KR20160059735A - Method for producing Chunggukjang with increased menaquinone-7 content and Chunggukjang produced by the same method - Google Patents

Abstract

The present invention relates to a method for producing cheonggukjang (fermented soybeans) with an increased menaquinone-7 content, the cheonggukjang with an increased menaquinone-7 content produced by the same method, and a healthy functional food composition containing the cheonggukjang for preventing and alleviating osteoporosis. The present invention provides the method for producing the cheonggukjang with a markedly increased menaquinone-7 content by optimizing fermentation time, the amount of a carbon source to be added, and the amount of bacteria to be inoculated. The cheonggukjang with an increased menaquinone-7 content can be effectively used as healthy functional food for preventing and alleviating osteoporosis.

Description

[0001] The present invention relates to a method for producing a cheonggukjang having an increased content of menaquinone-7 and a method for producing chungkukjang,

More particularly, the present invention relates to a method and a method for producing a fermented soy sauce having an increased content of menaquinone-7 by optimizing the fermentation time, And a health functional food composition for preventing or ameliorating osteoporosis, which comprises the cheonggukjang and the cheonggukjang which are increased in the content of menaquinone-7 produced by the above method.

Cheonggukjang is a traditional fermented food in Korea. Chungkukjang Soy beans are soaked in water, steamed and then inoculated with Bacillus spp. And fermented together. During fermentation, the biological compounds isoflavone, poly-gamma-glutamate, amino acid, fibrinolytic enzyme and menaquinone-7 (MK- 7) increased. Within these active elements, MK-7 is particularly effective in osteoporosis, cardiovascular disease, inflammation or cancer and is considered an attractive supplement to promote health. In addition, no side effects such as hypercoagulability were found in clinical trials when the dose of MK-7 was used in excess of 40 mg / day.

MK-7 is a kind of vitamin K ₂ and is mainly produced by fermentation by Bacillus species. In particular, Japanese soybean fermented food, natto, has been reported to have a relatively high content of MK-7 (8-9 μg / g), and Natto is a major source of vitamin K ₂ intake in Japan. However, the recommended daily intake of MK-7 is reported to be 180 μg / day, and thus the content of MK-7 in natto is not sufficient for the treatment of osteoporosis or cardiovascular disease. Therefore, research that can produce MK-7 to the recommended daily intake is necessary. The present inventors have attempted to isolate strains producing MK-7 by a large number of previous studies, to make genetic mutations, to optimize the fermentation medium and to control the production environment such as temperature and humidity. Although the production of MK-7 was significantly increased in the method using genetic mutations, the use of genetic mutations was limited for safety in the food industry, and in the liquid culture medium, the MK of Bacillus subtilis -7 production (62 / / mL) was significantly increased. However, since products in liquid medium can not be consumed as food materials, foods rich in MK-7 are still limited to Natto and Chungkukjang. However, the concentration of natto and chonggukjang is 12 ㎍ / g, which is still insufficient for daily intake.

Accordingly, it is known that the physiological characteristics of Chungkookjang are affected by fermentation conditions. Therefore, an optimized preparation method of Chungkookjang with increased content of MK-7 using Bacillus subtilis strain producing MK-7 Respectively.

Korean Patent Laid-Open Publication No. 2001-0036099 discloses 'a method for producing a vitamin B1-enriched chungkukjang', and Korean Patent Publication 2008-0018568 discloses a 'functionalized chungkukjang having enhanced antioxidant activity' There is no description about a method for producing the cheonggukjang with an increased content of menaquinone-7 of the present invention.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a fermentation broth of Bacillus subtilis strain having high productivity of menaquinone-7, And that the amount of menaquinone-7 was greatly increased compared with that of commercially available chungkukjang and natto, which were obtained through the optimum fermentation conditions. Thus, the present invention has been completed.

In order to solve the above problems, the present invention provides a method for producing chungkukjang wherein the content of menaquinone-7 is remarkably increased by optimizing optimum fermentation time, amount of carbon source and amount of inoculum.

In addition, the present invention provides an increased content of menaquinone-7 produced by the above method.

In addition, the present invention provides a health functional food composition for preventing or ameliorating osteoporosis including the above-mentioned chungkukjang.

The production method of soybean in accordance with the invention provides an optimized method for preparing a content of the menaquinone -7 form of vitamin K ₂ increase very Soybean Soybean than normal. In addition, the increased content of menaquinone-7 produced by the method described above is useful as a health functional food for many diseases related to prevention or improvement of osteoporosis, reduction of other cholesterol in blood, prevention of hypertension, antioxidant effect, It is expected that it can be used.

FIG. 1 shows the results of examining the effect of temperature (A) and relative humidity (B) on the production of menaquinone-7 during fermentation of Cheonggukjang.
FIG. 2 shows the results of confirming the effect of fermentation time on the production of menaquinone-7 during fermentation of Cheonggukjang.
FIG. 3 is a graph showing contour lines and reaction surface profiles of changes in the content of menaquinone-7 (MK-7) according to the combination of fermentation time and maltitol added during fermentation.
FIG. 4 is a graph showing contour lines and response surface profiles of changes in the content of menaquinone-7 (MK-7) according to the combination of fermentation time and mycobacterial inoculum during fermentation.
FIG. 5 is a graph showing contour lines and response surface diagrams of changes in the content of menaquinone-7 (MK-7) according to the combination of maltitol addition amount and mycobacterial inoculum during fermentation.
6 shows the results of confirming the thermal stability of menaquinone-7 (MK-7) in chungkukjang prepared according to the optimized fermentation conditions of the present invention.

In order to achieve the object of the present invention,

(a) washing soybeans and immersing them in water;

(b) boiling the soaked soybeans after removing the water of step (a);

(c) inoculating the yeast strain Bacillus subtilis BY07 (accession number: KCTC 12392BP) after boiling the soybeans in step (b); And

(d) fermenting soybeans inoculated with the strain of Bacillus subtilis in step (c), wherein the content of menaquinone-7 is increased.

In one embodiment of the present invention, the Bacillus subtilis strain of step (c) of the method for preparing a cheonggukjang can be inoculated at 3-4% of the total volume of soybean, preferably 3.5-3.9% , And more preferably 3.69%. However, the Bacillus subtilis strain of the present invention is a Bacillus subtilis BY07 strain (accession number: KCTC 12392BP) having excellent ability to produce menaquinone-7 Patent No. 2014-0010880).

The fermentation condition of step (d) may be performed at a temperature of 41 to 45 ° C under a relative humidity of 85 to 95% for 90 to 100 hours, preferably at 42 The reaction can be carried out at a temperature of ~ 44 ° C, at a relative humidity of 88-92% for 93-97 hours, more preferably at a temperature of 43 ° C, at a relative humidity of 90% for 96 hours, It is not limited. In the fermentation step, maltitol, glycerol, glucose, sucrose, maltose and oligosaccharide may be further added as a carbon source, preferably maltitol. The added amount is 7 To 8%, and more preferably, 7.51% of the total volume of soybean maltitol can be added to ferment, but the present invention is not limited thereto.

In the preliminary experiments of the present invention, the content of menaquinone-7 was superior to that of the control group in both of the cases using glycerol and maltitol, but the use of glycerol was restricted by the regulations of the United States Food and Drug Administration (FDA) The FDA has demonstrated that it is safe for maltitol to be used in food, and has disclosed that it can contain up to 75% of its use. In addition, maltitol has low calorie, it does not rot and does not increase blood sugar level, and it has an advantage of enhancing calcium absorption without stimulating insulin.

In one embodiment of the present invention, a preferred method of producing chinookugang with an increased content of menaquinone-

(a) washing soybeans and immersing them in water at 2 to 6 DEG C for 22 to 26 hours;

(b) removing the water in step (a) and boiling the soybeans at 110 to 130 ° C for 20 to 40 minutes;

(c) after boiling the soybeans in step (b), inoculating the strain Bacillus subtilis BY07 (accession no. KCTC 12392BP) with 3-4% of the total volume of boiled soybeans; And

(d) adding maltitol as a carbon source to soybeans inoculated with Bacillus subtilis BY07 strain in step (c) at 7 to 8% of the total volume of soybeans, and fermenting at 42 to 44 ° C for 90 to 100 hours; And most preferably < RTI ID = 0.0 >

(a) washing the soybeans and immersing them in water at 4 DEG C for 24 hours;

(b) boiling the soaked soybeans at 121 DEG C for 30 minutes after removing the water of step (a);

(c) after boiling the soybeans in step (b), inoculating the strain Bacillus subtilis BY07 (accession number: KCTC 12392BP) to 3.69% of the total volume of boiled soybeans; And

(d) adding maltitol as a carbon source to the soybeans inoculated with Bacillus subtilis BY07 strain of step (c) in an amount of 7.51% of the total volume of soybeans, followed by fermentation at 43 ° C for 96 hours, But is not limited thereto.

In addition, the present invention provides an increased content of menaquinone-7 produced by the above method.

In addition, the present invention provides a health functional food composition for preventing or ameliorating osteoporosis including the above-mentioned chungkukjang.

The content of menaquinone-7 was found to be at least 3 times to 46 times higher than that of the prior literature, commercially available chongkukjang or natto (Table 3), and the menaquinone- 7 is known to be effective for osteoporosis, the chongkukjang prepared by the method of the present invention can be usefully used for preventing or improving osteoporosis. There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

reagent

Pure MK-7 (menaquinone-7) was purchased from ChromaDex Inc. (USA). Glycerol, glucose, maltose and sucrose were purchased from Junsei Chemical Co. (Japan), 2-propanol, n-hexane, ethanol and methanol were purchased from JT Baker (USA). TSB (tryptic soy broth) was purchased from Difco (USA). All other compounds used in the invention were analytical grade.

Preparing Cheonggukjang

The soybeans used in the invention were purchased in the local market and were immersed in 4 ° C water for 24 hours after 3 washes. Then, the water was discarded and the beans were boiled for 30 minutes at 121 ° C in a high-pressure pot, cooled to about 40 ° C, and then inoculated with Bacillus subtilis BY07 strain (accession number: KCTC 12392BP) at 10 ⁸ cells / mL. Finally, the fermentation was carried out for 1 to 4 days at 43 ° C and a relative humidity of 90%.

MK-7 analysis using HPLC

Analysis of Chungkukjang extract and MK-7 was performed according to the prescribed procedure and some modifications were made. Briefly, 4.8 mL of 2-propanol and 9.6 mL of n-hexane were mixed with 4 g of chungkukjang and mixed for 1 minute. Thereafter, the mixture was kept at room temperature for 1 hour, followed by centrifugation at 3,200 rpm for 10 minutes. After centrifugation, the hexane layer was picked and evaporated with nitrogen gas. The remaining after evaporation was again dissolved in ethanol and filtered using a 0.45-μm syringe filter followed by HPLC analysis. MK-7 was measured using HPLC with a Waters Millennium system and a fluorescence detector (Waters). The column used was a Nova-Pak C18 column. The etector was measured using a fluorescence detector (λex: 320 nm, λem: 430 nm). The temperature was fixed at 40 占 폚. The mobile phase was 100% methanol and the concentration of MK-7 in the chungkukjang was expressed as the amount of content per gram of chongkukjang.

Optimization of fermentation conditions for increasing MK-7 content

The external environment of the fermentation process, including incubation temperature and relative humidity, to increase the content of MK-7 in chongkukjang has been previously studied. In addition to the external environment, the internal factors of the fermentation process include the amount of bacteria inoculated, the fermentation time, and the constituents of the substance, and other factors were constantly maintained in order to find the optimization condition of the single element.

First, the inoculum was tested at 0.4% to 0.5% and fermented at 36 hours fermentation time without any additional substances. In addition, previous studies have shown that the addition of carbon source during fermentation significantly increases the content of MK-7 in chungkukjang. Therefore, to investigate the content of MK-7 according to various concentrations of maltitol, one of the carbon sources, The optimum concentration of carbon source added to the weight was experimented. Finally, the fermentation time was varied from 24 hours to 96 hours, and the change of MK-7 content was confirmed. Three levels of each processing variable were determined by response surface method (RSM). The central composite design method (CCRD) was used for the three factors planned at the central point. The Central Composite Programming (CCRD) was used to analyze the interaction of these parameters and to determine the optimization level of each parameter. The Design Expert statistical package was used to compute the polynomial coefficients of the second instruction. And the change analysis (ANOVA) was performed to test the importance and appropriateness of the model.

Check the model

The optimal environment for MK-7 synthesis in Chungkookjang was obtained by solving the regression equation. Thereafter, the concentration of MK-7 was repeated three times by measuring the optimized amount of inoculum, fermentation time and carbon source, and the experimental and predictive values were compared to determine the validity of the model.

Confirmation of safety of MK-7 in Cheonggukjang

I checked the safety of MK-7's heat in Cheonggukjang by boiling Chongkukjang for various time. Briefly, 2 g of chungkukjang were mixed with 2 ml of distilled water and then boiled at 100 ° C for 15, 30, 45 and 60 minutes. Thereafter, MK-7 was extracted with a mixture of 2-propanol and n-hexane as described above and analyzed by HPLC.

Statistical analysis

Experimental results were analyzed using IBM SPSS statistical software, and significance analysis between samples was determined at p <0.05 using Duncan's multiple assays.

Example  1. Temperature and relative humidity during fermentation MK -7 Analysis of impact on production

The effect of temperature on the production of MK-7 was studied in the range of 37 to 46 캜, and it was confirmed that the concentration of MK-7 increased to 43 캜 similarly to the previous study (Fig. 1A). In addition, it was confirmed that the relative humidity, which is considered to be an important factor in the fermentation of chongkukjang, continuously increases the concentration of MK-7 as the relative humidity increases (Fig. 1B). Considering the cost efficiency and the possibility of realistic application, the optimum temperature of fermented soybean fermentation was selected as 43 ℃ and the relative humidity was 90%, and other internal factors were optimized.

Example 2. Selection of the number of experimental design variables

In fermentation of Cheonggukjang, influences such as incubation temperature, relative humidity, bacterial inoculation amount, addition of carbon source and fermentation time are expected to be independent and reciprocal. In order to optimize the MK-7 synthesis environment, the values of each variable were set to low, medium and high using the response surface analysis (RSM) in preliminary experiments. The results of preliminary experiments showed that the appropriate inoculum amount was about 3%, and the values of low, medium and high inoculum amounts were 1.5, 3 and 4.5%, respectively. Regarding the carbon source, considering that there is not a statistical difference between glycerol and maltitol used as a carbon source in promoting MK-7 synthesis by the strain Bacillus subtilis BY07 used in the present invention, maltitol was fermented The optimum amount of carbon source was determined to be 6% (v / v), and the response surface analysis showed low, medium and high choices of 4, 6 and 8% for maltitol addition, respectively. The fermentation time of Chungkukjang was about 2-3 days in the previous study. In the present invention, the fermentation time was prolonged from 24 hours to 96 hours, and as a result, it was confirmed that the concentration of MK-7 was continuously increased in proportion to the fermentation time (FIG. 2). However, as the fermentation time increases, the color of Chonggukjang becomes darker and unpleasant smell is produced. These phenomena were thought to hinder Chongkukjang consumption. Therefore, 48, 72 and 96 hours were selected and used to optimize the response surface analysis in consideration of the efficiency of consumption and cost.

Example 3. Model setting

The central composite rotatable design (CCRD) was used to analyze the effects of the above three factors (A: fermentation time, B:% maltitol addition, C: inoculum) Respectively. The values including the respective elements are shown in Table 1 with five values, and the temperature and the relative humidity were set to 43 ° C and 95%, respectively. The layout of the experiment was set at 3x and the three means are shown in Table 1. [

The response of the surface model set-up in the form of ANOVA is shown in Table 2 as the second result. The ANOVA of the secondary regression model was quite high, and its evidence was a very low F-value of 24.38. P-value is used as a tool to check each independent variable, and small p-value shows great importance in various variables. In the present invention, the P-value was less than 0.0001, and these values are values indicating that the model condition is significant.

In the present invention, A, AB, A2 and B2 were important model sources. In addition, a linear regression model (A) of fermentation time, a secondary regression model of fermentation time (A2) and% maltitol addition (B2) were significantly more important than other factors, -7 synthesis rate. A multiple regression analysis of the experimental data gives a mathematical model for the MK-7 content within the experimental range and this relationship can be represented by the following quadratic polynomial model.

Y = 41.77 + 9.46A + 1.38B + 1.21C + 2.07B - 0.046AC - 0.12BC - 2.62A ² - 2.25 B ² - 1.17 C ²

(Concentration of Y, MK-7; A, fermentation time; B, maltitol addition%; C,

The model decision coefficient is 0.9564, which is 96% of the variability of the target model. The value of the adjusted coefficient of determination was also sufficient to support the importance of the model. The lack-of-fit test replicates points by comparing the residual error with the pure error. The F-value of 1.99, the lack of conformance, is also not significant with the net error and lack of fit. In addition, the measured model is well suited to the experimental data. At the same time, the relatively low value of the coefficient of variation (CV = 6.87%) showed an increase in accuracy and reliability of the given experiment. In addition, "Adeq. Precision" is used to measure the noise ratio signal, and the signal ratio of the sufficient signal is greater than 4. In this analysis the ratio of 17.403 was a sufficient signal and this model could be used to guide the area. Determining the optimized values of each factor to investigate the interactions between variables and to maximize the production of MK-7 in fermentation of Chungkookjang fermentation can be accomplished by using the Bacillus subtilis BY07 strain and comparing the reaction of the surface and contour lines with the model equation Respectively. Each plot showed an infinite number of combinations of two test variables, the other two maintained their respective 0 values, Figure 3 depicted the effect of fermentation time, the interaction of maltitol addition, and the inoculum amount was maintained at 3% . When maltitol addition showed a secondary effect, fermentation time showed both straight and quadratic yields in the yield of MK-7. In addition, the interactions between fermentation time and maltitol were also found to be important in this study. 4, when the maltitol addition was maintained at 6%, the fermentation time caused a linear increase in the reaction under the level of the inoculum amount. However, the effect of inoculation dose on the production of MK-7 is not important. Figure 5 shows the interaction between the maltitol addition and the inoculum amount and obtained a similarity between these two variables. Fermentation time (A) And Fig. 4 and the model equation show that the interaction between maltitol addition and inoculum is not important. These results indicate that the synthesis of MK-7 in chungkukjang will be significantly affected by the fermentation time and the amount of maltitol added, while the inoculum dose is less effective. Fermentation time was dominant in the synthesis of MK-7, and increasing fermentation time in Chungkukjang resulted in increasing the concentration of MK-7. The optimized level of the three studies was obtained by solving the regression equation within the experimental area. The optimum fermentation time, amount of maltitol, and inoculum amount were increased to 96 hours, 7.51% and 3.69%, respectively, in order to increase the content of MK-7 in the production of chungkukjang. Under these optimization conditions, the maximum amount of MK-7 was 50.18 / / g and between 46.95 / / g and 53.41 / / g with 95% confidence.

Example 4. Model Identification

Based on the statistical results, confidence experiments were performed from the response surface analysis along with the predicted environment, and the concentration of MK-7 in the chrysanthemum was determined to be 51.73 μg / g, the maximum value of which was 95.1% did. A firm association between the predicted and experimental values confirmed the validity and validity of the predicted model. The concentration of MK-7 was carried out through a study comparing the commercially available Cheonggukjang and Natto, as in the previous study. The concentration of MK-7 in Chungkukjang was significantly higher than that of Chongkukjang and Natto (Table 3).

Example 5. Thermal stability of MK-7

Unlike Natto, which is consumed without processing, most of the Cheonggukjang is boiled. Therefore, it is believed that boiling chungkukjang may lower the content of MK-7, so that the thermal stability of MK-7 was confirmed in the present invention. The prepared fermented soybean curd of the present invention was boiled at 100 DEG C for 60 minutes. As a result, it was observed that the content of MK-7 in the fermented soy sauce simmered for 60 minutes was not substantially different from that of the control fermented soy sauce (Fig. 6), indicating that MK-7 in the fermented soy sauce had heat stability Respectively. Thus, the increased content of MK-7 produced by the method of the present invention will be a food that can be beneficially used to promote health to people who are deficient in vitamin K ₂ .

Claims (8)

(a) washing soybeans and immersing them in water;
(b) boiling the soaked soybeans after removing the water of step (a);
(c) inoculating the yeast strain Bacillus subtilis BY07 (accession number: KCTC 12392BP) after boiling the soybeans in step (b); And
(d) fermenting soybeans inoculated with the strain of Bacillus subtilis in step (c), wherein the content of menaquinone-7 is increased. The method according to claim 1, wherein the Bacillus subtilis strain of step (c) is inoculated with 3-4% of the total volume of soybeans. The method according to claim 1, wherein the fermentation in step (d) is carried out at 42 to 44 ° C under a relative humidity of 85 to 95% for 90 to 100 hours. Gt; [7] The method according to claim 1, wherein the fermentation in step (d) is performed by adding maltitol as a carbon source at 7 to 8% of the total volume of soybean. Gt; The method according to claim 1,
(a) washing soybeans and immersing them in water;
(b) boiling the soaked soybeans after removing the water of step (a);
(c) after boiling the soybeans in step (b), inoculating the strain Bacillus subtilis BY07 (accession no. KCTC 12392BP) with 3-4% of the total volume of boiled soybeans; And
(d) adding maltitol as a carbon source to soybeans inoculated with Bacillus subtilis BY07 strain of step (c) in an amount of 7 to 8% of the total volume of soybeans, measuring 85 to 95% relative humidity Wherein the menaquinone-7 is fermented for 90 to 100 hours. The method according to claim 1,
(a) washing soybeans and immersing them in water at 2 to 6 DEG C for 22 to 26 hours;
(b) removing the water in step (a) and boiling the soybeans at 110 to 130 ° C for 20 to 40 minutes;
(c) after boiling the soybeans in step (b), inoculating the strain Bacillus subtilis BY07 (accession no. KCTC 12392BP) with 3-4% of the total volume of boiled soybeans; And
(d) adding maltitol as a carbon source to soybeans inoculated with Bacillus subtilis BY07 strain of step (c) in an amount of 7 to 8% of the total volume of soybeans, measuring 85 to 95% relative humidity Wherein the menaquinone-7 is fermented for 90 to 100 hours. 6. Cheonggukjang produced by the method of any one of claims 1 to 6, wherein the content of menaquinone-7 is increased compared to the general cheonggukjang. A health functional food composition for preventing or ameliorating osteoporosis, which comprises the cheonggukjang of claim 7.

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