KR19990072468A - Nato products using black-soybean and process of preparation thereof - Google Patents

Abstract

The present invention relates to Cheonggukjang using black soybeans and a method of manufacturing the same, using black soybean as a main ingredient and inoculating strains having high enzymatic activity such as protease, amylase, and the like having excellent ability to produce viscous substances and thrombolytic ability, followed by addition of fruits and vegetables. Han Cheong-gukjang has the excellent scent of cheonggukjang's peculiar odor, and the sensory test shows excellent palatability. It has excellent antimicrobial effect against thrombolytic activity, peroxide inhibition, nitrite scavenging activity, food poisoning and decay bacteria.

Description

Cheonggukjang using black soybean and its manufacturing method {Nato products using black-soybean and process of preparation

The present invention provides a Cheonggukjang and a method of manufacturing the same using black beans. More specifically, the present invention relates to a cheonggukjang fermented by the addition of fruit and vegetables after inoculating a strain having high enzymatic activity such as protease, amylase and the like and excellent in the ability to produce viscous substances and thrombolytic ability to black soybeans and a method for producing the same.

Cheonggukjang is one of the representative fermented foods that Koreans have enjoyed since ancient times. Unlike soy sauce and soybean paste, it is not only very short and simple to manufacture, but also has a seasonal taste that is used from autumn to early spring.

Cheonggukjang contains more protein and fat than doenjang, but has a higher digestive absorption rate, an important source of calcium and vitamins A and B, the intestinal effect of Chongguk coli, the prevention of constipation of fiber, the extracorporeal release of carcinogens and cholesterol, and mucin. It has been shown that there is an effect on the rehabilitation by alcohol absorption, strengthening of blood vessels, promoting blood circulation and lactic acid degradation of saponin, and preventing brain aging of lecithin and prevention of dementia, hypertension and arteriosclerosis. Although Cheonggukjang is far superior to other foods, Cheonggukjang has been used directly in the traditional way because of the increase of national income, rapid industrial development, the conversion of the housewife's consciousness and participation in social activities, the change of the living environment and the westernization of dietary life. Rather than making, it is becoming a form of purchasing and consuming Cheonggukjang, which is mass-produced in factories. In addition, current small production facilities and non-automated hand-made manufacturing will have to lose marketability, especially children, young people and foreigners hate Cheonggukjang because of its peculiar smell. Without improving this and producing high-quality and hygienic Cheonggukjang, it will be difficult to establish the status of Cheongguk as an international commodity. Albon's napdu, which is similar to Korea's Cheonggukjang, is highly preferred in Japan, similar to the consumption of tofu in Japan, and the consumption of tempera, which is a traditional soybean fermented food from Indonesia, is increasing every year in Japan and the United States. Naphtdu uses Bacillus subtilis as the basic species like Cheonggukjang, but its raw materials and manufacturing method are slightly different. Naphtha uses small-sized soybeans, does not use salt, and has a smoky odor. On the other hand, Tempe is an unsalted, funky soybean fermentation style that has no smell or viscosity.

The study of Cheonggukjang includes the changes of Cheonggukjang ingredients during fermentation process based on soybean, changes in Cheonggukjang quality due to differences in microorganisms and limited fermentation conditions, bacterial characteristics, enzymatic properties, and fragrance components. . In addition, a study on the improved manufacturing method to remove some of the unpleasant odor of Cheonggukjang by using the yellow soup and the increase of proteolytic enzyme power, amino nitrogen content and vitamin content by the change of the target of Cheongguk coli using CaCO ₃ were reported. have. Recently, Cheonggukjang was prepared from Bacillus subtilis and Bacillus naphtha to produce processed foods in the form of spores and research reports on the improvement of the quality and palatability of Cheonggukjang by adding garlic, mugwort, pepper, chili oil, etc. The viscosity, color, and spreadability were examined, and there were reports of changes in quality characteristics over time and the manufacturing method and usage of Cheonggukjang with lyophilized instant Cheonggukjang stew at 25, 35 and 45 ° C. Cheonggukjang's functional studies were much lower than that of Japanese naphtha, but recently, enzymes produced by Bacillus sp. Isolated from Korea's Cheonggukjang (CK 11-4) have been reported to have higher thrombolytic capacity than naphtha kinases. In addition, Korean traditional soybean paste has better anti-cancer and immune enhancement effects than Japanese doenjang. Cheonggukjang has been reported to have lower physiological activity than these, but there are few studies on physiological activity with bioregulatory function. .

Black soybeans have been traditionally introduced as medicinal soybeans. Like soybeans, the main isoflavones are genistein and daidzein. Especially, genestane removes harmful active oxygen species to prevent antioxidant effects. Anticancer activity against breast, rectal and prostate cancers by inhibiting the production of stress proteins such as heat shook protein (HSP) and glucose-related protenes (GRPs) that help cancer cells survive attack by the immune system It is known to represent. It has also been reported that daizein inhibits the rapid rise in blood alcohol levels after drinking alcohol by delaying the rate of alcohol release in the stomach. Soy saponin of black soybean has been reported to be closely related to antioxidant activity, lowered serum cholesterol, selective toxicity to cancer cells and immune effects. Especially, isoflavones of antioxidant and anticancer substances have recently been detected from black areas of black soybean seedlings. Compared with soybeans, the darker the black color, the stronger the antioxidant effect, and the lower the serum cholesterol when fed black bean skin to rats. Compared to soybeans, these black beans have a thicker epidermis and a weaker tissue, resulting in lower swelling and lower protein degradation rate when increasing the weight, and low fermentation due to the presence of antimicrobial substances from the soybeans and flesh of black soybeans. Because it causes a change in the color and quality of food, it is mainly used as a rice bean or mixed with rice, barley, and grains. It is also used as rice bean, confectionery, and medicinal soybeans. It is rarely used in the manufacture of tofu.

The conventional production method of Cheonggukjang has been fermented by washing whiten or fermented soybeans and using it as a protein raw material, and then inoculating the Bacillus bacteria. The conventional conventional Cheonggukjang can be easily fermented by injecting Naphtha bacterium due to its raw material's characteristics as white soybean or Rung bean.However, if black bean is used as a raw material, it is impossible to produce excellent Cheonggukjang by inoculating naphtha bacteria. Could not eat.

Therefore, the present inventors investigated the treatment conditions of soybeans for the improvement of fermentation efficiency by using high quality black soybeans having excellent functionality as a main ingredient while reproducing the unique taste of Cheonggukjang, and isolated and identified strains suitable for fermentation of black soybeans. In addition, we searched the most suitable natural food materials to be added at the beginning of fermentation to suppress odor and improve quality of Cheonggukjang, and analyzed nutrients from Cheonggukjang made with black soybeans. Bioactivity was analyzed.

It is an object of the present invention to provide Cheonggukjang, which is excellent in antimicrobial activity, antioxidant activity, and thrombolytic activity using black soybean as a main material and significantly suppressed the squishy smell peculiar to Cheonggukjang. It is another object of the present invention to provide a method for producing the Chungkookjang.

The above object of the present invention is to isolate the strains with high enzyme activity such as protease, amylase from Chunggukjang or traditional doenjang and select strains excellent in the production of slime and thrombolytic activity among these inoculated this strain in black soybeans and kiwi and radish The fermentation was made by adding fermented soybean and prepared by examining the physicochemical properties, physical properties, viable cell counts and enzyme activities of cheonggukjang, sensory tests of panelists, and bioactivity of cheonggukjang.

Hereinafter, the configuration and operation of the present invention.

1 is a photograph showing the protease and α-amylase activity produced by SMY-212 strain isolated from Cheonggukjang.

Figure 2 is a graph showing the change in amino nitrogen content of Cheonggukjang prepared with alleles and small black soybeans over time.

Figure 3 is a graph showing the change in nitrogen content of ammonia nitrogen of Cheonggukjang prepared with alleles and small black soybeans over time.

Figure 4 is a graph showing the changes in the peptide-type nitrogen content of Cheonggukjang prepared with alleles and small black soybeans over time.

5 is a graph showing the change in the water-soluble protein content of Cheonggukjang prepared with alleles and small black soybeans over time.

Figure 6 is a graph showing the changes in the salt-soluble protein content of Cheonggukjang prepared with alleles and small black soybeans over time.

Figure 7 shows the chromatographic detection of free amino acids in Cheonggukjang prepared from allelic soybeans.

FIG. 8 shows HPLC chromatography detecting nucleic acids associated with compounds in Cheonggukjang prepared from allelic soybeans.

9 is a graph showing the change in brightness over the fermentation time of the cheonggukjang prepared using alleles and small black soybeans.

Figure 10 is a graph showing the change in redness over time fermentation of Cheonggukjang prepared using alleles and small black soybeans.

Figure 11 is a graph showing the change in yellowness over time fermentation of Cheonggukjang prepared using alleles and small black soybeans.

12 is a graph showing the change in the hardness (hardness) of the fermentation time of Cheonggukjang prepared using alleles and small black soybeans.

Figure 13 is a graph showing the change in cohesiveness (Cohesiveness) over time fermentation of Cheonggukjang prepared using alleles and small black soybeans.

Figure 14 is a graph showing the change in viable counts over time fermentation of Cheonggukjang prepared using alleles and small black soybeans.

15 is a photograph showing the thrombolytic ability of Cheonggukjang prepared using various black beans.

Figure 16 is a graph showing the change in the antioxidant power over the fermentation time of the cheonggukjang prepared using alleles and small black soybeans.

The present invention is to obtain a strain with high enzyme activity such as protease, amylase from black soybeans, commercial and home Cheonggukjang, traditional doenjang and inoculated and fermented again into black soybeans to select strains excellent in aromaticity, viscous production ability and thrombolytic ability; Identifying morphological, culture, physiological and biochemical properties of the selected strains; Preparing the cheonggukjang using the isolates and the identified strains and vegetables; Investigating the physicochemical properties by measuring the amount of protein, free amino acid, fatty acids, nucleic acids and minerals of the Cheonggukjang prepared with the black soybeans; Investigating physical properties by measuring the viscosity, chromaticity and texture of the Cheonggukjang prepared with the black soybeans; Measuring the bioburden and enzyme activity of the Cheonggukjang prepared with the black soybeans; Performing a sensory test on a panelist of the cheongukjang prepared with the black soybeans; It consists of the step of investigating the biological activity by measuring the antimicrobial activity against the thrombolytic ability, antioxidant power, nitrite scavenging ability and food poisoning causative bacteria and rot bacteria of Cheonggukjang prepared by the black soybean.

Cheongkuk-jang (Glycine max (L.) Merrill) used in the present invention was a soybean harvested from Gabo agricultural products in Naju, Jeonnam in 1997 as a control, black soybean 1 type (Seritae, genus blue beans) ) And one small grain (ratny bean, poly-bean, medicinal bean) were used. In addition, odor suppression and quality during the fermentation process of these black beans include vegetables and fruits, leaf vegetables (cabbage, cabbage, celery, green onions, leek, aloe), root vegetables (daikon radish, carrot, garlic, onion), fruit vegetables and berries (Cucumber, pumpkin, tomato, strawberry, kiwi, melon, watermelon), causal fruits (citrus fruits, pears, apples, persimmons), nuclear fruits (plums, peaches) and tropical fruits (bananas, pineapples), and other spices (ginger) , Mustard, red pepper, cinnamon) and wild edible plants (wormwood, pine needles, green tea) were used.

Hereinafter, the specific configuration and operation of the present invention will be described with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Preparation of Cheonggukjang with Black Beans

Step 1: Isolation of Cheonggukjang Strains

Add 1 g of black soybean, commercial and domestic Cheonggukjang, traditional soybean paste, etc. to 100 mL of sterile saline solution, suspend well, and leave for 10 minutes. Each plate was plated in% soluble starch-added LB plate medium (SM) and incubated at 30 ° C. for 2 days. Of the colonies on both media, only single colonies with large halozone size, clear and fast growth rate were selected. After inoculating the firstly isolated pure bacteria in LB medium (Yeast extract 0,5%, tryptone 1%, NaCl 1%) and shaking for 2 days, the protease and amylase activity of each isolate was measured. Strains with relatively high enzymatic activity were selected for the second time. Finally, each of the strains secondarily screened with small halo-zone and high enzyme activity was incubated in LB medium, and 1 mL of the culture solution was placed in an Eppendorf tube, centrifuged at 8,000 rpm for 5 minutes, and then the supernatant was removed. After discarding about 2/3, 1 mL of sterile water was added to resuspend the cells, and 100 μl was inoculated on the fibrin medium, and the size of the halo zone was observed over time to measure the thrombolytic ability. In addition, the remaining culture solution was washed and sterilized with sterile physiological saline solution, diluted to a constant concentration, inoculated into the cooked black soybean, and fermented at 42 ° C for 72 hours. The strain was selected. As a result, the fungal sample was suspended in sterile physiological saline, and 100 µl was smeared on CM medium and SM medium, and 437 strains forming a clear zone in each medium were selected first. As shown in FIG. 1, each of the first isolated strains was subjected to tooth picking on CM and SM media, and the second and second strains of the fast growing rate were relatively large and distinct in size of halozone simultaneously on two kinds of media. Shake culture was performed on LB medium for 2 days. Table 1 shows the protease, α-amylase and glucoamylase enzyme activities of the supernatant obtained by centrifuging the culture (8,000 rpm, 10 min). The size of the halozone was marked and relatively large in the isolate, but the enzyme activity showed a lot of difference according to the strain. Protease activity was higher in SMY-212, 117, and 038 strains, and α-amylase was higher in SMY-311, 096, and 029 strains, and glucoamylase activity was not significantly different among the strains, but SMY-095, 409, 029 and High in 212 strains. The thrombolytic activity of the secondary isolated strains was shown in Table 2 by SMY-212 (180%, relative activity against 1 unit of plasmin), 117 (147%), 38 (147%) and 94 strains (134%). ) Showed a larger size of halozone than other strains. Therefore, finally SMY-212 was selected.

Protease, α-amylase and Glucoamylase Enzyme Activities Strain number Halo john Enzyme Activity (U / mL) Source CM * SM ** Protease α-amylase glucoamylase SMY-026 +++ ++ 108.6 34.9 11.2 Chungkuk SMY-029 ++ +++ 77.2 89.2 14.2 Chungkuk SMY-038 +++ ++ 113.1 76.0 10.4 Chungkuk SMY-061 ++ ++ 87.2 53.4 9.2 Chungkuk SMY-066 +++ ++ 98.6 18.2 5.6 Chungkuk SMY-086 ++ ++ 45.7 45.1 8.7 Chungkuk SMY-094 ++ ++ 69.2 56.9 7.4 Chungkuk SMY-095 ++ +++ 54.6 54.2 14.9 Chungkuk SMY-096 ++ ++ 85.4 97.6 10.2 Chungkuk SMY-117 +++ ++ 115.7 54.2 7.2 Chungkuk SMY-164 ++ ++ 76.3 46.2 8.3 Chungkuk SMY-212 +++ +++ 124.8 78.4 13.9 Chungkuk SMY-216 ++ ++ 67.0 66.4 12.3 Chungkuk SMY-283 ++ ++ 87.8 78.2 8.2 Doenjang SMY-285 ++ ++ 64.2 83.1 7.6 Doenjang SMY-311 +++ ++ 90.1 99.2 9.7 Blacksoybean SMY-320 ++ +++ 76.2 45.7 11.4 Blacksoybean SMY-409 ++ +++ 83.2 72.1 14.5 Chungkuk Halo Size: + Small, ++: Normal, +++: Large CM: Casein badge, SM: Starch badge

Thrombolytic activity, viscosity, and odor control of strains isolated from various sources Strain number Odor Removal Viscosity Thrombolytic activity Source Clear Zone (mm) ^* % ^** SMY-026 ++ +++ 9.9 130 Chungkuk SMY-029 +++ + 6.2 82 Chungkuk SMY-038 ++ +++ 11.2 147 Chungkuk SMY-061 ++ +++ 5.2 68 Chungkuk SMY-066 ++++ +++ 7.5 99 Chungkuk SMY-086 ++ ++ 5.6 74 Chungkuk SMY-094 +++ + 10.2 134 Chungkuk SMY-095 ++ ++ 6.8 90 Chungkuk SMY-096 +++ ++ 8.7 115 Chungkuk SMY-117 ++ ++ 11.2 147 Chungkuk SMY-164 +++ ++ 7.1 93 Chungkuk SMY-212 ++ ++++ 13.7 180 Chungkuk SMY-216 +++ + 6.0 79 Chungkuk SMY-283 +++ ++ 4.6 61 Doenjang SMY-285 + ++ 9.4 124 Doenjang SMY-311 ++++ ++ 8.5 112 Blacksoybean SMY-320 ++ +++ 6.4 84 Blacksoybean SMY-409 + ++ 7.1 93 Chungkuk * Clear zone was measured for 5 hours at 35 ° C. ** Relationship activity (%) = clear zone (mm) / clear zone of flamin (1 unit) +: very weak, ++: weak, ++ +: Strong, ++++: very strong

Step 2: Identification of Cheonggukjang Strains

The strains selected in step 1 were identified according to Bergey's Manual of Systematic Bacteriology. Morphological characteristics were observed in colonies grown on NA medium containing 1.5 agar in NB medium, and endogenous spore formation was observed by optical microscopy by staining the strains by the Wirtz method by Barthlomew and Wittwer. Surface morphology was fixed in 3% glutaraldehyde phosphate (pH 7.2) 3 hours ago, washed with the same concentration of buffer and then fixed in 2% OsO ₄ -phosphate buffer 2 hours after alcohol 50, 70, 85, 90 , 95, 99, 100% in increasing concentration of dehydrated in acetone and re-dehydrated, and then adhered to the sample bed and coated with Au (200 ℃, vacuum deposition) was observed by scanning electron microscope (SEM, Akasi SX-40). The culture characteristics were characterized by the culture conditions and culture characteristics of purely isolated Cheonggukjang fermented strains by inoculating the test strains in NB medium by varying the temperature (25, 30, 35, 40, 45 ℃) and initial pH (5.5, 6.0, 6.5). , 7.0, 7.5) shaking for 20 hours (220rpm) and then measured by measuring the absorbance at 660nm. Physiological and biochemical properties were investigated for casein gelatine, starch hydrolysis, H ₂ S production, nitrate reduction and enzyme production. Fatty acid analysis of isolated strains was inoculated with TSBA (tryptic soy broth agar, Difco) and incubated for 24 hours, followed by extracting the bacterial fatty acid from colonies 40-50 mg in logarithmic growth and methanolysis (MIS, Hewlett-Packard 5890A). It was analyzed by GC and compared with a fatty acid library of aerobic bacteria. In addition, carbon atomization of the isolate strain was investigated using a GP microplate (BIOLOG, Inc., USA). As a result, isolated strains were bacillus (1.0 ~ 1.2 × 5.2 ~ 6.3㎛) and formed endogenous spores. Also, as aerobic bacteria with motility, starch, casein, gelatin were hydrolyzed and no H ₂ S gas was produced. The growth pH ranged from 5 to 8 neutral and the growth temperature was 25 ~ 45 ℃. Fatty acid composition and carbon source on GP microplates were dextrin, glycogen, cellulose, fructose, α-D-glucose, maltose, mannose, 3-methyl glucose, palatinose, sucrose, frannose and methyl pyruvate as organic acid Pyruvic acid. Some of the sugars used were genthiobiose, maltotriose, and cicos. The organic acids were L-malic acid, and other sugars, organic acids, and amino acids were not used well. In conclusion, the strain was identified as Bacillus megaterium. The growth of the disclosed strains was 2.5 hours after inoculation, and as the growth of bacteria increased in the logarithmic growth period, the enzymatic activity of α-amylase and protease increased rapidly.

Step 3: Manufacture Cheonggukjang

Bacillus subtilis (KFCC 11314) separated in the first step and various isolates such as black soybeans, Cheonggukjang and soy sauce were isolated and identified to use the strain SMY-212 strain of the genus Bacillus with the best cell growth and enzyme activity. The seed was inoculated with 250 mL of NB medium (Difco, nutrient broth: beef bxtract 0.3%, peptone 0.5%) added with 0.5% glucose, incubated at 37 ° C for 24 hours, and then the culture solution was sterilized by centrifugation. The cells were washed three times and then the cell concentration was adjusted (absorbance 580 nm = 2.0) and used as a seed solution. Soaked soybeans and black soybeans 3kg each was immersed in cold water, drained and placed in stainless steel, steamed for 30 minutes in a autoclave and cooled to 50 ℃. Cheonggukjang was prepared by fermenting 500g of black bean, two kinds of cooked allelic and small grains, with fermented for 3 days at 42 ℃ in constant temperature, and adding about 6% (w / w) of fruits and vegetables for the purpose of protein degradation and odor suppression. Fermented Cheonggukjang and pre-cultivated seed solution were prepared by dividing 1% (v / w) of the raw material into Cheonggukjang fermented under the same conditions. As a control, the soybeans were fermented as it was after adding soybeans.

Experimental Example 1: Physicochemical Properties of Cheonggukjang Prepared with Black Beans

The pH of the Cheonggukjang prepared in Example 1 was added to 40g of 10g of Cheonggukjang, distilled water from which CO ₂ was removed, and then triturated with a homogenizer (8,000rpm, 5 minutes), filtered to make 50mL, and then a portion of the solution was taken to measure a pH meter ( Fisher, US A). The total acidity was expressed as the amount (mL) of 0.1 N-NaOH taken until the pH was 8.3 by taking 20 mL of the previous filtrate. Nitrogen is micro-Kjeldahl method for total nitrogen, water-soluble nitrogen is 5g of Cheonggukjang, 200mL of distilled water, 1 drop of silicone antifoam, triturated for 3 minutes, and made into 250mL and centrifuged (3,000rpm, 30 minutes) Nitrogen dissolution rate was converted into SN / TN (%). Amino nitrogen (NH ₂ -N) was shown as the formal nitrogen content minus the ammonia nitrogen content. Determination of the porom molten nitrogen was added 5g Cheonggukjang in 250mL beaker and 100mL of distilled water and stirred for 1 hour and thoroughly mixed, and titrated to 8.4 with 0.1mL NaOH solution. At this time, 20 mL of neutral formalin solution was added, and when the pH was acidified, the solution was titrated to pH 8.4 with 0.1 N NaOH solution and calculated by the following equation.

A: Sample titration amount (mL) of 0.1N NaOH solution

B: Empty test titration amount of 0.1N NaOH solution (mL)

F: Urea of 0.1N NaOH

Ammonia nitrogen (NH ₃ -N) was added to 2 mL of 30% NaOH and 3 mL of silicone resin in 20 mL of the pretreatment extract, and the gas generated when distilling in a distillation apparatus for 5 minutes was collected with 3% boric acid, and then 0.02 using a pH meter. N-HCl was titrated to pH 4.04 to calculate HCl consumption, and nitrogen in the peptide was converted into TN- (NH ₂ -N + NH ₃ -N). Protein isolation was performed according to Wang's classification. That is, the water-soluble protein and the salt-soluble protein were extracted by adding 60 ml of sodium phosphate buffer (pH 7.0) containing 1 M NaCl to deionized water in 3 g of Cheonggukjang, stirring for 90 minutes at room temperature, and centrifuging at 3,500 rpm for 30 minutes. This supernatant was quantitated repeatedly by the Lowry method. For analysis of free amino acids, about 100 mL of distilled water was added to 5 g of Cheonggukjang, ground to 500 mL, and extracted at 60 ° C. for 30 minutes. The extract was filtered through a 0.2 μm membrane filter and Sep-pak C ₁₈ cartridge and analyzed by amino acid analyzer. Nucleic acid-related substances were centrifuged (4,000 x g, 10 min) by grinding 25 min of 10% cold perchloric acid solution to 10 g of crushed Cheonggukjang. The supernatant was taken and extracted again in the same manner. The supernatants were combined and adjusted to pH 6.5 by adding 5N-KOH solution to make 100 mL with cold perchloric acid solution. After allowing to stand for 30 minutes, the mixture was centrifuged (10,000 × g, 10 min), the supernatant was filtered through a 0.45 μm membrane filter, passed through a Sep-pak C ₁₈ cartridge, and analyzed by HPLC. After the preparation was converted. The analysis of minerals was carried out by adding 25 mL of a disintegrating agent (HClO ₄ : H ₂ SO ₄ : H ₂ O ₂ = 9: 2: 5, v / v) to ₂ g of Cheonggukjang, and slowly heating at low temperature to make the decomposition solution completely colorless. It was hydrolyzed until changed and filtered (Whatman No. 2) to make 100 mL, and analyzed by Inductively Coupled Plasma Spectrometer (ICP, Optima 300DV, Pekkin-Elmer, USA). Experimental results, the results of analyzing the moisture, crude protein, crude fat, crude ash of the soybean, soybean, alleles and small black soybeans are shown in Table 3. The water content of black soybean Chungkukjang was 58.03 ~ 69.72%, which was much lower than that of yellow soybean Chungkukjang (69.72%). Crude protein content ranged from 17.33 to 26.71%. Among them, the content of non-added allele black soybeans was highest, and the content of allele black soybeans inoculated with Bacillus subtilis strain was the lowest. Crude fat content was in the range of 9.73 ~ 12.47%, but there was no significant difference, but the highest content was found in non-added all black soybeans, and the smallest in black soybeans inoculated with Bacillus subtilis strain. As the fermentation time elapsed, the pH tended to increase rapidly up to 24 hours in most of Cheonggukjang, and then gradually increased, but there was no significant change. Particularly, in case of allele test, the pH and the range of change were slightly lower than those of Cheonggukjang inoculated with Bacillus subtilis strain after 72 hours of fermentation. . In the case of total acid, as the fermentation time passed, the total acid decreased sharply until 24 hours and then decreased gradually after the fermentation time. After 72 hours fermentation, 0.1N ~ NaOH consumption There was no significant difference in the range of 1.1 ~ 1.5mL. In the case of water-soluble nitrogen, the contents of black beans and small black beans added with the strain strain SMY-212 and Bacillus subtilis increased sharply after 48 hours of fermentation, especially when the isolate was added to the strain than the Bacillus subtilis strain. The content of Chungkookjang was higher and the other Cheonggukjang had little change. As in the case of water-soluble nitrogen, the nitrogen solubility rate increased rapidly after 24 hours of fermentation, and the increase of the cheonggukjang inoculated with the isolate was greater than that of the Bacillus subtilis strain. There was no change. Amino nitrogen content tended to increase gradually with the fermentation time, with the largest increase of Cheonggukjang added with isolated strain, followed by Cheonggukjang added with Bacillus subtilis, and nothing added as fermentation time elapsed. The amino nitrogen content of Cheonggukjang was higher than that of radish and kiwi. This is shown in FIG. The content of ammonia nitrogen tended to be the same as that of amino nitrogen. Especially, the fermentation time of the test inoculated with black soybean Cheonggukjang increased as the fermentation time increased. This is shown in FIG. 3. Peptide-branched content tended to decrease gradually with the fermentation time, with the largest decrease in the Cheonggukjang added with the isolated strain, followed by the Cheonggukjang added with Bacillus subtilis, and nothing was added as the fermentation time passed. Cheonggukjang added with radish and kiwi showed a lower nitrogen content. This is shown in FIG. 4. The water-soluble protein increased rapidly after 24 hours of fermentation and was highest at 48 hours, when the content of allele black soybeans was higher than that of soybean soybeans and also tended to be higher than that of small black soybeans. In the black soybeans, the inoculated Bacillus subtilis was the highest in the small black soybeans. This is shown in FIG. 5. The soluble protein content tended to increase continuously with fermentation time, with the greatest increase between 48 and 72 hours of fermentation, followed by small black soybeans, soybeans, and non-added black soybeans. Increased. This is as shown in FIG. The free amino acid of soybeans, alleles and small black soybeans was shown by the chromatographic results of the Cheonggukjang free amino acid of FIG. The total free amino acid content was 439.6 ~ 776.7 mg%. Among them, the allele Cheonggukjang inoculated with the isolated strain was the highest with 776.7mg%, and the lowest addition of the allele Cheonggukjang was 439.6mg%. Glutamic acid content was the highest in all samples, and black soybean chungkukjang was significantly higher than yellow soybean chungkukjang (107.1mg%). The highest percentage of allelic black soybeans were 214.6mg%. Nucleic acid-related substances of Chunggukjang using soybeans, alleles and small black soybeans are shown in Figure 8, and the contents of Urisil and UMP were higher than those of other nucleic acid-related substances. And 10.34 mg%. In addition, cytosine, guanine, CMP, GMP, IMP, AMP, ADP and ATP were also detected.

Analysis of Moisture, Crude Proteins, Crude Fats, and Crude Powders in Cheonggukjang Prepared with Allo and Small Black Bean Irradiation Big head Allele Black Beans Small Black Beans Black Bean (I-BO) Food Ingredients (I-BF) Strain (I-BS) Black Beans (Ⅱ-BO) Food Ingredients (Ⅱ-BF) Strain (II-BS) radish Kiwi B.Subtilis SMY-212 radish Kiwi B. subtils SMY-212 moisture 69.72 58.33 59.74 60.32 59.18 60.86 57.34 58.03 58.34 58.84 59.2 Crude protein 22.26 26.31 26.71 20.08 17.33 18.03 25.71 23.37 25.39 17.85 20.30 Crude fat 12.25 12.47 11.72 10.35 10.42 11.89 12.30 10.52 10.12 9.73 11.67 View minutes 3.00 3.44 2.32 2.11 2.05 2.18 2.67 2.79 3.42 2.23 2.69

Experimental Example 2: Physical Properties of Cheonggukjang Made with Black Beans

In this experimental example, the viscosity, chromaticity and texture of soybeans prepared with black soybeans were investigated. Viscosity is mixed with 10 g of crushed Cheonggukjang in 100 mL of distilled water, and then immersed in a cylindrical glass container with an inner diameter of 8 cm and kept in a constant temperature water bath maintained at 20 ° C., and a cylindrical spindle No. 2 (radius 0.159 cm, length 3.175 cm) is rotated with a viscometer Synchro-Lectic Viscometer (model LVT) and the sample surface were aligned with the spindle mark. After standing for 10 minutes, the speed was measured until the torque value reached equilibrium while rotating at 0.6 rpm (factor = 5000 for 2 minutes. Viscosity was calculated by dial reading × factor (centripoise). For chromaticity, 20g of Cheonggukjang was evenly placed on the measuring table of the Chroma Meter CR-200 (MINOLTA), and the L (Lightness), a (redness), and b (yellowness) values were repeated five times. The L, a, and b values of the standard white tube were 89.2, 0.921, and 0.78, respectively. The texture was crushed in Cheonggukjang and then Zig (3.5cm in diameter, 1.2cm in height) using a texture analyzer (TA-XT2i stable micro). Cohesiveness and hardness were measured by system Co., US A. As a result of the experiment, the viscosity tended to increase gradually as the fermentation time passed. Radish, kiwi and more than black beans It was found that the viscosity of the test plots added with the strain increased with the fermentation time, especially, after 72 hours of fermentation, the test plots containing the isolates had higher viscosity than the other test plots. It was found that soybeans fermented more easily than black soybeans, and the lightness L value (brightness) in color tended to decrease slightly as the fermentation time elapsed, as shown in Fig. 9. On the other hand, the a value (redness) tended to increase gradually as the fermentation time elapsed, as shown in Fig. 10, and the value of black soybean chungkukjang was higher than that of the control. As shown in FIG. 11, there was no significant change as the fermentation time elapsed. The hardness of the texture tended to be lower than that of the control, but the hardness of black soybean was higher than that of soybean. It was. In addition, in the case of allele black soybean, as the fermentation time elapsed, the addition of the isolate strain SMY-212 significantly decreased the hardness, and in the case of the small black soybean, the decrease was larger than that of the all black soybean. This is shown in FIG. The cohesiveness increased with the fermentation time, especially after 24 hours, the cohesiveness increased significantly, and black beans were lower than soybeans. In addition, the addition of kiwi was lower in the black soybean soybeans compared to the others, and the small black soybeans showed more cohesiveness than the non-added green soybeans containing the strains SMY-212 and Bacillus subtilis. This is shown in FIG.

Experimental Example 3: Viable cell count and enzyme activity of Cheonggukjang prepared with black soybean

Determination of the viable cell count of aerobic bacteria of Cheonggukjang prepared with black soybean of the present invention was diluted 1 g of sterilely ground Cheonggukjang with sterile physiological saline according to the three-step dilution method and then plate count agar medium (PCA: tryptone 0.5%, yeast extract 0.25). %, Glucose 0.1%, agar 1.5%) and plated, and the number of colonies shown after incubation for 2 days at 40 ± 1 ℃ was measured. During the enzyme activity, the protease was firstly prepared by mixing 10 g of crushed Chungkukjang and 100 mL of distilled water, shaking for 2 hours at room temperature, and centrifuging. The substrate solution was prepared by dissolving 1.8 g of milk casein in 300 mL of sodium phosphate buffer (pH 7.2) in a 0.6% casein solution. Enzyme activity was measured by adding 1 mL of substrate solution and 1 mL of distilled water into a test tube, pretreatment in a constant temperature water bath, adding 1 mL of crude enzyme solution, reacting at 35 ° C for 30 minutes, and adding 3 mL of acetic acid (TCA) with 0.4m TRICRO for 30 minutes at 35 ° C. The reaction was stopped by standing. The reaction solution was centrifuged to remove the precipitate, and then 5 mL of 0.5 M Na22CO3 and 1 mL of 1/3 polyline phenolic reagent were added to 2 mL of the supernatant, followed by color development in a 35 ° C. constant temperature water bath, and then cooled at room temperature. Then, UV / Vis Spectrophoto The optical density was measured at 660 nm as a meter, and protease activity was expressed in terms of tyrosine (µg / mL). One unit of the protease was the amount of enzyme that 1 mL of the crude enzyme solution produced 1 μg of tyrosine in 1 minute. Amylase activity was measured by adding 10 mL of 0.1 M acetate buffer (pH 48) containing 1% water soluble starch to 1 mL of crude enzyme as a substrate. After 10 minutes, 1 mL of the reaction solution is taken and 10 mL of 0.1 N-HCl is added thereto to identify the reaction. 0.5 mL of the solution was added with 10 mL of a coloring solution containing 0.005% urine and 0.05% potassium urine, and then the absorbance (OD) was measured at 660 nm. One unit of α-amylase was defined as an amount of enzyme in which the blue color of the amylose-yodine complex was reduced by 10%. Glucoamylase activity was kept at 30 ° C. for several minutes by adding 2 mL of the same buffer solution to 5 mL of 0.05 M acetate buffer (pH 4.8) containing 1% raw starch, using raw starch as a substrate. After adding 1 mL of crude enzyme solution and reacting for 30 minutes, 1 mL of the solution was taken to measure the reducing equivalents of liquid increase before and after the reaction according to the DNS method, and the reducing sugar produced in the enzyme reaction was calculated from the difference between the two. One unit of glucoamylase was defined as the amount of enzyme that produced 1 μmole of glucose per hour. As a result, the number of viable cells was changed from the beginning of fermentation, as shown in FIG. 14, two strains to which the strain culture medium was added were slower in the growth rate of bacteria than the others. In one test plot, the growth rate was very fast as the fermentation time elapsed, and the total number of viable cells showed a similar tendency to the total test bacteria at 72 hours. In addition, the protease activity during fermentation was increased in all the test zones. Especially, the enzyme activity of cheonggukjang inoculated with isolated strains was very high in alleles and small-species, and the order of fermentation time was As time passed, the increase in enzyme activity of Chungkookjang added with radish and Bacillus subtilis was greater than that without adding anything. α-amylase activity increased in all the test zones and increased until 48 hours of fermentation and then decreased. After that, the enzyme activity of Cheonggukjang added with S. aureus Bacillus subtilis was very high, and it was very characteristic that yellow soybean Cheonggukjang as a control, and the enzyme activity of Cheonggukjang inoculated with the isolate was not very strong unlike protease. As fermentation time elapsed, the addition of nothing to all black soybean showed a small increase in enzymatic activity, such as Cheonggukjang added with radish and kiwi.

Experimental Example 4 Viable Cell Number and Enzyme Activity of Cheonggukjang Prepared with Black Beans

The sensory test was carried out using five cheonggukjang fermented through five trained panelists as it was, and made with cheonggukjang stew (50g of cheonggukjang, 10g of tofu, 3g of salt, 10g of radish) in 500mL of water. In the case of the former, color, intrinsic odor, and formation of viscous substance in the former case were performed in the case of color, delicious taste, off-flavor, and overall preference in the latter case. As for the evaluation method, the four-point symbol grading method (+ very dislike, ++ slightly disliked, +++ slightly good, ++++ very good) was used. As a result, as shown in Table 4, the color of black soybeans was slightly worse than that of soybean soybean, but in case of discomfort, the soybeans added with radish and kiwi improved significantly compared to soybean and other black soybeans. Black soybeans were produced less than soybeans, but black soybeans were inoculated with the isolates. After boiling the Cheonggukjang, seasoning sensory test showed that the colors of the soybean and soybean black soybeans were not good because the soup had a little black light as compared to Soybean Cheonggukjang, but its color was unique. In the case of taste, Cheonggukjang, which added radish and kiwi to the black soybeans, was stronger than soybean. Also, in case of non-challenge, allele and small black soybeans were produced less than soybean soybeans, and especially allied black soybeans and small black soybeans added with radish, kiwi and isolate were reduced more. In terms of overall acceptability, the products of allied black soybeans were higher in radish and kiwi compared to the control of yellow soybeans, and the rest were similar, while those of small black soybeans were lower than those of non-black soybeans.

Sensory Evaluation of Cheonggukjang Made with Small and Alligated Black Beans Inspection items Big head Allied Black Bean (Ⅰ) Small Black Beans (Ⅱ) Black Bean (I-BO) Food Ingredients (I-BF) Strain (I-BS) Black Beans (Ⅱ-BO) Food Ingredients (Ⅱ-BF) Strain (II-BS) radish Kiwi B.subtilis SMY-212 radish Kiwi B.subtilis SMY-212 Color ++++ +++ +++ +++ ++ +++ ++ ++ ++ ++ + Odor suppression ++++ ++++ ++ + ++++ ++ ++ + + ++++ ++ Slime ++++ +++ +++ +++ +++ ++++ ++ +++ +++ +++ ++++ + Very hate, ++ a little hate, +++ a little good, ++++ very good

Food Sensory Evaluation of Cheonggukjang Made with Small and Alligated Black Beans Inspection items Big head Allied Black Bean (Ⅰ) Small Black Beans (Ⅱ) Black Bean (I-BO) Food Ingredients (I-BF) Strain (I-BS) Black Beans (Ⅱ-BO) Food Ingredients (Ⅱ-BF) Strain (II-BS) radish Kiwi B.subtilis SMY-212 radish Kiwi B.subtilis SMY-212 Color ++++ +++ +++ +++ +++ +++ ++ ++ ++ ++ ++ smell +++ +++ ++++ ++ +++ ++++ ++ +++ +++ ++ +++ Odor suppression ++++ +++ ++ +++ +++ ++ ++ ++ + +++ ++ Overall taste +++ +++ ++++ ++++ +++ +++ ++ +++ ++ ++ ++ + Very hate, ++ a little hate, +++ a little good, ++++ very good

Experimental Example 5: Biological Activity of Cheonggukjang Prepared with Black Beans

The sample solution for measuring the thrombolytic ability of Cheonggukjang was prepared by adding 100 mL of 0.85% NaCl solution in 0.1M phosphate buffer (pH 7.5) to 5 g of crushed Cheonggukjang, extracting at 5 ° C for 24 hours, and filtering. Fibrin plates were prepared by dissolving 0.17M borate containing 0.01M NaCl in the same buffer (pH 7.5) to a concentration of fibrinogen of 0.15%, and then dispensing 10 mL of this solution into a sterile sare 90 mm in diameter. Then, 0.5 mL of thrombin (20 units / mL) was added thereto, mixed well, and then prepared by leaving a cover and standing at room temperature for 1 hour. The measurement of nattokinase activity was compared by measuring the long axis diameter of fibrin soluble clear rings formed by incubating the sample solution and fibrin plate at 37 ° C. for 30 minutes and adding 10 μl of the sample solution to the fibrin plate and reacting at 37 ° C. for a certain time. . Standard plasmin solution was prepared in tris-NaCl buffer solution to 0.4, 0.8, 1.2, 1.6, 2.0 units / mL, and 10 μl of each was added dropwise to the fibrin plate, and the long axis diameter formed was measured and compared with the activity of the sample solution. The hydrogen donating ability of the black soybean Chungkukjang was measured by UV / Vis-spectrophotometer at 516 nm using the reducibility of α, α'-diphenyl-β-picrylhydrazine (DPPH). That is, 3 ml of 4 × 10 ^-4 M DPPH solution was added to 0.1 mL of each extract and 1 mL of 0.1% BHT used as a control, and then mixed with a vortex mixer for 5 seconds to measure the absorbance of distilled water. 1 mL was added to indicate the decrease in absorbance relative to the control.

The antioxidant effects of the extracts of Chungkookjang were observed in vitro by measuring the peroxide value (POV) of linolenic acid. That is, 1 mL of linolenic acid, 20 mL of ethanol without carbonyl, and 0.1 mL of Cheonggukjang extract were added to the Erlenmeyer flask, and then 25 mL of 0.2M phosphate buffer was added and stored at 37 ° C. for a period of time (1, 3, 5, 7 days). The solution was transferred to a separatory funnel, and 25 mL of chloroform was added thereto, followed by extraction two to three times. Next, 25 mL of acetic acid and 1 mL of saturated KI solution were added to the chloroform extract, which was allowed to stand in the dark for 5 minutes. Then, 50 mL of distilled water was added thereto, and titrated with 1/100 N Na ₂ S ₂ O ₃ solution using soluble starch as an indicator. Calculated by the formula.

Peroxidation inhibitory effect on hepatic lipids was observed in vitro using the liver homogenate in rats. That is, the livers of the rats were extracted and homogenized with phosphate buffer (pH 7.4), and then incubated at 37 ° C for 40 minutes by adding 0.05 mL of H ₂ O ₂ (1M), FeSO 4 (50 mM) and Cheonggukjang extract to the homogenate. Thiobarbituric acid reactive substances (TBARS) were measured. The scavenging effect of nitrite on the extract of Cheonggukjang was added to 1mL of 1mM NaNO2 in 0.2mL of Cheonggukjang Extract, and the pH of the reaction solution was adjusted to .2 using 0.1N HCl (pH 1.2). The volume of the solution was made into 10 mL, and it was made to react at 37 degreeC for 1 hour. 5 mL of 2% acetic acid solution was added to 1 mL of the reaction solution, 0.4 mL of Griess reagent was added thereto, mixed, and left for 15 minutes. The absorbance was measured at 520 nm using a spectrophotometer. The blanks were measured by adding distilled water instead of NaNO ₂ solution and the scavenging rate was expressed as the percentage of nitrite removed by the following equation.

R: nitrite scavenging rate

A: absorbance of 1 mM NaNO ₂ after reaction for 1 hour

B: absorbance of blank

C: Absorbance of Extracted Sample Additions

As antimicrobial activity, 8 species including general rot bacteria, Staphylococcus aureus (C. aeruginosa), cavities, and Bacillus subtilis were used as gram positive strains. Used. The test plate was used within two weeks while the LB soft agar containing 0.6% agar was layered and coagulated in a previously prepared LB medium containing 1.5% agar and immediately wrapped in a wrap and stored in a refrigerator. The antimicrobial activity was measured according to the agar diffusion method. After extracting the solvent of black soybean Chungkukjang, the concentrated solution which was concentrated under reduced pressure (60 ℃) to 10ml using a rotary evaporator was used as a sample solution for antimicrobial activity measurement. Spot the sample solution on a sterile filter paper disc, and then completely evaporate the solvent in a dry oven at 80 ° C, place it on the surface of the middle-layer test plate, place it in contact with the refrigerator for 1 hour, and incubate in incubation (30-35 ° C, 24 Time) to measure the size of the clear zone (diameter, mm) to compare the antimicrobial activity. As a result, as shown in Figure 15, the thrombolytic ability was found to be generally excellent in Cheonggukjang prepared by the addition of natural food materials and test strains. Especially, the addition of kiwi was higher than that of B. subtilis SMY-212. As shown in Table 6, the hydrogen donating ability of 0.1% BHT was lower than 93.1%, but all showed significant effects. In addition, compared to 67.3% of soybean soybean soup, the soybean and soybean black soybeans were 85.8 and 80.5% respectively. Figure 16 shows the antioxidant power of the chungkukjang methanol extract to linolenic acid, the control group without the chungkukjang methanol extract was 38, 84, 157 and 204 meq / kg peroxide value after 1, 3, 5 and 7 days of storage The added test plots had a significant antioxidant effect, even after 7 days of storage, at 30 meq / kg or less. Among the test plots, the allergenic and small black soybeans were lower in methanol extracts than the methanol extract of soybean soybeans. The lowest inoculation showed that their antioxidant effect was greater. The TBA values of liver lipids in rats were 3597 μmol MDA / g liver in the control group without the addition of the Cheonggukjang methanol extract, and the TBA values of the test broths added with the Cheongguk methanol extract were 1443 μmol MDA / g liver, all compared to the control. It appeared low and had an antioxidant effect. In addition, TBA was lower in test plots with methanol extract of Chungkookjang, and kiwi was added more than radish in functional Cheonggukjang, and Bacillus subtilis was inoculated lower than seed isolate (SMY-212). appear. Nitrite scavenging ability of the chungkukjang methanol extract showed more than 70% as shown in Table 8, but only 85.5, 74.5 and 72.8% of the ethanol extract of radish, Bacillus subtilis and isolates added to black soybean, respectively. All of the remaining Cheonggukjang extracts showed a high effect of more than 90%. The antimicrobial activity against food poisoning bacterium was almost the antimicrobial activity of soybean Chungcheongjang, and the two black soybeans showed the highest antimicrobial activity in methanol extract of Cheonggukjang added with isolate. The antimicrobial activity of the extract of Cheonggukjang fermented with radish rather than kiwi was high.

Hydrogen Donating Activity of Methanol Extracts from Cheonggukjang Prepared with Soybeans, Alleles, and Small Black Soybeans Big head Allele Black Bean (I) Small Black Beans (Ⅱ) Black Bean (I-BO) Food Ingredients (I-BF) Strain (I-BS) Black Beans (Ⅱ-BO) Food Ingredients (Ⅱ-BF) Strain (II-BS) radish Kiwi B. subtilis SMY-212 radish Kiwi B.subtilis SMY-212 67.3 76.4 85.8 87.8 79.2 84.4 75.5 84.8 85.9 78.9 83.4 [Unit] unit:%

Effect of Ethanol Extract of Cheonggukjang on TBA Value of Liver Big head Allele Black Bean (I) Small Black Beans (Ⅱ) Black Bean (I-BO) Food Ingredients (I-BF) Strain (I-BS) Black Beans (Ⅱ-BO) Food Ingredients (Ⅱ-BF) Strain (II-BS) radish Kiwi B. subtilis SMY-212 radish Kiwi B.subtilis SMY-212 3730 1620 1480 1210 1030 920 1820 1490 1010 830 910 [Note] Unit: μmol MDA / g liver

Nitrite Scavenging Effects of Methanol Extracts from Cheonggukjang Prepared with Soybeans, Alleles and Small Black Soybeans Big head Allele Black Bean (I) Small Black Beans (Ⅱ) Black Bean (I-BO) Food Ingredients (I-BF) Strain (I-BS) Black Beans (Ⅱ-BO) Food Ingredients (Ⅱ-BF) Strain (II-BS) radish Kiwi B. subtilis SMY-212 radish Kiwi B.subtilis SMY-212 96.8 97.2 99.6 98.3 93.4 92.6 92.8 85.5 92.6 74.5 72.8 [Unit] unit:%

Cheonggukjang prepared by adding black kiwi and radish after inoculating the disclosed strain identified as Bacillus mecaterium-like strain by using black soybean as a main ingredient and separating from Bacillus strain as described through the Examples and Experimental Examples Intrinsic squeaky odor is suppressed, sensory test results have excellent palatability effect, thrombolytic ability, peroxide inhibiting ability, nitrite scavenging ability, food poisoning and antimicrobial effect on the decay bacteria is very useful invention in the food industry.

Claims (2)

Cheonggukjang contains black beans as main ingredients and fruit vegetables as subsidiary materials. The selected black soybeans are immersed in cold water, drained, and then steamed and cooled in a autoclave, and fermented for 3 days. Cheonggukjang manufacturing method.