KR101925095B1 - Production method for easy digested food containing nuts, which are coated with grain fermented enzyme powder - Google Patents

Abstract

The present invention relates to a production method for food, which facilitates digestion since a grain fermentation enzyme is coated. Provided are a food coated with powder of a grain fermentation enzyme and a production method therefor, to provide a variety of essential nutrients contained in rice bran, bran, yeast extract, soybean meal and isolated soybean protein powder which are bases for a starter to promote fermentation of grains and supplement beneficial ingredients, and enable a user to intake various kinds of physiologically active ingredients such as fermented products, digestive enzymes and the like produced by fermentation of Bacillus coagulans.

Description

Technical Field [0001] The present invention relates to a method for producing a food containing nuts which are coated with a fermentation enzyme of grain and easy digestion,

The present invention relates to a method for producing a food product which is coated with a fermentation enzyme of grain and is easy to extinguish. More particularly, the present invention relates to a method for producing a food product, which comprises (a) a bacterium base comprising rice bran, wheat bran, soybean meal, isolated soybean protein and yeast extract powder, ( Bacillus coagulans ) strain to prepare a liquid culture medium for a liquid seed culture; (b) mixing and cultivating the present culture base comprising rice bran, wheat bran, soybean powder, isolated soybean protein and yeast extract powder; (c) adding the fermented liquid medium prepared in step (a) to the fermented cereal of step (b) and fermenting the same; (d) drying and pulverizing the fermented product fermented in the step (c); And (e) coating the outer surface of one of the group consisting of roasted nuts, unheated nuts, legumes, fruits and cereals with sugar and mixing with the powder of step (d) And a method for producing a food product coated with the fermentation enzyme powder.

An enzyme is an important protein that acts on the metabolism of the body and acts as a catalyst for the chemical action. It can be divided into food enzymes that are not made in the body and need to be consumed from the outside, digestive enzymes which are made by themselves in the body and play a necessary role in digestion, and metabolic enzymes which are necessary for other metabolic processes except digestion. Among them, food enzymes have been reported to be capable of physiological functions such as digestive absorption, digestion and excretion, anti-inflammatory / antibacterial, detoxification, blood purification and cell activation (Shin Hyun Jae, -41, 2013).

Enzymes are digestive enzymes (amylase, protease, cellulase) that are involved in digestion, excretion, absorption of nutrients, etc., based on the human body, and nutrients taken for life sustaining action. Metabolism (peripheral nerves, autonomic nerves, Etc.), and it is known that it is produced mainly in the process of culturing a fermenting microorganism. Fibrin causes the platelets to clump together to form a flaky mass that gradually stiffenes and shrinks, interfering with blood flow. When fibrinolytic enzyme (natokinase) dissolves fibrin, it inhibits platelet aggregation and helps smooth blood flow.

Enzymatic foods are referred to collectively by cultivating edible microorganisms in edible raw materials in order to enhance the function of these enzymes to contain a large amount of enzymes, extracting enzyme-containing portions from foods, or processing them. These enzyme foods contain various trace elements and physiologically active substances which help digestion and absorption through the fermentation and aging process of enzymes and microorganisms of food itself, through the generation of various physiologically active substances and nutrients and the proliferation of beneficial bacteria. (Huh, SH and Kim, MH The modern health and health food, 1997. Hongikjea press. Korea, p.35-36).

In addition, Bacillus coagulans not only plays a role of acid-resistant and heat-resistant lactic acid bacterium, but also possesses both characteristics of bacillus and lactobacillus that produce lactic acid as well as spore formation. In addition, it exhibits beneficial effects on human body and various physiologically active substances, It is known that it is effective for digestion, diarrhea, irritable bowel syndrome, constipation, cholesterol reduction, etc. by facilitating metabolism and promoting the body's physiological activity. Through fermentation, it is possible to digest various nutrients It is easy to absorb and disassemble so that our body can use it well.

Recently, methods of producing various fermented foods by combining brown rice and several major grains have been widely known. Most grains contain many nutrients in their shells, but their husks are hard and brittle, so they are usually used as foodstuffs in a stripped state. Using rice bran and wheat bran, which are components of grain in the grain, as a seed base, it is possible not only to ingest beneficial ingredients in the shell but also to take beneficial ingredients generated in the fermentation process.

Nuts include walnuts, peanuts, chestnuts, almonds, pine nuts, and banks, and are rich in unsaturated fatty acids such as oleic acid and linoleic acid. In particular, omega-3, one of the unsaturated fatty acids, has been reported to have various effects such as reduction of neutral lipids in the blood, improvement of ocular dryness, and prevention of dementia. For this reason, modern people have increased nuts consumption and consumption .

The recommended daily intake of nuts is 25g. People with weak digestive function feel discomfort during ingestion of nuts, and digestion and absorption of protein and fat, the major nutrients, are not progressing efficiently.

Therefore, nuts processed foods are necessary for consumers who feel discomfort in digestive function after eating nuts, so that nuts and their nutrients are effectively digested and absorbed.

Accordingly, the present inventors have established a manufacturing method of coating grain fermentation enzymes with various saccharides on amylase (protease, lipase) activity, and provide nuts that are easy to digest and absorb nut ingredients of nuts .

One object of the present application

(a) preparing a culture medium for a liquid seed culture by inoculating a strain of Bacillus coagulans to a seed base comprising rice bran, wheat bran, soybean powder, isolated soybean protein and yeast extract powder;

(b) mixing and cultivating the present culture base comprising rice bran, wheat bran, soybean powder, isolated soybean protein and yeast extract powder;

(c) adding the fermented liquid medium prepared in step (a) to the fermented cereal of step (b) and fermenting the same;

(d) drying and pulverizing the fermented product fermented in the step (c); And

(e) coating the outer surface of one of the group consisting of roasted nuts, unheated nuts, legumes, fruits and grains with sugar and mixing with the powder of step (d) and drying, And a method of producing a food product coated with the enzyme powder.

Another object of the present invention is to provide a food having increased activity of at least one enzyme selected from the group consisting of amylase, protease, lipase, cellulase and fibrin degrading enzyme produced by the above method.

In order to achieve the object of the present invention described above,

(a) preparing a culture medium for a liquid seed culture by inoculating a strain of Bacillus coagulans to a seed base comprising rice bran, wheat bran, soybean powder, isolated soybean protein and yeast extract powder;

(b) mixing and cultivating the present culture base comprising rice bran, wheat bran, soybean powder, isolated soybean protein and yeast extract powder;

(c) adding the fermented liquid medium prepared in step (a) to the fermented cereal of step (b) and fermenting the same;

(d) drying and pulverizing the fermented product fermented in the step (c); And

(e) coating the outer surface of one of the group consisting of roasted nuts, unheated nuts, legumes, fruits and grains with sugar and mixing with the powder of step (d) and drying, And a method of producing a food product coated with the enzyme powder.

The present invention provides a food having increased activity of at least one enzyme selected from the group consisting of amylase, protease, lipase, cellulase, and fibrin degrading enzyme produced by the above method.

Hereinafter, the present invention will be described in detail.

The present invention

(a) Rice bran , Bran, soybean powder, Isolated soy protein  And a yeast extract powder Bacillus Coagulant ( Bacillus coagulans ) Strain to produce a culture medium for liquid seed culture;

(b) on grain Rice bran , Bran, soybean powder, Isolated soy protein  And yeast extract powder For culture  Mixing and growing the base;

(c) adding the fermented liquid medium prepared in step (a) to the fermented cereal of step (b) and fermenting the same;

(d) mixing the fermented product obtained in the step (c) The fermented product  after drying Powdered  step; And

(e) coating the outer surface of one of the group consisting of roasted nuts, unheated nuts, legumes, fruits and grains with sugar and mixing with the powder of step (d) and drying, And a method of producing a food product coated with the enzyme powder.

In step (a) of the present invention, Bacillus coagulans is inoculated into a seed base of rice bran, wheat bran, soybean powder, isolated soybean protein, and yeast extract powder to produce a liquid culture medium .

The 'seed bases' may include soybean powder, rice bran, wheat bran, isolated soybean protein, and yeast extract. Specifically, the seed base is prepared by mixing 1 to 5 parts by weight of soybean powder, 1 to 5 parts by weight of rice bran, 1 to 5 parts by weight of wheat bran, 1 to 5 parts by weight of isolated soybean protein, 3 parts by weight.

Bacillus coagulans strain is inoculated to the seed base.

The inoculated Bacillus coaguerans strain may preferably be a strain deposited with Accession No. KCTC13284BP. The strains were selected from strains derived from traditional fermented foods having the best ability to produce starch, protein, fat and fibrinolytic enzymes.

In the inoculation, the pre-culture broth in which the strain is cultured may be used as it is, or the isolated strain may be used, or the strain may be pulverized in the form of lyophilization or a culture thereof.

The inoculum amount of the Bacillus coaguerans strain may be inoculated in an amount of 5 to 20 parts by weight, preferably 10 parts by weight, based on 100 parts by weight of the mixture.

The liquid seed culture may be specifically liquid cultured. The incubation temperature may be 30 ° C to 40 ° C, and the incubation time may be 10 hours to 30 hours.

As demonstrated in the following Examples, by seeding and culturing the seed base into a grain, excellent starch hydrolyzing enzyme activity and proteolytic enzyme of the strain, protein in the cereal fermentation enzyme obtained by the cellulase activity can be made low molecular weight, And a fibrinolytic enzyme activity effect that helps blood circulation can be obtained.

In the step (b), the present culture base composed of rice bran, wheat bran, soybean powder, isolated soybean protein, and yeast extract powder is mixed with the cereal.

The 'grain' used in the step (b) may be one of brown rice, soybean, barley, mixed grain (wheat, corn, rice). The grain may have undergone an immersion process called water after having undergone proper screening and cleaning processes. At this time, depending on the type of grain, it is called for 4 to 24 hours soaking in water. Since the characteristics are different according to the kind of grain, the time to immerse in water can be controlled within the range according to the kind. Specifically, 14 to 16 hours of brown rice, 5 to 9 hours of soybeans, 3 to 5 hours of barley, and 14 to 16 hours of mixed grains.

The temperature of the water at the time of immersion may be specifically 20 ° C to 35 ° C, preferably 25 ° C to 30 ° C. This is because there is a difference in the moisture content due to the temperature difference at the time of immersion. The washed and soaked grains can be used in the form of a pulverized form, a powder form, or without pulverization.

The culture base used in step (b) may be a soybean powder, rice bran, wheat bran, isolated soybean protein, or yeast extract. Specifically, the mixture may contain 5 to 10 parts by weight of soybean powder, 1 to 5 parts by weight of rice bran, 1 to 5 parts by weight of wheat bran, 1 to 5 parts by weight of isolated soybean protein, 1 to 5 parts by weight of yeast extract powder, Weight portion.

Although it is possible to cultivate the strain in the grain without the step of addition, it can provide an environment in which the microorganism can vigorously grow by destroying the grain cell wall and denaturing the glutathione and protein while killing the bacteria in the grain by heat treatment The cost can be reduced by lowering the amount of strain inoculated.

The addition may be carried out using various methods known in the art, for example, using steam or superheated steam.

Specifically, the capital increase can be carried out in two stages. Step 1 may include a step of preliminarily growing at a pre-growing step at 60 ° C to 110 ° C for 10 minutes to 60 minutes. Preferably 80 ° C to 120 ° C, for 20 minutes to 40 minutes. The second step may be carried out at 90 ° C to 140 ° C for 10 minutes to 60 minutes, and preferably at 100 ° C to 120 ° C for 30 minutes to 50 minutes.

During the capital increase process, the amount of moisture is controlled by confirming the state of the spread of the grain. Preferably, the grain that is grown at this stage and does not spread and remains in the form may be from 50 to 90% of the total weight, more preferably from 60 to 80%. This is to allow the grain to retain its shape and to form a space between the crocks to ensure breathability.

The method may further include cooling the additionally-grown grain after the addition. The cooling can proceed naturally after the addition is over, or the cooling rate can be increased to prevent overheating and to cool uniformly. Specifically, the cooling temperature may be 35 캜 to 50 캜, and preferably 40 캜 to 45 캜. It hardens when cooled below the above range, and is difficult to mix, so that it cools to the above temperature range.

The step (c) is a step of adding the liquid culture medium prepared in the step (a) to the cereal of the step (b) and fermenting the same.

The amount of the liquid cultivation medium may be inoculated in an amount of 5 to 20 parts by weight, preferably 10 parts by weight, based on 100 parts by weight of the cereal containing the mixture.

The 'fermentation' is not limited to a culture method, but can be performed using, for example, a liquid culture tank, a rotary drum fermentor, or a tray fermentor. In addition to the above-mentioned fermenter, any method useful for fermentation of cereals or a mixture thereof can be used in the method of the present application without limitation, and an appropriate device can be selected according to the production scale.

The incubation temperature may be 20 ° C to 50 ° C, 30 ° C to 45 ° C, and the humidity at the time of culturing may be 40% to 90%, preferably 50% to 85%. The incubation time can be 1-72 hours, 12-72 hours, 36-50 hours or 48 hours.

The step (d) is a step of drying and pulverizing the fermented material fermented in the step (c).

The drying may be carried out by various methods known in the art. However, when dried at an excessively high temperature, viable bacteria among cereal fermented products may be killed and enzyme activity may be decreased. Specifically, the presently filed strain should be dried at a low temperature where the enzyme activity is maintained without being killed. The drying temperature may be 30 ° C to 60 ° C, preferably 35 ° C to 45 ° C.

The drying time may be from 12 hours to 48 hours, preferably from 20 hours to 28 hours.

The dried fermented product may be dried to have a moisture content of 3% to 20%, preferably 3% to 7%. This is the preferred moisture content for the enzyme activity and shelf stability of the product.

The pulverization process may be carried out in various sizes depending on the intended use of the fermented cereal product. Examples of the pulverization process include crushing, grinding, milling, abrating, pounding, Smashing, grating, or any other means used to reduce the food material to powder (powder) or fine particles. Specifically, a hammer mill or the like can be used.

In the step (e), sugar is coated on the outer surface of one of roasted nuts, unheated nuts, legumes, fruits and cereals, and the mixture is mixed with the powder of step (d) and dried.

The nuts represent ordinary nuts. Specifically, it includes almonds, cashew nuts, pistachios, walnuts, chestnuts, peanuts, pine nuts, banks, coconut, Hegel, macadamia nuts and the like.

Typically, the nuts have a double-wrapped structure of a hard shell and a soft shell. In the present invention, the nuts are not particularly limited as far as they are provided as mats with easy teeth. However, the nuts are not particularly limited in terms of the state in which both the outer and outer shells of the nuts are removed, That is, a state in which only the crust which is hard and is not edible is removed) may be used. Preferably, the nuts of the present invention can be used without removing the inner skin, thereby allowing nutrients and functional ingredients contained in the inner skin to be ingested at once.

The nuts may be roasted and heated to 170 to 200 DEG C for 40 to 60 minutes in a roaster, a drum or a fan. The heated nuts are more flavorful than the unheated nuts.

The legumes include soybeans, soya beans, kidney beans, red beans, peanuts, bean curds, peas, black beans, lentils, chickpeas and dried legumes.

The legumes may be roasted.

The fruits may be selected from the group consisting of cherry, cherry, black cherry, cherry, raisin, raspberry, strawberry, strawberry, blueberry, youngberry ), Loganberry, Tayberry, Olallieberry, Boysenberry, Marionberry, Elderberry, Bilberry, Raspberry, Blackberry, Blackberry, Dewberry, Gooseberry, Cranberry, Cranberry, Lingonberry, Grapevine, Bilberry Trees, Vendor, Trefoil, Black Corinth, Black It is also possible to use various flavors such as black currant, red currant, white currant, grape, scallop, mulberry, prosciutto, kiwi fruit, citron, lemon, Orange, tangerine, grapefruit, kumquat, oval kumquat, mandarin orange, mandarin orange, tangerine, pomegranate, Mulberry, Mulberry Mulberry, Mulberry Mulberry, Pineapple, Pineapple, Melon, Papaya, Cucumber, Melon, Watermelon, Tomato, Pumpkin, Jujube, Peach, apple, pear, tobacco, mulberry, persimmon, sorghum, Acerola, Damson plum, plum, mesyl, apricot, fig, mango, , Borealis fruit, geonjae, mountain oil, mountain sorghum, kizija, omiza, gabeon, joseongil, Namjeongsil, Includes tree fruit (water cherries), acacia fruit, rowanberry, corvus nuts, madam berries, young pine cones, young fruit of the fir or young fruit of the pine tree.

The fruit may be dried.

Rice, rice, brown rice, barley, glory, yulmu, whole wheat, sorghum, quinoa, sweet potato, potato, chestnut or pine nuts.

The grain may be roasted.

Roasting the above-mentioned nuts, legumes or cereals, and then further screening the burnt products. Specifically, it may include a process of selecting burrs at an illuminance of 540 Lux or more.

After the screening process, sugar can be coated on nuts, legumes, fruits or cereals. It is intended to express "sugar-dressing work" or "sugar-coating", in which the sugar can prevent the deterioration of the product and is a pre-work to make the grain fermentation enzyme powder adhere well to the nuts.

The sugar coating process of the present invention can be performed using a general mixer such as a screw type ribbon mixer. At this time, the coated sugar may be 8 to 15 parts by weight based on 100 parts by weight of nuts. The sugar coating process of the present invention can be carried out at 35 ° C to 45 ° C for 10 minutes to 30 minutes.

 Specifically, the sugar of the present invention may be a fructooligosaccharide, a sugar, an isomaltooligosaccharide, or a syrup, preferably a fructooligosaccharide.

The sugar-coated nuts, legumes, fruits or grains are coated with the grain fermentation enzyme powder prepared in steps (a) to (d) of the present invention. Specifically, brown rice fermented enzyme powder; Barley fermentation enzyme powder; Soybean fermentation enzyme powder; Corn, wheat, and yulmu.

The grain fermentation enzyme may be coated on only one kind of nuts, or two or more kinds of them may be mixed and coated.

Specifically, 1 to 7 parts by weight of a brown rice fermentation enzyme powder, 1 to 7 parts by weight of a soybean fermentation enzyme powder, 1 to 7 parts by weight of a barley fermentation enzyme powder and a mixed grain fermentation enzyme consisting of corn, 1 to 7 parts by weight of powder may be coated on the mixed grain fermentation enzyme powder.

The grain fermentation enzyme powder coating process may be performed at 35 ° C to 55 ° C for 10 minutes to 30 minutes.

The nuts coated with the sugar and grain fermentation enzyme powder can be dried at room temperature for 5 minutes to 15 minutes. This is to prevent desorption of the powder during further work where it is mixed with lower volume unheated nuts or grains or legumes or dried fruits.

The nuts, legumes, fruits or grains of which step (e) has been completed may further comprise non-nut mixing.

The non-nuts may be dry cranberries, dried blueberries, dried apples, dried bananas, roasted chickpeas, roasted chickpeas, creamy oatmeal, fried lentil beans.

The step of mixing with the non-nuts may be carried out at room temperature for 10 minutes to 20 minutes.

After the processing step, packaging may further be included.

The present invention provides a food having increased activity of at least one enzyme selected from the group consisting of amylase, protease, lipase, cellulase, and fibrin degrading enzyme produced by the above method.

In the examples of the present invention, the enzymatic activity of the cereal fermentation enzyme powder containing the liquid culture medium and the culture base for each kind of grain was measured. As a result, it was confirmed that the protease activity was 3000 - 15000 U / g, the lipase activity was 5 - 30 U / g, and the fibrinolytic enzyme activity was 50 - 200 U / g. This is a marked increase in enzyme activity compared to grains that do not contain this culture base.

In another embodiment of the present invention, the grain fermentation enzyme powder obtained by adding the seedling base 2 and the main culture base to each grain of brown rice, soybean, barley and mixed cereals (corn, wheat, %, Barley 25%, mixed grains (corn, wheat, yulmu) 20%. As a result, it was confirmed that the protease activity was 3000 - 15000 U / g, the lipase activity was 5 - 30 U / g, and the fibrinolytic enzyme activity was 50 - 200 U / g. This is a marked increase in enzyme activity compared to grains that do not contain this culture base.

In another embodiment of the present invention, the activity of digestive enzymes of nuts coated with fermented soybean syrup, fructooligosaccharide, isomaltooligosaccharide and sugar at 40 ° C for 20 minutes was compared. As a result, it was confirmed that the amylase, protease and lipase activities of the nuts coated with the mixed grain fermentation enzyme to fructooligosaccharide were 870.0 U / g, 890.3 U / g and 1.36 U / g, respectively, Respectively.

The method of the present invention can provide a food coated with a grain fermentation enzyme powder. The nut is easy to extinguish because the grain fermentation enzyme powder having excellent digestive enzyme activity such as alpha-amylase, protease, and lipase is coated.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a simplified process diagram of a method of manufacturing a food product coated with a fermented grain powder of the present invention. FIG.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<Experimental Example 1>

<Experimental Example 1-1>

Method for measuring amylase activity

The test substance was dissolved in RO water at a concentration of 5 g / 100 ml and shaking incubated at 30 ° C and 200 rpm for 30 minutes. The sample solution was centrifuged at 12,000 rpm for 3 minutes and the supernatant was filtered through a 0.45-μm filter. The filtrate was appropriately diluted using RO water. For the blank test, the filtrate was deactivated by heating at 100 占 폚 for 30 minutes. 5 ml of 1% soluble starch, 13 ml of Mcllvaine buffer, and 1 ml of 0.1% calcium chloride were prepared in a glass test tube. The substrate solution was preincubated at 37 ° C for 10 minutes. 1 ml of test solution or blank test solution was added and reacted at 37 ° C for 30 minutes. 10 ml of iodine solution was prepared in a 15 ml test tube, 200 ul of test / blank test reaction solution was added, and absorbance was measured at 660 nm with distilled water as a reference solution.

Potency  Calculation method

* Amylase Titer Definition of unit / g: value that 1 mg of starch contained in 1 g of sample reacts with enzyme for 30 minutes to decompose 1 mg of starch.

<Experimental Example 1-2>

Method for measuring protease activity

The test substance is dissolved in RO water at a concentration of 5 g / 100 ml and shaking incubated at 30 ° C and 200 rpm for 30 minutes. The supernatant obtained by centrifugation of the sample solution at 12,000 rpm for 3 minutes was filtered with a 0.45-μm filter. The filtrate was diluted with an appropriate amount using RO water. 1 ml of 0.6% casein was prepared in a test tube and pre-incubated at 37 ° C for 3 minutes. 1 ml of the test solution was added and the mixture was reacted at 37 ° C for 10 minutes. 2 ml of 0.4 M trichloroacetic acid was added, incubated at 37 ° C for 25 minutes, and filtered through a 0.45-μm filter. 1 ml of the supernatant was taken in a 15 ml conical tube. To this solution, 5 ml of 0.4 M sodium carbonate and 1 ml of folin soil were added, pipetted, and incubated at 37 ° C for 20 minutes. Absorbance was measured at 660 nm. 1 ml of the test solution was added and incubated at 37 ° C for 10 minutes. (Blank test solution) After adding 2 ml of 0.4 M Trichloroacetic acid, 1 ml of 0.6% casein was added and incubated at 37 ° C for 25 minutes. Filtered through a 0.45 um filter. 1 ml of the supernatant was taken in a 15 ml conical tube. To this solution, 5 ml of 0.4 M sodium carbonate and 1 ml of Folin soil were added, and the mixture was incubated at 37 ° C for 20 minutes. Absorbance was measured at 660 nm.

* Protease Titer Unit / g Definition: The value calculated by converting the amount of L-tyrosine produced by the reaction of the enzyme with the standard product for 10 minutes in the reverse direction containing 1 g of the sample.

< Experimental Example  1-3>

Method for measuring lipase activity

5 g of the test substance was diluted with glycine buffer, and then this solution was adjusted to 100 mL by using water. 0.05N sodium hydroxide solution was previously added to the buret of the titration apparatus, and the scale of the apparatus was adjusted, and the temperature was adjusted to 30 ° C and the pH to 7.0. 15.0 mL of the substrate emulsion was taken out, placed in a reaction vessel of the titration apparatus, and stirred magnet was added. The reaction vessel was placed in an autoclave and 1.0 mL of the test solution was added. The titrator was activated and reacted while adjusting the pH to 7.0 with 0.05 N sodium hydroxide solution. The titration curve per minute was plotted against the consumption mL of the 0.05 N sodium hydroxide solution .

(※ Note: When titrating for 5 minutes, the reaction rate shown on the recorder should be straight line)

  Calculate the activity of the enzyme according to the following formula.

R: mL / min of the appropriate consumption per minute in a straight line section.

N: Specification of sodium hydroxide solution

1,000: Coefficient to convert mmol of acid to μmol

W: Amount of specimen (g) contained in 1 mL of Test Solution

* Definition of activity: 1 Lipase unit (LU) is the amount of enzyme that liberates 1 μmol of butyric acid per minute from the substrate under the test conditions.

<Experimental Example 1-4>

How to measure fibrinolytic enzyme activity

* Amount

-Sodium borate buffer (50 mM) solution: 19.07 g of sodium tetraborate decahydride and 9.0 g of NaCl were dissolved in about 900 mL of distilled water, adjusted to pH 8.5 with HCl, and made up to 1,000 mL with distilled water.

-Trichloracetic acid [TCA] (0.2M) solution: Trichloroacetic acid 32.678 was added to distilled water to make 1000 mL.

-Fibrinogen solution (0.72%): 10 ml of 50 mM sodium borate buffer was added to an Erlenmeyer flask, and 96 mg of fibrinogen (Sigma-aldrich, F8630) was added to dissolve and then filtered with filter paper (Advantech No.6) )

-Thrombin solution: Thrombin (Sigma-aldrich, T6634) was dissolved in 50 mM sodium borate buffer, and the concentration was adjusted to 1,000 U / mL. In the experiment, 50-fold dilution was used in 50 mM sodium borate buffer.

* Way

- Preparation of test solution: The sample was diluted with 50 mM sodium borate buffer to a concentration of 0.67 ~ 1.33 FU / g to prepare a test solution. 1.4 mL of sodium borate buffer and 0.4 mL of fibrinogen solution were placed in two test tubes, and the mixture was heated in a constant temperature water bath at 37 ° C for 5 minutes. Then, 0.1 mL of thrombin solution was added thereto and stirred. The solution was allowed to stand in a constant temperature water bath at 37 ° C for 10 minutes, and then 0.1 mL of the test solution was added to only one test tube (AT), followed by stirring for 5 seconds and left for 1 hour (after 20 minutes, To the solution, 2 mL of 0.2 M TCA solution was added and stirred. The test solution was put in a test tube (AB) containing no test solution, stirred and left in a constant temperature water bath at 37 캜 for 20 minutes. The supernatant was placed on an e-tube and centrifuged at 15,000 x g for 5 minutes. The absorbance of 1 mL of the supernatant was measured at 275 nm.

* Calculation

Fibrinolytic enzyme activity (FU / g) = (AT-AB / 0.01) x (1/60) x (1 / 0.1) x D

0.01: FU conversion factor (/ min)

60: reaction time (minute)

0.1: Amount of test solution added (mL)

* Definition of activity: 1 Unit (1FU) is the amount of enzyme that increases the absorbance of 0.01 sample per minute at the wavelength of 275nm

<Experimental Example 1-5>

Method for measuring cellulase activity

* Preparation of Test Solution: 1 mL of the final dilution was diluted with water to produce a relative fluidity change between 0.18 and 0.22 within 5 minutes under the conditions specified in this test method. A certain amount of the sample was taken in a glass grinder and water was added. This was transferred to a suitable volumetric flask, diluted with water to a predetermined amount, and filtered through Whatman No. 1 or the same kind of filter paper before use.

Test Procedure: The calibrated viscometer was immersed in a sufficient amount of detergent in advance, then wiped clean with water, and then mounted vertically and precisely in a glass bath of 40 ± 0.1 ° C. 20 mL of the substrate solution and 4 mL of the acetate buffer (pH 4.5) were taken in a 50 mL-flask Erlenmeyer flask, and two for the enzyme test and one for the substrate test were prepared per one sample. The flask was placed in an enzyme test flask and allowed to stand for 15 minutes in a water bath to equilibrate the temperature. Then, 1 mL of the test solution was precisely taken and the time was started to be measured and the solution was well mixed. Immediately, 10 mL of this mixture was added to the large branch of the viscometer, and after about 2 minutes, the reaction solution was attached to the narrow inlet of the viscometer by rubber tube and sucked up to the upper scale. The following procedure was repeated four times in total within 15 minutes to "suck up the reaction solution again on the upper scale" with respect to the reaction time (T _R ). The solution is by measuring the time (in minutes) to reach the upper grid, and it measures the time in seconds to pass through the lower grid and an upper grid, again, as it is referred to as T _R T _T. The reaction solution was again sucked onto the upper scale and the time (minutes) until the surface of the solution reached the upper scale was measured immediately, and this was measured as T _R and the time (sec) from the upper scale to the lower scale was measured This is called T _T. For the substrate blank test, add 1 mL of water to a mixed solution of 20 mL of substrate solution and 4 mL of acetate buffer (pH 4.5), immediately take 10 mL of the mixed solution, place it in the large branch of the viscometer, and measure the time T _s (Seconds) were measured five times repeatedly and the average value was defined as T _S. For the water ball test, 10 mL of water equilibrated at 40 ° C in advance was placed in a large branch of the viscometer _and the time T _W (sec) between the two graduations was measured five times to obtain the average value T _W.

Relative fluidity (F _R ) and T _N values were calculated for each of the four outflow times (T _T ) and reaction times (T _R ) according to the following formula.

Calibration curves were generated using four relative mobility (F _R ) for four reaction times (T _N ). At this point, a straight line should be drawn. The slope of the line corresponds to the relative flow change per minute and was proportional to the amount of enzyme. The best slope through a series of test points became a criterion for a better enzyme titer than a single relative fluidity value. Measure F _R from 10 minutes and 5 minutes from the calibration curve. The difference in fluidity should be between 0.18 and 0.22.

* Calculation

* Definition of activity: 1 Cellulase unit (CU) is the amount of enzyme that produces a one-relative flow change over 5 minutes on a carboxymethylcellulose substrate under the test conditions.

* Device

- Viscometer: Canon-Fensk Type Viscometer with scale calibration of -100 or equivalent was used.

- Glass water bath: A glass constant temperature water bath of 40 ± 0.1 ° C or equivalent was used.

* Reagents and reagents

- Acetate buffer solution (pH 4.5): The pH of the solution is adjusted to 4.5 by adding 0.4N sodium acetate solution to 400 mL of 0.4N acetic acid while stirring continuously.

- Carboxymethylcellulose sodium: Carboxymethylcellulose sodium [Cellulose gum, Hercules (Aqualon) Type 7HF or equivalent] was used.

- Substrate solution: 200 mL of water was added to the mixing bowl and the mixer was set to a low speed. 1 g of carboxymethylcellulose sodium as a dried material was slowly dispersed in a vessel so as not to protrude in the vessel. Using only rubber poles, the vessel wall was washed with hot water, then the vessel was capped and mixed at high speed for 1 minute. This was transferred to a 500 mL volumetric flask and water was added to make 500 mL. The mixture was filtered through gauze before use.

<Experimental Example 2>

How to measure total bacteria

 1) Test procedure: Test sample is mixed well with sterilized glass rod and sterilized reagent spoon, and a certain amount (10 ~ 25 g) is taken in a sterilized container and mixed with 9 times diluted solution. 1 mL of the test solution and 1 mL of the 10-fold dilution are aseptically taken aseptically in two or more sterilized Petri dishes and approximately 15 mL of the standard agar medium (medium 1) maintained at about 43 to 45 ° C is aseptically dispensed, While being careful not to attach to the lid, rotate gently and tilt to the left and right to mix the specimen and medium well and coagulate. To prevent the spread of colonization, add 3-5 mL of standard agar medium again to overlay. In this case, the time until the sample is taken and the medium is added should not exceed 20 minutes. The solidified Petri dish is inverted and cultured at 35 ± 1 ° C for 48 ± 2 hours (30 ± 1 ° C or 35 ± 1 ° C for 72 ± 3 hours depending on the sample). The calculation of the number of colonies is based on the principle that the number of colonies is calculated by taking the reputation that has no spread colonies and has generated 15 to 300 colonies per plate. 1 mL of the same diluted solution to which no sample solution has been added is used as a control test solution to confirm the sterility of the test procedure.

2) Estimation of the number of colonies: Immediately after incubation, the number of colonies generated using the colonization calculator is calculated. If unavoidable, store at 5 ° C and calculate within 24 hours. The calculation of the number of colonies is based on the principle that the number of colonies should be calculated by choosing a flat plate that produces 15 to 300 colonies per flat without any spreading colonies (it does not interfere with half of the colonies). If more than 300 colonies occur on the entire plate, close to 300 plates shall be calculated according to the dense plate method. If only 15 or fewer colonies are obtained on the plate, the lowest dilution factor should be measured.

&Lt; Example 1 >

Selection of strain

Brown rice, soybean, barley, mixed grain (wheat, corn, yulmu) One grain was selected to prepare 100g of grain. At this time, brown rice, soybean, barley mixed grain (wheat, corn, yulmu) was used after washing and sorting in advance. The amount of water that can be completely immersed in the grains was prepared, and the grains were immersed for 24 hours, and the grains were transferred to a tray. Then, the culture base 3 was added, and the tray was transferred to a pottery machine. The tray was kept at 121 ° C for 40 minutes and then cooled to 40-45 ° C in the pot. Bacillus coagulans (KCTC13284BP), Bacillus amyloliquefaciens (KCTC 3002), Bacillus natto (KCTC 3239) and Lactobacillus fermentum (KCTC 13097) were inoculated with 10% of the grain in the well - cooled grain trays. The temperature was 32 ~ 40 ℃ and the humidity was 50 ~ 85%. Dried and aged at 40 ~ 60 ℃ to maintain the final moisture content of 3 ~ 7%.

The above examples show that Bacillus coagulans has the best amylase, protease and lipase activity.

Strain setting grain zymogen Amylase activity (U / g) Protease activity (U / g) Lipase
Active (U / g) Brown rice Bacillus coagulans 4,071 6,222 12 Bacillus amyloliquefaciens 3,452 5,521 14 Bacillus natto 2,120 4,821 10 Lactobacillus fermentum 424 332 0 barley Bacillus coagulans 3,584 6,113 14 Bacillus amyloliquefaciens 1,995 5,976 11 Bacillus natto 2,412 4,942 5 Lactobacillus fermentum 256 282 One Big head Bacillus coagulans 3,252 3,521 12 Bacillus amyloliquefaciens 2,413 2321 9 Bacillus natto 1,120 1,299 2 Lactobacillus fermentum 314 124 0 mix Bacillus coagulans 3,374 3,547 8 Bacillus amyloliquefaciens 3,245 2,412 5 Bacillus natto 3,302 1,214 2 Lactobacillus fermentum 214 101 0

< Example 2>

Setting base composition for seed bacterium

The seed culture was adapted to the seed culture to establish the seed culture for optimum fermentation.

Bacterial base component Seed bacterium Composition (based on 100 parts by weight of purified water) Bacterial base 1 Soybean powder 1%, rice bran 1%, wheat bran 1%, isolated soybean protein 1%, yeast extract 0.5% Bacteria base 2 Soybean powder 2%, rice bran 2%, wheat bran 2%, isolated soybean protein 1%, yeast extract 0.5% Bacteria base 3 Soybean powder 5%, rice bran 5%, wheat bran 5%, isolated soybean protein 1%, yeast extract 0.5%

Bacillus coagulans cultured on LB broth (Luria Bertani; tryptone 1%, yeast extract 0.5%, NaCl 1%) was inoculated in 10% of the culture medium and incubated at 37 ° C, The cells were incubated at 180 rpm for 15 hours to confirm the total number of bacteria.

Prepare 100g of brown rice, soybeans, barley, mixed grains (wheat, corn, yulmu) in advance. At this time, brown rice, soybean, barley mixed grain (wheat, corn, yulmu) was used after washing and sorting in advance. The amount of water that can be completely immersed in the grains is prepared, and then the grains are soaked for immersion time. The grains were transferred to a tray, transferred to a tray, pre-heated at 100 ° C for 15 minutes, and stored at 121 ° C for 30 minutes, and then cooled to 40-45 ° C. The seeds of the above-mentioned seedlings 1, 2 and 3 were inoculated into each of the cooled cereals by inoculating 10% of the seeds. The temperature was maintained at 30 to 40 ° C and the humidity was maintained at 50 to 85%. After 48 hours of fermentation in the fermentation chamber Completed. After drying and aging at 40 ~ 60 ℃, final 5 ~ 10% moisture was obtained, and the total number of bacteria was measured after pulverization with 40 mesh.

As a result of checking the liquid culture medium and the total bacterial counts of the final grain fermentation enzyme powders, it was confirmed that the seed bacterium base 2, seed base 3 and total bacterium were the most similar, and the seed base 2 was the most economical and efficient composition (See Table 3).

Total number of bacteria in the final powder per liquid culture medium By kind of seed bacterium Total number of bacteria Final grain fermentation enzyme powder Total number of bacteria LB medium
(Liquid seed culture is not added) 6.8 x 10 ⁸ Brown rice 4.2 × 10 ⁵ Big head 2.3 × 10 ⁶ barley 3.7 × 10 ⁵ Mixed grain 3.1 × 10 ⁵ Bacterial base 1 4.2 × 10 ⁶ Brown rice 1.5 × 10 ⁶ Big head 4.7 × 10 ⁶ barley 2.0 × 10 ⁶ Mixed grain 8.4 × 10 ⁵ Bacteria base 2 2.2 x 10 ⁸ Brown rice 2.7 × 10 ⁷ Big head 2.7 × 10 ⁷ barley 4.2 × 10 ⁷ Mixed grain 7.6 × 10 ⁶ Bacteria base 3 2.7 × 10 ⁸ Brown rice 2.9 × 10 ⁷ Big head 3.2 × 10 ⁷ barley 4.0 × 10 ⁷ Mixed grain 8.1 × 10 ⁶

&Lt; Example 3 >

Set immersion time

In order to establish the immersion time for the optimum fermentation condition for each grain in the above example, water having an amount of completely immersing the grain is prepared and then 100 g of each grain is dipped for 4 hours, 7 hours, 15 hours and 24 hours . Transfer the grain to the tray. Transfer the tray to the growing machine, add it for 40 minutes at 121 ° C, and cool it to 40-45 ° C in a thickener. Bacillus coagulans were inoculated at the rate of 10% relative to the grains. The temperature is kept at 32 ~ 40 ℃ and the humidity is 50 ~ 85%, and it is completed after fermentation in the fermentation room for 48 hours. It is aged at 40 ~ 60 ℃ to maintain final 5 ~ 10% moisture.

Amylase activity, protease activity and lipase activity were found to be the best when immersed for 7 hours in soybean, immersed in barley for 4 hours, immersed in brown rice for 15 hours and immersed in mixed cereals (corn, wheat, rice bran) for 15 hours 4)

Set immersion time grain Immersion time (hr) Amylase activity (U / g) Protease activity (U / g) Lipase
Active (U / g) Brown rice 4 3724 5715 5 7 5641 6924 6 15 6124 7315 11 24 4212 6243 10 barley 4 4521 8012 5 7 4152 7851 4 15 3921 7051 4 24 3120 6224 3 Big head 4 3712 3358 2 7 5021 4752 10 15 3502 3653 4 24 3201 3552 4 mix 4 3325 2921 5 7 3752 3214 5 15 4212 3922 7 24 3524 3721 6

<Example 4>

Set advance advance time

For optimal fermentation conditions for each grain, prepare an amount of water to completely immerse the grain in order to establish the increase time. Then, 100 g of grain is immersed for 7 hours in soybean, immersed for 4 hours in barley, immersed in brown rice for 15 hours, , Wheat, yulmu) and soaked for 15 hours. Transfer the grain to the tray. The culture bases were put in, and the trays were transferred to a thickener. After 10 minutes, 30 minutes, 60 minutes at 100 ° C, 10 minutes, 30 minutes and 60 minutes at 80 ° C, After the increase, cool to 40 ~ 45 ℃ in the boiler. Bacillus coagulans were inoculated at the rate of 10% of the grains in each well. The temperature is kept at 32 ~ 40 ℃ and the humidity is 50 ~ 85%, and it is completed after fermentation in the fermentation room for 48 hours. It is aged at 40 ~ 60 ℃ to maintain final 5 ~ 10% moisture.

The amylase activity, protease activity and lipase activity were found to be the best during the 30 minute pre-heating at 100 ° C. (see Table 5).

Set advance advance time grain Preliminary casting temperature ( 캜 ) Prospective capital increase time
(min) Amylase activity (U / g) Protease activity (U / g) Lipase
Active (U / g) Brown rice 80 10 5524 6715 5 30 6112 7224 6 60 4953 7101 10 100 10 6024 6915 11 30 7112 7951 11 60 6253 7195 10 barley 80 10 4469 7562 5 30 4981 7861 9 60 4251 7354 6 100 10 5006 8051 10 30 5701 8961 13 60 5614 8624 13 Big head 80 10 5342 5114 9 30 5551 5234 11 60 5342 5142 10 100 10 5124 5221 14 30 5912 5512 14 60 5341 5142 12 mix 80 10 4625 3725 4 30 4875 3952 6 60 4752 3967 5 100 10 4998 4126 7 30 5129 4215 8 60 5075 4200 8

< Example 5>

Setting fermentation conditions

Fermentation temperature of 30 ℃, 37 ℃, 40 ℃, humidity 50%, 60%, 70%, 85%, fermentation time 12 hours, 24 hours, 36 hours, 48 hours by using brown rice The enzyme activity was measured after fermentation at different times, 60 hours, and 72 hours.

The amylase activity, protease activity and lipase activity were the best at 37 ° C. and 48 hours of water fermentation at 70 ° C. (see Table 6, Table 7 and Table 8).

Setting fermentation temperature grain Fermentation temperature Amylase activity (U / g) Protease activity (U / g) Lipase
Active (U / g) Brown rice 30 ℃ 4123 4125 4 37 ℃ 7712 8124 13 40 ℃ 5124 7124 12

Setting fermentation humidity grain Fermentation Humidity (%) Amylase activity (U / g) Protease activity (U / g) Lipase
Active (U / g) Brown rice 50 5472 4278 4 60 7006 6024 9 70 7170 7991 12 85 6945 7214 10

Set fermentation time grain Fermentation time (hr) Amylase activity (U / g) Protease activity (U / g) Lipase
Active (U / g) Brown rice 12 2543 1264 One 24 4582 3459 3 36 6410 7991 12 48 7561 8124 16 60 7256 7952 10 72 5142 5461 6

&Lt; Example 6 >

Setting aging drying conditions

In order to establish the optimal aging conditions for grain, the fermentation was carried out at 30 ℃, 40 ℃, 50 ℃ and 60 ℃ for 12 hours, 24 hours and 48 hours after fermentation using brown rice and soybean. Respectively.

It was found through the above examples that both the moisture content of 5% and the amylase, protease and lipase activities are both satisfactory when aged and dried at 40 ° C. (see Table 9).

grain Aging Drying Temperature ( 캜 ) Drying time (hr) Humidity
(%) Amylase activity (U / g) Protease activity (U / g) Lipase
Active (U / g) Brown rice 30 12 13.6 7115 7964 14 24 9.2 7015 7836 13 48 6.7 7169 7761 12 40 12 11 7115 7864 11 24 4.6 7152 7731 10 48 2.3 6582 7554 10 50 12 8.4 7052 7634 8 24 4.1 6786 7441 7 48 2.7 6852 6942 5 60 12 6.1 6524 6742 5 24 2.6 6745 6475 3 48 2.1 6451 6273 2 Big head 30 12 14.8 6121 5625 15 24 10.3 6058 5687 15 48 7.6 6007 5576 14 40 12 12.1 6122 5623 15 24 5.3 6025 5512 13 48 2.7 5996 5424 11 50 12 9.1 6077 5586 10 24 4.1 5796 4985 7 48 2.9 5119 4786 6 60 12 6.7 4998 5099 6 24 2.8 4752 4116 4 48 2.3 4218 3925 4

< Example  7>

For culture  Base composition setting

100 g of brown rice, soybean, barley, mixed grain (wheat, corn, yulmu) were prepared. At this time, brown rice, soybean, barley mixed grain (wheat, corn, yulmu) was used after washing and sorting in advance. The amount of water that can be completely immersed in the grains is prepared, and then the grains are soaked for immersion time, and the grains are transferred to the tray.

Base composition for culture The present culture base Composition (based on 100 parts by weight of grain) This culture base 1 Soybean powder 5%, rice bran 5%, wheat bran 5%, isolated soybean protein 1%, yeast extract 1% This culture base 2 Soybean powder 10%, rice bran 5%, bran 5%, isolated soybean protein 1%, yeast extract 1% This culture base 3 Soybean powder 10%, rice bran 5%, bran 5%, isolated soybean protein 2%, yeast extract 1% This culture base 4 Soybean powder 10%, rice bran 5%, bran 5%, isolated soybean protein 5%, yeast extract 2%

The culture bases 1 to 4 were added to a tray containing cereals in an amount of 100 parts by weight of the cereals, respectively. The tray was transferred to a pottery machine, preliminarily heated at 100 ° C for 15 minutes, and kept at 121 ° C for 30 minutes, and then cooled to 40 to 45 ° C in a pot. The culture bases were added to the cereal tray in an amount of 10% relative to the cereal in a 10% amount. The mixture was kept at a temperature of 30 to 40 ° C and a humidity of 50 to 85%, and was completed after fermentation for 48 hours in a fermentation chamber. Dried and aged at 40 to 60 캜 to maintain a final moisture of 5 to 10%.

The total bacterial counts and enzymatic activity of the final grain fermentation enzyme powders of each culture base were found to be higher than that of the culture bases without addition of the culture base, Table 11)

Enzyme activity according to the type of culture base For culture  Base grain Amylase
activation
(U / g) Protease
activation
(U / g) Lipase
activation
(U / g) Fibrinolysis
Enzyme activity
(FU / g) Cellulase
activation
(CU / g) Total number of bacteria For culture  No base additive Brown rice 713 3129 5 25 4 2.4 × 10 ⁷ Big head 1354 853 2 45 2 1.7 × 10 ⁷ barley 1147 905 One 23 3 3.5 × 10 ⁷ Mixed grain 953 1012 4 45 3 7.2 × 10 ⁶ For culture  Base 1 Brown rice 3334 7829 9 92 4 5.5 × 10 ⁸ Big head 4754 4623 11 125 5 3.9 × 10 ⁹ barley 4316 5124 12 113 4 1.0 × 10 ⁹ Mixed grain 3952 4517 8 95 2 4.1 × 10 ⁸ For culture  Base 2 Brown rice 5998 9565 11 126 5 1.7 x 10 ⁹ Big head 7653 6622 16 125 5 8.6 × 10 ⁹ barley 6012 6823 17 143 5 2.5 x 10 ⁹ Mixed grain 4689 5541 13 105 4 7.3 × 10 ⁸ For culture  Base 3 Brown rice 8625 11525 14 137 7 3.2 × 10 ⁹ Big head 10521 7855 20 142 5 1.9 × 10 ¹⁰ barley 7521 7256 15 132 7 5.4 × 10 ⁹ Mixed grain 7066 6971 13 124 4 1.3 x 10 ⁹ For culture  Base 4 Brown rice 5625 9226 11 111 3 2.6 x 10 ⁹ Big head 6521 6526 14 132 4 2.7 × 10 ¹⁰ barley 5521 6521 12 101 6 7.5 × 10 ⁹ Mixed grain 3689 4584 11 109 5 1.8 × 10 ⁹

< Example  8>

Of fermented enzyme powder by addition of fermentation base Enzymatic ability

< Example  8-1>

Liquid culture medium for liquid seed and For culture  Enzyme activity of cereal fermented enzyme powder without added base

10% of Bacillus coagulans cultured in LB broth (Luria Bertani: tryptone 1%, yeast extract 0.5%, Nacl 1%) was added to six pre-soaked brown rice, barley, soybean, mixed cereals (corn, wheat, And maintained at a temperature of 30 to 40 ° C and a humidity of 50 to 85%, respectively, and completed after fermentation in a fermentation chamber for 48 hours. After 40 ~ 60 ℃ of dryness, the mixture is pulverized to 40 ~ 50mesh with 5 ~ 10% moisture content. After 20 ~ 60% of brown rice, 20% soybeans, 25% barley and 20% mixed grains (corn, . Thereafter, the activity of digestive enzymes (amylase, protease, lipoase) was measured (see Table 12).

&Lt; Example 8-2 >

Enzymatic Activity of Grain Fermentation Enzyme Powder Added with Liquid Culture Medium

The liquid culture medium was prepared by fermenting the seed medium 3 (soybean powder 2%, rice bran 2%, bran 2%, isolated soybean protein 1%, yeast extract 0.5%), Mixed grains (corn, wheat, yulmu) are inoculated to each and maintained at a temperature of 30 ~ 40 ℃ and a humidity of 50 ~ 85%. After 40 ~ 60 ℃ of dryness, the mixture is pulverized to 40 ~ 50mesh with 5 ~ 10% moisture content. After 20 ~ 60% of brown rice, 20% soybeans, 25% barley and 20% mixed grains (corn, . Thereafter, the activity of digestive enzymes (amylase, protease, lipoase) was measured (see Table 12).

< Example  8-3>

Liquid culture medium for liquid seed and For culture  Enzyme Activity of Grain Fermentation Enzyme Powder with Added Bases

A liquid culture medium was prepared by fermenting base 3 (soybean powder 2%, rice bran 2%, bran 2%, isolated soybean protein 1%, yeast extract 0.5%). The liquid culture medium was prepared by mixing six kinds of cereals including brown rice, barley, soybean, and mixed grains including base 3 (10% of soybean powder, 5% of rice bran, 5% of wheat bran, 2% of isolated soybean protein and 1% of yeast extract) (Corn, wheat, yulmu) was added, and 10% inoculation was carried out, and fermentation was carried out in a fermentation room for 48 hours while maintaining a temperature of 30 to 40 ° C and a humidity of 50 to 85%. After 40 ~ 60 ℃ of dryness, the mixture was pulverized to 40 ~ 70mesh with 3 ~ 7% moisture content. After the pulverization, grain fermentation enzyme powder was added at a ratio of 35% of brown rice, 20% of soybeans, 25% of barley, 20% of mixed grains (corn, . Thereafter, the activity of digestive enzymes (amylase, protease, lipoase) was measured (see Table 12).

Digestive Enzyme Activity of Grain Fermented Enzyme Powder with Addition of Fermentation Base Digestive enzyme Example  8-1 Example  8-2 Example  8-3 Amylase (U / g) 349 711 8752 Protease (U / g) 588 1347 8931 Lipase (U / g) One 3 14

&Lt; Example 9 >

Selection of sugars and temperature to coat nuts

Nutrients and saccharides were mixed for 20 minutes at the temperature (30 ± 5 ° C, 40 ± 5 ° C, 50 ± 5 ° C, 60 ± 5 ° C) for 20 minutes using syrup, fructooligosaccharide, isomaltooligosaccharide, After that, mixed grain fermentation enzyme powder was added and completely coated at 40 ± 5 ° C for 20 ± 5 minutes. After the mixed grain fermentation enzyme powder was coated on the nuts and dried at room temperature for 10 minutes, the mixed grain fermentation enzyme powder was found to have adsorption rate and rejection rate to nuts.

When the syrup, fructooligosaccharide, isomaltooligosaccharide and sucrose were coated at 40 ± 5 ° C for 20 ± 5 minutes, the highest adsorption rate was obtained, and the fructooligosaccharide adsorption rate was 92% And the dropout rate was 8%. [Refer to Table 13]

Adsorption Rate and Dropout Rate According to Saccharide and Temperature Selection for Coating of Grain Fermentation Enzyme Powder Using Bacillus coagulans 30 ℃ 40 ℃ 50 ℃ 60 ° C corn syrup Adsorption rate 75 85 69 54 Dropout rate 25 15 31 46 Fructooligosaccharide Adsorption rate 78 92 75 55 Dropout rate 22 8 25 45 Isomaltooligosaccharide Adsorption rate 73 87 65 49 Dropout rate 23 13 25 51 Sugar Adsorption rate 65 83 54 39 Dropout rate 35 17 46 61

< Example  10>

Comparison of nuts digest enzyme enzyme coated with grain fermented enzyme powder

On the basis of the results of Example 9, the digestive enzyme titer of the nuts coated with the mixed grain fermentation enzyme at 40 ± 5 ° C for 20 ± 5 minutes at 40 ° C after the starch, fructooligosaccharide, isomaltooligosaccharide and sugar were coated at 40 ° C for 20 minutes (See Table 14).

The amylase, protease and lipase activities of fructooligosaccharides coated with the mixed grain fermentation enzyme at 40 ± 5 ° C for 20 ± 5 minutes were 870.0 U / g, 890.3 U / g and 1.36 U / g, respectively, And it was confirmed that this is the best result compared to other conditions.

Digestive enzyme activity measurements of nuts coated with fermented enzyme powder according to saccharides sugars/
Temperature corn syrup Fructooligosaccharide Isomaltooligosaccharide Sugar amylase 400.1 870.0 600.5 429.3 protease 299.5 890.3 511.0 331.0 lipase 0.06 1.36 0.46 0.08

Various essential nutrients contained in rice bran, wheat bran, yeast extract, soybean powder, and isolated soybean protein powder and various fermented products and digestive enzymes produced by fermentation of Bacillus coagulans in the seed base and the culture base of the present invention The physiologically active ingredient can be taken evenly.

Therefore, the method for producing the food coated with the granulated fermented enzyme powder of the present invention and the food prepared by the method are industrially applicable inventions.

Korea Biotechnology Research Institute KCTC13284BP 20170612

Claims (15)

(a) preparing a culture medium for a liquid seed culture by inoculating a strain of Bacillus coagulans to a seed base comprising rice bran, wheat bran, soybean powder, isolated soybean protein and yeast extract powder;
(b) mixing and cultivating the present culture base comprising rice bran, wheat bran, soybean powder, isolated soybean protein and yeast extract powder;
(c) adding the fermented liquid medium prepared in step (a) to the fermented cereal of step (b) and fermenting the same;
(d) drying and pulverizing the fermented product fermented in the step (c); And
(e) coating the outer surface of one of the group consisting of roasted nuts, unheated nuts, legumes, fruits and grains with sugar and mixing with the powder of step (d) and drying, A method for producing food coated with enzyme powder.
The method according to claim 1, wherein the grain in step (b) is selected from the group consisting of mixed grains consisting of corn, wheat, and yulmu, brown rice, barley and soybean .
The method according to claim 1, wherein the grains of step (b) are immersed for 3 to 16 hours.
The method according to claim 1, wherein the seed base of step (a) comprises 1 to 5 parts by weight of soybean powder, 1 to 5 parts by weight of rice bran, 1 to 5 parts by weight of wheat bran, 1 to 5 parts by weight of isolated soybean protein, 5 parts by weight of a yeast extract powder, and 0.1 to 3 parts by weight of a yeast extract powder.
The method according to claim 1, wherein the culture base of step (b) comprises 5 to 10 parts by weight of soybean powder, 1 to 5 parts by weight of rice bran, 1 to 5 parts by weight of wheat bran, 1 to 5 parts by weight of isolated soybean protein 1 To 5 parts by weight of the yeast extract powder, and 1 to 5 parts by weight of the yeast extract powder.
The method according to claim 1, wherein the Bacillus coaguerans strain of step (a) is deposited with the deposit number KCTC13284BP.
The method according to claim 1, wherein the step (b) is carried out at 70 ° C to 140 ° C for 10 minutes to 60 minutes.
The method according to claim 1, wherein the step (b) further comprises the step of preliminarily growing at 60 ° C to 110 ° C for 10 minutes to 60 minutes.
The method according to claim 1, wherein the fermentation of step (c) is carried out at a temperature of 30 ° C to 40 ° C or a humidity of 50% to 85%.
The method according to claim 1, wherein the drying of step (d) is carried out at a temperature of 35 ° C to 45 ° C for 20 hours to 28 hours.
The method according to claim 1, wherein the sugar of step (e) is selected from the group consisting of fructooligosaccharide, sugar, isomaltooligosaccharide and syrup.
The method according to claim 1, wherein the coating of step (e) is performed at 35 ° C to 45 ° C for 10 minutes to 30 minutes.
The method according to claim 1, wherein the step (c) comprises adding 5 to 20 parts by weight of the liquid culture medium to 100 parts by weight of the cereal of step (b) .
The method according to claim 1, wherein the mixing of step (e) is performed at a ratio of 5 to 20 parts by weight of the powder of step (d) to 100 parts by weight of roasted nuts. Gt;
A food product coated with a grain fermentation enzyme powder characterized in that the activity of at least one enzyme selected from the group consisting of amylase, protease, lipase, cellulase and fibrin degrading enzyme produced by the method of claim 1 is increased.

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