CN116634890A

CN116634890A - Bioconversion process development and application of functional black bean flour by biotransformation of enzyme group derived from bacillus

Info

Publication number: CN116634890A
Application number: CN202180077992.6A
Authority: CN
Inventors: 成文喜
Original assignee: National Biological Corp
Current assignee: National Biological Corp
Priority date: 2020-09-21
Filing date: 2021-09-23
Publication date: 2023-08-22

Abstract

The present application relates to a method for preparing bioconversion powders having various improved physiological active functions by treating with an enzyme solution derived from strain Bacillus polyfermenticus, and a method for preparing bioconversion powders using a fermentation broth of strain Bacillus polyfermenticus by confirming that a low molecular weight amino acid, peptide and various functional components are formed by treating whole soybean milk with a fermentation broth mixed with various degrading enzymes and peptide synthetases derived from strain Bacillus polyfermenticus.

Description

Bioconversion process development and application of functional black bean flour by biotransformation of enzyme group derived from bacillus

Technical Field

The present disclosure relates to a method of preparing black soybean (black soybean) bioconversion powder having enhanced functional and nutritional characteristics, and more particularly, to a method of preparing black soybean bioconversion powder having enhanced various physiological functions by treatment with an enzyme solution derived from Bacillus polyfermenticus strain.

Background

Soybeans contain proteins, fats and various functional components beneficial to the body, and are ideal foods that are nutritionally excellent and important and necessary in the diet. In addition, in recent years, with recently known physiological functions (such as anticancer properties and immune enhancement), the nutritional value of soybean as a functional food is gradually increasing. However, while soybeans play an important role as a nutritional source of protein and fat, dense tissue makes digestion and absorption very difficult. In addition, problems associated with digestion and absorption may occur in case of improper processing due to special odors, nondigestible carbohydrates and physiological inhibitors (e.g., trypsin inhibitors), causing side effects (e.g., diarrhea).

Soy milk is one of the main processed foods of soybean, a representative processed product of soybean having an improved utilization ratio of soybean protein, and is known as a functional nutritional drink because it is rich in soybean protein, essential amino acids and essential fatty acids, and contains a large amount of minerals such as iron, phosphorus and potassium, and physiologically active substances such as isoflavone, saponin and phytic acid as functional components. Recently, studies have been actively conducted to enhance food functionality and develop food-derived functional materials by elucidating the biological regulatory functions of food ingredients.

In the case of soy milk, by treating soy milk with proteolytic enzymes, degrading soy proteins, studies have been conducted to improve functionality by producing peptides having nutritional functions that promote digestion and absorption and physiological activities such as enhancing blood pressure, promoting calcium absorption, anti-allergy, and lowering serum cholesterol. However, there is still insufficient research on the development of functional regulatory biomaterials suitable for enzymatic hydrolysis processing by soy proteins and the industrialization stage of the related materials.

In addition, fermented soybean milk is prepared from soybean as a raw material by subjecting it to fermentation using microorganisms available in foods. And when only soybean is consumed in its original state, only about 30% is absorbed, and the remaining 70% is excreted; while when soybeans are fermented, more than 90% of the nutrients in the soybeans can be absorbed into the body, providing an advantage in nutrient absorption. Among them, fermented soybean milk contains not only the nutrition of soybean but also enzymes produced by microorganisms (such as amylase, protease, lipase and thrombolytic enzyme), and various physiologically active substances produced by the decomposition of these enzymes such as peptides, amino acids, oligosaccharides, fatty acids, active isoflavones, phytosterols, lecithins, saponins and the like. However, soy milk still has problems in terms of: beany flavors (which are special characteristic odors), and unpleasant mouthfeel due to the harsh texture, the soy milk losing its unique flavor due to fermentation and not meeting consumer preference in terms of taste, and the product coagulating or having a strange taste due to heat treatment.

[ Prior Art literature ]

[ patent literature ]

Korean patent application laid-open No. 10-2011-0027247 (published on 16 days of 3 months of 2011).

Disclosure of Invention

Technical object

In order to solve the above problems, an object of the present disclosure is to provide a method of preparing bioconversion powder having enhanced functionality, and a method of preparing bioconversion powder using a fermentation broth of Bacillus polyfermenticus strain by treating whole soybean milk with a fermentation broth in which various degrading enzymes derived from Bacillus polyfermenticus strain and peptide synthase are mixed, thereby determining formation of low molecular weight amino acids, peptides and various functional components.

Technical proposal

The present disclosure provides a method of preparing a bioconversion powder, the method comprising: (1) pulverizing soybeans soaked by immersing in water; (2) Heat-treating the crushed soybeans to obtain whole soybean milk; and (3) treating whole soybean milk with Bacillus polyfermenticus KMU01 (accession number: KCTC 11751 BP) strain, a culture thereof, a fermentation product thereof or a mixture thereof.

Furthermore, the present disclosure provides a health functional food composition comprising a bioconversion powder prepared by the method of preparing the bioconversion powder as an active ingredient.

Advantageous effects

According to the present disclosure, a method for preparing bioconversion powders using the fermentation broth of strain Bacillus polyfermenticus may be provided by treating whole soybean milk with a fermentation broth having mixed therein various degrading enzymes and peptide synthetases derived from strain Bacillus polyfermenticus to determine the formation of low molecular weight amino acids, peptides and various functional components.

Drawings

FIG. 1 shows the genetic map of the Bacillus polyfermenticus KMU01 strain and the genes of the enzyme group.

Fig. 2 shows the results of determining the extent of soy protein degradation and soy peptide production of whole soy milk and bioconversion powders.

FIG. 3 is a graph showing the results of evaluating the antioxidant activity of whole soybean milk and bioconversion powders.

Detailed Description

The terms used herein have been selected as much as possible from general terms currently widely used in view of the functions herein, but these terms may vary according to the intention or precedent of those skilled in the art, the appearance of new technologies, etc. Furthermore, in a specific case, there are terms arbitrarily selected by the applicant, and in this case, the meaning thereof will be described in detail in the specification of the present disclosure. Accordingly, the terms used herein should not be defined as simple names of the terms, but should be based on the meanings of the terms and the entire contents of the present disclosure.

Unless otherwise defined, all terms (including technical or scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Terms (such as those defined in commonly used dictionaries) should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Numerical ranges include values defined within the ranges. All maximum numerical limits set forth herein include all lower numerical limits as best described for the lower numerical limits. All minimum numerical limits set forth herein include all higher numerical limits as best described for the higher numerical limits. All numerical limits set forth herein will include all better numerical ranges within the broader numerical range as best illustrated for the narrower numerical limits.

Hereinafter, the present disclosure will be described in more detail.

Thus, as a result of an effort to develop a method of preparing bioconversion powders with enhanced functional and nutritional properties, the present inventors have completed the present disclosure by optimizing the enzymatic hydrolysis conditions of whole soybean milk using an enzyme group in which various proteolytic enzymes and peptide synthetases secreted and produced by GRAS-fermented food-derived microorganisms isolated from fermented foods are mixed, and studying the functionality of whole soybean milk hydrolysates to develop bioconversion powders with enhanced functional and nutritional properties and a differential preparation method allowing application in powder form.

Bioconversion refers to a technique of converting an existing material (substrate) by using a microorganism or a biological reaction of an enzyme produced by the microorganism, specifically bioconversion refers to conversion of soybean or black soybean (substrate) using an enzyme group which is a culture supernatant containing therein various enzymes produced by the Bacillus polyfermenticus KMU (accession: KCTC 11751 BP) strain, and a product obtained by the above bioconversion is referred to as bioconversion powder.

Bacillus polyfermenticus KMU01 (accession number: KCTC 11751 BP) strain was registered in Korean type culture Collection (KCTC) under the name of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Kimchi at 8.2010, but was renamed to Bacillus polyfermenticus KMU01 at 27.2018 because its exact seed name was identified as Bacillus polyfermenticus.

The soy milk may include all soy milk obtained by a conventional method, and soy milk on the commercial market may be used. For example, a liquid obtained by grinding steamed soybeans that are swelled by immersing peeled soybeans or defatted soybeans in water; and a liquid obtained by removing dregs from the liquid may be used; but is not limited thereto. In addition, a solution in which whole soybean flour or defatted soybean flour is dissolved may be used; according to JAS standards, soybean milk having a soybean solids content of 8.0% or more can be used; the soybean milk with the soybean solid content of more than 6.0% can be used for preparing soybean milk; or soybean milk having a soybean solids content of 4.0% or more may be used as the soybean milk drink, but is not limited in terms of soybean solids content.

Whole soybean milk may be treated with Bacillus polyfermenticus KMU (accession No: KCTC 11751 BP) strain, its culture, its fermentation product, or a mixture thereof at a concentration of 3% (v/v) to 7% (v/v), preferably 5% (v/v).

Bacillus polyfermenticus KMU01 (accession number: KCTC 11751 BP) strain, its culture, its fermentation product or a mixture thereof can be treated at 35℃to 40℃for 3 hours to 5 hours, preferably at 37℃for 4 hours.

The culture may be an artificial medium obtained by culturing the Bacillus polyfermenticus KMU (accession number: KCTC 11751 BP) strain, and the fermentation product may be a natural medium fermented using the Bacillus polyfermenticus KMU (accession number: KCTC 11751 BP) strain.

The artificial medium may be a commercially prepared synthetic medium capable of culturing Bacillus polyfermenticus and bacteria, such as trypsin soybean broth (TBS), trypsin Soybean Broth (TSB), nutrient Broth (NB), and Luria-Bertani broth (LB), but is not limited thereto.

The natural medium refers to a natural product that can be fermented by bacteria, and may be a medium using natural products such as potato, tomato, and milk, but is not limited thereto.

Cultures and fermentation products may exhibit protease, gamma-glutamyl transferase (GGT) and nattokinase activity.

Proteases are enzymes that hydrolyze peptide bonds between amino acids constituting proteins with proteolytic enzymes, some of which include exopeptidases that cleave the amino-terminus (aminopeptidase) or the carboxy-terminus (carboxypeptidase) of the protein, and endopeptidases that cleave the middle of the protein (e.g., trypsin, chymotrypsin, pepsin, papain, elastase). Gamma-glutamyl transferase (GGT) is an enzyme that transfers the glutamyl group in gamma-glutamyl compounds to the appropriate acceptor (amine) and is a transamidase. Nattokinase is a thrombolytic enzyme produced during growth by Bacillus natto (Bacillus natto) upon fermentation of soybeans by ingestion of nutrients of the soybeans, and includes vitamin B complex and a large amount of antioxidant enzyme.

The bioconversion powder may exhibit antioxidant activity.

Furthermore, the present disclosure provides a health functional food composition comprising as an active ingredient a bioconversion powder prepared by the method of preparing the bioconversion powder.

The present disclosure is generally applicable as a general food product.

The food composition of the present disclosure is useful as a health functional food. The term "health functional food" as used herein refers to a food manufactured and processed according to the korean health functional food law with raw materials or ingredients having useful functionalities for the human body, and the term "functional" as used herein refers to the intake of the effectiveness of obtaining health care (e.g., physiological effects of human body structure and function or regulation of nutrients).

The food composition of the present disclosure may contain common food additives, and the applicability as "food additives" is determined by specifications and standards related to respective projects according to general rules and general test methods of the korean food additives code approved by the korean food and drug safety department, unless otherwise specified.

The items listed in the korean food additive code may include, for example: chemically synthesized compounds such as ketones, glycine, potassium citrate, nicotinic acid and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sorghum pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparation, noodle-added alkaline agent, antiseptic and tar colorant.

The food compositions of the present disclosure may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills.

For example, a hard capsule formulation in a health functional food in the form of a capsule may be prepared by mixing and filling a composition according to the present disclosure with additives (e.g., excipients) in a conventional hard capsule, while a soft capsule formulation may be prepared by mixing a composition according to the present disclosure with additives (e.g., excipients) and then filling it in a capsule matrix (e.g., gelatin). If necessary, the soft capsule formulation may contain a plasticizer (such as glycerin or sorbitol), a colorant, and a preservative.

The definition of terms for excipients, binders, disintegrants, lubricants, flavoring agents and flavoring agents are described in the literature known in the art and include those having the same or similar functions. The type of food is not particularly limited and includes all health functional foods in a general sense.

Examples

Hereinafter, exemplary embodiments will be described in detail to aid in understanding the present disclosure. However, the following exemplary embodiments are merely illustrative of the present disclosure, and the scope of the present disclosure is not limited to the following examples. Exemplary embodiments of the present disclosure are provided to more fully explain the present disclosure to those of ordinary skill in the art.

Example 1 evaluation of various Activity of enzymes derived from fermented food microorganisms

To prepare a functional fermented whole soybean milk, various enzyme activities of the fermentation strain Bacillus polyfermenticus KMU01 (accession: KCTC 11751 BP) strain were evaluated.

First, activities of protease, gamma-glutamyl transferase (GGT) and nattokinase (nattokinase) were evaluated. The fermentation strain was inoculated in 50mL of Trypsin Soybean Broth (TSB) medium and cultured at 37 ℃ for 24 hours, and the culture supernatant was collected and centrifuged at 8000rpm for 20 minutes. The supernatant of the centrifuged culture was used to evaluate the activity of each enzyme.

For protease activity, 0.1mL of 0.5% azocasein solution and 0.1mL coenzyme solution were added as substrates to an Eppendorf tube, and the reaction was carried out at 37℃in a thermostatic water bath for 1 hour, followed by adding 0.4mL of 10% trichloroacetic acid solution to stop the reaction. The reaction solution was centrifuged at 13,000rpm for 5 minutes to collect a supernatant, and then the supernatant was neutralized by adding 0.6mL 0.525N NaOH solution to 0.6mL of the supernatant, absorbance was measured at 420nm, and the amount of enzyme releasing 1 μg tyrosine at 1 minute under the reaction condition was set as 1 unit to evaluate the protease activity.

For gamma-glutamyl transferase (GGT) activity, 0.01mL of coenzyme solution and 0.09mL of 50mM phosphate buffer solution (pH 7.0) containing 0.1mM gamma-L-glutamyl paranitroaniline (p-NA-Glu, sigma-Aldrich) were mixed, reacted at 40℃for 30 minutes, and then 0.01mL of 3.5N acetic acid was added to stop the reaction. The amount of free paranitroaniline was measured at 410 nm. The enzyme activity was calculated by plotting a standard curve using paranitroaniline as a standard solution. For 1 unit of enzyme activity of GGT, the degree of enzyme activity of GGT was evaluated by calculating the amount of enzyme that releases 1 mole of p-nitroaniline from p-NA-Glu per minute.

For nattokinase activity, 350. Mu.L of 50mM boric acid buffer (pH 8.5), 100. Mu.L of 1% fibrinogen solution and 25. Mu.L of 10 unit thrombin solution were mixed, reacted at 37℃for 10 minutes, then 25. Mu.L of coenzyme solution was added, followed by reaction at 37℃for 1 hour. To the reaction solution was added 500. Mu.L of 0.2M TCA solution to stop the reaction, and then the mixture was allowed to stand at 37℃for 10 minutes. After collecting the supernatant by centrifuging the reaction solution at 8,000rpm for 20 minutes, the absorbance of the collected supernatant was measured at 275nm, and the enzyme activity was calculated according to the following calculation formula to evaluate the degree of thrombolytic activity.

Thrombolytic Activity level (FU/mL) =A1-A0/0.01X1/60X 1/0.025 XD

A1: absorbance value of sample

A0: absorbance value (blank) of blank test sample prepared without coenzyme solution

0.01: enzyme activity increased by 0.01 per minute in absorbance

60: enzyme reaction time (min)

0.025: enzyme dosage

D: dilution ratio of sample

As shown in Table 1 below, it was found that the protease activity was 78U/mL, the GGT activity was 3500mU/mL, and the nattokinase activity indicating thrombolytic activity was 24U/mL.

TABLE 1

In addition, the genome of Bacillus polyfermenticus KMU01 (accession number: KCTC 11751 BP) strain was analyzed using a PacBio_20K sequencer and SMRT 2.3.0 (HGAP 2) assembler to identify genes of various functional enzymes. As a result, as shown in FIG. 1, it was confirmed that Bacillus polyfermenticus KMU01 (accession No: KCTC 11751 BP) strain had 61 peptidase genes, 23 protease genes, 8 glucosidase genes, 6 lipase genes, 2 gamma-glutamyl transpeptidase (GGT) genes, 2 cellulase genes, amylase genes and nattokinase genes.

EXAMPLE 2 preparation of functional bioconversion powders Using fermentation strains

The following were used as enzyme solutions for bioconversion of whole soy milk: supernatant obtained by culturing a Bacillus polyfermenticus KMU (accession number: KCTC 11751 BP) strain in Trypsin Soybean Broth (TSB) medium at 37℃for 24 hours. To prepare whole soybean milk, cv.socheongja from Yishan was washed and immersed in water for 14 hours, then water was removed, followed by grinding using a grinder while removing water. The ground sample was boiled at 100 ℃ for 30 minutes, and then whole soybean milk was obtained. The whole soybean milk obtained was treated with an enzyme solution at a ratio of 5% (v/v), subjected to a reaction at 37℃for 4 hours for bioconversion, and subjected to freeze-drying to prepare bioconversion powder.

TABLE 2

Category(s)	Bioconversion conditions
		Whole soybean milk (mL)	95
Enzyme solution (mL)	5
		Reaction temperature (. Degree. C.)	37
Reaction time (hr)	4

EXAMPLE 3 evaluation of degree of hydrolysis of bioconversion powders

The degree of hydrolysis of proteins was evaluated on the bioconversion powders prepared in example 2. From each sample, 2mL of the hydrolysate was taken, added to a test tube containing 2mL of 20% (w/v) trichloroacetic acid (TCA), and then centrifuged (3000 Xg, 10 min) after mixing, and a certain amount of the centrifuged supernatant was taken to measure the amount of protein and calculate the degree of hydrolysis. The calculation result revealed that the degree of hydrolysis of the bioconversion powder was 53.8%.

Further, as a result of identifying the molecular weight difference of black bean protein by performing 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as shown in fig. 2, the content of soybean peptide of 10,000da or less in the bioconversion powder (enzyme-treated black soybean milk) was increased by 1.23 times as compared with the control group (whole black soybean milk).

EXAMPLE 4 compositional analysis of amino acids in bioconversion powders

The bioconversion powders were analyzed for functional amino acids using an automated amino acid analyzer (biochrom30+). As shown in table 3 below, the content of functional amino acids, such as Branched Chain Amino Acids (BCAA) and aromatic amino acids, which are neurotransmitter precursor amino acids, was increased.

TABLE 3 Table 3

EXAMPLE 5 compositional analysis of bioconversion powders

In order to identify the functional components of the bioconversion powder, analyses of soybean dietary fiber, soybean oligosaccharide, isoflavone, flavone and anthocyanin were performed by making requests to korean functional food institute, korean analytical test institute and korean basic science institute. As shown in table 4 below, soy dietary fiber, soy oligosaccharides (raffinose, stachyose), aglycone (aglycon) isoflavones, flavones and anthocyanins were identified as components of bioconversion powders.

TABLE 4 Table 4

Furthermore, as a result of analysis of G-peptide by korean basic science institute, as shown in table 5 below, in enzyme-treated soybean milk, the contents of γ -glutamyl glycine (γ -Glu-Gly), γ -glutamyl-valine (γ -Glu-Val), γ -glutamyl-cysteine (γ -Glu-Cys), γ -glutamyl-leucine (γ -Glu-Leu) and γ -glutamyl-glutamine (γ -Glu-gin) have been increased, and their functions of increasing taste (kokumi) and relieving inflammatory bowel disease and inflammation have been reported.

TABLE 5

EXAMPLE 6 evaluation of antioxidant Activity of bioconversion powders

DPPH radical scavenging activity was analyzed to evaluate the antioxidant activity of bioconversion powders. For DPPH radical scavenging ability, as a method of measuring the degree of DPPH radical reduction using a spectrophotometer by reacting stable free radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) with a predetermined sample solution, 50 μl of a sample and 50 μl of 0.1mM DPPH solution were mixed, the mixture was allowed to stand in a dark room at room temperature for 30 minutes, and then absorbance was measured at 517nm to calculate the degree of radical reduction compared with the control group. Blank absorbance was measured by mixing 50 μl of water and 50 μl of 0.1mM DPPH solution, and control absorbance of each sample was measured by mixing 50 μl of sample with 95% ethanol. Ascorbic acid was used as a sample for the positive control group. As shown in fig. 3, the bioconversion powder showed 74% high DPPH radical scavenging activity compared to the control group (whole black soy milk).

As described above, a specific portion of the present disclosure is described in detail, and it is apparent to those skilled in the art that the detailed description is merely of a preferred embodiment, and the scope of the present disclosure is not limited thereto. In other words, the true scope of the disclosure may be defined by the following claims and their equivalents.

(translation)

Proof of original preservation

And (3) the following steps: chinese character' Chengwenxi

The glossy privet fruit-tomb hole 861-1 in the north district of the city of korea, university of national, 136-702,

the translated text is not different from the original text

(translation)

Later demonstration and revision of scientific description and/or taxonomic localization resulted in: chinese character' Chengwenxi

Korea first-hand city, northern area Zhen Ling Lu 77, national university, 02707

Accessories: notification of later specification and revision of scientific description and taxonomic localization according to clause 8.1

The translated text is not different from the original text

PCT/RO/134 table

Claims

1. A method of preparing a bioconversion powder, the method comprising:

(1) Pulverizing soybeans soaked by immersing in water;

(2) Heat-treating the crushed soybeans to obtain whole soybean milk; and

(3) With accession number: the whole soybean milk is treated with the Bacillus polyfermenticus KMU strain of KCTC 11751BP, a culture thereof, a fermentation product thereof or a mixture thereof.

2. The method of claim 1, wherein the accession number is used at a concentration of 3% (v/v) to 7% (v/v): the Bacillus polyfermenticus KMU strain of KCTC 11751BP, a culture thereof, a fermentation product thereof or a mixture thereof is treated.

3. The method of claim 1, wherein the accession number is used at 35 ℃ to 40 ℃): the Bacillus polyfermenticus KMU strain of KCTC 11751BP, its culture, its fermentation product or its mixture is treated for 3 to 8 hours.

4. The method of claim 1, wherein the culture is obtained by culturing the accession number: an artificial medium obtained from strain Bacillus polyfermenticus KMU01 of KCTC 11751 BP.

5. The method of claim 1, wherein the fermentation product is produced using the accession number: a native culture medium fermented by the Bacillus polyfermenticus KMU strain Bacillus polyfermenticus KMU of KCTC 11751 BP.

6. The method of claim 1, wherein the culture and the fermentation product exhibit protease, gamma-glutamyl transferase (GGT) and nattokinase activity.

7. The method of claim 1, wherein the bioconversion powder exhibits antioxidant activity.

8. A health functional food composition comprising as an active ingredient the bioconversion powder prepared by the method of any one of claims 1-7.