KR20190018204A - Lactobacillus brevis WCP02 strain having high gastric acid tolerance and bile acid, and high GABA productivity, and probiotics and fermented food by using the same - Google Patents

Abstract

In the present invention, provided are a Lactobacillus brevis WCP02 strain, which has excellent resistance to gastric acid and bile acid, is highly utilizable as a probiotic agent, also has remarkably high GABA productivity, and thus can be used as a fermentation seed of a functional food material; and the probiotic agent and fermented food using the same. The soybean fermented food prepared by fermenting with the Lactobacillus brevis WCP02 strain of the present invention increases not only a content of GABA, phenolics and isoflavone aglycone, but also alpha-glucosidase and pancreatic-lipase inhibitory activity, thereby capable of being used as a material for functional foods and medicines, which exhibits effects of lowering cholesterol, alleviating diabetes, obesity, hyperlipidemia and arteriosclerosis.

Description

[0001] The present invention relates to a lactobacillus brevis WCP02 strain having excellent gastric acid tolerance and bile acid resistance and having high GABA productivity, a probiotic preparation and a fermented food using the same, food by using the same}

The present invention relates to a Lactobacillus brevis WCP02 strain excellent in gastric acid tolerance and bile acid resistance and having excellent GABA productivity, a probiotic agent using the same, and a fermented food. More particularly, the present invention relates to a lactobacillus brevis WCP02 strain which can be used as a fermentation seed of a functional food material because of its excellent gastric acid tolerance and bile acid resistance, .

As the diet is becoming westernized, the intake of animal foods is on the rise, and metabolic syndrome such as various cardiovascular diseases, various cancers and diabetes is increasing. Although the treatment of these diseases has been highly dependent on medical technology, recent efforts and studies to find solutions for improvement and prevention using microbial-based functional foods such as probiotics and fermented foods are increasing.

Probiotics refers to 'active live bacteria culture', meaning 'live bacteria', ie live bacteria, which keep the host's gastrointestinal tract healthy, such as humans and animals. In 2002, FAO-WHO recommended Bifidobacterium and Lactobacillus strains as representative strains of probiotics. These strains are classified as lactic acid bacteria. They are known as Gram-positive bacteria that decompose glucose to produce lactic acid. They form dominant species on the intestinal surface, and by their antibacterial proteins (bacteriocin) inhibit harmful bacteria, stabilize intestinal flora, It is known to have a great effect on the improvement of colon diseases such as irritable bowel syndrome.

In order to obtain useful effects of such probiotics (probiotics), lactic acid bacteria should survive to the large intestine and small intestine through the host's digestive tract and live in the intestinal and epithelial cells in any state. However, more than 90% of normal lactic acid bacteria consumed by gastric acid are killed and even if they are alive, they are killed again by bile acid, and the probability of reaching the colon and small intestine is reported to be within about 5%. Therefore, lactic acid bacteria resistant to gastric acid and bile acid, which have high utility as a probiotic agent, have been extensively studied and studied.

Gamma-aminobutyric acid (GABA) is a non-monoclonal amino acid composed of four carbon atoms, which is released from glutamic acid (GA) by glutamate decarboxylase (GADase) . In addition to its role as a neurotransmitter in the brain, GABA has a wide variety of physiological functions such as brain function promotion, mental stabilization action, blood pressure lowering action, diuretic action, liver function improvement action, obesity prevention action, alcohol metabolism acceleration action, It is also used as a medicine to treat headaches, tinnitus, and decreased motivation due to stroke or cerebral artery sequelae. Generally, it causes the generation of GABA in the body from aging due to excessive intake of inappropriate food or additives, insufficient intake of vitamins and minerals, and so on. Therefore, the necessity and usefulness of probiotics with excellent GABA productivity are greatly increased.

In the prior art, some lactic acid bacteria having GABA producibility have been discovered (Patent No. 10-1461116, Patent No. 10-1131069). However, since the GABA productivity is not high or the gastric tolerance and bile acid resistance are not sufficient, .

Accordingly, the present inventors have found that a novel Lactobacillus brevis WCP02 strain having a remarkably superior GABA production, gastric juice resistance and bile acid resistance is isolated and identified in kimchi, and this strain is used as a live- And the present invention has been completed.

Accordingly, an object of the present invention is to provide a novel Lactobacillus brevis WCP02 strain which can be used as a fermentation seed of a functional food material because of its excellent gastric acid tolerance and bile acid resistance, its high utility as a probiotic agent, and its high productivity.

It is still another object of the present invention to provide a probiotic agent comprising Lactobacillus brevis WCP02 strain and / or a culture thereof as an active ingredient.

It is still another object of the present invention to provide a fermented soybean food which is fermented with Lactobacillus brevis WCP02 strain to remarkably increase the amount of GABA, and to which antioxidative activity, phenolics and non-glycoside isoflavones are promoted.

In order to achieve the above object, the present invention provides a novel Lactobacillus brevis WCP02 strain having excellent gastric acid tolerance, bile acid tolerance and excellent GABA productivity and a culture thereof.

The present inventors isolated / identified a strain having excellent prophylactic properties (acid resistance, gastric tolerance, response to saponin resistance) and excellent gas production ability from kimchi and named this strain Lactobacillus brevis WCP02, (KACC) on December 12, 2016, and received the accession number KACC 92159P (Example 1).

Gram stain and electron microscopic observation were used for morphological observation of Lactobacillus brevis WCP02 strain. API kit, which is a method for assaying glucose tolerance, was used for identification of physiological and biochemical characteristics. Analysis of cell wall fatty acids (MIDI analysis) and 16S rDNA sequence analysis. The Lactobacillus brevis WCP02 strain of the present invention based on the 16S rRNA base sequence belonged to the genus Lactobacillus (Figs. 1 to 3).

The Lactobacillus brevis WCP02 showed a survival rate of 39% or more after 4 hours of culture at pH 2, and survival rate of 86% or more after 48 hours of culture in bile acid at pH 3.0, and 0.1% (w / v) monosodium glutamic acid (monosodium glutatmate, MSG) (Table 1 to Table 3).

The Lactobacillus brevis WCP02 strain according to the present invention can be used as a bacterium for the production of a fermented food in which an active ingredient such as GABA is promoted.

According to yet another object of the present invention, probiotics comprising Lactobacillus brevis WCP02 strain and / or a culture thereof as an active ingredient are provided.

Since the probiotics according to the present invention are selected from safe fermented foods such as kimchi, the strains can be used safely as a non-toxic probiotic agent. Therefore, the probiotics according to the present invention can be used for the edible use of animals including humans, and specifically, they can be used as food or pharmaceutical materials.

The probiotics can be supplied in various forms such as dried cell forms or fermented products. For example, fermented food, beverage, or kimchi.

According to another object of the present invention, there is provided a soybean fermented foodstuff, which is fermented with Lactobacillus brevis WCP02 strain to remarkably increase the amount of GABA, and to which antioxidative activity, phenolics and non-glycoside isoflavones are promoted.

In particular, the soybean fermented food of the present invention includes soy milk-yogurt, soybean fermented seasoning, and the like.

The soybean fermented food fermented with the Lactobacillus brevis WCP02 strain according to the present invention exhibited remarkable increase in the content of GABA and enhanced antioxidative activity, phenolic content, and non-glycosylated isoflavine content, and showed alpha-glucosidase inhibitory activity and pancreatic -Lipase inhibitory activity is enhanced and diabetic and obesity are alleviated and improved.

 The novel Lactobacillus brevis WCP02 strain of the present invention has excellent GABA productivity and is excellent in both gastric acid resistance and bile acid resistance and thus is highly utilizable as a probiotic agent. It can also be used as a seed for the production of a fermented food in which the active ingredient of guava and the like is enhanced.

The probiotics according to the present invention not only enhance the survival rate in the body due to excellent gastric acid tolerance and bile acid resistance but also can enhance the metabolism, antioxidant activity, antidiabetic activity and anti-obesity activity.

The soybean fermented food prepared by fermenting with the Lactobacillus brevis WCP02 strain of the present invention not only promoted the content of GABA, phenolicx and non-glycosylated isoflavone but also enhanced the alpha-glucosidase inhibitory activity and the pancreatic-lipase inhibitory activity Cholesterol lowering, diabetes, obesity, hyperlipidemia, and atherosclerosis, and can be used as a material for functional foods and medicines.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph showing the selection process of Lactobacillus brevis WCP02 strain on the basis of the Gaba production by thin-layer chromatography (TLC).
2 is a morphological photograph of Lactobacillus brevis WCP02 strain. a is the morphological photograph on the MRS solid medium, and b is the photograph observed with the electron microscope after the Gram stain.
3 shows a 16S rRNA base sequence of Lactobacillus brevis WCP02 strain.
4 is a phylogenetic relationship diagram of Lactobacillus brevis WCP02 strain.
5 shows the antioxidant activity of Lactobacillus brevis WCP02 strain (lyophilized cell extract). a represents a DPPH radical scavenging activity, b represents an ABTS radical scavenging activity and c represents a hydroxyl radical scavenging activity.
Fig. 6 shows digestive enzyme inhibitory activity of Lactobacillus brevis WCP02 strain. a indicates an alpha-glucosidase inhibitory activity and b indicates a pancreatic-lipase inhibitory activity.
Figure 7 shows the biochemical properties of Lactobacillus brevis WCP02 strain.
Fig. 8 shows the composition of the microbial fatty acid of Lactobacillus brevis WCP02 strain.

The present invention will be described in more detail by the following examples. These examples are for illustrating the present invention, and the scope of the present invention should not be limited by them.

Example

Example 1. Isolation and Selection of Strain

<Strain isolation>

1 g or 1 ml of cabbage Kimchi, Dongchimi and radish Kimchi collected in Gyeongnam area (Jinju, Hamyang, etc.) was suspended in 9 ml of sterile physiological saline, strongly shaken, left for 5 minutes, .

The sample was diluted 10 ⁶ to 10 ⁸ with 10 steps of dilution with sterilized physiological saline to isolate about 500 kinds of lactic acid bacteria pure. The purely isolated lactic acid bacteria were inoculated into MRS liquid medium and cultured at 30 ° C for 2 days For colonies, 20% (v / v) glycerol was added and stored in a 70 ° C freezer and used as needed for activation.

&Lt; Selection of strain based on GABA production >

The purely isolated lactic acid bacteria were inoculated into an MRS liquid medium containing 5% (w / v) MSG (monosodium glutamic acid) and cultured at 30 ° C for 2 days. The culture broth was centrifuged and the supernatant was applied to a thin layer chromatograph, TLC). After incubation for 20 min in a development tank containing 5% MSG, GABA, and 2 μl of each culture, the cells were placed in a development bath containing a developing solvent (butanol, acetic acid, and water 4: 1: 1 / v / v / v) Dried, immersed in a 1% ninhydrin coloring solution, and dried at 100 ° C for about 10 seconds to confirm the presence or absence of the production of lactic acid bacteria. Thus, the final 10 kinds of lactic acid bacteria having excellent GABA production were firstly selected and each WCP02 , LAB28, LAB40, LAB48, LAB62, LAB112, LAB224, BMK41, BMK184, BMK484 (Fig. The control strain L. brevis KCTC3320 was cultivated in the same manner and developed on TLC (FIG. 1).

Example 2: Gabor conversion rate test

For the 10 selected strains of lactic acid bacteria and the strains isolated from the above, the conversion of GABA to MSG was investigated.

A single colony on the MRS solid medium was inoculated to the MRS liquid medium supplemented with 0.05% (w / v) and 0.1% (w / v) MSG and incubated at 30 ° C for 2 days. (GA) and GABA content and conversion rate were calculated by free amino acid pretreatment by culturing 10 kinds of lactic acid bacteria and the culture of the isolates at 2.5% (v / v) respectively and culturing at 30 ° C for 60 hours And the results are shown in Table 1. As a reference, non-inoculated culture medium (NIM) was cultured in the same manner and measured in the same manner, and the results are shown in Table 1:

(%) = [Glutamic acid ÷ (Glutamic acid + Gabar)] × 100

Free amino acid analysis for the determination of glutamic acid and guar gum content was carried out using an automatic amino acid analyzer (Hitachi, L-8900, Japan). Specifically, 1 ml of the sample was precisely weighed in a test tube, and 4 ml of distilled water was added, followed by hydrolysis at 60 ° C for 1 hour. Then, 1 ml of 10% 5-sulfosalicylic acid was added, and the mixture was allowed to stand at 4 ° C for 2 hours to precipitate proteins. The filtrate was filtered through a glass filter, and the filtrate was concentrated under reduced pressure at 60 ° C to completely evaporate the water. The concentrated sample was dissolved in 2 ml of lithium citrate buffer (pH 2.2), filtered through a 0.45 μm membrane filter (Dismic-25CS, Toyoroshikaisha Ltd), and analyzed by an automatic amino acid analyzer.

Strain Content (mg / 100 ml) / conversion rate (%) 0.05% MSG 0.1% MSG GA GABA Conversion Rate
(%) GA GABA Conversion Rate
(%)  NIM 844.58 42.86 - 1182.30 35.30 - KCTC3320 66.65 581.43 89.72 39.27 560.12 93.45 WCP02 43.72 629.79 93.51 29.58 572.23 95.08  LAB28 236.62 479.56 66.96 192.33 474.53 71.16  LAB40 97.17 586.30 85.78 52.91 602.76 91.93  LAB48 81.08 588.88 87.90 48.66 531.00 91.61  LAB62 98.73 577.21 85.39 129.48 596.46 82.16  LAB112 107.39 609.92 85.03 80.61 573.88 87.68  LAB224 101.58 578.41 85.06 48.74 613.36 92.64  BMK41 120.97 576.27 82.65 124.77 583.66 82.39  BMK184 131.69 558.92 80.93 132.68 512.82 79.44  BMK484 78.65 530.70 87.09 83.03 597.54 87.80 GA, glutamic acid, GABA, gamma -aminobutyric acid
NIM: Not vaccinated

As shown in Table 1, the conversion rate of glutamic acid (GA) to GABA was the highest at 93.51% and 95.06% at the concentration of MSG 0.05% (w / v) and 0.1% (w / v) , Even higher than the conversion rates of the disclosure strain KCTC3320 (89.72%, 93.45%, respectively).

Example  3. Strain Probiotics  black

The probiotics test of the first 10 selected strains of lactic acid bacteria was carried out by measuring gastric acid tolerance and bile acid tolerance.

<Measurement of gastric acid tolerance>

 After addition of 1% pepsin in the MRS liquid medium with pH (2.0, 3.0) adjusted, artificial gastric juice acid was prepared, and then 10 kinds of lactic acid bacteria and the culture broth of the first selection were inoculated at 5% Lt; 0 &gt; C for 4 hours. The viable cell counts were confirmed by the stepwise dilution method in the MRS solid medium immediately after culturing and at 2 hours and 4 hours after the culture, respectively. The survival rate was the number of viable cells at the initial stage of culture and the number of colonies formed on the MRS solid medium at 2 hours and 4 hours The survival rate (%) was calculated and shown in Table 2.

 Survival rate (%) = (number of initial live bacteria ÷ number of living cells after cultivation) × 100

Strain Survival rate (%) / incubation time (h) pH 2.0 pH 3.0 2 4 2 4 KCTC 3320 15.27 nd 94.05 94.86 WCP02 44.43 39.64 98.86 102.24 LAB 28 42.52 38.27 94.74 96.65 LAB 40 36.59 32.54 95.65 92.77 LAB 48 nd nd 91.64 88.62 LAB 62 18.30 11.42 94.14 90.56 LAB 112 nd nd 96.91 91.46 LAB 224 nd nd 97.52 95.32 BMK 41 nd nd 105.65 94.29 BMK 184 nd nd 101.32 85.42 BMK 484 24.30 nd 93.91 105.19 nd = not detected

As shown in Table 2, in the case of the test strain, it died at 4 hours in the gastric juice of pH 2.0, but the survival rate of WCP02 was the highest at 39.64%, and WCP02 showed the highest survival rate at pH 3.0 The survival rate of the stomach acid was 102.24% at 4 hours after culture and was higher than that of the published strain (94.86%).

<Bile acid resistance>

The bile acids were prepared by adding 1% pancreatin and bile salts to the pH-adjusted MRS liquid medium, and 10 kinds of the first lactic acid bacteria and the culture broth were inoculated at 5% After 48 hours of incubation at 37 ° C, viable cell counts formed on the MRS solid medium were calculated as% compared with the number of viable cells in the initial stage of culture.

Survival rate (%) = [number of viable cells per day ÷ number of viable cells after culture] × 100

Strain Survival rate (%) pH 2.5 pH 3.0 KCTC3320 49.58 79.59 WCP02 51.26 86.25 LAB28 38.98 74.54 LAB40 42.10 61.22 LAB48 nd 60.09 LAB62 nd 59.25 LAB112 nd 49.65 LAB224 nd 66.66 BMK41 nd nd BMK184 nd nd BMK484 nd nd nd = not detected

As shown in Table 3, the WCP02 strain showed the highest resistance to bile acid resistance at pH 2.5 and pH 3.0, respectively, and was higher than that of the published strain (49.58%, 79.59%).

Example  4. Lactobacillus Brevis WCP02  Identification of strain

In the above examples, WCP02 strains having excellent GABA production, gastric acid tolerance, and bile acid resistance were selected and their morphological, biochemical, folate composition and molecular genetic characteristics were identified.

<Morphological Characteristics>

The morphological characteristics of the WCP02 strain were confirmed by single-colony formation and Gram staining using an optical microscope on a MRS solid medium by a quadruple method, and the result is shown in FIG.

As shown in Fig. 2, the cell form had a white oval shape (Fig. 2 (a)), and as a result of Gram staining, it appeared to be a gram-positive bacterium with a dark blue and violet system color in the form of a bacterium ).

<Biochemical Properties>

Biochemical characterization was carried out by assaying the growth of WCP02 strain and controlling the sugar availability by adjusting the culture temperature, pH and NaCl concentration.

Specifically, the pH was adjusted to 3, 6, 9, 11 by using HCl and NaOH in the MRS liquid medium, and 2.5% of the WCP02 strain was inoculated and cultured at 37 ° C. for 48 hours. The color of the culture was visually confirmed And the sensitivity to pH was measured by confirming the remaining bacteria by the flat plate smear method.

NaCl was added to the MRS broth by 0, 2, 4, 8, and 10%, followed by 2.5% inoculation of WCP02 culture medium at 37 ° C, followed by plate culture.

In order to confirm whether the growth was dependent on the incubation temperature, the number of viable cells was measured by flat plate coating method after 2.5% inoculation of WCP02 culture medium in MRS liquid medium and incubation at 10, 20, 30, 40, 50 ℃ for 48 hours.

Glucose availability was measured by using API kit (For Growth Promotion Tests, BB coporation, Korea).

As a result, the growth characteristics of WCP02 strain were poor at 10 ℃ and 50 ℃, but growth was possible at temperatures between 20 ℃ and 40 ℃, and salt tolerance was up to 6%. Growth activity was weak at pH 3.0, but increased at pH 5, 7, 9 and 11. Glucose availability can be assessed using L-arabinose, ribose, D-xylose, galactose, D-fructose, D-glucose, alpha-methyl-D-glucoside, esculine, maltose, melibos, And the remaining parties were not available (Fig. 7).

&Lt; Analysis of fatty acid composition of fungi &

For the analysis of the fatty acid composition, 1 ml of WCP02 culture broth was placed in a test tube, 4 ml of 0.5 N methanolic NaOH was added, and the mixture was heated for about 10 minutes using a heating block at 100 ° C to hydrolyze the fatty acid and glycerol. Then, 2 ml of boron trifluoride (BF ₃ ) was added and stirred, and the mixture was heated again for 30 minutes to proceed the methyl esterification of the fatty acid. After completion of the reaction, 1 ml of isooctane was added, and the mixture was vigorously shaken. Then, centrifugation and only the isooctane layer were collected, dehydrated with anhydrous sodium sulfate, and then filtered through a 0.45 μm membrane filter (Dismic-25CS) to obtain GC and phenylmethyl silicone fused silica capillary Fatty acids were detected using a column (2.5 mm, Hewlett Packard) and finally analyzed using a MIDI program.

In the saturated fatty acids, palmitoic acid (C16: 0) was the most abundant with 32.37% and 11,12-mithylene hexadecanoic acid (C19: 0CYCLO) was 17.16% Respectively. In unsaturated fatty acids, oleic acid w 9 c ( C 18: 1) accounted for 4.14%, and unsaturated fatty acids accounted for 0.26-0.95% (Fig. 8).

<Molecular Genetic Characterization: 16S rRNA Sequence Analysis>

Molecular genetic identification of WCP02 strain was carried out by shaking culture in MRS liquid medium for 2 days, and genomic DNA was isolated using Intron Genomic DNA purification kit (Intron Botechnology Co.) and 16S rDNA base sequence was determined by PCR amplification with this template.

The PCR reaction was denatured at 95 ° C for 5 minutes, DNA annealed at 49 ° C for 30 seconds, and elongated at 72 ° C for 1 minute. This reaction was repeated 30 times. The primer production was made to be about 1,500 bp in 16S rDNA preservation area of bacteria. The forward primer used was 5'-CGGAGAGTTTGATCCTGG-3 ', and the reverse primer used was 5'-TACGGCTACCTTACGAC-3'. After completion of the PCR reaction, the 16S rRNA fragment was confirmed by electrophoresis and DNA was separated from the agarose gel according to the method described in the Total Fragment DNA Purification Kit (Intron Biotechnology). The nucleotide sequence of the purified 16S rRNA fragment was determined as a template. The nucleotide sequence was determined by the dideoxy chain termination method using a PRISM Ready Reaction Dye terminator / primer cycle sequencing kit (Perkin-Elmer Corp., Norwalk, Conn., USA), resulting in a 1,520 bp base sequence ).

The determined nucleotide sequence was compared with 16s rDNA of another bacterium. The similarity value was calculated from the alignment and evolution distance using a DNAMAN analysis system (Lynnon Biosoft), and a phylogenetic tree was created. 4.

As shown in Fig. 4, Lac . 99% similarity to brevis 100D8 (CP015338) strain.

The WCP02 strain was designated as Lactobacillus brevis WCP02 in consideration of the morphological, biochemical, fats composition and molecular genetic characteristics. The strain was named as WCP02 by the National Institute of Agricultural Science and Technology (KACC) on Dec. 12, 2016 And deposited the accession number KACC 92159P as a patented strain.

Example  5. Lactobacillus Brevis WCP02  Antioxidant activity of strains

The antioxidant activity of Lactobacillus brevis WCP02 was determined by DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2'-azino-bis-3-ethylbenzthiazoline- The scavenging activity of the radical was measured and assayed.

<Sample Preparation>

The Lactobacillus brevis WCP02 strain and the control strain were each inoculated 2.5% in MRS liquid medium (50 ml) and then primary cultured at 30 ° C for 48 hours. Subsequently, a second subculture was performed in a new MRS liquid medium (500 ml × 4) After conducting the centrifugation, the supernatant was discarded and the lactic acid bacterium cells were recovered. Then, the cells were washed three times with sterilized distilled water and lyophilized to finally obtain lyophilized powders of pure lactic acid bacterium cells.

The lyophilized microbial cells were dissolved in 10 ml of 50% methanol, extracted for 12 hours, filtered through a 0.45 μm filter, concentrated, and then prepared at a concentration of 1 mg / ml.

&Lt; DPPH radical scavenging activity >

The DPPH radical scavenging activity was determined by adding 0.8 ml of DPPH solution (1.5 ^-4 M) to 0.2 ml of each of the samples prepared above, allowing to stand for 30 minutes while uniformly mixing and measuring the absorbance at 525 nm. The negative control of the DPPH radical scavenging activity proceeded in the same manner using distilled water instead of the sample, and the difference in absorbance was calculated as a percentage (%) by the following equation, and the result is shown in FIG.

Radical scavenging activity (%) = [1- (negative control absorbance / experiment absorbance)] × 100

As shown in FIG. 5 (a), it was confirmed that the DPPH radical scavenging activity of the WCP02 strain of the present invention was significantly higher than that of the published strain.

<ABTS radical scavenging activity>

The ABTS radical scavenging activity was obtained by mixing 5 ml of 7 mM ABTS reagent and 5 ml of 140 mM K ₂ S ₂ O ₈ (FW 270.3, Sigma 9392) in a dark place for 14 to 16 hours to produce cation radicals. The ABTS solution adjusted to an absorbance value of 0.7 was used. 0.1 ml of each sample and 0.9 ml of ABTS solution were mixed, reacted for 3 minutes, and absorbance was measured at 732 nm. The ABTS radical inhibitory activity was also measured in the same manner using distilled water instead of the sample as the negative control, and the difference in absorbance was calculated as a percentage (%) by the above formula. The results are shown in FIG.

As shown in Fig. 5 (b), it was confirmed that the ABTS radical scavenging activity of the WCP02 strain of the present invention was as high as that of the published strain.

<Hydroxyl radical scavenging activity>

Hydroxyl radical scavenging activity was measured 10mM FeSO _{4. 7H 2 0-EDTA 0.2ml, 10mM} 2- deoxyribose After reacting for 4 hours at _{0.2ml, 10mM H 2 O 2 0.2ml} , 1.4ml extract was mixed after 37 ℃ made of a mixture of 1% in this mixed solution, thio 1 ml of each of barbitic acid (in DW) and 2.8% trichloroacetic acid (in DW) was added thereto, followed by color development at 100 ° C for 20 minutes. After cooling, the absorbance was measured at 520 nm. As a negative control, PBS (8.76 g of 1 L NaCl, 0.11 g of NaH ₂ PO ₄ , 0.596 g of Na ₂ HPO ₄ ) was used instead of the sample. The hydroxyl radical scavenging activity was calculated by the above equation using the above equation, and the results are shown in FIG. 5, where the difference in absorbance between the addition of the sample solution and the no addition was calculated.

As shown in Fig. 5C, it is confirmed that the DPPH radical scavenging activity of the WCP02 strain of the present invention is higher than that of the published strain.

From these results, it can be seen that the probiotics containing Lactobacillus brevis WCP02 strain and the culture thereof have an enhanced antioxidative activity.

Example  6. Lactobacillus Brevis WCP02  Effectiveness of diabetes and obesity improvement of strain

The antidiabetic effect of the Lactobacillus brevis WCP02 strain was evaluated by measuring the alpha-glucosidase inhibitory activity and the anti-obesity effect was evaluated by measuring the pancreatic-lipase inhibitory activity.

&Lt; Alpha-Glucosidase Inhibitory Activity >

50 μl of 0.5 U / ml α-glucosidease enzyme solution was added to 50 μl of each sample prepared in Example 5, and 50 μl of 200 mM sodium phosphate buffer (pH 6.8) was mixed and preliminarily cultured at 37 ° C. for 10 minutes And sodium phosphate buffer (pH 6.8) were added, and the mixture was reacted at 37 占 폚 for 10 minutes. The reaction was stopped by adding 0.75 ml of 100 mM Na ₂ CO ₃ to the reaction solution, and the absorbance at 420 nm was measured. Negative control was carried out in the same manner using distilled water instead of the sample, and the difference in absorbance was expressed as a percentage (% ), And the results are shown in Fig.

Alpha-glucosidase inhibitory activity (%) =

 [1- (negative control absorbance / experimental absorbance)] × 100

As shown in Fig. 6 (a), it was confirmed that the alpha-glucosidase inhibitory activity of the WCP02 strain of the present invention is higher than that of the published strain.

<Pancreatic Lipase Inhibitory Activity>

50 占 퐇 of each sample prepared in Example 5, 50 占 퐇 of 1.0 U / ml pancreatic lipase enzyme solution and 50 占 퐇 of 200 mM sodium phosphate buffer (pH 6.8) were preliminarily cultured at 37 占 폚 for 10 minutes and then diluted with sodium phosphate buffer 6.8) was added and reacted at 37 占 폚 for 10 minutes. The reaction was stopped by adding 0.75 ml of 100 mM Na ₂ CO ₃ to the reaction solution, and the absorbance at 420 nm was measured. Negative control was carried out in the same manner using distilled water, and the difference in absorbance was expressed as a percentage (%) by the following equation: The results are shown in FIG.

Pancreatic-lipase inhibitory activity (%) =

[1 - (negative control absorbance / experimental absorbance)] × 100

As shown in Fig. 6 (b), it was confirmed that the pancreatic-lipase inhibitory activity of the WCP02 strain of the present invention was significantly enhanced as compared with the published strain.

From the above results, it was found that the probiotics containing the Lactobacillus brevis WCP02 strain and the culture thereof had an antidiabetic and anti-obesity effect by imparting enhanced alpha-glucosidase inhibitory activity and pancreatic-lipase inhibitory activity have.

Example  7. Lactobacillus Brevis WCP02  Manufacture of Used Soy Milk-Yogurt

<Preparation of soybean powder>

Raw soybeans (product name: Woram bean) Water was added so that it was about twice as much as the weight of the raw soybeans, and the water was soaked for 12 hours at room temperature. The water was then removed from the wafers and the rice was appropriately stacked in a soybean sprout grower (model HANCELL, hi green, Korea) Germination was carried out so that the germinated body was twice as much as the raw bean grains while supplying water. The germinated soybeans were treated at 121 ° C for 30 minutes, dried at 55 ° C for 2 to 3 days with hot air, and pulverized to prepare soybean powder.

10 g of soybean powder and 100 ml of purified water were added to a 250 ml Erlenmeyer flask, which was then stirred and sterilized at 121 ° C for 15 minutes to prepare soybean powder soybean milk. Soybean - powder soy milk was cooled at room temperature (25 ℃), and Lactobacillus brevis WCP02 medium was inoculated 2.5% and fermented for 72 hours to produce soybean milk - yogurt.

<Physicochemical properties>

Soybean milk powder and soy milk yogurt were measured by pH meter. Total acidity was measured by neutralization titration. The amount of 0.1 N NaOH consumed in neutralizing 1 ml of sample to pH 8.2 was calculated and converted into the amount of lactic acid. The number of viable cells was measured by dilution with sterilized water, followed by plating on MRS plate medium, followed by incubation at 30 ° C. for 48 hours. The results are shown in Table 4.

Physicochemical properties pH Acidity (%) Viable cell count
(log cfu / ml) Soybean - powdered soy milk 6.49 0.32 nd Soy milk - yogurt 5.57 0.45 11.01 nd: not detected

As shown in Table 4, soybean milk-yogurt was suitable as a fermented food having a pH of 5.57, an acidity of 0.45%, and a viable cell count of about 10 log cfu / ml. In general, yogurt has a pH of 5.0 or less and an acidity of 0.5% or more. The sweetness of the yogurt is stronger than that of the yoghurt (eg, sugar) Sour taste, sweet taste, and the like can be obtained.

<Sample Preparation>

Soybean-powdered soybean milk and soybean milk-yogurt prepared above were lyophilized and powdered. To 1 g of each lyophilized powder was added 10 times 50% methanol and extracted at room temperature for 12 hours. The extract was centrifuged for 30 minutes, and the supernatant was filtered with a 0.45 μm membrane filter (Dismic-25CS) to measure total phenolics and isoflavone content and physiological activity.

 <Active ingredient content test: Gabba, total phenolics and isoflavone derivatives>

 The contents of gabas, total phenolics and isoflavone derivatives as active ingredients were analyzed for soy milk - yogurt. For comparison, active ingredient analysis was also performed on soybean soybean milk before fermentation.

In the same manner as in Example 2, glutamic acid and GABA content were measured by free amino acid analysis. The total phenolic content was determined by the Folin Denis method (1952). Specifically, 0.5 ml of the sample is dispensed into a test tube, and 25% Na ₂ CO ₃ 0.5 ml was added and allowed to stand for 3 minutes. After addition of 0.25 ml of 2 N Folin-Ciocalteu phenol reagent, the mixture was incubated at 30 ° C for 1 hour to develop color. The color developed blue was measured at 750 nm using a spectrophotometer (Spectronic 2D). The content of total phenolics was determined from the standard curves prepared using gallic acid. The isoflavones were analyzed by HPLC. The mobile phase solvent was 0.2% glacial acetic acid (in water) (solution A) and 0.2% acetonitrile (in glacial acetic acid) (solution B) using a Lichrophore 100 RP C18 column (4.6 mm, Respectively. The mobile phase conditions were maintained at 0 min -100%, 15 min-90%, 25 min-80%, 35 min-75%, 45 min-65% and 50 min-65% on the A solvent basis. 20 μl of sample was injected and the mobile phase velocity was kept at 1 ml / min at 30 ° C. The detector was detected at 254 nm using a diode array detector (DAD). All analytical results are shown in Table 5

Analysis item sample Soybean powder soy milk Soy milk - yogurt GA & GABA (mg / 100 g d.w.)  Glutamic acid (GA) 175.59 11.04  Gamma-aminobutyric acid (GABA) 57.86 135.89 Total phenolic content (mg / g d.w.) 0.42 0.66 Isoflavone ([mu] g / g d.w.)  Glycosides (glycoside isoflavones) 1290.93 59.49   Daidzin 485.06 25.06   Glycitin 198.29 21.23   Genistin 607.58 13.20  Aglycones (non-glycoside isoflavones) 59.26 487.29   Daidzein 26.85 236.21   Glycitein 16.19 57.92   Genistein 16.22 193.16

As shown in Table 5, the content of soybean milk-yogurt, total phenolics and non-glycoside isoflavones was 135.89 mg / 100 g, 0.66 mg / g and 487.29 μg / g, respectively, All were greatly improved.

<Physiological activity test: antioxidant activity, antidiabetic activity and anti-obesity effect>

Antioxidant activity, antidiabetic effect and anti - obesity effect of soybean milk - yogurt were tested. For comparison, physiological activity assays were also performed on soybean soybean milk before fermentation.

Antioxidant activity was measured in the same manner as in Example 5, antidiabetic effect and anti-obesity effect were measured in the same manner as in Example 6, and the results are shown in Table 6.

Analysis item sample Soybean - powdered soy milk Soy milk - yogurt Radical scavenging activity (%)   DPPH 47.25 63.65   ABTS 53.80 98.82   Hydroxyl 15.83 62.52 Enzyme inhibitory activity (%)   alpha -glucosidase 6.49 55.11   Pancreatic lipase 15.02 41.12

As shown in Table 6, soybean milk-yogurt according to the present invention had higher DPPH, ABTS and hydroxyl radical scavenging activity than soybean-soybean soybean milk before fermentation, thereby enhancing antioxidative activity. In addition, the alpha-glucosidase inhibitory activity was increased about 10-fold and the pancreatic-lipase inhibitory activity was enhanced about 3-fold.

From the above results, it can be confirmed that the soybean milk-yogurt fermented with the strain of the present invention has abundant and enhanced antioxidant activity, anti-diabetic activity and anti-obesity effect.

Example  8. Lactobacillus Brevis WCP02  Used Soybean fermentation  Seasoning manufacture

Soybean fermented seasoning was prepared using the strain of the present invention.

<Preparation of soybean hydrolyzate>

The beans were soaked in water for 12 hours, and then the water was removed and the mixture was incubated at 121 ° C for 1 hour. Bacillus subtilis 2TDJ15) culture medium was inoculated at 5%, fermented at 37 ° C for 3 days, dried at 55 ° C for two days, and pulverized by a pulverizer to prepare bean meju powder. To 400 g of meju powder, 1,600 ml of 6% brine was added to hydrate, and 100 ml (5%, v / v) of kiwifruit juice was added. The mixture was reacted at 40 ° C for 24 hours and sterilized at 120 ° C for 15 minutes to obtain soybean hydrolyzate And prepared.

<Preparation of soybean fermented seasoning>

The prepared soybean hydrolyzate was inoculated with 5% (v / v) of the WCP02 culture medium of the present invention and fermented at 25 캜 for 30 days to prepare a soybean fermented seasoning.

<Physicochemical properties>

The physicochemical properties of soybean fermented seasoning were measured before and after fermentation in the same manner as in Example 6. Salinity was measured with a salinity meter, and protein quantification was measured with a Biuret method. The results are shown in Table 7.

pH Acidity
(%) Salinity
(%) protein
(mg / ml) Soybean hydrolyzate (before fermentation) 6.11 0.87 6.11 29.09 Soybean fermented seasoning (after fermentation) 5.34 1.12 6.30 27.35

As shown in Table 7, the soybean fermented seasoning was suitable as a fermented food with a pH of 5.34, an acidity of 1.12, a salt content of 6.3% and a protein content of 27.35 mg / ml. The seasoning of the present invention contains appropriate acidity and salinity through fermentation and can be used as a natural seasoning applicable to various dishes.

<Sample Preparation>

The same amount (50 ml) of 100% methanol was added to 50 ml of each of the soybean hydrolyzate (before fermentation) and soybean fermentation seasoning prepared above, and the mixture was extracted at room temperature for 12 hours. The extract was centrifuged for 30 minutes, and the supernatant was filtered with a 0.45 μm membrane filter (Dismic-25CS) to measure total phenolics and isoflavone content and physiological activity.

<Active ingredient content test: Gabba, total phenolics and isoflavone derivatives>

In the same manner as in Example 7, the soybean fermented seasoning was analyzed for the content of the active ingredient, isoflavone derivative, and for comparison, the soybean hydrolyzate before fermentation was also analyzed for active ingredients.

Analysis item Soybean hydrolyzate Soybean fermented seasoning GA & GABA (mg / 100 ml)  Glutamic acid (GA) 1,709.39 422.01  Gamma-aminobutyric acid (GABA) 31.57 1,365.24

As shown in Table 8, the crude content of the soybean fermented seaweed was 1,365.24 mg / 100 ml, which was significantly increased by about 43 times as compared with the soybean hydrolyzate before fermentation.

<Physiological Activity Test: Antioxidant, Antidiabetic and Anti-obesity Effects>

Antioxidant activity, antidiabetic effect and antiobesity effect of soybean fermented seasoning were tested. For comparison, physiological activity assays were also performed on soybean hydrolyzate before fermentation.

Antioxidant activity was measured in the same manner as in Example 5, antidiabetic effect and anti-obesity effect were measured in the same manner as in Example 6, and the results are shown in Table 9.

Analysis item sample Soybean hydrolyzate Soybean fermented seasoning Radical scavenging activity (%)   DPPH 79.66 92.61   ABTS 61.93 77.04   Hydroxyl 54.32 65.38 Enzyme inhibitory activity (%)  alpha -glucosidase 14.95 25.31  Pancreatic lipase 15.41 23.77

As shown in Table 9, the soybean fermented seasoning according to the present invention had higher DPPH, ABTS and hydroxyl radical scavenging activity than the soybean hydrolyzate before fermentation, and thus the antioxidative activity was enhanced, and the alpha-glucosidase inhibitory activity and the pancreatic- The lipase inhibitory activity was increased by 50% or more.

From the above results, it can be seen that the soybean fermented seasoning fermented with the strain of the present invention has an enhanced antioxidant activity, antidiabetic activity and anti-obesity effect.

Agricultural Biotechnology Research Institute KACC92159P 20161212

<110> Gyeongnam National University of Science and Technology Industry-Academic Cooperation Foundation <120> Lactobacillus brevis WCP02 strain having high gastric acid          tolerance and even acid, and high GABA productivity, and          probiotics and fermented food by using the same <130> p10188 <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 1520 <212> RNA <213> Lactobacillus brevis <220> <221> rRNA &Lt; 222 > (1) <223> 16s rRNA <400> 1 cggagagttt gatcctggct cttgacgaac gctggcggca tgcctaatac atgcaagtcg 60 aacgagcttc cgttgaatga cgtgcttgca ctgatttcaa caatgaagcg agtggcgaac 120 tggtgagtaa cacgtgggga atctgcccag aagcagggga taacacttgg aaacaggtgc 180 taataccgta taacaacaaa atccgcatgg attttgtttg aaaggtggct tcggctatca 240 cttctggatg atcccgcggc gtattagtta gttggtgagg taaaggccca ccaagacgat 300 gatacgtagc cgacctgaga gggtaatcgg ccacattggg actgagacac ggcccaaact 360 cctacgggag gcagcagtag ggaatcttcc acaatggacg aaagtctgat ggagcaatgc 420 cgcgtgagtg aagaagggtt tcggctcgta aaactctgtt gttaaagaag agcacctttg 480 agagtaactg ttcaagggtt gacggtattt aaccagaaag ccacggctaa ctacgtgcca 540 gcagccgcgg taatacgtag gtggcaagcg ttgtccggat ttattgggcg taaagcgagc 600 gcaggcggtt ttttaagtct gatgtgaaag ccttcggctt aaccggagaa gtgcatcgga 660 aactgggaga cttgagtgca gaagaggaca gtggaactcc atgtgttgcg gtggaatgcg 720 tagatatatg gaagaacacc agtggcgaag gcggctgtct agtctgtaac tgacgctgag 780 gctcgaaagc atgggtagcg aacaggatta gataccctgg tagtccatgc cgtaaacgat 840 gagtgctaag tgttggaggg tttccgccct tcagtgctgc agctaacgca ttaagcactc 900 cgcctggggg agtacgaccg caaggttgaa actcaaagga attgacgggg gcccgcacaa 960 gcggtggagc atgtggttta attcgaagct acgcgaagaa ccttaccagg tcttgacatc 1020 ttctgccaat cttagagata agacgttccc ttcggggaca gaatgacagg tggtgcatgg 1080 ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttat 1140 tatcagttgc cagcattcag ttgggcactc tggtgagact gccggtgaca aaccggagga 1200 cgtgctacaa tggacggtac aacgagtcgc aaagtcgtga ggctaagcta atctcttaaa gccgttctca 1320 gttcggattg taggctgcaa ctcgcctaca tgaagttgga atcgctagta atcgcggatc 1380 agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgagag 1440 tttgtaacac ccaaagccgg tgagataacc ttcgggagtc agccgtctaa ggtgggacag 1500 atgattaggg tgaagtcgta 1520 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 cggagagttt gatcctgg 18 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 tacggctacc ttacgac 17

Claims (9)

Lactobacillus brevis WCP02 strain which is excellent in gastric acid tolerance and bile acid resistance, excellent in GABA productivity, and deposited under Accession No. KACC92159P.
A culture of Lactobacillus brevis WCP02 strain according to claim 1.
The method according to claim 1, wherein the gastric acid tolerance exhibits a survival rate of 39% or more after culturing in gastric acid at pH 2 for 4 hours, the survival rate of bile acid is 86% or more after culturing in bile acid at pH 3.0 for 48 hours, Lactobacillus brevis WCP02 strain characterized in that it exhibits a 95% or higher Gabar conversion at a concentration of monosodium glutamic acid (MSG) of% (w / v).
A prophylactic agent comprising the strain of claim 1 or the culture of claim 2 as an active ingredient.
The prophylactic or therapeutic agent according to claim 4, wherein the probiotics are provided in a form of dried cells or any one selected from the group consisting of fermented foods, beverages, and kimchi.
A fermented soybean fermented product obtained by fermenting with the Lactobacillus brevis WCP02 strain according to claim 1 and having an improved antioxidative activity, phenolic compound and non-glycosylated isoflavone content and enhanced alpha-glucosidase inhibitory activity and pancreatic-lipase inhibitory activity .
The soybean fermented food according to claim 6, wherein the soybean fermented food is soy milk-yogurt or soybean fermented seasoning.
A soybean milk yogurt produced by inoculating a germinated soybean milk with the Lactobacillus brevis strain WCP02 according to claim 1,
Wherein the soy milk-yogurt contains enhanced gabas, phenolics and non-glycoside isoflavones and has enhanced antioxidant activity, enhanced anti-diabetic activity and anti-obesity activity.
A soybean fermented seasoning prepared by fermenting soybean hydrolyzate with Lactobacillus brevis WCP02 according to claim 1,
Wherein the soybean fermented seasoning has an enhanced content of goverberry.

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