SOYBEAN OLIGOPEPTIDE WITH LOW ALLERGENICITY AND LITTLE BITTERNESS AND PREPARATION METHOD AND APPLICATION THEREOF

Abstract

The present invention provides a soybean oligopeptide with low allergenicity and little bitterness and its preparation method and application. The preparation method includes the following steps: 1) mixing a soybean protein powder with water to obtain a soybean protein solution, and then performing thermal denaturation on the soybean protein solution, to prepare a denatured protein solution; 2) adjusting pH value of the denatured protein solution to 6-9, and then adding a neutral protease and papain to conduct a first enzymolysis, to obtain a first enzymatic hydrolysate; 3) adding an alkaline protease and a flavor protease into the first enzymatic hydrolysate to conduct a second enzymolysis, and after performing enzyme inactivation, to obtain a second enzymatic hydrolysate; and 4) centrifuging the second enzymatic hydrolysate, and performing membrane filtration on centrifuged supernatant liquid, to obtain the soybean oligopeptide with low allergenicity and little bitterness.

Claims

1. A method for preparation of a soybean oligopeptide with low allergenicity and little bitterness, comprising the following steps: 1) mixing a soybean protein powder with water to obtain a soybean protein solution, and performing thermal denaturation on the soybean protein solution to obtain a denatured protein solution; 2) adjusting pH value of the denatured protein solution to 6-9, and then adding a neutral protease and papain to conduct a first enzymolysis to obtain a first enzymatic hydrolysate; 3) adding an alkaline protease and a flavor protease into the first enzymatic hydrolysate to conduct a second enzymolysis, and after performing enzyme inactivation, to obtain a second enzymatic hydrolysate; and 4) centrifuging the second enzymatic hydrolysate, and performing membrane filtration on centrifuged supernatant liquid, to obtain the soybean oligopeptide with low allergenicity and little bitterness.

2. The method in accordance with claim 1, wherein a mass to volume ratio of the soybean protein powder and water is 1: (5-10).

3. The method in accordance with claim 1, wherein the performing thermal denaturation comprises: heating the soybean protein solution to 70-90 C., maintaining this temperature and continuously stirring for 20-60min.

4. The method in accordance with claim 1, wherein an amount of the neutral protease is 10-100 U/g, an amount of the papain is 10-100 U/g, the first enzymolysis is conducted at 30-60 C., and time of the first enzymolysis is controlled to be 1-3 h.

5. The method in accordance with claim 1, wherein an amount of the alkaline protease is 10-100 U/g, an amount of the flavor protease is 10-100U/g, and the second enzymolysis is conducted at 30-60 C., and time of the second enzymolysis is controlled to be 1-3 h.

6. The method in accordance with claim 1, wherein the enzyme inactivation is performed at 110-120 C., and time of the enzyme inactivation is controlled to be 10-30 min.

7. The method in accordance with claim 1, wherein the membrane filtration is performed using a filtration membrane with a pore diameter of 1-200 nm.

8. A soybean oligopeptide with low allergenicity and little bitterness prepared by the method according to claim 1, wherein in the soybean oligopeptide with low allergenicity and little bitterness, content of glycinin is less than 200 mg/kg, content of -conglycinin is less than 150 mg/kg, and content of soybean trypsin inhibitor is less than 100 mg/kg.

9. The soybean oligopeptide with low allergenicity and little bitterness in accordance with claim 8, wherein content of peptides with a molecular weight of less than 5000 Da in the soybean oligopeptide with low allergenicity and little bitterness is greater than 85% by weight, and content of peptides with a molecular weight of less than 1000 Da is greater than 60% by weight.

10. Use of the soybean oligopeptide with low allergenicity and little bitterness in accordance with claim 8 in milk powder or health food.

Description

DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described in conjunction with the examples of the present invention, and obviously, the described examples are merely part rather than all of the examples of the present invention. Based on the examples of the present invention, all other examples obtained by one with ordinary skill in the art without creative efforts shall fall into the protection scope of the present invention.

[0027] All proteases used in the present invention were bought from Novozymes Biotechnology Co., Ltd.

EXAMPLE 1

[0028] 1. Thermal Denaturation

[0029] 500 kg of a soybean protein powder with protein content of about 60% and then 4000 L of water were added into a reactor and stirred for mixing uniformly, to prepare a soybean protein solution. The soybean protein solution was heated to about 80 C., maintaining this temperature and continuously stirring for about 40 min, to prepare a denatured protein solution.

[0030] 2. First Enzymolysis

[0031] The denatured protein solution was cooled to about 50 C., and pH value thereof was adjusted to about 7. A neutral protease and papain were added into the denatured protein solution, where amounts of the neutral protease and the papain were both about 50 U per gram of soybean protein powder. Remaining at the temperature of about 50 C., a first enzymolysis was performed for about 3 h, thereby preparing a first enzymatic hydrolysate.

[0032] 3. Second Enzymolysis

[0033] To the first enzymatic hydrolysate obtained above was added an alkaline protease and a flavor protease, where an amount of the alkaline protease was about 50 U per gram of soybean protein powder, an amount of the flavor protease was about 100 U per gram of soybean protein powder. Remaining at the temperature of about 50 C., a second enzymolysis was performed for about 2 h. The resulting enzymatic hydrolysate was heated to 120 C. and subjected to enzyme inactivation for 20 min, thereby preparing a second enzymatic hydrolysate.

[0034] 4. Centrifugation and Membrane Filtration

[0035] The second enzymatic hydrolysate was centrifuged at a rotation speed of 4000 r/min, and the centrifuged supernatant liquid was collected for later use;

[0036] The centrifuged supernatant liquid was filtered with a ceramic membrane having a pore diameter of about 50 nm, where the absolute pressure during filtration was controlled at about 0.3 MPa and the temperature at about 50 C., thereby obtaining a filtrate.

[0037] 5. Decolorization, Concentration and Sterilization

[0038] To the filtrate was added an activated carbon powder in a mass ratio of 10:100 of the activated carbon powder to the filtrate. Then decolorization was performed at about 80 C. for 30 min under stirring, and after the decolorization, the activated carbon powder was removed via a plate and frame filter, to obtain a decolorized solution.

[0039] The decolorized solution was concentrated by evaporation to half of original volume thereof, where the vapor pressure was controlled at about 0.1 MPa and the evaporation temperature at about 60 C. Sterilization and spray-drying were conducted on the concentrated solution, thus preparing a soybean oligopeptide with low allergenicity and little bitterness.

[0040] 6. Performing a Quality Detection and Taste Evaluation

[0041] A Glycincin ELISA Kit (from the Unibiotest Company) and a -conglycinin ELISA Kit (from the Unibiotest Company) were used for detecting contents of glycinin and -conglycinin in the soybean oligopeptide with low allergenicity and little bitterness, respectively, a Soy Allergens reagent kit (from the ELISASYSTEM Company) was used for detecting content of the soybean trypsin inhibitor in the soybean oligopeptide with low allergenicity and little bitterness, while a soybean protein solution without any treatment was used as a blank control. Quality detection results were shown in table 1.

[0042] The molecular weight distribution of various components in the soybean oligopeptide with low allergenicity and little bitterness, as prepared above, was detected in accordance with GB/T 22729-2008. The results were shown in table 2.

[0043] The soybean oligopeptide with low allergenicity and little bitterness prepared above was dissolved in water to prepare a solution containing 10% by weight of the soybean oligopeptide with low allergenicity and little bitterness; and establishing an evaluation group of 20 people (half men and half women) for bitterness evaluation of the solution of the soybean oligopeptide with low allergenicity and little bitterness, and the evaluation method is as follows: taking 1mL of the solution of the soybean oligopeptide with low allergenicity and little bitterness, and conducting a gradient dilution on the solution, until a bitterness was just tasted, and calculating an average bitterness value of the 20 people with the dilution multiple as the bitterness value. The results were shown in table 3.

EXAMPLE 2

[0044] 1. Thermal Denaturation

[0045] 500 kg of a soybean protein powder with protein content of about 65% and then 5000 L of water were added into a reactor, and stirred for mixing uniformly, to prepare a soybean protein solution. The soybean protein solution was heated to about 90 C., maintaining this temperature and continuously stirring for about 20 min, to prepare a denatured protein solution.

[0046] 2. First Enzymolysis

[0047] The denatured protein solution was cooled to about 40 C., and pH value thereof was adjusted to about 8. A neutral protease and papain were added into the denatured protein solution, where an amount of the neutral protease was about 10 U per gram of soybean protein powder, and an amount of the papain was about 30 U per gram of soybean protein powder. Remaining at the temperature of about 40 C., a first enzymolysis was performed for about 2 h, thus preparing a first enzymatic hydrolysate.

[0048] 3. Second Enzymolysis

[0049] To the first enzymatic hydrolysate obtained above was added an alkaline protease and a flavor protease, where amounts of the alkaline protease and the flavor protease were both about 75 U per gram of soybean protein powder. Remaining at the temperature of about 40 C., a second enzymolysis was performed for about 3 h. The resulting enzymatic hydrolysate was heated to 110 C., performing enzyme inactivation for 30 min, to prepare a second enzymatic hydrolysate.

[0050] 4. Centrifugation and Membrane Filtration

[0051] The second enzymatic hydrolysate was centrifuged at a rotation speed of 3500 r/min, and the centrifuged supernatant liquid was collected for later use;

[0052] The centrifuged supernatant liquid was filtered with a filtration membrane having a pore diameter of about 200 nm, where the absolute pressure during the filtration was controlled at about 0.4 MPa and the temperature at about 80 C., thereby obtaining a filtrate.

[0053] 5. Decolorization, concentration and sterilization

[0054] To the filtrate was added an activated carbon powder in a mass ratio of 5:100 of the activated carbon powder to the filtrate. Then decolorization was conducted at about 80 C. for about 30 min under stirring, and after the decolorization, the activated carbon powder was removed via a plate and frame filter, to obtain a decolorized solution;

[0055] The decolorized solution was concentrated by evaporation to of original volume thereof, where the vapor pressure was controlled at about 0.1 MPa and the evaporation temperature at about 80 C. Sterilization and spray-drying were conducted on the concentrated solution, thereby preparing a soybean oligopeptide with low allergenicity and little bitterness. The quality detection results, molecular weight distribution and taste evaluation results of the soybean oligopeptide with low allergenicity and little bitterness were respectively shown in table 1 to table 3.

EXAMPLE 3

[0056] 1. Thermal Denaturation

[0057] 500 kg of a soybean protein powder with protein content of about 70% and then 2500 L of water were added to a reactor and stirred for mixing uniformly, to prepare a soybean protein solution. The soybean protein solution was heated to about 80 C., maintaining this temperature and continuously stirring for about 60min, to prepare a denatured protein solution.

[0058] 2. First Enzymolysis

[0059] The denatured protein solution was cooled to about 60 C., and pH value thereof was adjusted to about 6. A neutral protease and papain were added into the denatured protein solution, where an amount of the neutral protease was about 50 U per gram of soybean protein powder, and an amount of the papain was about 100 U per gram of soybean protein powder. Remaining at the temperature of about 60 C., a first enzymolysis was conducted for about 1 h, thus preparing a first enzymatic hydrolysate.

[0060] 3. Second Enzymolysis

[0061] To the first enzymatic hydrolysate obtained above was added an alkaline protease and a flavor protease, where an amount of the alkaline protease was about 40 U per gram of soybean protein powder, and an amount of the flavor protease was about 160 U per gram of soybean protein powder. Remaining at the temperature of about 60 C., a second enzymolysis was conducted for about lh. The resulting enzymatic hydrolysate was heated to 120 C. and subjected to enzyme inactivation for 20 min, thus preparing a second enzymatic hydrolysate.

[0062] 4. Centrifugation and Membrane Filtration

[0063] The second enzymatic hydrolysate was centrifuged at a rotation speed of 4000 r/min, and the centrifuged supernatant liquid was collected for later use;

[0064] The centrifuged supernatant liquid was filtered with a filtration membrane having a pore diameter of about 50 nm, where the absolute pressure during filtration was controlled at about 0.2 MPa and the temperature at about 30 C., thereby obtaining a filtrate.

[0065] 5. Decolorization, Concentration and Sterilization

[0066] To the filtrate was added an activated carbon powder in a mass ratio of 8:100 of the activated carbon powder to the filtrate. Then decolorization was conducted at about 80 C. for about 30 min under stirring, and after the decolorization, the activated carbon powder was removed via a plate and frame filter, to obtain a decolorized solution;

[0067] The decolorized solution was concentrated by evaporation to of original volume thereof, where the vapor pressure was controlled at about 0.1 MPa and the evaporation temperature at about 60 C. Sterilization and spray-drying were conducted on the concentrated solution, thus preparing a soybean oligopeptide with low allergenicity and little bitterness. The quality detection results, molecular weight distribution and taste evaluation results of the soybean oligopeptide with low allergenicity and little bitterness were respectively shown in table 1 to table 3.

COMPARATIVE EXAMPLE 1

[0068] The denatured protein solution prepared in Example 1 was cooled to about 40 C., and pH value thereof was adjusted to about 8. A neutral protease was added into the denatured protein solution in an amount of about 100 U per gram of soybean protein powder.

[0069] Maintaining at the temperature of about 40 C., an enzymolysis was performed for about 5 h, and the resulting enzymatic hydrolysate was centrifuged, concentrated, sterilized, and dried in sequence, in accordance with the method of Example 1, thus preparing a soybean peptide. The quality detection results and taste evaluation results thereof were respectively shown in table 1 and table 3.

COMPARATIVE EXAMPLE 2

[0070] The denatured protein solution prepared in Example 1 was cooled to about 50 C., and pH value thereof was adjusted to about 7. Bromelain was added into the denatured protein solution in an amount of about 250 U per gram of soybean protein powder.

[0071] Maintaining at the temperature of about 50 C., an enzymolysis was performed for about 5 h, and the resulting enzymatic hydrolysate was centrifuged, concentrated, sterilized, and dried in sequence, in accordance with the method of Example 1, thus preparing a soybean peptide. The quality detection results and taste evaluation results thereof were respectively shown in table 1 and table 3.

COMPARTIVE EXAMPLE 3

[0072] The second enzymatic hydrolysate prepared in Example 1 was directly centrifuged, concentrated, sterilized and dried in sequence to in accordance with the method of Example 1 without going through membrane filtration and decolorization, to prepare a soybean peptide. The quality detection results and taste evaluation results thereof were respectively shown in table 1 and table 3.

TABLE-US-00001 Quality detection results of soybean peptides in table 1 Content of Experimental Content of Content of soybean trypsin example glycinin -conglycinin inhibitor Blank control 3.78 10.sup.5 mg/kg 2.98 10.sup.5 mg/kg 1.57 10.sup.4 mg/kg Example 1 124.72 mg/kg 79.95 mg/kg 46.74 mg/kg Example 2 56.84 mg/kg 68.69 mg/kg 31.97 mg/kg Example 3 117.48 mg/kg 85.85 mg/kg 46.53 mg/kg Comparative 6.78 10.sup.4 mg/kg 4.11 10.sup.4 mg/kg 8.24 10.sup.3 mg/kg Example 1 Comparative 3.52 10.sup.4 mg/kg 2.45 10.sup.4 mg/kg 5.42 10.sup.3 mg/kg Example 2 Comparative Example 3 6.80 10.sup.3 mg/kg 5.82 10.sup.3 mg/kg 708.42 mg/kg

[0073] It can be concluded from the results of table 1:

[0074] 1. In the soybean oligopeptide with low allergenicity and little bitterness prepared by the present invention, the contents of allergenic proteins, namely, glycinin, -conglycinin and soybean trypsin inhibitor were significantly reduced, and the total content of the three proteins may be reduced by 99% by weight or more. This showed that the method of the present invention was able to completely eliminate allergenicity of soybean proteins and had excellent deallergization effect.

[0075] 2. The deallergization effect of the soybean proteins was not obvious when using bromelain for treating the soybean proteins; and in the case of adopting a neutral protease for treating the soybean proteins, the allergenicity of the soybean proteins was able to be eliminated to a certain extent, but the deallergization effect was not very good.

[0076] 3. The allergenic protein components of soybean were unable to be eliminated completely just through enzymolysis technology, and the soybean allergen can be eliminated to the maximum extent only using complex enzymolysis technology of the present invention in combination with specific processes such as membrane filtration and decolorization.

[0077] This showed that not arbitrary proteases or combinations thereof were able to reduce or eliminate allergenicity of soybean proteins when being used to treat the soybean proteins, and only the adoption of proteases with specific composition and specific processes (for example, pre-denaturation, stepwise enzymolysis, membrane filtration, decolorization and so on) were able to completely eliminate the allergenicity of soybean proteins.

TABLE-US-00002 TABLE 2 Molecular weight distribution of the soybean oligopeptide with low allergenicity and little bitterness Range of molecular weight Example 1 Example 2 Example 3 More than 5000 2.34 1.13 2.23 1000-5000 5.20 10.24 9.69 500-1000 22.78 27.02 29.64 140-500 65.32 56.51 54.85 Less than 140 4.22 5.09 3.49 5000 or less 97.66 98.87 97.77 1000 or less 92.32 88.62 87.98

[0078] It can be concluded from the results of table 2:

[0079] The contents of peptides with a molecular weight of less than 5000 Da in the soybean oligopeptide with low allergenicity and little bitterness prepared by the present invention were greater than 95% by weight, and the contents of peptides with a molecular weight of less than 1000 Da were greater than 85% by weight.

TABLE-US-00003 TABLE 3 Taste evaluation results of the soybean peptides Experimental examples Average bitterness value Example 1 3 Example 2 2 Example 3 2 Comparative Example 1 6 Comparative Example 2 7 Comparative Example 3 6

[0080] It can be concluded from the results of table 3:

[0081] The soybean oligopeptide with low allergenicity and little bitterness prepared by the present invention had a low amount of bitter components and thus had a good flavor, showing that the method of the present invention was able to efficiently inhibit production of bitter substances in the enzymolysis products; and the adoption of proteases such as bromelain, neutral protease, etc., for processing the soybean proteins fails to efficiently avoid the release of bitter and astringent components from the soybean proteins.

[0082] At last, it should be stated that, the above examples are merely intented to illustrate rather than limit the technical solutions of the present invention; although the present invention has been described in detail in accordance with the above examples, one with ordinary skill in the art should understand, that modifications can still be made to the technical solutions recorded in the above examples, or equivalent replacements can still be made to part or all the technical features therein; and neither these modifications nor these replacements shall make essence of the corresponding technical solutions depart from the scope of the technical solutions of these examples of the present invention.