A puffed grain fermentation enzyme having superoxide dismutase activity and catalase activity, a method for producing the same, and a food composition containing the same.

Abstract

This invention relates to a puffed grain fermentation enzyme with excellent superoxide dismutase activity and catalase activity, a method for producing the same, and a food composition containing the same, obtained by fermenting puffed grain with the Bacillus amyloliquefaciens strain. According to this invention, since no food additive enzymes or coating agents are added, it has the advantage of providing a puffed grain fermentation enzyme that is superior in taste and cost-effectiveness, as well as having excellent superoxide dismutase activity and catalase activity.

Description

Technical Field

[0001] The present invention relates to an expanded cereal fermentation enzyme having superoxide dismutase (SOD) activity and catalase activity, a method for producing the same, and a food composition containing the same. More specifically, the present invention relates to an expanded cereal fermentation enzyme having excellent superoxide dismutase activity and catalase activity, a method for producing the same, and a food composition containing the same by fermenting expanded cereals with a Bacillus amyloliquefaciens strain.

Background Art

[0002] One of the most important elements of our body is an enzyme, and the enzyme is necessary for our body to function normally. If the enzyme does not exist in the body, many body functions such as breathing, digestion, and synthesis of nutrients will be lost.

[0003] When our body is young, it can synthesize and supply abundant enzymes in the body. However, modern bad eating habits such as smoking, drinking alcohol, high-fat, and excessive sugar intake stress cells and cause cell death, reducing the synthesis of enzymes in the body. Therefore, the remaining cells have to produce more digestive enzymes, and ultimately, a vicious cycle of reduced durability is repeated.

[0004] Therefore, taking enzyme agents for improving digestion can help decompose the ingested diet, reduce the synthesis of enzymes in the body, not only maintain the health of the stomach, but also improve the biological rhythm.

[0005] “Enzyme food” refers to a product obtained by culturing edible microorganisms in plant raw materials to contain a large amount of enzymes, or extracting an enzyme-containing part from food, or processing this as a main raw material.

[0006] Such enzyme-rich foods are known to contain various trace elements and physiologically active substances that aid digestion and absorption through the production of various physiologically active substances and nutrients, as well as the proliferation of beneficial bacteria, via the enzymes and fermentation and maturation processes of microorganisms within the food itself.

[0007] Grains fermented enzymes are a general term for enzyme foods made by cultivating edible microorganisms on grain raw materials to produce a large amount of enzymes. Prior art has been disclosed that the grain raw material may be one of the following grains: brown rice, soybeans, barley, or mixed grains (wheat, corn, and Job's tears) (Korean Registered Patent Publication No. 10-1855125), or grains such as rice, barley, wheat, rye, oats, or other grains, or miscellaneous grains such as millet, corn, foxtail millet, barnyard millet, buckwheat, and Job's tears (Korean Registered Patent Publication No. 10-2088758).

[0008] However, as in the present invention, there is currently no known method for fermenting puffed grains with a strain of Bacillus amyloliquefaciens to obtain a puffed grain fermentation enzyme with excellent superoxide dismutase activity and catalase activity. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Korean Registered Patent Publication No. 10-1855125 [Patent Document 2] Korean Registered Patent Publication No. 10-2088758 [Overview of the project] [Problems that the invention aims to solve]

[0010] The object of the present invention is to provide a puffed grain fermentation enzyme having superoxide dismutase activity and catalase activity by fermenting puffed grain with a Bacillus strain.

[0011] Another object of the present invention is to provide a method for producing a puffed grain fermentation enzyme having superoxide dismutase activity and catalase activity by fermenting puffed grain with a Bacillus strain.

[0012] Another object of the present invention is to provide a food composition containing a puffed grain fermentation enzyme having superoxide dismutase activity and catalase activity, obtained by fermenting puffed grain with a Bacillus strain. [Means for solving the problem]

[0013] To achieve the above objective, the present invention provides a puffed grain fermentation enzyme that is fermented by the Bacillus amyloliquefaciens strain and has superoxide dismutase activity and catalase activity.

[0014] In one embodiment of the present invention, the grain may be one or more selected from the group consisting of defatted soybeans, oats, brown rice, white soybeans, barley, sorghum, flaxseed, rice germ, sword beans, sword bean pods, wheat, rye, and millet.

[0015] In one embodiment of the present invention, the strain may be Bacillus amyloliquefaciens NPKE6 deposited under accession number KCCM13257P.

[0016] In one embodiment of the present invention, the puffed grain fermentation enzyme has a superoxide dismutase activity of 37-88% at a concentration of 10-100 mg / mL and a catalase activity of 1439-3276 mU / mL at a concentration of 5-100 mg / mL, wherein 1 unit of catalase activity may be the amount that decomposes 1 μmole of H2O2 in 1 minute at pH 7.0 and 25°C.

[0017] As one embodiment of the present invention, the cereal may be at least one selected from the group consisting of defatted soybeans, oats, brown rice, white soybeans, barley, sorghum, flaxseeds, rice germ, jack beans, jack bean pods, wheat, rye, and millet.

[0018] To achieve the above object, the present invention provides a method for producing an expanded cereal fermentation enzyme, comprising: (a) a step of pulverizing expanded cereal; (b) a step of immersing the expanded cereal in water; (c) a step of inoculating Bacillus amyloliquefaciens strain into the cereal and then mixing; and (d) a step of fermenting the cereal inoculated with the strain.

[0019] As one embodiment of the present invention, the production method may further include, before the step (a), a step of putting the cereal into an extrusion expander and expanding it under the conditions of a temperature of 140 to 160°C and a pressure of 80 to 120 bar to obtain expanded cereal, and may further include, after the step (d), steps of hot air drying and pulverizing.

[0020] As one embodiment of the present invention, the cereal may be at least one selected from the group consisting of defatted soybeans, oats, brown rice, white soybeans, barley, sorghum, flaxseeds, rice germ, jack beans, jack bean pods, wheat, rye, and millet.

[0021] As one embodiment of the present invention, the strain may be Bacillus amyloliquefaciens NPKE6 deposited under the accession number KCCM13257P.

[0022] To achieve the above object, the present invention provides a food composition containing the expanded cereal fermentation enzyme.

[0023] As one embodiment of the present invention, the food composition may be an enzyme food.

Advantages of the Invention

[0024] According to the present invention, since no food additive enzymes or coating agents are added, it has the advantage of being superior in taste and cost-effectiveness, and it can provide a puffed grain fermentation enzyme that has superoxide dismutase activity and catalase activity.

[0025] Furthermore, according to the present invention, by adding only the aforementioned expanded grain fermentation enzyme and plant-based raw materials, it complies with the recommendations of the Korea Food and Drug Administration, and by using grain raw materials, it has the advantage of being able to provide a health-oriented superfood. [Brief explanation of the drawing]

[0026] [Figure 1] This shows the 16S rRNA base sequence of the Bacillus amyloliquefaciens NPKE6 strain (KCCM13257P) of the present invention. [Figure 2] This is a diagram illustrating the manufacturing process of a puffed grain fermentation enzyme according to one embodiment of the present invention. [Figure 3] This figure shows the DPPH radical scavenging ability of a puffed grain fermentation enzyme according to one embodiment of the present invention. [Figure 4] This figure shows the SOD activity of a puffed grain fermentation enzyme according to one embodiment of the present invention. [Figure 5] This figure shows the catalase activity of a puffed grain fermentation enzyme according to one embodiment of the present invention. [Modes for carrying out the invention]

[0027] A first embodiment of the present invention relates to a puffed grain fermentation enzyme fermented by the Bacillus amyloliquefaciens strain and possessing superoxide dismutase activity and catalase activity.

[0028] The aforementioned strain may be Bacillus amyloliquefaciens NPKE6 deposited under accession number KCCM13257P, and this strain may possess superoxide dismutase (SOD) activity and catalase activity.

[0029] The aforementioned Bacillus amyloliquefaciens NPKE6 strain may, but is not limited to, possess DPPH radical scavenging ability.

[0030] Furthermore, the puffed grain fermentation enzyme of the present invention has a superoxide dismutase activity of 37-88% at a concentration of 10-100 mg / mL and a catalase activity of 1439-3276 mU / mL at a concentration of 5-100 mg / mL, wherein 1 unit of catalase activity is the amount that decomposes 1 μmole of H2O2 in 1 minute at pH 7.0 and 25°C.

[0031] In one embodiment of the present invention, the grain may be one or more selected from the group consisting of defatted soybeans, oats, brown rice, white soybeans, barley, sorghum, flaxseed, rice germ, sword beans, sword bean pods, wheat, rye, and millet, but is not limited thereto.

[0032] A second embodiment of the present invention relates to a method for producing a fermented enzyme for puffed grains, comprising the steps of (a) crushing puffed grains, (b) immersing the puffed grains in water, (c) inoculating the grains with a strain of Bacillus amyloricephasiensis and then mixing them, and (d) fermenting the grains inoculated with the strain.

[0033] The method for producing the puffed grain fermentation enzyme of the present invention may further include, before step (a), a step of putting grain into an extruder and puffing it at a temperature of 140 to 160°C, preferably 145 to 155°C, more preferably 150°C, at a pressure of 80 to 120 bar, preferably 90 to 110 bar, more preferably 100 bar to obtain puffed grain.

[0034] Furthermore, the step after step (d) may further include a step of hot air drying and powdering, but the powdering can be used to form other dosage forms as needed.

[0035] In the method for producing the expanded grain fermentation enzyme of the present invention, the grain may be one or more selected from the group consisting of defatted soybeans, oats, brown rice, white soybeans, barley, sorghum, flaxseed, rice germ, sword beans, sword bean pods, wheat, rye, and millet, but is not limited thereto.

[0036] In the method for producing the puffed grain fermentation enzyme of the present invention, the bacterial strain may be, but is not limited to, Bacillus amyloliquefaciens NPKE6 deposited under accession number KCCM13257P.

[0037] A third embodiment of the present invention relates to a food composition containing the puffed grain fermentation enzyme.

[0038] The food composition may preferably be an enzyme food, but is not limited thereto. In addition to the puffed grain fermentation enzyme, the food composition may further contain, but is not limited thereto, two or more fermented mixed grains selected from the group consisting of brown rice, white soybeans, sorghum, and barley; two or more mixed grain powders selected from the group consisting of roasted brown rice, roasted soybeans, and papaya extract; and potato extract powder.

[0039] The food composition may contain, but is not limited to, 20-40% by weight of the fermented grain product, 50-70% by weight of the fermented mixed grain, 1-5% by weight of the mixed grain powder, and 1-5% by weight of the potato extract powder, based on the total weight.

[0040] The food composition may preferably contain, but is not limited to, 30-35% by weight of the puffed grain fermentation enzyme, 55-65% by weight of the fermented mixed grain, 2-4% by weight of the mixed grain powder, and 2-4% by weight of the potato extract powder, based on the total weight.

[0041] The food composition may more preferably contain, but is not limited to, 33-34% by weight of the puffed grain fermentation enzyme, 60-62% by weight of the fermented mixed grain, 2.5-3.5% by weight of the mixed grain powder, and 2.5-3.5% by weight of the potato extract powder, based on the total weight.

[0042] The brown rice that may be included in the aforementioned fermented mixed grains is rice that has been dried and threshed after harvesting, and then had its husks removed using a machine consisting of rubber rollers. It is known to have an effect in preventing lifestyle-related diseases.

[0043] White soybeans, which may be included in the aforementioned fermented mixed grains, are also called miso beans, and are particularly rich in nutrients such as lecithin, saponins, isoflavones, and trypsin inhibitors. These components have anti-cancer effects, lower blood cholesterol, prevent obesity by suppressing fat synthesis, and prevent constipation by stimulating bowel movements through intestinal regulation.

[0044] The sorghum that may be included in the aforementioned fermented mixed grains is low in fat and calories, making it suitable as a diet food. It has the effect of improving cholesterol levels and breaking down residues in blood vessels. The proanthocyanidins contained in sorghum strengthen the immune function of the bladder and play a role in reducing oxidative stress on cells and alleviating inflammation.

[0045] The wheat that may be included in the aforementioned fermented mixed grains helps to improve constipation due to its abundant dietary fiber, and further contributes to antioxidant effects and improved immunity due to its rich content of polyphenol compounds. It also contains tocotrienols, which can lower cholesterol levels and help prevent vascular diseases.

[0046] The papaya extract that may be included in the aforementioned grain mixture powder contains papain, a type of proteolytic enzyme extracted from papaya fruit. Papain is known to be one of the most potent digestive enzymes found in nature, helping to activate digestive function and having various effects such as suppressing gastric distension, promoting diuresis, and anti-inflammatory effects.

[0047] In the potato extract powder contained in the aforementioned food composition, the potato is an alkaline food that protects and improves ulcers and mucous membranes caused by excessive stomach acid, thus protecting the stomach, contains a large amount of vitamin C (similar to the amount found in spinach and oranges), the vitamin C in potatoes is trapped in starch and is relatively heat-resistant, contains a high amount of potassium (410-485 mg per 100 g) compared to other vegetables and foods, and the starch in potatoes contains a large amount of pectin, a type of dietary fiber, which has the effect of facilitating bowel function and stool.

[0048] The present invention will be described in detail below with reference to examples and other details to aid in understanding the present invention. However, the examples of the present invention can be modified into a variety of other forms and should not be construed as limiting the scope of the present invention. The examples of the present invention are provided to give a more complete explanation of the present invention to those who are ordinary skill in the art.

[0049] <Example 1> Identification of Bacillus amyloricephaciens strain NPKE6 1. Analysis of the nucleotide sequence of Bacillus amyloricephaciens strain NPKE6 DNA extraction and 16S rRNA sequencing analysis were performed by Macrogen Corporation for identification. Bacterial identification was performed using the Basic Local Alignment Search Tool (BLAST) search engine of the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov) based on the 16S rRNA sequence.

[0050] Analysis of the 16S rRNA sequences of the isolated strains for identification and classification yielded the sequence of Sequence ID No. 1.

[0051] 2. Confirmation of the biochemical characteristics of the Bacillus amyloricephasiens NPKE6 strain. The biochemical characteristics of the Bacillus amyloricephaciens NPKE6 strain were investigated using API 50CH (bioMerieux Co., France), which is used for Bacillus identification. The API kit was used according to the manufacturer's guidelines, and the results are shown in Table 1 below.

[0052] Bacillus amyloricephaciens NPKE6 strain was inoculated into liquid medium of TSB (17.0 g casein, 3.0 g soybean meal, 5.0 g NaCl, 2.5 g dipotassium phosphate, 2.5 g dextrose, final pH 7.3 ± 0.2 at 25°C), cultured at 37°C, then dispensed into Eppendorf tubes and centrifuged at 10,000 rpm for 5 minutes. Subsequently, the cells were harvested and washed once with sterile physiological saline (0.85%). The cells were suspended in sterile physiological saline, inoculated into ampoules of aseptically disrupted API 50CH medium, pipetted to homogeneous mixture, and dispensed 150 μL into microtubes for each test strip.

[0053] After dispensing, the API kits were incubated in a 37°C incubator for 24 hours, and the color changes were observed. The results are shown in Table 1. In Table 1 below, "+" indicates the use of each enzyme, and "-" indicates the absence of each enzyme.

[0054] [Table 1]

[0055] Based on the identification results and a comprehensive evaluation of the biochemical characteristics, the selected NPKE6 strain was identified as Bacillus amyloliquefaciens and named Bacillus amyloliquefaciens NPKE6. It was deposited with the Korean Culture Center of Microorganisms (KCCM) on October 31, 2022, and assigned accession number KCCM13257P.

[0056] <Example 2> Production of grain fermentation enzyme using Bacillus amyloricephaciens NPKE6 strain 1. Culturing of bacterial strains Bacillus amyloliquefaciens NPKE6 was inoculated (1.0% v / v) into BM medium (D-glucose monohydrate (13.5 g / L), hydrolyzed soy protein (3 g / L), yeast extract (3 g / L), sodium carbonate (0.3 g / L), magnesium sulfate heptahydrate (0.2 g / L)) and incubated at 30°C for 24 hours.

[0057] 2. Production of puffed grains After weighing the grain, 18% water was added and mixed. The mixture was expanded into 2.5 cm x 2.5 cm particles at 150°C and 100 bar, and cooled with 30°C water.

[0058] 3. Production of expanded grain fermentation enzyme The puffed grains were weighed, 9 times the amount of sterile water at 70°C was added, and the grains were soaked for 20 minutes. The unabsorbed sterile water was then removed. 2.5 kg of the soaked grains were placed in each tray, and the aforementioned Bacillus amyloliquefaciens NPKE6 strain, which had been cultured in BM medium at 30°C and 150 rpm for 24 hours, was inoculated at a 2% (v / w) inoculation rate relative to the soaked grains.

[0059] The grain was fermented at 30°C and over 80% relative humidity for 24 hours, then hot-air dried at 70°C for 24 hours, and finally ground to a size of 60 mesh using a multi-purpose pulverizer to produce grain fermentation enzymes.

[0060] <Example 3> Analysis of the enzymatic activity of the fermentation enzyme of expanded grains 1. Confirmation of DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging ability. The expanded grain fermentation enzyme produced in Example 2 was diluted to different concentrations at sterile temperatures, centrifuged at 4000 rpm for 20 minutes, and then filtered through a 0.2 μm filter to produce an expanded grain fermentation enzyme filtrate.

[0061] 80 μL of each diluted sample was mixed with 500 μL of 0.2 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) solution and 420 μL of ethanol. After measuring the absorbance at 517 nm, the DPPH radical scavenging ability was calculated using the following formula, and the results are shown in Figure 3.

[0062] DPPH radical scavenging ability (%) = {1 - (absorbance of sample-added group / absorbance of negative control group)} * 100

[0063] As shown in Figure 3, it can be confirmed that the DPPH radical scavenging ability of the expanded grain fermentation enzyme increases in a concentration-dependent manner.

[0064] 2.SOD activity Using the EZ-SOD Assay Kit (DoGen, Korea), the SOD activity of the puffed grain fermentation enzyme produced in Example 2 was measured according to the manufacturer's manual.

[0065] Specifically, the expanded grain fermentation enzyme was diluted to different concentrations in a sterile number of solutions, centrifuged at 4000 rpm for 20 minutes, and then filtered through a 0.2 μm filter to produce the expanded grain fermentation enzyme filtrate. As shown in Table 2 below, 20 μL of each diluted sample was added to each sample well and to the well of Blank 2. At this time, 20 μL of ddH2O was added to the wells of Blank 1 and Blank 3, respectively.

[0066] 200 μL of WST working solution was added to each well. 20 μL of dilution buffer was added to the wells of Blank 2 and Blank 3. Using a multichannel pipette, 20 μL of Enzyme working solution was added to the wells of Blank 1 and the sample, and then carefully mixed. The 96-well plate was incubated at 37°C for 20 minutes.

[0067] [Table 2]

[0068] Figure 4 shows the SOD activity of the puffed grain fermentation enzyme, calculated using the following formula after measuring the absorbance at 450 nm.

[0069] SOD activity={(OD blank1 -OD blank3 )-(OD sample -OD blank2 )} / (OD blank1 -OD blank3 )*100

[0070] As shown in Figure 4, it can be confirmed that the SOD activity of the expanded grain fermentation enzyme increases in a concentration-dependent manner.

[0071] 3. Catalase activity Using the EZ-Catalase Assay Kit (Dogen, Korea), the catalase activity of the puffed grain fermentation enzyme produced in Example 2 was measured according to the manufacturer's manual.

[0072] Specifically, the expanded grain fermentation enzyme was diluted to sterile concentrations, centrifuged at 4000 rpm for 20 minutes, and then filtered through a 0.2 μm filter to produce the expanded grain fermentation enzyme filtrate. 25 μL of each diluted sample and 25 μL of 40 μM H2O2 solution were added to a 96-well plate.

[0073] After allowing the plate to react at room temperature in a light-free environment for 30 minutes, 50 μL of an Oxi-Probe / HRP Working solution, prepared by mixing 30 μL of 10 mM Oxi-Probe, 12 μL of 100 U / mL HRP (horseradish-peroxide), and 3 mL of 1X reaction buffer, was added to each well of the reacted plate.

[0074] After reacting for 30 minutes in a light-blocked environment at 37°C, the absorbance of the reacted plates was measured at 560 nm. A standard calibration curve (y=23673x-224.98, R) was prepared using catalase measured at different concentrations as standard substances. 2 I found the answer by substituting (=0.999) into the formula.

[0075] The catalase activity (unit: mU / mL) of the expanded grain fermentation enzyme is shown in Figure 5. As shown in Figure 5, it can be confirmed that the catalase activity of the expanded grain fermentation enzyme increases in a concentration-dependent manner.

[0076] <Example 4> Measurement of Bacillus amyloricephaciens viable bacteria count in expanded grain fermentation enzyme The samples were analyzed using dilutions prepared by diluting them 10-fold with physiological saline. Viable cell counts were determined by spreading 100 μL of the prepared dilution onto sterile and dried PCA solid medium, then incubating it in a 37°C incubator for 16 hours and calculating the number of colonies.

[0077] The measured number of viable Bacillus amyloricephaciens bacteria is shown in Table 3 below.

[0078] [Table 3]

[0079] Although specific parts of the present invention have been described in detail above, it will be clear to those with ordinary skill in the art that such specific techniques are merely preferred embodiments and that the scope of the present invention is not limited by these embodiments.

[0080] Therefore, the substantial scope of the present invention can be defined by the appended claims and their equivalents. Simple modifications or alterations of the present invention can be readily used by those with ordinary skill in the art, and any such modifications or alterations can be understood to fall within the scope of the present invention.

[0081] [Accession Number] Depository name: Korea Center for Microbial Conservation (KCCM) Depository address: Yurim Building, 45 Hongjaenae 2-gil, Seodaemun-gu, Seoul Deposit date: October 31, 2022 Deposit number: KCCM13257P

Claims

1. A puffed grain fermentation enzyme produced by the Bacillus amyloliquefaciens strain, possessing superoxide dismutase activity and catalase activity.

2. The puffing grain fermentation enzyme according to claim 1, characterized in that the strain is Bacillus amyloricefaciens NPKE6 deposited under accession number KCCM13257P.

3. The aforementioned puffed grain fermentation enzyme exhibits a superoxide dismutase activity of 37-88% at a concentration of 10-100 mg / mL, and The aforementioned puffed grain fermentation enzyme has a catalase activity of 1439 to 3276 mU / mL at concentrations of 5 to 100 mg / mL. One unit of the catalase activity was measured at pH 7.0, 25°C for 1 minute. ２ O ２ The puffed grain fermentation enzyme according to claim 1, characterized in that it is in an amount that decomposes 1 μmol.

4. The puffing grain fermentation enzyme according to claim 1, wherein the grain is one or more selected from the group consisting of defatted soybeans, oats, brown rice, white soybeans, barley, sorghum, flaxseed, rice germ, sword beans, sword bean pods, wheat, rye, and millet.

5. (a) A step of crushing the puffed grain, (b) The step of soaking the puffed grain in water, (c) The step of inoculating the grain with the Bacillus amyloricephasiens strain and then mixing it, (d) A method for producing a puffed grain fermentation enzyme, comprising the step of fermenting grain inoculated with the strain.

6. The process further includes, prior to step (a), feeding grain into an extruder and expanding it at a temperature of 140 to 160°C under conditions of 80 to 120 bar to obtain expanded grain, The method for producing a puffed grain fermentation enzyme according to claim 5, further comprising the steps of hot air drying and powdering after step (d).

7. The method for producing a puffed grain fermentation enzyme according to claim 5, characterized in that the grain is one or more selected from the group consisting of defatted soybeans, oats, brown rice, white soybeans, barley, sorghum, flaxseed, rice germ, sword beans, sword bean pods, wheat, rye, and millet.

8. The method for producing a puffed grain fermentation enzyme according to claim 5, characterized in that the strain is Bacillus amyloliquefaciens NPKE6 deposited under accession number KCCM13257P.

9. A food composition comprising the puffing grain fermentation enzyme according to any one of claims 1 to 4.

10. The food composition according to claim 9, characterized in that the food composition is an enzyme food.

Images