KR101787546B1 - Unpolished rice of solid-phase fermented by complex microorganisms and method for manufacturing the same - Google Patents

Abstract

The present invention provides a solid brown solid fermented by fermentation with one or more strains of an Aspergillus sp. Strain and a Rhizopus sp. Strain. The solid fermented product of the present invention exhibits enhanced enzyme activity in the brown rice, and in the case of the liquid fermented product, it is possible to provide a high stability by solving the problem of loss of enzymatic activity and deterioration of the useful ingredient during the distribution process.

Description

[0001] The present invention relates to a solid fermentation product of a complex strain of brown rice and a method for producing the solid fermentation product,

The present invention relates to a brown rice fermentation product solid-phase fermented with a complex strain and a production method thereof.

The rice is composed of husks, rice husks, pericarp, seed coat, horny layer, embryo, and endosperm. The rice husk is called brown rice, and rice husk is removed from brown rice. And the embryo is removed. The more rice, the less protein, minerals, vitamins, and lipids, and the carbohydrate content increases. The standard chemical composition of brown rice was 15.5% of moisture, 7.4% of protein, 3.0% of lipid, 71.8% of carbohydrate, 1.0% of fiber, 1.3% of ash, and 0.54mg of 100g of vitamin B1. E is more than four times, vitamin B2 is two times, fat, iron and phosphorus more than two times, dietary fiber is three times more.

Brown rice can prevent arteriosclerosis and aging due to the prevention of constipation and the emission of harmful substances as well as the increase of useful components. As the interest in health increases, the preference for brown rice is increasing.

Korean Patent No. 10-1441790

It is an object of the present invention to simultaneously provide a large amount of three enzymes, glucoamylase, alpha-amalase and protease, by fermentation in a solid phase using a brown rice as a substrate.

In order to solve the above-mentioned problems, one aspect of the present invention is to provide a method for producing a fermented brown rice fermented by a strain comprising at least one of Aspergillus sp . Strain and Rhizopus sp. Solid-phase fermentation product, and a food comprising the same.

According to another aspect of the present invention, there is provided a method for producing a solid fermentation product of brown rice, which comprises culturing a substrate containing a solid brown rice having a water content of 33.5 to 35.5% by weight and a strain of Aspergillus sp . , And culturing the strain inoculated with the solid phase brown rice at a temperature ranging from 28 to 45 DEG C for 30 to 46 hours with time, wherein the strain is inoculated with at least one strain of Rhizopus sp. A method for producing a solid fermentation product of brown rice.

The fermented product of brown rice according to the present invention can be obtained by hydrolyzing the protein with glucoamylase and alpha-amylase, which catalyze the hydrolysis reaction of starch, by solid phase fermentation with one or more strains of Aspergillus sp. Thereby increasing the production of the catalytic protease. When the enzymatic product is used as a coenzyme and a liquid enzyme decomposition is carried out by adding a certain amount of steam (rice grown) and water, the free sugar, peptide / amino acid and the like are produced from the substrate. In addition, the present invention can provide a high stability by solving the problem of loss of enzymatic activity and deterioration of useful components in the course of liquid fermentation by solid phase fermentation of brown rice. The above-mentioned solid fermented product of brown rice can be used not only as a food material itself, but also as an enzyme product such as brewing, organic acid fermentation, starch decomposing enzyme, health supplement, food additive and the like.

FIG. 1 is a photograph showing the degree of penetration of hyphae of fermentation strains in solid phase fermentation of Examples 1 to 3. FIG.
Fig. 2 is a graph showing moisture content (%) according to elapsed fermentation time during solid phase fermentation in Examples 1 to 3; Fig.
FIG. 3 is a graph showing the concentration (U / g) of glucoamylase in a substrate according to the elapsed time of fermentation in solid phase fermentation of Examples 1 to 3; FIG.
FIG. 4 is a graph showing the concentration (U / g) of alpha-amylase in the substrate according to the fermentation time elapse during solid phase fermentation in Examples 1 to 3.
FIG. 5 is a graph showing the concentration (U / g) of the protease in the substrate according to the elapsed time of fermentation in the solid phase fermentation of Examples 1 to 3.
FIG. 6 is a diagram showing conditions of solid phase fermentation temperatures according to fermentation time elapsed (emulsion accumulation time) in Examples 4 and 5; FIG.
FIG. 7 is a graph showing moisture content (%) of fermentation time of solid phase fermentation in Examples 4 and 5. FIG.
8 is a graph showing the concentration (U / g) of glucoamylase in the substrate according to the elapsed time of fermentation in solid phase fermentation in Examples 4 and 5.
FIG. 9 is a graph showing the concentration (U / g) of alpha-amylase in the substrate according to the fermentation time elapse during solid phase fermentation in Examples 4 and 5.
10 is a graph showing the concentration (U / g) of the protease in the substrate according to the elapsed time of fermentation in solid phase fermentation in Examples 4 and 5.
FIG. 11 is a graph showing the amounts of cells (mg / g) in the substrate according to the elapsed time of fermentation in solid phase fermentation in Examples 4 and 5.
Fig. 12 is a photograph showing the degree of penetration of hyphae of fermentation strains in solid phase fermentation in Examples 4 and 5.
FIG. 13 is a graph showing the analysis of water content (%) according to elapsed fermentation time during solid phase fermentation in Examples 6 to 8. FIG.
FIG. 14 is a graph showing the concentration (U / g) of glucoamylase in a substrate according to the elapse of fermentation time during solid phase fermentation in Examples 6 to 8; FIG.
FIG. 15 is a graph showing the concentration (U / g) of alpha-amylase in the substrate according to the elapsed time of fermentation in solid phase fermentation in Examples 6 to 8; FIG.
FIG. 16 is a graph showing the concentration (U / g) of protease in a substrate according to the elapsed time of fermentation in solid phase fermentation in Examples 6 to 8; FIG.

Hereinafter, the present invention will be described in detail.

One embodiment of the present invention is a solid-phase fermentation product of brown rice fermented by a strain comprising at least one of the strains Aspergillus sp . And Rhizopus sp. .

In the present specification, the term "brown rice" includes all rice husks of rice, and is not limited to the kind of rice, the cultivation conditions and the environment.

In one embodiment, the brown rice may comprise one or more of brown rice, brown rice crushed and brown rice extrudates.

In the present specification, the term " tongue brown rice "means a brown rice of a whole grain not processed or crushed, and it is the best meaning as long as it has an original grain size.

The pulverized product of the brown rice may be, for example, pulverized product pulverized to 15 to 35% by volume based on the total volume of the brown rice flour as an example, and may have an average diameter of 1.0 to 2.5 mm, for example. Specifically, the crushed product of brown rice may be crushed to a size of about 1/6 to 1/2, more specifically, crushed to 1/5 to 1/3, or about 1/4 size But it is not limited thereto.

In this specification, the term "extrudate of brown rice" means that the rice husk rice is squeezed by pushing the rice husk rice to a die having various shapes at a constant pressure. In one embodiment, the extrusion is not accompanied by a heat treatment, or may be accompanied by a heat treatment before and / or after extrusion. In one embodiment, the average size of the extrudates of brown rice may range from 170 to 220 袖 m.

In the present specification, the term "solid fermentation" may be described as "solid fermentation" or "solid culture", and means a method of culturing a fermentation strain on a solid substrate. The solid phase fermentation is performed by inoculating and infiltrating a fermentation strain into a solid substrate having a low water content without culturing it in a liquid. Thus, the volume of the substrate per unit volume is large, and the possibility of contamination of bacteria during fermentation and distribution is low. The problem of loss of enzyme activity when powdered in liquid fermentation can be solved. In addition, solid phase fermentation facilitates separation of fermentation products. However, since the solid phase has a lower moisture content than that of the liquid phase and is difficult to be stirred, the control of the incubation temperature and the selection of the fermentation strain are important factors for the quality of the fermented product.

The fermenting bacteria according to an embodiment of the present invention may include one or more strains of the Aspergillus sp . Strain and the Rhizopus sp. Strain. Specifically, the Aspergillus sp . Strain may include Aspergillus oryzae , and the Rhizopus sp. Strain may include Rhizopus delemar . have.

The strain may produce an enzyme comprising at least one of gluco-amylase, alpha-amylase, and protease, which catalyze the hydrolysis reaction of proteins, which catalyze the hydrolysis reaction of starch.

Examples of the strains usable as an embodiment of the present invention include Aspergillus oryzae SMF-138 (KCTC 11989BP), Aspergillus oryzae strains, Rhizopus delemar ) SMF-136 (KCTC 11990BP). These strains have a high activity of at least one of alpha-amylase and glucoamylase.

In one embodiment, the Aspergillus genus that zymogen in accordance with one embodiment of the present invention strains and separation tank crispus sp is 4 (Aspergillu s sp.) ( Rhizopus sp.): 6 to 6: a compound mixed in the ratio of 4 And may include strains. More specifically, the mixing ratio of the two strains may be 1: 1. When the pH is out of the above range, the activity of the Aspergillus sp . Strain having a high alpha-amylase activity is not high and it is not suitable for the solid phase fermentation, or the solidification of the substrate (brown rice) Can not be obtained.

Starch degrading enzyme refers to an enzyme that completely degrades starch. Amylase is a representative starch hydrolyzing enzyme and is classified into α-amylase, β-amylase and glucoamylase depending on its mode of action. Amylases differ in their degradation products according to the species and are classified according to their properties. Among the starch degrading enzymes, three stages of starch degradation by amylase are carried out: liquefaction, resorption and saccharification. Particularly, liquefying and resorptive power is called liquefying amylase (α-amylase) Gluco-amylase ". Alpha-amylase is predominantly present in saliva and irregularly hydrolyzes only the alpha-1,4 linkage. β-amylase is present in cereals, beans, peas, and saliva, and hydrolyses only α-1,4 bonds sequentially from the end of the starch chain to the maltose unit. The glucoamylase hydrolyzes not only the α-1,4 bond in order, but also the α-1,6 bond.

Protease, a protease, means an enzyme that hydrolyzes a peptide bond. It is largely divided into exopeptidase (endopeptidase) and endopeptidase (endopeptidase) according to the degradation mode. The exopeptidase acts on only the terminal end of the peptide chain and in turn liberates the amino acid. And examples thereof include leucine aminopeptidase and carboxypeptidase. Endopeptidase acts on the inside rather than the end to liberate peptides of various sizes, such as pepsin, trypsin, chymotrypsin, and the like.

In one embodiment of the present invention, the solid fermented product of the brown rice contains an enzyme product (coenzyme) such as glucoamylase, alpha-amylase, or protease, and is used as a coenzyme to increase (increase rice) Etc. can be added in a certain amount so that the liquid enzyme can be decomposed. The liquid enzyme degradation products may include free sugars, peptides / amino acids, and the like.

Another embodiment of the present invention can provide a food comprising the solid fermented product of the brown rice. Examples of the food include brewing, organic acid fermentation, preparation of enzyme products such as starch degrading enzyme, health supplement, food additives, and the like.

One embodiment of the present invention is a method for producing a solid fermented product of brown rice as described above, which comprises culturing a substrate containing a solid brown rice having a water content of 33.5 to 35.5% by weight, and a strain of Aspergillus sp . A strain of Rhizopus sp .; And culturing the strain inoculated with the solid phase brown rice at a temperature ranging from 28 to 45 DEG C for 30 to 46 hours with time. The solid fermented product of brown rice produced according to the above method has enzymatic activity to decompose starch and protein components of brown rice.

In one embodiment, the method may further include pre-treating a solid brown rice before the step of inoculating the strain. Specifically, the step of pre-treating the brown rice of the solid phase comprises the steps of immersing the untreated rice brown rice in water and drying and boiling it; And then milling the brown rice flour to a size of 15 to 35% by volume based on the total volume of the brown rice flour, and raising the brown rice flour; And then extruding the mixture so as to have an average diameter of 170 to 220 mu m, and drying the extrudate of the brown rice; ≪ / RTI >

More specifically, the step of pre-treating the solid brown rice may include washing of the brown rice, immersion in distilled water, removal of distilled water, and a boiling step. The step of pretreating the solid brown rice may include washing of the brown rice, immersion in distilled water, removal of distilled water, and grinding and boiling to a size of 1/3, 1/4 or 1/5. The step of pre-treating the solid brown rice may include washing of the brown rice, immersion in distilled water, extrusion and drying.

In one embodiment, the solid brown rice having a water content of 33.5 to 35.5% by weight may include the brown rice is immersed in distilled water or the water content is adjusted by adding distilled water to brown rice. The moisture content is the content (% by weight) of water in brown rice with respect to the total weight of brown rice. If the moisture content is less than 33.5 wt%, the microorganism culture is slow and the enzyme activity is low, and if the moisture content exceeds 35.5 wt%, microbial contamination and enzyme activity may be low.

In one embodiment, the step of culturing the strain inoculated with the solid brown rice at a temperature ranging from 28 to 45 ° C for 30 to 46 hours with time may include a fermentation inducing step in which the strain is cultured at 30 to 34 ° C for 7 to 11 hours ; A germ stage for culturing at 30 to 38 DEG C for 9 to 13 hours; Culturing the cells at 32 to 42 DEG C for 8 to 12 hours; And an enzyme producing step of culturing at 34 to 44 DEG C for 8 to 10 hours.

In one embodiment, the fermentation inducer may proceed for about 8-10 hours from the time of entry, and the germinating and growing step may be performed for a total of 17 hours to 25 hours. The enzyme-producing step may be carried out for about 8-10 hours until departure.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

As a factor for maximizing the useful ingredient of the solid fermented product of brown rice according to one embodiment of the present invention, the rate of bacterial growth rate according to the mycelial infiltration ability of the strain to the inside of the substrate (brown rice), the pretreatment condition of brown rice and the particle size The relationship between the enzyme activity and the amount of enzyme, and the temperature program setting and cycle establishment according to the life cycle of the strain (germination period, germination period, bacterial growth period, enzyme production period) Respectively.

[Experimental Example 1] Comparison of the quality of solid fermented product of brown rice according to the size of brown rice grain

First, as raw material pretreatment conditions, brown rice was prepared under the following three conditions.

Solid substrate Manufacturing method Particle mean diameter (占 퐉) Example 1 Tong Hyeonmi Washing → immersion → water drain → high water 3 to 5 mesh (width: 5 to 8 mm / length: 2 to 4 mm) Example 2 Brown rice powder Washing → dipping → water removal → crushing (1/4 size) → high water 1.0 to 2.5 mm Example 3 Brown rice extrudate Washing → dipping → extrusion → drying 150 to 220 μm

Using a mixture of SMF-138 (KCTC 11989BP) and Risofus delarema SMF-136 (KCTC 11990BP) at a ratio of 1: 1 to Aspergillus oryzae using each of the brown rice samples obtained in Examples 1 to 3 as a solid substrate, And 0.15-0.35% by weight based on the weight of the substrate. At this time, the water content of the brown rice is 33.5 to 35.5% by weight based on the total weight of brown rice, and the water content of the brown rice means the moisture content after immersion in brown rice.

Then, all of the above Examples 1-3 were fermented under the same conditions as described below.

A fermentation inducing step of culturing the strain inoculated in the solid brown rice at 30 to 34 占 폚 for 7 to 11 hours; A germ stage for culturing at 30 to 38 DEG C for 9 to 13 hours; Culturing the cells at 32 to 42 DEG C for 8 to 12 hours; And an enzyme-producing step of culturing at 34 to 44 DEG C for 8 to 10 hours.

Since microorganisms proliferate, fever occurs. In case of solid-phase fermentation, the temperature of the culture should be controlled by heat generation by blowing cool air in the lower part or by shuffling. In the germination stage and the cell growth stage, shaking was performed to dissipate heat to maintain the desired temperature.

The quality of each brown solid fermentation product of Example 1-3 was analyzed as follows.

(1) Comparison of the degree of mycelial penetration according to the particle size of brown rice

A predetermined amount of each of the brown rice solid fermentations of Examples 1 to 3 prepared above was taken and the degree of hyphae penetration was measured with an optical microscope (Olympus BX41).

As a result, as shown in Fig. 1, it can be confirmed that the mycelium of the fermentation strain is spread more in the raw material in the brown rice powder of Example 2 than those of Examples 1 to 3. 1 is a photograph of a microscopic observation of a solid fermentation product at a magnification of 40 times and observing the degree of mycelial infiltration according to a pre-treatment method of brown rice. The second line of FIG. 1 shows a visual observation image immediately after incubation of the brown rice solid fermentation product by the brown rice pretreatment method.

(2) pH comparison

First, the pH change of the culture step in the fermentation step of the strain was analyzed by the following method.

A certain amount of each of the brown rice solid fermentations of Examples 1 to 3 was sampled, and after taking it in a blender, 5 g was taken. After adding 100 mL of 2% NaCl, extract at 30 ~ 1 hour at 25 ~ 30 ℃ and 100 ~ 200 rpm, and measure the pH by filtration.

pH change 0 hours
(Entrance) 9 hours 20 hours 39 hours
(Departure) Example 1 (by Tong Yong Mei) 6.87 6.25 5.53 4.58 Example 2 (brown rice powder) 6.82 5.75 5.64 4.67 Example 3 (brown rice extrudate) 6.79 6.38 5.88 5.73

As a result, the pH of the fermented product tended to decrease gradually as the solid phase fermentation step proceeded.

Then, the change in water content in the substrate (brown rice) over time in the fermentation step of the strain was analyzed by the following method.

Moisture content was measured by the General Test Method in Food Code, 1.1 General Component Test Method atmospheric pressure heating drying method. Specifically, the above Examples 1 to 3 were dried at a temperature higher than the boiling point of water by 105 ° C at normal pressure, and the decreased weight was referred to as a moisture change amount.

Moisture change
(Unit: weight / weight%) 0 hours
(Entrance) 9 hours 20 hours 39 hours
(Departure) Example 1 (by Tong Yong Mei) 34.19 32.90 32.10 30.14 Example 2 (brown rice powder) 35.44 31.07 31.07 30.87 Example 3 (brown rice extrudate) 34.02 31.53 30.39 29.09

As a result, as shown in the above table and Fig. 2, in each of Examples 1 to 3, the moisture content tended to decrease gradually with the progress of solid phase fermentation. Of these, the brown rice powder of Example 2 maintained the highest moisture content in the fermentation stage and maintained the most suitable moisture for growth of the strain and enzyme production.

Then enzyme titer and activity of glucoamylase, alpha-amylase and protease in the substrate (brown rice) over time in the fermentation step of the strain were analyzed by the following method.

Glucoamylase

The 200 mM sodium acetate buffer (pH 4.5) reagent and the Trizma base (pH 8.5) reagent were used in the laboratory for the determination of glucoamylase activity using a Sigma product ≪ / RTI > 200 쨉 l of the enzyme extract of the cultured strain was added to each 200 쨉 l of the substrate, followed by reaction at 40 째 C for 10 minutes. The reaction was then stopped by adding 3 ml of Trizma base and the absorbance was measured at 400 nm.

Alpha-amylase

The measurement of alpha-amylase activity was carried out using an assay kit (Bioassay, DAMY-100) which provided a substrate, a stop reagent and a calibrator. After incubation for 5 minutes at 37 ° C, 80 μl of stopping reagent was added, and after stopping the reaction, the reaction mixture was centrifuged at 14,000 rpm for 5 minutes to obtain 200 μl of the supernatant, and the absorbance at OD 595 nm Respectively.

Protease

A certain amount of samples (Example 1-3) was sampled by a sample pretreatment, and after taking the sample in a blender, 5 g was taken. After adding 100 mL of 2% NaCl, the mixture was extracted at 25 to 30 DEG C and 100 to 200 rpm for 30 to 1 hour. The extract was filtered through NO.2 Whatman filter paper to obtain a filtrate (enzyme solution). The reaction was carried out by adding 1000 효 of the enzyme of each of the cultured strains and 2000 기 of the substrate and reacting at 30 캜 for 20 minutes. 0.4 M TCA 5 ml of the reaction mixture was reacted at 30 ° C for 20 minutes, filtered through NO.4 Whatman filter paper, then treated with 1 ml of filtrate, 5 ml of 0.4 M Na2CO3 and ₁ ml of Foline reagent, and after 20 minutes of reaction at 30 ° C after OD measurement at 660 nm .

The unit "U / g" in the table below means the enzyme activity (U), ie the enzyme activity unit, for the amount (g) of starch or protein hydrolyzed by the enzyme. Herein, "1U" represents the amount of starch that dextrins 1 mg of soluble starch per minute under standard assay conditions for glucoamylase and alpha-amylase, the amount of tyrosine (μg) produced per minute under standard analytical conditions for protease, .

Glucoamylase
(Unit: U / g) 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 1 (by Tong Yong Mei) 0.0 6.80 24.4 581.2 Example 2 (brown rice powder) 0.0 7.5 47.2 746.7 Example 3 (brown rice extrudate) 0.0 24.1 113.8 137.9

α-amylase
(Unit: U / g) 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 1 (by Tong Yong Mei) 0.0 11.3 43.2 101.8 Example 2 (brown rice powder) 0.0 20.5 62.5 232.9 Example 3 (brown rice extrudate) 0.0 20.8 53.1 226.7

Protease
(Unit: U / g) 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 1 (by Tong Yong Mei) 0.0 1.5 2.5 39.5 Example 2 (brown rice powder) 0.0 1.3 10.5 68.9 Example 3 (brown rice extrudate) 0.0 0.0 3.6 15.9

As a result, as can be seen from Tables 4 to 6 and Figs. 3 to 5, the concentrations of glucoamylase, alpha-amylase and protease were the highest in the fermentation of the brown rice crumbs, indicating excellent enzyme activity .

[Experimental Example 2] Comparison of the quality of solid fermented product of brown rice with fermentation temperature

The temperature program according to the life cycle of the fermentation strains during the solid phase fermentation of brown rice was set as shown below, and the enzymatic activities were compared and analyzed.

As the brown rice according to one embodiment of the present invention, SMF-138 (KCTC 11989BP) and Rizofus delemma SMF-136 (KCTC 11990BP) were added to Aspergillus oryzae at a ratio of 1: 1 Was inoculated to the substrate in an amount of 0.15 to 0.35% by weight based on the total weight of the substrate.

Then, the fermentation time was set to be the induction period (9 hours to enter), germination period and cell growth period (9 hours to 30 hours) and enzyme generator (30 hours to departure time = 39 hours) Respectively. Example 4 was set based on the optimum temperature for enzyme activity of protease at 30 to 36 ° C, and Example 5 was set at 35 to 42 ° C, which is the optimum temperature for enzyme activity of glucoamylase and alpha-amylase.

Since microorganisms proliferate, fever occurs. In case of solid-phase fermentation, the temperature of the culture should be controlled by heat generation by blowing cool air in the lower part or by shuffling. In the germination stage and the cell growth stage, shaking was performed to dissipate heat to maintain the desired temperature.

Temperature condition (℃) Item 0 hours
(Entrance) 9 hours
(Germination) 20 hours
(Cell growth period) 30 hours
(Enzyme generator) 39 hours
(Departure) Example 4 32 32 36 36 36 Example 5 33 35 40 42 35

The quality of each brown solid fermentation product according to Examples 4 and 5 was analyzed as follows.

(1) pH comparison

The pH change over time in the fermentation stage of the strain was analyzed by the following method.

A certain amount of a sample (brown rice powder of Examples 4 and 5) is sampled, and after taking it in a blender, take 5 g. After adding 100 mL of 2% NaCl, extract at 30 ~ 1 hour at 25 ~ 30 ℃ and 100 ~ 200 rpm, and measure the pH by filtration.

pH change 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 4 6.87 6.25 5.53 4.58 Example 5 6.82 5.75 5.64 4.67

As can be seen from the above Table 8, the pH of Examples 4 and 5 tended to gradually decrease with the passage of the solid fermentation culture time. This is because the pH of the strain is decreased due to the generation of organic acids and the like as the strain grows.

Then, the change in water content in the substrate (brown rice) over time in the fermentation step of the strain was analyzed by the following method.

Moisture content was measured using the General Test Methods in Food Code, 1.1 General Component Test Method atmospheric pressure drying method. The sample was dried at atmospheric pressure at a temperature slightly higher than the boiling point of water at 105 ° C, and the reduced amount was used as the water content.

Moisture change
(Unit: weight / weight%) 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 4 35.6 34.2 30.1 30.4 Example 5 34.7 34.1 30.2 29.1

As a result, as shown in the above table and Fig. 7, in each of Examples 4 and 5, the water content tended to decrease with time in solid fermentation culture. The highest moisture content was maintained in the fermentation step under the temperature condition of Example 4. It can be confirmed that proper moisture content is maintained for fungal growth and enzyme production, and enzyme activity can be maintained well.

Then, the changes in the amounts of production of glucoamylase, alpha-amylase and protease in the substrate (Examples 4 and 5) over time in the fermentation step of the strain were analyzed by the following method.

Glucoamylase

The 200 mM sodium acetate buffer (pH 4.5) reagent and the Trizma base (pH 8.5) reagent were used in the laboratory for the determination of glucoamylase activity using a Sigma product ≪ / RTI > 200 쨉 l of the enzyme extract of the cultured strain was added to each 200 쨉 l of the substrate, followed by reaction at 40 째 C for 10 minutes. The reaction was then stopped by adding 3 ml of Trizma base and the absorbance was measured at 400 nm.

Alpha-amylase

The measurement of alpha-amylase activity was carried out using an assay kit (Bioassay, DAMY-100) which provided a substrate, a stop reagent and a calibrator. After incubation for 5 minutes at 37 ° C, 80 μl of stopping reagent was added, and after stopping the reaction, the reaction mixture was centrifuged at 14,000 rpm for 5 minutes to obtain 200 μl of the supernatant, and the absorbance at OD 595 nm Respectively.

Protease

A certain amount of the sample was sampled by the pretreatment of the sample, and after taking the sample in the blender, 5 g was taken. After adding 100 mL of 2% NaCl, the mixture was extracted at 25 to 30 DEG C and 100 to 200 rpm for 30 to 1 hour. The extract was filtered through NO.2 Whatman filter paper to obtain a filtrate (enzyme solution). The reaction was carried out by adding 1000 효 of the enzyme of each of the cultured strains and 2000 기 of the substrate and reacting at 30 캜 for 20 minutes. The reaction was carried out in 5 ml of 0.4 M TCA for 20 min, filtered through NO.4 Whatman filter paper, and then treated with 1 ml of filtrate, 5 ml of 0.4 M Na2CO3 and ₁ ml of Foline reagent, followed by 20 min at 30 ° C and OD after 660 nm.

Glucoamylase
(Unit: U / g) 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 4 0.5 7.6 25.9 757.6 Example 5 0.6 6.8 14.5 507.9

α-amylase
(Unit: U / g) 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 4 0.1 18.8 75.1 242.9 Example 5 .2 23.5 61.5 58.6

Protease
(Unit: U / g) 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 4 0.0 2.0 12.5 70.9 Example 5 0.0 1.5 7.5 29.5

As a result, as can be seen from Tables 10 to 12 and FIGS. 8 to 10, the concentration of glucoamylase, alpha-amylase and protease was the highest at fermentation at the temperature condition of Example 4, .

(2) Comparison of cell mass

90 g of 0.85% NaCl diluted water was added to 10 g of each of the solid-phase fermentation samples of Examples 4 and 5, and then sufficiently treated so that cells (mycelium and spores) could be dropped from the raw material using a stomacher. It was repeated 3 to 5 times per sample, filtered through filter paper, and weighed before and after drying.

Amount of bacterium
(Unit: mg / g) 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 4 - 20.5 33.6 54.6 Example 5 - 14.1 23.2 29.3

As a result, as shown in the above table and Fig. 11, the cell mass of Example 4, in which the temperature condition at the time of solid phase fermentation was adjusted to the protease, was measured to be about two times higher than that of Example 5. Brown rice has a part of rice bran, so it can not easily infiltrate the fungus and its enzyme production is weak. Therefore, high cell mass in the solid fermented medium means that the microorganism penetrates into the brown rice substrate well and grows well, and thus the desired enzyme production is also excellent.

(3) Comparison of degree of penetration of hypha according to fermentation temperature

A predetermined amount of each of the brown rice solid fermentations of Examples 4 and 5 prepared above was collected and the degree of hyphae penetration was measured with an optical microscope (Olympus (BX41)).

As a result, as shown in Fig. 12, it can be confirmed that the hyphae of the fermentation strain spread more in the raw material in the solid fermented product in the solid state of Example 4. [

[Experimental Example 3] Comparison of the quality of solid fermented products of brown rice with fermentation strains

As the brown rice according to one embodiment of the present invention, Rizopus delarema SMF-136 (KCTC 11990BP) and Aspergillus oryzae SMF-138 (KCTC 11989BP) strains were mixed at a ratio of 7: 3, 7: 5, and 3: 7 (weight ratio) was inoculated to the substrate in an amount of 0.15-0.35 wt% based on the total weight of the substrate.

Then, the fermentation time was set to be the induction period (9 hours for entry), germination period and cell growth period (9 hours to 30 hours), enzyme generator (30 hours to departure time = 39 hours) Respectively.

At this time, since the microorganisms proliferate, a heat is generated. For the solid fermentation, the temperature of the culture should be controlled by heating by blowing cool air or by shaking the lower part. In the germination stage and the cell growth stage, shaking was performed to dissipate heat to maintain the desired temperature.

Finally set blend ratio Item Risofus delerma SMF-136: Aspergillus oryzae SMF-138 Example 6 7: 3 Example 7 5: 5 Example 8 3: 7

(1) pH comparison

First, in order to analyze the enzyme activity, the pH change over time in the fermentation step of the strain was analyzed by the following method.

A certain amount of each of the samples of Examples 6 to 8 was sampled and taken in a blender to take 5 g. After adding 100 mL of 2% NaCl, extract at 30 ~ 1 hour at 25 ~ 30 ℃ and 100 ~ 200 rpm, and measure the pH by filtration.

Moisture change
(Unit: weight / weight%) 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 6 (7: 3) 34.19 32.90 32.10 30.14 Example 7 (5: 5) 35.44 31.07 31.07 30.87 Example 8 (3: 7) 34.02 31.53 30.39 29.09

As a result, as shown in the above table and FIG. 13, it can be confirmed that the enzyme activity can be maintained well by maintaining the highest moisture content in the fermentation step in the mixed strains mixed at the ratio of Example 7.

Then, the changes in the production amounts of glucoamylase, alpha-amylase and protease in the substrate (Examples 6-8) over time in the fermentation step of the strain were analyzed by the following method.

Glucoamylase

The 200 mM sodium acetate buffer (pH 4.5) reagent and the Trizma base (pH 8.5) reagent were used in the laboratory for the determination of glucoamylase activity using a Sigma product ≪ / RTI > 200 쨉 l of the enzyme extract of the cultured strain was added to each 200 쨉 l of the substrate, followed by reaction at 40 째 C for 10 minutes. The reaction was then stopped by adding 3 ml of Trizma base and the absorbance was measured at 400 nm.

Alpha-amylase

The measurement of alpha-amylase activity was carried out using an assay kit (Bioassay, DAMY-100) which provided a substrate, a stop reagent and a calibrator. After incubation for 5 minutes at 37 ° C, 80 μl of stopping reagent was added, and after stopping the reaction, the reaction mixture was centrifuged at 14,000 rpm for 5 minutes to obtain 200 μl of the supernatant, and the absorbance at OD 595 nm Respectively.

Protease

A certain amount of the sample was sampled by the pretreatment of the sample, and after taking the sample in the blender, 5 g was taken. After adding 100 mL of 2% NaCl, the mixture was extracted at 25 to 30 DEG C and 100 to 200 rpm for 30 to 1 hour. The extract was filtered through NO.2 Whatman filter paper to obtain a filtrate (enzyme solution). The reaction was carried out by adding 1000 효 of the enzyme of each of the cultured strains and 2000 기 of the substrate and reacting at 30 캜 for 20 minutes. 0.4 M TCA 5 ml of the reaction mixture was reacted at 30 ° C for 20 minutes, filtered through NO.4 Whatman filter paper, then treated with 1 ml of filtrate, 5 ml of 0.4 M Na2CO3 and ₁ ml of Foline reagent, and after 20 minutes of reaction at 30 ° C after OD measurement at 660 nm .

Glucoamylase
(Unit: U / g) 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 6 (7: 3) 0.0 12.8 50.4 981.1 Example 7 (5: 5) 0.0 10.5 52.7 800.7 Example 8 (3: 7) 0.0 11.1 32.1 105.1

α-amylase
(Unit: U / g) 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 6 (7: 3) 0.0 9.2 32.3 91.5 Example 7 (5: 5) 0.0 32.5 73.2 322.9 Example 8 (3: 7) 0.0 120.8 53.1 23.1

Protease
(Unit: U / g) 0 hours
(Entrance) 9 hours
20 hours
39 hours
(Departure) Example 6 (7: 3) 0.0 0.5 2.5 19.5 Example 7 (5: 5) 0.0 3.3 25.5 88.9 Example 8 (3: 7) 0.0 0.0 2.6 5.9

As a result, as can be seen from Tables 16 to 18 and Figs. 14 to 16, the three strains showed a high concentration when fermenting with the complex strain mixed at the ratio of Example 7, and in particular, alpha-amylase and protease And the enzyme activity was excellent.

The results are summarized as follows. In Example 6, in which the strain was mixed with Risofus delema and Aspergillus oryzae at a ratio of 7: 3, pH was lowered at the time of departure due to the effect of the Risofus delrema strain producing the organic acid, Although the glucoamylase activity was highly derived, it was not suitable for the conditions for optimizing the solid fermentation of the brown rice fermentation because it did not meet the purpose of the complex enzyme activity that the three enzyme activities of glucoamylase, alpha-amylase and protease were expected to be high.

In Example 8 where the two strains were mixed at a ratio of 3: 7, the emulsion process proceeded from 20 hours after the solid phase fermentation due to the activity of the strain in Aspergillus oryzae having high alpha-amylase activity, and the liquefaction of the brown rice It was difficult to expect a solid fermented product in the form of a complete form at the time of going out. In addition, since the enzymatic activity of alpha - amylase appeared during the solid fermentation process, enzymes were not active at the completion of fermentation 39 hours after the entry, and were not suitable for solid fermentation conditions of brown rice.

Example 7, which was fermented by mixing the above two strains at a ratio of 5: 5, showed a fermentation result of Aspergillus oryzae which produces protease and α-amylase external cell wall degrading enzyme, Rizopus delarema strain producing organic acid and glucoamylase , The activities of the desired enzymes glucoamylase, alpha-amylase and protease are shown to be high in the solid fermentation product, which is suitable for solid phase fermentation.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Biotechnology Research Institute KCTC11989BP, KCTC11990BP 20110728

Claims (10)

A solid brown rice powder having an average particle diameter of 1.0 to 2.5 mm is fermented at 30 to 36 占 폚 by a complex strain comprising an Aspergillus sp . Strain and a Rhizopus sp. Lt; / RTI &
The complex strain is a solid-phase fermentation product of brown rice, which is a complex strain in which Aspergillus oryzae and Rhizopus delemar are mixed at a weight ratio of 4: 6 to 6: 4 . delete The method of claim 1, wherein the Aspergillus spp and Rhizopus crispus in strain glucoamylase (glucoamylase), alpha-one amylase (α-amylase) and protease (protease) (Aspergillu s sp. ) (Rhizopus sp.) The solid fermented product of brown rice, which is a strain that produces the above. delete The method of claim 1, wherein the Aspergillus genus (Aspergillu s sp.) Strains are Aspergillus and the scan duck chair (Aspergillus oryzae) SMF-138 ( KCTC 11989BP), the separation tank crispus in (Rhizopus sp.) Strain is Rhizopus A solid fermented product of brown rice, which is a strain of Rhizopus delemar SMF-136 (KCTC 11990BP). delete The method for producing a solid fermented product of brown rice according to any one of claims 1, 3 and 5,
A step of inoculating Aspergillus oryzae and Rhizopus delemar strains to a substrate comprising a solid phase brown rice powder having a water content of 33.5 to 35.5% by weight; And
Culturing the strain inoculated with the solid phase brown rice powder at 30 to 36 DEG C for 30 to 46 hours with varying temperature over time;
≪ / RTI > 8. The method according to claim 7, wherein, before the step of inoculating the strain,
And a step of pulverizing the brown rice flour into a size of 15 to 35% by volume based on the total volume of the brown rice flour, and then heating the brown rice flour to pre-treat the solid brown rice to prepare a solid brown rice flour ≪ RTI ID = 0.0 > 1, < / RTI > 8. The method of claim 7, wherein the step of varying and varying the temperature over time comprises:
A fermentation inducing step of culturing at 30 to 34 DEG C for 7 to 11 hours;
A germic step of culturing at 30 to 36 DEG C for 9 to 13 hours;
Culturing the cells at 32 to 36 DEG C for 8 to 12 hours; And
An enzyme producing step of culturing at 34 to 36 DEG C for 8 to 10 hours;
≪ / RTI > A food comprising a solid fermented product of brown rice according to any one of claims 1, 3 and 5.

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