KR20110120802A - A novel b. subtilis sp. strain and fermented soymilk for anti-gastric cancer by using the same - Google Patents

Abstract

PURPOSE: A novel bacillus subtilis strain, and functional fermented soymilk using thereof having an anti-gastric cancer effect is provided to offer an anti-oxidant effect of the soymilk to users, and to reduce the unique smell of the soymilk by fermenting. CONSTITUTION: Functional fermented soymilk using a novel bacillus subtilis strain is obtained by the following steps: heating conventional soymilk at 121 deg C for 15 minutes using a high pressure sterilizer; cooling the soymilk at 30 deg C, and adding 1%(v/w) of glucose into the soymilk; injecting the novel bacillus subtilis strain obtained from traditional fermented soybeans cultivated for 24 hours at 37 deg C into the soymilk; and fermenting the mixture in shaking water bath.

Description

New iii. Ｓｕｂｔｉｌｉｓ ｓｐ. Strain and gastric cancer inhibiting functional fermented soy milk using the same {A novel B. Subtilis sp. strain and fermented soymilk for anti-gastric cancer by using the same}

The present invention is a novel B. Subtilis sp . It relates to a strain and its use for suppressing human gastric cancer.

Soybean, the main ingredient of soy milk, is rich in functional ingredients such as isoflavone, saponin, phytic acid, trypsin inhibitor, oligosaccharides, and it is rich in high quality protein and fat. It is known to have various physiological activities such as effects, blood cholesterol lowering ability. Soybeans are fermented by enzymes produced by strains to break down soy protein, which contains aglycone, free amino acids, and peptides of isoflavone. Non-fermented foods such as soy milk and tofu have less than 10% of aglycone, but fermented foods such as Cheonggukjang and Doenjang increase by about 70-80%. Aglycone form has higher physiological activity and faster absorption than glycoside form, so fermented soybean food is more effective in disease prevention because it has many physiological activities than non-fermented soybean food.

Soy milk has a problem in that the soy milk is in the form of a drink made by grinding soybeans, which is nutritionally superior due to high content of water-soluble protein, unsaturated fatty acids, and essential amino acids, but its availability is lowered due to its unique soy fishy smell. Fermented soy milk has the advantage that it can give a new flavor or remove odor through fermentation and increase digestibility. Fermented soy milk on the market is Lactobacillus acidophilus , Lab . There are many products and cases manufactured using lactic acid bacteria such as bulgaricus , but Bacillus derived from soy milk, a raw material of soy milk. Few strains have been applied to milk beverages using the genus strains. B. subtilis isolated from Cheonggukjang has been reported as an excellent strain that produces thrombolytic enzymes and produces bioactive substance, poly-γ-glutamic acid (PGA), and is known as indole and carcinogens produced by intestinal rot. To reduce and adsorb and excrete.

We believe that B. Subtilis Kyuju isolated from Cheonggukjang in Sunchang, Jeonnam, Korea, identified as KC-1 strain (KCCM No. 42923), and attempted to produce fermented soymilk using this strain. The conventional Cheonggukjang improves the physicochemical component and γ-GTP activity according to the fermentation degree. The purpose of the present invention is to determine whether the ingredients and functionality of the fermented soymilk fermented with the present invention Cheonggukjang-derived bacteria change according to the fermentation degree. Therefore, the present invention is to examine the physicochemical changes of fermented soymilk of the strain B. genus, and further to determine the component changes and functional differences of fermented soymilk according to the antioxidant capacity and the degree of fermentation (pH reduction).

The object of the present invention Bacillus By identifying new strains of the genus, pure cultured it was achieved by producing fermented soymilk and confirming its anticancer function.

Inventive B. Subtilis KC- 1 strain has excellent DDPH, hydroxy scavenging activity SOD activity, excellent antioxidant effect and AGS human gastric cancer cell growth inhibitory effect, fermented soy milk using this strain can be used as a functional food composition for gastric cancer prevention.

1 is a graph showing the change in cell number (A) and acidity and viscosity (B) of fermented soymilk by the strain of the present invention.
Figure 2 is a graph showing DPPH group scavenging ability (A) and OH group scavenging ability (B) of fermented soy milk by the strain of the present invention.
Figure 3 is a graph showing the SOD-like activity of fermented soy milk by the strain of the present invention.
Figure 4 is a graph showing the growth inhibitory effect of AGS human gastric cancer cells of fermented soy milk by the strain of the present invention.

Example  1. Preparation of Cheonggukjang Sample

In this example, 6 kinds of representative Cheonggukjang products (K-, M-, Mn-, O-, Os-, H-Cheongkukjang) produced and marketed in the traditional method in Sunchang Gochujang Folk Village, Jeonbuk, Korea were purchased and tested for physical and chemical performance. After the evaluation, selected K-, M- and H-Cheongkukjang were used for the experiment. Cheonggukjang samples were lyophilized and crushed to add 20 times (v / v) methanol to the samples. The mixture was stirred three times for 12 hours, filtered and concentrated using a rotary vacuum concentrator (EYELA, Tokyo Rikakikai Co., Japan) to obtain a methanol extract. These extracts were diluted in dimethyl sulfoxide (DMSO) and used for the experiment.

Example  2. Bacillus Isolation and Identification of Bacteria

In this example, a sample of Cheonggukjang diluted 10-fold in sterile distilled water was taken as platinum, and plated on a nutrient agar plate (beef extract 3 g / L, peptone 5 g / L, agar 15 g / L) by a flat plate method and 37 ° C. Incubated for 24 hours. The colonies were selected from the resulting colonies with clear shapes and then inoculated in nutrient broth, followed by secondary culture. Finally, three single strains were isolated from K-Cheonggukjang (Kim Yong-soon traditional food in Soonchang Gochujang Folk Village) and named KC-1, KC-2, and KC-3, respectively. In order to identify them, each sequence was obtained by amplifying the recA gene by PCR (SEQ ID NOs: 1-3), and comparing the sequence homology with other strains through BLAST homology search. I identified it.

As a result, KC-1 is B. subtilis 98% concordance with the recA gene was finally identified as B. subtilis . KC-2 is B. amyloliquefaciens FZB42 illustrates a base sequence homology of bacteria and 99% were identified as B. amyloliquefaciens, B. cereus ATCC 14579 KC -3 is shown a homology of bacteria and 99% of the final identified as B. cereus It became . Among them , B. Subtilis , the present disclosure material The KC- 1 strain was deposited as KCCM 42923 with the Korea Microorganism Conservation Center on March 26, 2009 .

Therefore, three representative bacteria isolated from K-Cheonggukjang are B. subtilis , B. amyloliquefaciens, and B. cereus , and these bacteria were all present in the range of 10 ⁷ ~ 10 ⁸ / g, which are the microorganisms that are central to fermentation of K-Cheonggukjang. It is thought to be.

Example  3. Fermented Soymilk  And extract manufacturer

Soy milk used in the experiment was purchased from Donghwa Food Co., Ltd. (Yangsan, Gyeongnam) and is a soybean soybean milk made from US soybeans containing no additives. The soy milk purchased was sterilized by heating at 121 ° C. for 15 minutes using an autoclave and then cooled to 30 ° C. for use in experiments. B. subtilis inoculated with soymilk KC-1 KCCM 42923 was isolated from traditional Cheonggukjang and incubated for 24 hours at 37 ℃ in nutrient broth medium. Sterilized soymilk was added with 1% (w / w) of glucose for fermentation and 1% (v / w) of the incubated bacteria at about 107 / g. Fermented.

After fermentation, the soy milk was lyophilized, and then ground. Then, 20 times (w / v) methanol was added to the sample, and the mixture was filtered three times for 24 hours and then filtered. The filtrate was concentrated with a rotary vacuum concentrator (EYELA, Tokyo Rikakikai Co., Japan) to obtain an extract and diluted with dimethyl sulfoxide (DMSO).

Experimental Example  1. Measure general characteristics

B. Subtilis : Determination of the number of B. Subtilis : Immediately after fermentation is completed, 1 mL of sample is aseptically taken and diluted to 10-fold, inoculated in plate count agar (Difco Co., Detroit, USA) medium and incubated at 37 ° C for 48 hours. Colony counts were counted.

Experimental Example  2. pH  And acidity measurements

The pH of fermented soymilk was measured using a pH meter (TP-93, Toko Chemical Laboratories, tokyo, Japan). The acidity was taken in 1 mL of soymilk, diluted in 19 mL of distilled water, titrated with 0.1 N NaOH until pH 8.4, and the mL number of NaOH consumed was converted into%.

As a result of examining the number of bacteria and acidity according to pH change of fermented soy milk (Fig. 1A), 8.5 × 104 cfu / mL of B. Subtilis added for fermentation sp . The strains were 3.4 × 10 6 cfu / mL at pH 5.5, 7.2 × 10 6 cfu / mL at pH 5.25, and 7.5 × 10 7 cfu / mL at pH 5.0. That is, the present invention B. Subtilis inoculated in soymilk sp . It was found that the number of strains increased with decreasing pH and increasing fermentation time. In the case of acidity, as the fermentation progressed, the pH increased from 0.1% before fermentation to 0.5% at pH 5.0, but the acidity was insufficient compared to yogurt, which is a representative fermented milk. This is due to the fact that the content of lactic acid and citric acid in the fermented soybean milk is significantly lower than that of the milk fermented product. In general, for the fermented milk fermented with lactic acid bacteria indicates a pH 4.0 ~ 4.2 pH from 1.0 to 1.1% the most preferred quality represents the present time by B. Subtilis sp . By strain When fermented soy milk is commercialized, it is more suitable to mix with lactic acid bacteria to lower pH and increase acidity.

Experimental Example  3. Viscosity Measurement

Viscosity was measured after rotating at 100 rpm for 60 seconds with spindle No. 3 at room temperature using a Brookfield viscometer (Viscometer, Model DV-I, Brookfield Engineering Lab. Inc., Stoughton, MA, USA).

In general, fermented milk products such as yoghurt depend on the viscosity of consumers, so the viscosity of soymilk was 127 cps, but fermented soymilk was 325 at pH 5.5, 5.25, and 5.0, respectively. cps, 621 cps, 712 cps (Fig. 1B). Viscosity increased significantly between pH 5.5 and pH 5.25, resulting in slight curd formation and change in titratable acidity, and in sensory evaluation, sour taste increased. Rasic and Kurmann reported that the viscosity of yogurt was affected by the total solid content of the yogurt mixture, the degree of protein hydrolysis, the slime production capacity and the acid production capacity. Viscosity of milk fermented with lactic acid bacteria showed high viscosity (130 cps ~ 1930 cps) due to the production of large amounts of organic acids and aggregation of casein and whey protein, but relatively low acid production of the present invention B. cereus sp . The strain showed a low viscosity.

Experimental Example  4. Amino  And ammonia nitrogen content

The amino nitrogen content was measured by the formol method. 18 mL of distilled water was added to 2 mL of the sample, stirred at room temperature for 1 hour, 20 mL of neutral formalin (pH 8.4) solution was added, titrated with 0.1 N NaOH until pH 8.4, and the amount of amino nitrogen The content was measured. Ammonia nitrogen was measured by Indophenol method by AM505-K (Asan Pharmaceuticals).

The amino nitrogen content indicates the degree of protein degradation in the form of amino acids. It is used to evaluate the fermentation degree due to the action of protease. As shown in [Table 4], the amino nitrogen content of soymilk was 542.5 mg / dL, but it increased rapidly as the fermentation began, ranging from pH 5.5 to 882 mg / dL, and then slowly increasing to pH 5.0. Amino nitrogen content of 1190 mg / dL was shown. Thus, the present invention B. cereus sp . Fermentation of soy milk by the strain results in the production of proteolytic enzymes, and it can be seen that the protein hydrolyzate increases as the fermentation time increases. Ammonia nitrogen is produced by deamination reactions in the process of protein decomposition, and is used as an indicator of decay or decay when excess ammonia nitrogen is generated. The soymilk's ammonia nitrogen content was 129.1 μg / dL, while as fermentation progressed, it increased to 508.3 μg / dL at pH 5.0.

Experimental Example  5. Antioxidant Effect Measurement

1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging effect: DPPH scavenging effect of fermented soybean milk was measured by the hydrogen donating effect of DPPH according to the method of Blois. 100 μL of the sample dissolved in methanol and 100 μL of the 150 μM DPPH solution were mixed in a 96-well plate and allowed to stand at room temperature for 30 minutes, and then the absorbance was measured at 540 nm using a UV / VIS Spectrophotometer (Jasco, Japan). It was. The difference in absorbance of fermented soymilk according to pH is expressed as a percentage (%) compared to the control without the sample.

DPPH, which is a representative method of measuring hydrogen donating ability, is a relatively stable free radical. It has a deep purple color.

As a result of measuring the antioxidant effect of fermented soybean milk by fermentation using DPPH radical scavenging method, fermented soymilk showed scavenging ability in a concentration-dependent manner [FIG. 2 (A)]. In particular, soymilk showed 50% scavenging activity at 2 mg / mL, while fermented soymilk showed 56% activating activity.

Experimental Example  6. Hydroxy radical  ( ㆍ OH Scavenging activity

According to the method of Chung et al., 10 mM FeSO ₄ ㆍ 7H ₂ O-ethylenediaminetetraacetic acid disodium salt (EDTA) was mixed with 10 mM 2-deoxyribose and the sample solution by concentration, and then 10 mM H ₂ O ₂ was added to Fenton. Incubate at 37 ° C. for 4 hours according to the reaction. 1.0 mL of 2.8% trichloroacetic acid (TCA) and 1.0 mL of 1.0% thiobarbituric acid (TBA) were added to the mixture, reacted in boiling water for 10 minutes, cooled, and the absorbance was measured at 520 nm using a UV-VIS spectrophotometer. .

Hydroxy radicals are the most chemically reactive among the active oxygen and cause oxidation in vivo and are known to cause DNA damage or mutations. ㆍ The antioxidant effects of fermented soymilk by OH scavenging activity were examined [Fig. 2 (B) ]. As fermented soybean milk was fermented, OH scavenging activity increased rapidly from 23% to 78% at 2 mg / mL. This is similar to the result that Choi showed more than 80% ㆍ OH scavenging ability in various soymilk fermented with Bacillus .

Experimental Example  7. Superoxide anion dismutase  ( SOD ) Pseudo-activity

According to Marklund and Marklund's method, add 3 mL of pH 8.5-corrected Tris-HCl buffer (50 mM Tris + 10 mM EDTA, pH 8.5) and 0.2 mL of 7.2 mM pyrogallol to 0.2 mL of sample at 10 ° C. After reacting for a minute, the reaction is stopped with 1 N HCl (0.1 mL). The absorbance of oxidized pyrogallol in the reaction solution was measured at 420 nm, and the difference in absorbance between the sample addition group and the no addition group was expressed as a percentage (%).

SOD-like activity of methanol extracts of soymilk and fermented soymilk was measured by autooxidation reaction against pyrogallol. Fermented soymilk with a pH of 5.0 showed 50.6% of activity at a concentration of 2 mg / mL, which was about three times higher than that of 17.3% of soymilk (FIG. 3). The SOD-like activity was reported to increase with the addition of flavonoids and phenolic compounds. The SOD-like activity of fermented soybean milk increased as the compound increased with fermentation.

Experimental Example  8. In in vitro  Anticancer effect

Cancer cell culture: AGS human gasrtic adenocarcinoma cells were used for the experiment while being cultured by the Korea Cell Line Bank (Seoul National University of Medicine). Cancer cells were cultured at 37 ° C and 5% CO ₂ incubator (model 311, Forma, USA) using RPMI 1640 medium containing 100 units / mL penicillin-streptomycin (GIBCO, USA) and 10% fetal bovine serum (FBS). Incubated at. Each cultured cancer cells were refeeded 2-3 times a week, washed with PBS in 6-7 days, and then centrifuged by detaching cells attached with 0.05% trypsin-0.02% EDTA. Then, the culture medium was added to the integrated cancer cells, and the pipettes were mixed well so that the cancer cells were evenly distributed and used in the experiment while passaged in a cell culture flask.

3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetra-zolium bromide (MTT) assay: Cultured cancer cells are divided into 180 μL in 1-104 cells / well in a 96-well plate and sampled. Was diluted to an appropriate concentration and 20 μL was added thereto, followed by incubation for 72 hours at 37 ° C. and 5% CO ₂ incubator. To this was added 20 μL of a MTT solution prepared at a concentration of 5 mg / mL to Phosphate buffer saline (PBS) and further incubated for 4 hours under the same culture conditions. Then, the supernatant was removed, DMSO 150 μL was added to each well, stirred for 30 minutes, and the absorbance was measured at 540 nm with an ELISA reader.

Bacillus used in the fermentation of the invention Subtilis sp . ( Bacillus The genus KC-1) strain is compared with other B. subtilis AGS human gastric cancer cells and the present invention confirmed the difference of cancer cell growth inhibition effect according to the fermentation degree of fermented soy milk using AGS human gastric cancer cells (Fig. 4), pH Inhibition rate of 76%, 93%, and 95% at 2 mg / mL of fermented soymilk at 5.5, pH 5.25, and pH 5.0, respectively, showed that the soymilk had the effect of inhibiting human gastric cancer cell growth as fermentation progressed. Therefore, according to the strain of the present invention, soymilk was determined to have an effect of enhancing cancer cell growth inhibition as the fermentation time elapsed.

Statistical analysis

In order to test the significance of the experimental results obtained from the control group and each sample, Duncan's multiple range test was performed at the p <0.05 level after ANOVA. The results were expressed as mean ± standard deviation. Indicated. All statistical analyzes were performed using the Statistic Analysis System (v9.1 SAS Institute Inc., NC, USA) statistical program.

Inventive strain B. Subtilis sp . Strain (KCCM 42923) is a very useful invention in the health fermented food industry because it has an excellent effect of increasing the antioxidant and gastric cancer cell growth inhibitory ability during soymilk fermentation.

Korea Microorganism Conservation Center (overseas) KCCM42923 20090326

Claims (1)

B. Subtilis in Soymilk sp . Fermented soy milk for antioxidant and gastric cancer suppression, characterized in that fermented by inoculating Bacillus [KCCM 42923] strain.

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