Grain fermentation enzyme with gluten-decomposing ability, method for producing the same, and food composition containing the same

Abstract

The present invention relates to a grain fermentation enzyme having α-amylase activity, protease activity, gluten-degrading activity, and fibrinolytic enzyme activity, which is obtained by fermenting grains with a Bacillus amyloliquefaciens strain, a method for producing the same, a food composition containing the same, and a novel Bacillus amyloliquefaciens strain with gluten-degrading ability.The present invention has the advantage of being excellent in taste and economy because no food additive enzyme preparations are added, and of being able to provide a grain fermentation enzyme with gluten-degrading ability.

Description

[Technical Field]

[0001] The present invention relates to a grain fermentation enzyme having gluten-degrading ability, a method for producing the same, and a food composition containing the same. More specifically, the present invention relates to a grain fermentation enzyme having α-amylase activity, protease activity, gluten-degrading activity, and fibrinolytic enzyme activity, which is obtained by fermenting grain with a Bacillus amyloliquefaciens strain, a method for producing the same, a food composition containing the same, and a novel Bacillus amyloliquefaciens strain having α-amylase activity, protease activity, gluten-degrading activity, and fibrinolytic enzyme activity. [Background technology]

[0002] One of the most important components of our body is enzymes, which are necessary for our body to perform its functions well, and if enzymes are not present in the body, many bodily functions such as respiration, digestion, nutrient synthesis, etc. can be lost.

[0003] When we are young, our bodies are able to synthesize and supply an abundant supply of enzymes. However, modern people's poor eating habits, such as smoking, drinking, and excessive intake of fat and sugar, stress and kill cells, which reduces enzyme synthesis in the body and forces the remaining cells to produce more digestive enzymes, ultimately resulting in a vicious cycle of reduced endurance.

[0004] Therefore, by taking enzyme supplements to improve digestion, helping to break down ingested food and reducing enzyme synthesis in the body, it is possible to not only maintain stomach health but also improve biological rhythms.

[0005] "Enzyme food" refers to a food made by cultivating edible microorganisms in plant-based ingredients to make them contain large amounts of enzymes, or by extracting enzyme-containing parts from foods, or by processing these as the main ingredients.

[0006] These enzyme foods are known to contain various trace elements and bioactive substances that aid digestion and absorption through the production of various bioactive substances and nutrients and the proliferation of beneficial bacteria through the fermentation and aging process of the food's own enzymes and microorganisms.

[0007] Grain fermented enzymes are a general term for enzyme foods made by cultivating edible microorganisms in grain raw materials to produce a large amount of enzymes. Conventional technologies have been disclosed that use one of brown rice, soybeans, barley, and mixed grains (wheat, corn, and Job's tears) as the grain raw material (Korean Patent Registered No. 10-1855125), or rice grains such as rice, barley, wheat, rye, and oats, or miscellaneous grains such as foxtail millet, corn, millet, barnyard millet, buckwheat, and Job's tears (Korean Patent Registered No. 10-2088758).

[0008] However, there is currently no known grain fermentation enzyme that has α-amylase activity, protease activity, gluten-degrading activity, and fibrinolytic activity, which can be obtained by fermenting grains with a Bacillus amyloliquefaciens strain, as in the present invention. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Korean Patent Registration No. 10-1855125 [Patent Document 2] Korean Patent Registration No. 10-2088758 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a grain fermentation enzyme having gluten-decomposing activity by fermenting grain with a Bacillus strain.

[0011] Another object of the present invention is to provide a method for producing a grain fermentation enzyme having gluten-decomposing activity by fermenting grain with a Bacillus strain.

[0012] Another object of the present invention is to provide a food composition containing a grain fermentation enzyme having gluten-degrading activity by fermenting grain with a Bacillus strain.

[0013] Another object of the present invention is to provide a novel Bacillus amyloliquefaciens strain for use in producing a grain fermentation enzyme having gluten-degrading activity. [Means for solving the problem]

[0014] To achieve the above object, the present invention provides a grain fermentation enzyme fermented by a Bacillus amyloliquefaciens strain, wherein the grain is one or more selected from the group consisting of defatted soybeans, oats, brown rice, soybeans, barley, sorghum, flaxseed, rice germ, jack beans, jack bean pods, wheat, rye, and foxtail 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 grain fermentation enzyme may have α-amylase activity, protease activity, gluten degrading activity and fibrinolytic enzyme activity.

[0017] To achieve the above object, the present invention provides a method for producing a grain fermentation enzyme, comprising the steps of: (a) soaking grain in water; (b) removing the soaking water; (c) steaming and sterilizing the grain; (d) dividing the sterilized grain into small portions and cooling them; (e) inoculating the divided grain with a Bacillus amyloliquefaciens strain and then mixing them; and (f) fermenting the grain inoculated with the strain.

[0018] In one embodiment of the present invention, the method for producing a grain fermentation enzyme may further include the steps of hot air drying and powdering after step (f).

[0019] 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, soybeans, barley, sorghum, flaxseed, rice germ, jack beans, jack bean pods, wheat, rye, and foxtail millet.

[0020] In one embodiment of the present invention, the strain may be Bacillus amyloliquefaciens NPKE6 deposited under accession number KCCM13257P.

[0021] In order to achieve the above object, the present invention provides a food composition containing the grain fermentation enzyme. .

[0022] In one embodiment of the present invention, the food composition may be an enzyme food.

[0023] To achieve the above object, the present invention provides the Bacillus amyloliquefaciens NPKE6 strain, deposited under accession number KCCM13257P.

[0024] In one embodiment of the present invention, the strain may have α-amylase activity, protease activity, gluten degrading activity and fibrinolytic activity. [Effects of the Invention]

[0025] The present invention has the advantage of being economical and capable of providing a grain fermentation enzyme having α-amylase activity, protease activity, gluten decomposition activity and fibrinolytic enzyme activity.

[0026] In addition, according to the present invention, by adding only the grain fermentation enzyme and plant-based ingredients, it is possible to comply with the recommendations of the Ministry of Food and Drug Safety, and by using grain ingredients, it is possible to provide a health-conscious superfood. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 2 is a diagram showing the 16S rRNA base sequence of the Bacillus amyloliquefaciens NPKE6 strain (KCCM13257P) of the present invention. [Figure 2] 1 is a process diagram for producing a grain fermentation enzyme according to an embodiment of the present invention. [Figure 3] FIG. 1 shows the results of confirming the gluten-decomposing ability of the NPKE6 strain according to the present invention by inoculating the strain into an aqueous solution containing 0.1% glucose and 0.5% gluten, and then checking the transparency 24 hours later. [Figure 4] FIG. 1 shows the results of confirming the gluten-degrading ability of the Bacillus amyloliquefaciens gluten NPKE6 strain using a Gliadin kit. [Figure 5] This figure shows the results of confirming the gluten-decomposing ability of the grain fermentation enzyme of the present invention by inoculating a 0.1% glucose and 0.3% gluten aqueous solution with a strain filtrate and a grain fermentation enzyme filtrate, and then checking the transparency 24 hours later. [Figure 6]FIG. 1 shows the results of confirming the gluten-decomposing ability of a grain fermentation enzyme using a Gliadin kit. DETAILED DESCRIPTION OF THE INVENTION

[0028] A first embodiment of the present invention relates to a grain fermentation enzyme fermented by a Bacillus amyloliquefaciens strain, wherein the grain is one or more selected from the group consisting of defatted soybeans, oats, brown rice, soybeans, barley, sorghum, flaxseed, rice germ, jack beans, jack bean pods, wheat, rye, and foxtail millet.

[0029] The strain may be Bacillus amyloliquefaciens NPKE6 deposited under accession number KCCM13257P, and the strain may have one or more activities selected from the group consisting of α-amylase activity, protease activity, gluten degradation activity, and fibrinolytic activity, but is not limited thereto.

[0030] A second embodiment of the present invention relates to a method for producing a grain fermentation enzyme, comprising: (a) soaking grain in water; (b) removing the soaking water; (c) steaming and sterilizing the grain; (d) dividing the sterilized grain into small portions and cooling them; (e) inoculating the divided and cooled grain with a Bacillus amyloliquefaciens strain and then mixing them; and (f) fermenting the grain inoculated with the strain.

[0031] The method for producing the grain fermentation enzyme of the present invention may further include, but is not limited to, the steps of hot air drying and powdering after step (f).

[0032] The hot air drying can be carried out for, but is not limited to, for example, 20 to 30 hours, preferably 22 to 26 hours, at 65 to 75° C. The powder can be formulated into different dosage forms as needed.

[0033] The grain may be one or more selected from the group consisting of defatted soybeans, oats, brown rice, soybeans, barley, sorghum, flaxseed, rice germ, jack beans, jack bean pods, wheat, rye, and foxtail millet, but is not limited thereto.

[0034] The strain may be Bacillus amyloliquefaciens NPKE6 deposited under accession number KCCM13257P, and the strain may have one or more activities selected from the group consisting of α-amylase activity, protease activity, gluten degradation activity, and fibrinolytic activity, but is not limited thereto.

[0035] The third embodiment of the present invention relates to a food composition containing the grain fermentation enzyme. The food composition may be preferably, but not limited to, an enzyme food.

[0036] In addition to the grain fermentation enzyme, the food composition may further contain, but is not limited to, two or more fermented mixed grains selected from the group consisting of brown rice, 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.

[0037] The food composition may contain, based on the total weight, 20 to 40 wt % of the grain fermentation enzyme, 50 to 70 wt % of the fermented mixed grain, 1 to 5 wt % of the mixed grain powder, and 1 to 5 wt % of the potato extract powder, but is not limited thereto.

[0038] The food composition may preferably contain, based on the total weight, 30 to 35 wt % of the grain fermentation enzyme, 55 to 65 wt % of the fermented mixed grain, 2 to 4 wt % of the mixed grain powder, and 2 to 4 wt % of the potato extract powder, but is not limited thereto.

[0039] More preferably, the food composition may contain, based on the total weight, 33 to 34 wt % of the grain fermentation enzyme, 60 to 62 wt % of the fermented mixed grain, 2.5 to 3.5 wt % of the mixed grain powder, and 2.5 to 3.5 wt % of the potato extract powder, but is not limited thereto.

[0040] The brown rice that can be included in the fermented mixed grains is harvested rice that has been dried and threshed, and then the husks removed using a machine made of rubber rollers. The brown rice is known to have the effect of preventing lifestyle-related diseases.

[0041] Soybeans that can be included in the fermented mixed grains are also called miso beans, but nutritionally they are It is particularly rich in compounds such as hydroxypropyl methylcellulose, saponins, isoflavones, and trypsin inhibitors. These compounds have anti-cancer properties, as well as the effects of lowering blood cholesterol, preventing obesity by suppressing fat synthesis, activating intestinal motility by regulating the intestines, and preventing constipation by facilitating bowel movements.

[0042] Sorghum, which can be included in the fermented mixed grains, is low in fat and calories, making it an easy diet food. It also has the effect of improving cholesterol levels and breaking down blood clots in blood vessels. The proanthocyanidins contained in sorghum strengthen the immune function of the bladder, reduce oxidative stress in cells, and relieve inflammation.

[0043] Barley, which can be included in the fermented mixed grains, is rich in dietary fiber and is effective against constipation, and its abundant polyphenol compounds help improve antioxidant effects and immunity, and its tocotrienol content can lower cholesterol levels and prevent vascular-related diseases.

[0044] The papaya extract that may be contained in the mixed grain powder contains papain, a type of proteolytic enzyme extracted from papaya fruit. Papain is one of the powerful digestive enzymes found in nature and is known to have various effects such as helping to activate digestive function, suppressing stomach distension, promoting diuretic action, and anti-inflammatory properties.

[0045] In the potato extract powder contained in the food composition, the potato is an alkaline food that protects the stomach by protecting and improving ulcers and mucous membranes caused by excess stomach acid, contains a large amount of vitamin C (similar to that found in spinach and mandarin oranges), and the vitamin C in potatoes is trapped in the starch, making them relatively heat-resistant. Potatoes also contain a high amount of potassium, 410 to 485 mg per 100 g, compared to other vegetables or foods. Potato starch also contains a large amount of pectin, a type of dietary fiber, which has the effect of facilitating intestinal function and bowel movements.

[0046] A fourth embodiment of the present invention relates to the Bacillus amyloliquefaciens NPKE6 strain deposited under accession number KCCM13257P.

[0047] The strain may have, but is not limited to, α-amylase activity, protease activity, gluten degrading activity, and fibrinolytic activity.

[0048] The present invention will be described in detail below with reference to examples to aid in understanding the present invention. However, the examples according to the present invention can be modified into various different forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

[0049] Example 1: Identification of Bacillus amyloliquefaciens NPKE6 strain

[0050] 1. Nucleotide Sequence Analysis of Bacillus amyloliquefaciens Strain NPKE6

[0051] DNA extraction and 16S rRNA sequence analysis were performed by Macrogen, Inc. 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) for the 16S rRNA sequence.

[0052] The 16S rRNA base sequence was analyzed for the identification and classification of the isolated strains. The base sequence of sequence number 1 was obtained.

[0053] 2. Biochemical Characterization of Bacillus amyloliquefaciens NPKE6

[0054] The biochemical characteristics of the Bacillus amyloliquefaciens NPKE6 strain were investigated using API 50CH (bioMérieux Co., France), a kit used to identify bacilli. The API kit was used according to the manufacturer's instructions, and the results are shown in Table 1.

[0055] Bacillus amyloliquefaciens NPKE6 strain was inoculated into 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) liquid medium and cultured at 37°C. The culture was then dispensed into Eppendorf tubes and centrifuged at 10,000 rpm for 5 minutes. The cells were then harvested and washed once with sterile saline (0.85%). The cells were suspended in sterile saline and inoculated into aseptically broken ampoules of API 50CH medium. The mixture was mixed uniformly by pipetting, and 150 μl of the suspension was dispensed into each microtube for each test strip.

[0056] After dispensing, the API kit was incubated in a 37°C incubator for 24 hours, and the color change was observed. The results are shown in Table 1 below. In Table 1 below, the use of each enzyme is indicated by "+", and the absence of each enzyme is indicated by "-".

[0057] [Table 1]

[0058] As a result of the identification and the biochemical characteristics, the selected NPKE6 strain was identified as Bacillus amyloliquefaciens and named Bacillus amyloliquefaciens NPKE6. It was deposited at the Korean Culture Center of Microorganisms (KCCM) on October 31, 2022, and assigned the accession number KCCM13257P.

[0059] Example 2: Production of grain fermentation enzymes using Bacillus amyloliquefaciens NPKE6 strain

[0060] 1. Cultivation of Bacterial Strains

[0061] Bacillus amyloliquefaciens NPKE6 strain 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 cultured at 30°C for 24 hours.

[0062] 2. Production of grain fermentation enzymes

[0063] The oats were soaked in one volume of water for 16 hours, and then the water was removed. The defatted soybean meal and one volume of water were added and mixed. The mixture was steam sterilized at 100°C for 3 hours, then divided into 2.5 kg portions on each tray and allowed to cool.

[0064] The seed culture of Bacillus amyloliquefaciens NPKE6, which was cultured at 30°C and 150 rpm, was inoculated into BM medium at a 2% (v / w) inoculation rate, and then fermented for 24 hours under conditions of a relative humidity of 80% or more.

[0065] After the fermentation was completed, each tray was dried with hot air at 70°C for 24 hours and then crushed to a size of 60 mesh using a multipurpose crusher to produce a grain fermentation enzyme.

[0066] Example 3: Analysis of enzyme activity of cereal fermentation enzymes

[0067] 1. Confirmation of gluten decomposition ability of NPKE6 strain

[0068] Bacillus amyloliquefaciens NPKE6 strain was inoculated into BM medium (1.0% v / v) and cultured at 30°C for 24 hours. As shown in Figure 3, the culture medium was inoculated at 5% with a 0.1% glucose and 0.5% gluten solution. After 24 hours, the degree of transparency of the gluten medium was confirmed by visual inspection. As shown in Figure 4, gluten degradation ability in the gluten medium was confirmed using the RIDASCREEN Gliadin kit (R-Biopharm AG, Germany). It was confirmed that the gluten content was reduced by approximately 77% when inoculated with NPKE6 strain compared to the control.

[0069] 2. Confirmation of gluten-decomposing ability of NPKE6 culture filtrate and grain fermentation enzyme filtrate

[0070] Bacillus amyloliquefaciens NPKE6 strain was inoculated into BM medium (1.0% v / v) and cultured at 30°C for 24 hours. The culture broth and the grain fermentation enzyme diluted with 10 mL of sterile water were centrifuged at 4000 rpm for 20 minutes and filtered through a 0.2 μm filter to separate the culture filtrate and the grain fermentation enzyme filtrate. Manufactured.

[0071] As shown in FIG. 5, the filtered culture filtrate and grain fermentation enzyme filtrate were inoculated into 0.1% glucose and 0.3% gluten aqueous solutions at 5% concentration, respectively. After 24 hours, the degree to which the gluten medium became transparent was visually confirmed. As shown in FIG. 6, the gluten decomposition ability in the gluten medium was confirmed using a RIDASCREEN Gliadin kit (R-Biopharm AG, Germany). It was confirmed that the gluten content was reduced by approximately 89% compared to the control group when the strain filtrate was inoculated, and by approximately 98% compared to the control group when the grain fermentation enzyme filtrate was inoculated.

[0072] Although specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific techniques are merely preferred embodiments and do not limit the scope of the present invention.

[0073] Therefore, it should be understood that the true scope of the present invention is defined by the appended claims and their equivalents. Simple modifications or variations of the present invention can be easily utilized by those having ordinary skill in the art, and all such modifications or variations can be considered to be included in the scope of the present invention. [Accession number]

[0074] Depository institution: Korea Center for Microorganisms (KCCM) Depository address: Yurim Building, 45 Hongsenae 2-ga-gil, Seodaemun-gu, Seoul, South Korea Deposit date: October 31, 2022 Accession number: KCCM13257P

[0075] [Table 2]

Claims

1. A grain fermentation enzyme fermented by a strain of Bacillus amyloliquefaciens, The grain fermentation enzyme is one or more grains selected from the group consisting of defatted soybeans, oats, brown rice, soybeans, barley, sorghum, flaxseed, rice germ, jack beans, jack bean pods, wheat, rye and foxtail millet.

2. The grain fermentation enzyme according to claim 1, wherein the strain is Bacillus amyloliquefaciens NPKE6 deposited under accession number KCCM13257P.

3. The grain fermentation enzyme according to claim 1, characterized in that the grain fermentation enzyme has α-amylase activity, protease activity, gluten decomposition activity and fibrinolytic enzyme activity.

4. (a) soaking grain in water; (b) removing the soaking water; (c) steaming the grain and then sterilizing it; (d) portioning the sterilized grain and then allowing it to cool; (e) inoculating the cooled portioned grain with a Bacillus amyloliquefaciens strain and then mixing; (f) fermenting the grain inoculated with the strain.

5. The method for producing a grain fermentation enzyme according to claim 4, further comprising the steps of hot air drying and powdering after step (f).

6. The method for producing a grain fermentation enzyme according to claim 4, characterized in that the grain is one or more selected from the group consisting of defatted soybeans, oats, brown rice, soybeans, barley, sorghum, flaxseed, rice germ, jack beans, jack bean pods, wheat, rye and foxtail millet.

7. The method for producing a grain fermentation enzyme according to claim 4, wherein the strain is Bacillus amyloliquefaciens NPKE6 deposited under accession number KCCM13257P.

8. A food composition comprising the grain fermentation enzyme according to any one of claims 1 to 3.

9. The food composition according to claim 8, characterized in that the food composition is an enzyme food.

10. Bacillus amyloliquefaciens strain NPKE6 deposited under accession number KCCM13257P.

11. The Bacillus amyloliquefaciens NPKE6 strain according to claim 10, characterized in that the strain has α-amylase activity, protease activity, gluten decomposition activity and fibrinolytic enzyme activity.

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