KR20230153168A - Manufacturing method of black soldier fly protein hydrolysate using protease and antioxidants containing its - Google Patents

Abstract

The present invention includes the steps of preparing raw materials by adding a solvent to defatted powder for Dongae, etc.; Hydrolyzing the protein in black soldier flies by adding a proteolytic enzyme to the raw material and hydrolyzing it so that a hydrolysis reaction occurs; and obtaining a protein hydrolyzate from the hydrolyzed black soldier fly.

Description

Method for producing protein hydrolyzate from black soldier flies using proteolytic enzymes and antioxidant composition containing protein hydrolyzate from black soldier fly {MANUFACTURING METHOD OF BLACK SOLDIER FLY PROTEIN HYDROLYSATE USING PROTEASE AND ANTIOXIDANTS CONTAINING ITS}

The present invention relates to a method for producing a protein hydrolyzate from black soldier flies using a proteolytic enzyme and an antioxidant composition containing a protein hydrolyzate from black soldier flies, etc. More specifically, the raw materials are prepared by adding a solvent to defatted powder of black soldier flies, etc.; It relates to a method of producing a protein hydrolyzate from black soldier flies by adding a proteolytic enzyme to raw materials and hydrolyzing the protein to cause a hydrolysis reaction, and to an antioxidant composition prepared therefrom.

Black Soldier Fly: Hermetia illucens (Diptera: Black Soldier Fly) larvae grow at very high densities even in organic waste, which can solve various environmental problems. They have excellent nutritional value and economic feasibility as commercial feed ingredients, so they can be used after oil recovery. Dry powder is a functional feed ingredient and is provided as feed for cattle, pigs, poultry, and fish with high value-added properties such as antibacterial properties, immune enhancement, and improved digestive ability. Some of it is also used as feed for companion animals.

The nutritional content of black soldier fly defatted powder is about 49% crude protein, 11% crude fat, and 7% crude fiber. Among the amino acids that make up protein, it has a high content of nine essential amino acids, including leucine, valine, and lysine, which are essential for animal growth. It also contains a lot of functional amino acids such as glutamic acid and aspartic acid, so it is possible to obtain an antioxidant composition containing a large amount of low-molecular-weight peptides and free amino acids that are expected to have antioxidant activity through efficient protein hydrolysis of black soldier fly.

Recently, many attempts have been made to produce bioactive peptides through hydrolysis of various insect proteins and utilize their functions. Antioxidant compositions through protein hydrolysis from black soldier flies are commercially manufactured to be used in various functional feeds such as pet food. It is expected to be used as an additive.

Publication of Patent No. 10-2022-0039195; Antioxidant composition containing enzyme decomposition products of edible insects

The purpose of the present invention is to provide a method for producing a hydrolyzate of black soldier fly, which has improved antioxidant functionality, by using the defatted powder of black soldier fly, which is currently commercially available, under optimal protein hydrolysis conditions.

Furthermore, the purpose of the present invention is to provide an antioxidant composition containing protein hydrolyzate of black soldier fly, etc., as a pet food additive, etc.

The method for producing a protein hydrolyzate using a proteolytic enzyme according to the present invention includes the steps of preparing raw materials by adding a solvent to a defatted powder of black soldier flies, etc.; Hydrolyzing the protein in black soldier flies by adding a proteolytic enzyme to the raw material and hydrolyzing it so that a hydrolysis reaction occurs; and obtaining a protein hydrolyzate from the hydrolyzed Dongae, etc.

Additionally, in the present invention, the solvent is water.

In addition, in the present invention, the proteolytic enzyme is characterized in that it is at least one selected from the group consisting of Protamax, Alcalase, and Flavorzyme.

In addition, in the present invention, the hydrolysis treatment is characterized in that the treatment is performed for 120 to 240 minutes under the conditions of pH 6 to 9 and temperature 40 to 60 ° C.

In addition, in the present invention, the amount of water added to the defatted powder for Dongae, etc. is 1:1 to 1:5 in weight ratio, and the amount of the proteolytic enzyme added is 0.1 to 5 parts by weight based on 100 parts by weight of the defatted powder for Dongae, etc. It is characterized by

In addition, in the present invention, the hydrolytic enzyme is Protamax:Alkalase, Protamax:Flaverzyme or Alkalase:Flaverzyme mixed at a ratio of 2:1 to 1:2, respectively, or Protamax:Flaverzyme. It is characterized by being selected from the group consisting of :alkalase:flavorzyme mixed in a ratio of 1:1:1.

In addition, the present invention is characterized by further comprising the step of inactivating the hydrolytic enzyme at 80 to 90° C. for 15 to 25 minutes after the hydrolysis step.

In addition, in the present invention, the step of obtaining a protein hydrolyzate from the hydrolyzed black soldier flies is characterized by taking the supernatant after hydrolyzing the protein in the black soldier flies, followed by centrifugation to separate the supernatant and the precipitate.

In addition, the antioxidant composition containing a protein hydrolyzate of black soldier flies according to the present invention is characterized in that it contains a protein hydrolyzate of black soldier flies prepared by the method for producing a protein hydrolyzate of black soldier flies using the above proteolytic enzyme as an active ingredient. do.

The method for producing protein hydrolyzate from black soldier flies according to the present invention can recover the protein hydrolyzate from black soldier flies in high yield by hydrolyzing the protein hydrolyzate under optimal conditions using a hydrolytic enzyme.

In addition, the protein hydrolyzate of Dongae et al. according to the present invention can increase the content of antioxidant functional ingredients, and the protein hydrolyzate of Dongae et al. prepared from the production method of the present invention can be used as a health functional food composition or pet food with improved antioxidant functionality. There are useful effects provided by additives, etc.

Figure 1 is a process diagram for manufacturing a protein hydrolyzate from black soldier flies using a proteolytic enzyme according to the present invention.
Figure 2 is a diagram showing the protein hydrolysis process and function of a hydrolytic enzyme.
Figure 3 shows the hydrolysis degree of each major enzyme of Novozyme (based on ground beef)
Figure 4 shows the results of component analysis of Dongae et al. powder used in the present invention.
Figure 5 is a test report showing the results of amino acid analysis of the powder of Dongae, etc. according to the present invention.
Figure 6 is a test report showing the results of analysis of constituent amino acids after hydrolysis according to the present invention.
Figure 7 is a test report showing the results of free amino acid analysis after hydrolysis according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention includes the steps of preparing raw materials by adding a solvent to defatted powder for Dongae, etc.; Hydrolyzing the protein in black soldier flies by adding a proteolytic enzyme to the raw material and hydrolyzing it so that a hydrolysis reaction occurs; and obtaining a protein hydrolyzate from the hydrolyzed Dongae, etc.

First, raw materials and hydrolytic enzymes were selected and prepared for implementation of the present invention.

The defatted powder for Dongae, etc. used in the present invention used the feed supply product of Company C as a raw material, and the analysis results are shown in Table 1 (Sweet Feed Association Sample Research Institute, Figure 4).

ingredient unit Test result Test method crude protein % 49.0 Feed standard analysis method crude fat % 11.03 Feed standard analysis method crude fiber % 7.10 Feed standard analysis method views % 22.64 Feed standard analysis method moisture % 5.48 Feed standard analysis method

The amino acid content of the black soldier fly skim powder protein is shown in Table 2 (Korea Food Research Institute amino acid automatic analyzer analysis, Figure 5).

item result item result 1. Aspartic acid 3,891.3mg/100g 9. Methionine 756.8mg/100g 2. Threonine 1,709.7mg/100g 10. Isoleusin 1,502.1mg/100g 3. Serine 1,929.7mg/100g 11. Roisin 2,707.4mg/100g 4. Glutamic acid 5,685.4mg/100g 12. Tyrosine 2,629.8mg/100g 5. Proline 2,106.2mg/100g 13. Phenylalanine 1,643.7mg/100g 6. Glycine 2,214.6mg/100g 14. Ricin 2,999.4mg/100g 7. Alanine 2,655.1mg/100g 15. Arginine 2,040.7mg/100g 8. Valine 3,065.2mg/100g Sum 38,764.1mg/100g

The proteolytic enzyme was purchased from Novozyme, and the enzymes mainly used to decompose meat proteins include Endo-type Protamex, Alcalase 2.4L, and Exo-type Flavourzyme. selected.

Proteolytic enzymes are broadly divided into two types in terms of function: Endo-type (Endoprotease) and Exo-type (Exopetidase). Endo-type enzymes randomly cleave the inside of the peptide of a protein, while Exo-type cleaves amino acid units at both ends of the peptide bond. Therefore, an appropriate combination of Exo-type enzymes is needed to resolve issues such as hydrolysis efficiency and bitter taste that may occur after treatment with Endo-type enzymes. The protein hydrolysis process and function are shown in Figure 2.

As shown in Figure 2, when hydrolyzed with Endo protease, short peptides are formed and some hydrophobic amino acids contained in them produce a bitter taste, so Exo peptide is added to decompose the bitter peptide (data from Novozyme).

The selection of proteolytic enzymes is shown in Table 3.

Enzyme type Enzyme name manufacture company form Functions and Characteristics Endoprotease Alcalase 2.4L Novozyme liquid Excellent hydrolysis, produces bitter taste Protamex Novozyme granule Excellent hydrolysis, slightly bitter taste Exopedidase Flavorzyme Novozyme granule Delayed hydrolysis, reduced bitterness

The manufacturer's recommended usage amount for proteolytic enzyme is 0.5 to 2.0 parts by weight based on protein weight, and 0.1 to 5.0 parts by weight based on 100 parts by weight of skim powder was used for Dongae, etc., depending on the administration combination of enzymes.

Raw materials are prepared by adding defatted powder and solvent to Dongae, etc. In the present invention, water was used as a solvent, and the mixing ratio of defatted powder and water in Dongae, etc. was in the range of 1:1 to 1:5 by weight.

During hydrolysis, the pH standard was 6.0~9.0, the temperature condition was 40~60℃, the stirring speed was 80~120rpm, and the hydrolysis time was 120~240 minutes.

After completion of the hydrolysis, the enzyme activity can be stopped by adjusting the pH or high temperature, but in the present invention, the enzyme function was inactivated by heating at a temperature of 80-90°C for 15-25 minutes.

In the step of obtaining the hydrolyzate, known recovery methods can be used without limitation. For example, protein hydrolyzate of Dongae et al. can be recovered by centrifuging to separate the supernatant and precipitate and then taking the supernatant.

In an embodiment of the present invention, in order to examine the quality characteristics of protein hydrolyzate, protein constituent amino acids and free amino acids at each stage were measured and analyzed using an automatic amino acid analyzer commissioned by the Korea Food Research Institute. The results of this analysis are shown in Figures 6 and 7.

In addition, the antioxidant efficacy (DPPH, ABTS) was analyzed at the Jeonju Agricultural and Life Materials Research Institute to examine the sulfation activity of protein hydrolysates obtained under each treatment condition.

Example

Example 1: Preparation of protein hydrolyzate from Dongae, etc.

Into a fermentation stirrer with a capacity of 2L, add 300g of skim powder and 700g of purified water to the prepared Dongae etc., then add 0.5 parts by weight of Alcalase and 1.0 parts by weight of Flavourzyme based on 100 parts by weight of skim powder in the Dongae etc. After adjusting the pH to 7 by adding water, temporary decomposition treatment was performed to cause a hydrolysis reaction while stirring at 100 rpm for 150 minutes at 50°C. After completion of hydrolysis, heat at 90°C for 20 minutes to inactivate enzyme activity. The supernatant and precipitate were separated through centrifugation, and the supernatant was used as a sample.

Example 2: Preparation of protein hydrolyzate from Dongae, etc.

Into a fermentation stirrer with a capacity of 2L, add 300g of skim powder and 700g of purified water to the prepared Dongae etc., then adjust the pH to 7 by adding 0.5 parts by weight of Protamex and 1.0 parts by weight of Flavourzyme based on 100 parts by weight of skim powder to the Dongae etc. Temporary decomposition treatment was performed to allow the hydrolysis reaction to occur under 50°C conditions and stirring at 100 rpm for 150 minutes. After hydrolysis is complete, the enzyme is inactivated by heating at 90°C for 20 minutes. The supernatant and precipitate were separated through centrifugation, and the supernatant was used as a sample.

Example 3 Preparation of protein hydrolyzate from Dongae et al.

Into a fermentation stirrer with a capacity of 2L, add 300g of skim powder and 700g of purified water to the prepared Dongae etc., then add 0.5 parts by weight of Alcalase, 0.5 parts by weight of Protamex, and 1.0 part by weight of Flavourzyme based on 100 parts by weight of skim powder to the Dongae etc. to pH 7. After adjusting to , temporary decomposition treatment was performed to cause a hydrolysis reaction while stirring at 100 rpm for 180 minutes at 50°C. After hydrolysis is complete, the enzyme is inactivated by heating at 90°C for 20 minutes. The supernatant and precipitate were separated through centrifugation, and the supernatant was used as a sample.

The hydrolysis reaction conditions performed in Examples 1, 2, and 3 are shown in Table 4 below.

experimental group Applicable enzyme and amount used (parts by weight) Reaction conditions (pH, temperature, time) Example 1 A(0.5), F(1.0) pH 7, 50℃, 150 minutes Example 2 P(0.5), F(1.0) pH 7, 50℃, 150 minutes Example 3 A(0.5), P(0.5), F(1.0) pH 7, 50℃, 150 minutes

In the table above, A: Alcalase 2.4L, P: Protamex, F: Flavourzyme

Experimental Example 1: Degree of hydrolysis (DH) of protein in Dongae, etc.

For the samples obtained in each of the above examples, bound amino acids were measured using an amino acid automatic analyzer (Korea Testing & Research Institute) to derive the degree of hydrolysis (DH%) (Equation 1), and the results are shown in the table below. It is shown in 5.

For reference, Novozyme's general degree of hydrolysis for each major enzyme is shown in Figure 3.

Experimental Example 2: Free amino acid content of hydrolyzate of Dongae, etc.

After the hydrolysis, free amino acids separated from the protein are produced together with the peptides decomposed from the protein. Free amino acids will be an important factor in the taste of food, etc., and the free amino acid content (%) obtained in each example is also automatically determined by amino acids. It was measured using an analyzer and is shown in Table 5 below.

experimental group Degree of hydrolysis (%) Uring amino acid content (%) Example 1 27.6 14.7 Example 2 23.8 13.8 Example 3 32.3 16.8

Experimental Example 3: Antioxidant activity assay of protein hydrolyzate from Dongae, etc.

Experimental Example 3-1: ABTS radical scavenging activity assay

The results of measuring the ABTS radical scavenging activity of the hydrolyzate of Dongae, etc. are shown in Table 6. As a result of measuring the ABTS radical scavenging activity of the hydrolyzate of Dongae et al. for 5 hours, the sample hydrolyzed in Example 3 showed the highest inhibition rate at 74.21%.

Experimental Example 3-2: DPPH radical scavenging activity assay

Table 6 shows the results of measuring the DPPH radical scavenging activity of hydrolysates of Dongae et al. As a result of measuring the DPPH radical scavenging activity of the hydrolyzate of Dongae et al. for 5 hours, the sample hydrolyzed in Example 3 showed the highest inhibition rate of 43.70%.

division Comparative example Example 1 Example 2 Example 3 ABTS radical scavenging activity 36.83±1.56 64.65±3.12 69.19±3.41 74.21±3.71 DPPH radical scavenging activity 21.35±0.46 41.64±0.23 41.75±1.00 43.70±0.53

Claims (9)

Preparing raw materials by adding a solvent to defatted powder for Dongae, etc.;
Hydrolyzing the protein in black soldier flies by adding a proteolytic enzyme to the raw material and hydrolyzing it so that a hydrolysis reaction occurs; and
A method for producing a protein hydrolyzate using a proteolytic enzyme, comprising the step of obtaining a protein hydrolyzate from the hydrolyzed black soldier fly.
The method of claim 1, wherein the solvent is water,
The method of claim 1, wherein the proteolytic enzyme is at least one selected from the group consisting of Protamax, Alcalase, and Flavourzyme. Method for producing protein hydrolyzate from Dongae, etc.
The method of claim 1, wherein the hydrolysis treatment is performed for 120 to 240 minutes under conditions of pH 6 to 9 and temperature of 40 to 60°C.
The method of claim 2, wherein the amount of water added to the defatted powder for the Dongae etc. is 1:1 to 1:5 in weight ratio,
A method for producing a protein hydrolyzate using a proteolytic enzyme, characterized in that the amount of the proteolytic enzyme added is 0.1 to 5 parts by weight based on 100 parts by weight of the defatted black soldier fly powder.
The method of claim 3, wherein the hydrolytic enzyme is Protamax:Alkalase, Protamax:Flaverzyme, or Alkalase:Flaverzyme, respectively, mixed in a ratio of 2:1 to 1:2,
Or a method for producing a protein hydrolyzate using a proteolytic enzyme selected from the group consisting of mixing Protamax: Alkalase: Flavorzyme in a ratio of 1:1:1.
The method of claim 1, further comprising the step of inactivating the hydrolytic enzyme at 80 to 90° C. for 15 to 25 minutes after the hydrolyzing step. method.
The proteolytic enzyme according to claim 1, wherein the step of obtaining a protein hydrolyzate from the hydrolyzed Dongae, etc. includes hydrolyzing the protein in the Dongae, etc., centrifuging the supernatant and the precipitate to separate the supernatant, and then taking the supernatant. Method for producing protein hydrolyzate from Dongae, etc. using .
A black soldier fly protein hydrolyzate produced by a method for producing a black soldier fly protein hydrolyzate using the proteolytic enzyme according to any one of claims 1 to 8, characterized in that it contains a black soldier fly protein hydrolyzate as an active ingredient. A composition for antioxidants containing.