.15 hydrochloric acid, 0.1N hydrochloric acid. The result is shown as FIG. 6 as 1.78 mg / m1 in 1.6 mg / ml and 0.2N hydrochloric acid.

The average peptide chain length of the hydrolyzate was estimated from the degree of hydrolysis and is shown in FIG. 7. Hydrolysis with alcalase for 3 hours confirmed 3-5 amino acids. Enzymatic digestion with the above combined enzymes identified 3-5 amino acids, as shown in FIG. 7, and the molecular weight distribution was determined by gel permeation chromatography. Most of the nitrogen compounds in the hydrolyzate were peptides with a molecular weight of 360-2,000 daltons.

As enzymatic digestion was added, peptides having a molecular weight of 2,000 Daltons or more decreased, and those having a molecular weight of 360 or less increased. Half of the proteins in the hydrolysates were free amino acids, dipeptides, tripeptides and 40% of the proteins were oligopeptides.

The present invention is a chlorohydrin such as monochloropropanediol (MCPD: 3-chloro-1,2-propanediol) or dichloropropanol (DCP: 1,3-dichloro-2-propanol) which is a carcinogen. Soy protein hydrolyzate free of) is high in protein content as high as 12% and does not gel during enzymatic degradation. In particular, the hydrolyzate has high nitrogen solubility and high degree of hydrolysis and uniform amino nitrogen and molecular weight distribution. In addition, there is no unpleasant odor peculiar to soybean odor, fluidity is good, and the color is light yellow, so the appearance is good, so it is easy to apply to the production of new soy sauce or nutritional food.

Claims (2)

The soy protein was dispersed in water, heated to 50 ° C., hydrolyzed at 95 ° C. for 40 minutes with hydrochloric acid, cooled to 50 ° C. and neutralized to pH 6.0-6.2, and then added to the endo-protease. The first enzymatic digestion for a time, the second enzymatic digestion for 21 hours by mixing the endo-protease and exo-protease, and the enzyme treatment solution cooled by sterilizing the hydrolyzate at 105 ℃ for 15 minutes to 50 ℃ A method for producing a high concentration protein hydrolyzate using hydrochloric acid and enzyme, characterized in that the step of filtration with a membrane filter to remove the cells, and the hydrolyzate is centrifuged to produce an enzymatically purified filtrate. The method of claim 1, wherein the concentration of hydrochloric acid is 0.05-1.ON.