KR100544683B1 - Korean traditional soy sauce containing sea tangle, its preparation method and ethanol extract thereof - Google Patents

Abstract

Functional miso with enhanced anticancer and antimutagenic properties. The doenjang of the present invention is a functional health food containing the kelp in traditional doenjang, and the mineral content is increased, and the bioactive function such as anticancer and antimutacity is enhanced. In addition, it is also provided with a method of preparing a traditional miso containing kelp and ethanol extract.

Traditional miso, kelp

Description

Traditional soybean paste containing kelp, preparation method thereof and ethanol extract thereof {KOREAN TRADITIONAL SOY SAUCE CONTAINING SEA TANGLE, ITS PREPARATION METHOD AND ETHANOL EXTRACT THEREOF}

1 is a graph showing the antimutagenic effect of Doenjang ethanol extract on MNNG (0.4 μg / plate) in Salmonella typhimurium TA100 strain. (●: control group (no additives), ○: 5% kelp, ▼: 10% kelp, ▽: 15% kelp)

Figure 2 is a graph showing the antimutagenic effect of soybean ethanol extract on 4NQO (0.15μg / plate) in Salmonella typhimurium TA98 and TA100 strains. (●: control group (no additives), ○: 5% kelp, ▼: 10% kelp, ▽: 15% kelp)

3 is a graph showing the antimutagenic effect of Doenjang ethanol extract on B (α) P (10 μg / plate) in Salmonella typhimurium TA98 and TA100 strains. (●: control group (no additives), ○: 5% kelp, ▼: 10% kelp, ▽: 15% kelp)

Figure 4 is a graph showing the antimutagenic effect of Doenjang ethanol extract on Trp-P-1 (0.5 μg / plate) in Salmonella typhimurium TA98 and TA100 strains. (●: control group (no additives), ○: 5% kelp, ▼: 10% kelp, ▽: 15% kelp)

Figure 5 is a graph showing the growth inhibitory effect of doenjang ethanol extract on human normal cells 293. (□: control group (no addition), ▨: 5% kelp added, ▩: 10% kelp added, ▧: 15% kelp added)

Figure 6 is a graph showing the inhibitory effect on human lung cancer cells A549 ethanol extract of Doenjang added with kelp powder. (□: control group (no addition), ▨: 5% kelp added, ▩: 10% kelp added, ▧: 15% kelp added)

Figure 7 is a graph showing the growth inhibitory effect of Doenjang ethanol extract on human liver cancer cells HepG2. (□: control group (no addition), ▨: 5% kelp added, ▩: 10% kelp added, ▧: 15% kelp added)

8 is a graph showing the growth inhibitory effect of Doenjang ethanol extract on human gastric cancer cell KATO III. (□: control group (no addition), ▨: 5% kelp added, ▩: 10% kelp added, ▧: 15% kelp added)

The present invention relates to a traditional doenjang, its preparation method and ethanol extract having functionalities such as anti-cancer, anti-mutagenicity, more specifically, doenjang prepared by adding kelp during the preparation of doenjang and its manufacturing method and ethanol extract therefrom It's about

Korean traditional soybean paste, including miso and red pepper paste, are not only basic foods that taste Korean food, but also fermented foods that contain nutrients such as protein, carbohydrates, and fat. It is also a nutritious food with an important position as a source.

Doenjang is especially known for its anti-mutagenicity, antioxidant activity, and other physiological effects such as preventing healthy diseases as well as preventing adult diseases. In addition, traditional miso has recently been newly recognized for its nutritional value in relation to its fermentation properties, thus offering not only a simple daily food but also a functional food for health promotion.

Among these physiological activities, anticancer effects are estimated to be caused by ingredients derived from soybeans and decomposed or newly made during fermentation. Anti-cancer actives derived from soybean include protease inhibitors (Kennedy AR et al. Cancer Res 43 : 2454s-2459s, 1981; Yavelow J. et al., Cancer Res 43: 2454s-2459s, 1983; St Clair et al., Cancer Res 50: 580-. 586, 1990), phytic acid (Shamsuddin AM et al., Carcinogenesis 10 : 1461-1463, 1989; Shamsuddin AM et al., Carcinogenesis 9 : 577-580, 1988) and isoflavones (Tiisala S. et al . , Biochim. Biophys Res Commum 203 : 443-449, 1994; Okura A. et al . , Biochim. Biophys Res Commum 157 : 183-189, 1988; Peterson G. et al . , Biochim. Biophys Res Commum 179 : 661-667, 1991). In particular, studies on the anticancer effects of soy isoflavones, including Genistein, are still under great interest (see Messina M. et al., J Am Diet Assoc 91 : 836-840, 1991).

As such, in recent years, research has been conducted to improve the functionality by adding various functional materials in addition to the efforts to transform traditional foods to fit the tastes of modern people.

On the other hand, Laminaria longissima is a genus of seaweeds belonging to the brown algae, which is inhabited in the Far East of Korea such as Korea, Japan, and China. Ito K., Tsuchiya Y., In Proc. Of 7th Int.Seaweed Symp.Nishizawa K. Univ.Tokyo Press, Japan, 558-561, 1972). In addition, the brown algae contain a large amount of neutral polysaccharide laminaran and acidic polysaccharide containing sulfuric acid, and representative examples are sulfuric acid polysaccharides fucoidan and alginate (Kim DS et al., J. Korean). Fish Soc. 18 : 29-36, 1985).

So far, there are known foods that add green tea and mushrooms to doenjang, but no systematic studies have been conducted on foods that combine doenjang and kelp ingredients.

Therefore, one object of the present invention is to focus on the functions of the traditional doenjang and kelp as described above, by using kelp under the optimum conditions, as well as nutritional characteristics, physiological functions such as anti-cancer, antimutagenicity, kelp Is to provide added traditional miso.

That is, one object of the present invention is to provide a traditional miso added with kelp to satisfy the taste and commerciality of consumers by harmonizing the unique functionality, taste components and nutrition and functionality of traditional Korean miso.

Still another object of the present invention is to provide a method of preparing traditional doenjang, in which kelp is added as described above.

Still another object of the present invention is to increase the applicability as a useful physiologically active substance by providing an ethanol extract of traditional doenjang added with kelp as described above.

Other objects of the present invention will become more apparent from the following description.

As a result of intensive research to achieve the above object, the present inventors have found that when the traditional miso is contained in an amount of 2 to 10% by weight, more preferably 3 to 7% by weight, especially 5% by weight, The present invention has been completed by discovering that the physiological functions such as anticancer and antimutagenicity of doenjang can be enhanced. More specifically, the kelp miso of the present invention is obtained by mixing meju, barley rice, salt and kelp in a weight percent of 50:25 to 33: 15: 2 to 10 and aged for 3 to 6 months. Hereinafter, the present invention will be described in detail.

Doenjang used in the present invention was prepared as follows. However, it will be apparent to those skilled in the art that the miso usable in the present invention is not limited to those obtained by the following manufacturing method, considering that there are differences in ingredients of fermented soybeans and fermentation conditions for each fat.

material

The kelp used in the present invention was used in 2000, Goseong-gun, Gangwon-do, and was directly used to grind the kelp to 80 mesh. However, the characteristics of kelp added to the kelp containing miso of the present invention is not limited to such powders, and various types of kelp such as kelp extract may be used. On the other hand, meju, barley, salt, etc., which are the main raw materials of soybean paste, were used commercially.

Doenjang was prepared according to the traditional natural fermentation method as follows.

Manufacture of miso

Floating well and dried meju in the sun was used as a miso material.

Put the meju in the clean jar and remove the meju in the brine so that the concentration of meju floats, press it with bamboo, and press the meju so that it does not float and cook it for 40 to 70 days depending on the time of soaking. At this time, charcoal and dried red peppers are put in to remove impurities and prevent germ breeding, and open the jar lid during the day to let enough sun. When fermentation is complete, sift meju and miso. When the kelp content is 5% by weight, barley rice, salt, and kelp powder are added to the meju at a ratio of 50: 30: 15: 5 and mix well. Toss well with soybean paste in a jar and sometimes inverted and aged for 3 to 6 months to prepare a doenjang added to the sea tangle according to the present invention. To find out the amount of the kelp powder that shows the optimum effect, kelp powder was prepared by varying the amount of kelp powder in 10% and 15%. In this case, by increasing the amount of kelp to 10% and 15%, the barley rice content was reduced to 25% and 10%, respectively.

The bioactive function of the kelp miso thus prepared was tested using each ethanol extract prepared by the following method.

Preparation of Ethanol Extract

10 times 70% ethanol was added to the kelp miso prepared as described above, extracted three times at 80 ° C. for 8 hours, filtered under reduced pressure and concentrated under reduced pressure to remove the extraction solvent, and then freeze-dried. It was dried and used for the experiment. Ethanol, the extraction solvent used, used a special reagent.

Using the ethanol extract prepared as described above, the biological activity effects such as mutagenicity, antimutagenicity and cytotoxicity of kelp miso.

The reagents and assays used in the experiments are described in detail below. In the following description, the kelp content is based on weight percent.

reagent

4-nitroquinoline-1-oxide (4NQO: 4-nitroquinoline-1-oxide), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG: N-methyl-N) from Sigma, USA as direct mutagens '-nitro-N-nitrosoguanidine) and glucose-6-phosphate were used.

Indirect mutagens include benzo (α) pyrene (B (α) P) and 3-amino-1,4-dimethyl-5H-pyrido- (4,3-b) Indole (Trp-P-1), and L-histidine were used.

As a medium for cell culture, RPMI 1640, Hepes buffer, fetal bovine serum (FBS) and trypsin-EDTA were purchased from Gibco (USA).

In addition, human lung cancer cells A549, human liver cancer cells HepG2, human gastric cancer cells KATO III, and normal cells 293 (transformed primary human embryonal kidney) used in the experiment were purchased from KCLB (Korea Cell Line Bank) and used for the experiment. .

1. General Analysis

The general composition analysis of kelp miso is carried out by the AOAC method (Official methods for analysis, 16th ed.Association of official analytical chemists, Washington, DC CH 27, p. 31, 1995) and Food Code (Food and Drug Administration 2000, Food Code (separate book)). Moon Young-sa, Seoul, p. 69-72, p. 273-277) was analyzed three times to obtain the average value. In other words, moisture was analyzed by 105 ° C atmospheric drying, crude ash by dry calcification, crude fat by ether extraction, and crude protein by Kjeldahl method. Salinity was analyzed by AgNO3 titration method, and carbohydrate was 100 minus water, crude ash, crude protein, crude fat, and salt. Minerals were analyzed by Food Code.

First, the results of kelp, meju, and general component analysis and mineral component analysis of kelp miso added with kelp at different concentrations are shown in Table 1 and Table 2, respectively.

TABLE 1

TABLE 2

Compared with the general ingredient analysis result of Table 1 compared to the food ingredient table (National Institute of Agriculture and Life Food Ingredient Table, 5th Revision, 1996), there are slight differences in the harvest time, cultivation conditions, climate, varieties. It is believed that this is due to various conditions such as manufacturing method and manufacturing method.

As can be seen from the results of Table 1, in the case of kelp miso added with 5, 10, and 15% by weight of kelp, respectively, crude protein content was reduced by 2.5% or more compared with the kelp-free miso, and crude fat and carbohydrates were also reduced. Reductions were 1.4% and 1.3% respectively. In comparison, the content of water and salt was increased in the doenjang added with kelp more than the doenjang.

In addition, as can be seen from the results of Table 2, the mineral content of kelp miso is increased in the case of Fe, Mn, P, Ca, Mg, K, especially in the case of K increased 119.7 mg% more than the doenjang without added kelp The contents of Cu and Zn were decreased by 0.2 mg% and 0.5 mg%, respectively, compared to the seaweed doenjang added with seaweed.

2. Mutagenicity experiment

Mutagenesis experiments of doenjang with different concentrations of kelp were carried out by preincubation with an improved Ames test using TA98 and TA100 strains of Salmonella typhimurium (Yahagi T. et al . , Mutation Res. 48: 121-130). 50 μg / plate of each sample was added to the dry heat sterilized glass cap tube, and 100 μL of the precultured culture solution was added thereto, and the total amount was adjusted to 700 μL with 0.2 M sodium phosphate buffer (pH 7.4). Shake incubate at 37 ° C for 20 minutes, mix well by adding 2 mL of histidine / biotin-added top agar (45 ° C), plate onto pre-prepared minimal glucose agar plate, and fix plate to 48 ° C at 37 ° C. The presence of mutagenicity was determined by measuring the number of hiss + revertant colonies resulting from time incubation.

As a result of the test, the number of mutants in the negative control group was 17 ± 3 in TA98 and 174 ± 7 in TA100. The ethanol extract of doenjang added with sea tangle was tested at various concentrations of 50, 100, 150 and 200 μg / plate, and the colony number did not show a large change in colony number according to the concentration change compared to the negative control group. From this, it was judged that ethanol extract of doenjang added with kelp did not show mutagenicity (data omitted).

3. Antimutagenicity Experiment

Antimutagenicity was tested according to the pre-incubation method which improved the Ames test. Mutants are positive in the Ames test, and the substances themselves are 4NQO and MNNG, which are direct mutagens, and B (α) P and Trp-P, which are indirect mutagens that require metabolic activity. -1 was used. 50 μL of each sample, ie, ethanol extract of kelp miso, was added to the dry heat sterilized glass cap tube, followed by 50 μL of the mutant material. In the case of indirect mutagenes, 250 μL of 10% S-9 mix was added. 100 μL of the precultured bacteria solution was added thereto, followed by addition of 0.2 M sodium phosphate buffer to a final volume of 700 μL. After shaking for 20 minutes at 37 ℃ experiment was carried out by the same method as the above mutagenicity experiment to determine the number of the resulting back mutations to determine the presence of anti-mutagenicity. The concentrations of extracts and mutagens of Doenjang added with sea tangle were determined by preliminary experiments. The antimutagenic activity was expressed as% inhibition of the mutagen. The inhibition rate shown in the figure is the average value of three repeated experiments.

Inhibition Rate (%) = [(MS ₁ ) / (MS ₀ )] X 100

In the formula,

M: number of return mutations when only mutants are present

S ₀ : number of spontaneous return mutations

S ₁ : number of return mutations when sample is added

The analysis results are as follows.

The ethanol extract of seaweed with miso added, in the case of MNNG (0.4μg / plate) was used to direct mutations won a powerful carcinogens that occur among the cooking process of foods Salmonella typhimurium (S. typhimurium) in the sample strain TA100 Soybean paste added with 5% kelp showed a high inhibitory effect of 97.0% at 200 μg / plate and 59.3 kelp at 89.3%, 83.6%, and 82.1%, respectively. It showed a lower inhibitory effect compared to the added doenjang (Fig. 1).

Salmonella typhimurium (S. typhimurium) higher inhibitory effect than the TA98 and results also in both cases 5% seaweed powder added is added to the group or other non-addition group of soybean TA100 also for the direct transition circle 4NQO (0.15μg / plate) Indicated. That is, in the case of TA98, 5% kelp-added soybean ethanol extract showed 60.2% at a sample concentration of 200 μg / plate, whereas no additive, 10% and 15% kelp-added miso ethanol extracts were 49.5%, 43.7%, and 45.8, respectively. In the case of TA100 strain, 59.1%, no addition, 10% and 15% kelp-added miso, respectively, of 59.1%, 63.4%, and 57.2%, Inhibition rate was shown, the effect of the concentration of 5% kelp was found to be the most excellent (Fig. 2).

On the other hand, when using a polycyclic aromatic hydrocarbon B (α) P (10μg / plate) as an indirect mutant exhibiting mutagenicity only by the microsomal enzyme metabolic activity (Fig. 3) ), The inhibitory effect of Salmonella typhimurium TA98 and TA100 increased with increasing sample concentration. In the 5% kelp powder added group, the sample concentration of 200 μg / plate in the TA98 strain was 71.7%, which was somewhat higher than the other added groups. In case of TA100, soybean paste containing 5% kelp powder showed 87.3% higher inhibitory effect than other groups. In Trp-P-1 (0.15μg / plate), sample concentration 200 μg in TA98 strain At the / plate concentration, the 5% kelp-added group was 66.6%, and the kelp-free, 10%, and 15% -added groups showed 57.7%, 61.3%, and 58.1%, respectively. In addition, the TA100 strain showed an inhibitory effect of 80.8% at the concentration of 200 μg / plate of the kelp 5% added group of kelp, and 63.5% at the same concentration without the kelp, 10%, and 15% kelp at the same concentration. , 71.4% and 73.8% showed slightly lower inhibition than the 5% addition group (FIG. 4).

As can be seen from the above results, the doenjang itself showed some degree of antimutagenicity, but it was found that the addition of kelp shows a higher antimutagenic effect. Thus, the addition of kelp showed a synergistic effect with soybean paste, showing a higher bioactive effect.

In the present invention, the synergistic effect of the addition of kelp to soybean paste is thought to act in concert with the bioactive components of soybean paste, such as fucoidan and alginate, which are sulfur-containing acidic polysaccharides of kelp. In addition, the addition of kelp-derived dietary fiber, alginate, and fucoidan-containing physiologically active components and soybean physiologically active components, such as isoflavones, interact with various components and produce unknown compounds. It is estimated to promote synergistic effects.

4. Cytotoxicity Experiment

Mutations play a very important role in the early stages of cancer and the commonality that most carcinogens are mutagens suggests that antimutagenic substances may have anticancer activity. In this experiment, we tried to investigate the cytotoxicity of various kinds of cancer cells of ethanol extract of Doenjang added with kelp. Experiments were performed using human lung cancer cells (A549), human liver cancer cells (HepG2) and human gastric cancer cells (KATO III) as cancer cells, and 293 as normal cells as follows.

The SRB [sulforhodamine B] assay is a method of measuring cell proliferation or toxicity using cellular protein staining (Scudiero, DA et al., Cancer Res ., 48, 4827-4836, 1988) and 10% fetal bovine serum (FBS). RPMI 1640 medium containing A549, HepG2, and normal cells 293, each cancer cell, was incubated in each well for 100 μl at a concentration of 5 × 10 4 cells / mL and cultured for one day (37 ° C., 5% CO 2), followed by PBS (phosphate). Extracts dissolved in buffered saline) were added to 0.25, 0.5, 0.75, and 1.0 mg / mL, respectively, and incubated again for 48 hours. The supernatant was then carefully removed with an aspirator and cells were fixed at the bottom of the well by adding 50 μL of cold 10% TCA (trichloroacetic acid, 4 ° C.) solution. After incubation at 4 ° C. for one hour, it was washed five times with distilled water to remove TCA and media. The plate was dried and salined for 30 minutes by adding 0.4% SRB dissolved in 1% acetic acid and washed four times with 1% acetic acid solution to remove unbound SRB stain. The plate dried in the dryer was sufficiently dissolved in 100 μL of 10 mM Tris buffer, and then absorbance was measured with a microplate reader at 540 nm.

The MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide) assay is a method for measuring cell growth and differentiation (Martin, A. et al., Cytotechnology , 11, 49-54, 1997), this experiment focuses on the dehydrogenase in mitochondria of living cells producing dark blue formazan by MTT, a yellow water-soluble substance. Incubate for 24 hours by adding 100 μL of human gastric cancer cells (KATO III) and RPMI 1640 medium containing 10% fetal bovine serum to each well at a concentration of 5 × 10 4 cells / mL (37 ° C., 5% CO 2) Each sample was incubated again for 48 hours by adding 100 μL at 0.25, 0.50, 0.75 and 1.0 mg / mL concentrations. 20 μL of MTT (5 μg / μL) solution was added thereto, followed by incubation for 4 hours to form formazan, and then the supernatant was carefully removed by an aspirator. Then, 150 μL of DMSO (dimethyl sulfoxide) was added to dissolve formazan, and then absorbance was measured using a microplate reader at 540 nm.

The analysis results are as follows.

The toxic effect on human normal cells 293 shows a low inhibitory effect of less than 30% in both the no-tangled and added groups at the highest concentration of 1.0 mg / mL, which is considered safe for normal cells (FIG. 5).

In FIG. 6, the ethanol extract of Doenjang added with kelp showed an inhibitory effect on human lung cancer cells A549. At concentrations of 0.5, 0.75, and 1.0 mg / mL, the 5% group of kelp showed a slightly higher inhibitory effect of 36.7%, 42.6%, and 56.4% than the other groups.

On the other hand, in the inhibitory effect on HepG2 of human liver cancer cells, ethanol extracts of 5% kelp-added doenjang were more active than those of no-tangle, 10% and 15% kelp-added doenjang at 0.5, 0.75, and 1.0 mg / mL sample concentrations. High inhibitory effect of 53.2%, 74.4%, and 87.6% (FIG. 7).

FIG. 8 shows the cancer cell growth inhibitory effect of kelp-added miso ethanol extract on human gastric cancer cell KATOIII cells, and when 0.5, 0.75, and 1.0 mg / mL were added, doenjang added 5% of kelp powder was relatively higher than other groups. 55.4%, 78.6%, and 90.5% showed cancer cell growth inhibitory effect.

The optimum content of kelp to be added is 2 to 10% by weight, preferably 5% by weight of miso weight. If the kelp content is less than 2% by weight, the synergistic effect of the desired anti-cancer, anti-mutant effect, etc. is insignificant, and even if it exceeds 10% by weight, this increase is not observed. Did.

In other words, in the group without addition and addition of kelp, hepG2 and KATOIII showed very high cancer cell growth inhibitory effect, especially in the group with 5% kelp addition (p <0.05). The addition of 5% showed the greatest synergistic effect of enhancing physiological activity. In addition, as the sample concentration increases from 0.5 to 1.0 mg / mL, the inhibition rate also appears to be high, and thus the sample concentration and the inhibition rate may be correlated. As such, it can be seen that the physiological activity of the traditional miso containing kelp at an appropriate concentration shows the physiological activity and synergy of soybeans and soybean-related foods, which are the raw materials of traditional soybean paste. It is expected to be usable as a material for bioactive substances.

When the concentration of kelp was added to the fermented soybean paste prepared by natural fermentation method, most of the mineral content was increased than before adding kelp. In addition, the antimutagenic effect and cancer cell growth effect also appeared to be increased than before adding the kelp powder, it is possible to enhance the nutritional and physiological functions of traditional doenjang by adding kelp to traditional doenjang.

Claims (6)

A traditional miso containing kelp obtained by mixing kelp in a traditional miso made mainly of meju, barley rice, and salt in an amount of 5% by weight based on the total weight of miso. The traditional miso containing kelp according to claim 1, comprising 50% by weight of meju, 30% by weight of barley rice, 15% by weight of salt, and 5% by weight of kelp. delete delete delete delete

Images