KR100978415B1 - Leuconostoc pseudomesenteroides ????? having high activity of ??galactosidase and ??glucosidase and Isoflavon concentrates using thereof - Google Patents

Abstract

The present invention relates to Leuconostoc pseudomesenteroides K-AP3 ( Leuconostoc pseudomesenteroides K-AP3, Accession No. KFCC 11409P). Α-galactosidase, which breaks down the glycoside-formed isoflavones into non-glycosides, and breaks down the indigestible oligosaccharides of stachyose and raffinose into monosaccharides. As a strain having a very high activity, it is excellent in useful value as a raw material of health functional food or medicine using the present invention.

Leukonostock Pseudomecentroides, α-galactosidase, β-glucosidase

Description

Leuconostoke pseudometh centroides VII-AP3 having high activity of α-galactosidase and β-glucosidase and isoflavone concentrates using the same.Leuconostoc pseudomesenteroides VII-AP3 having high activity of α-galactosidase and β-glucosidase and Isoflavon concentrates using pretty}

The present invention relates to Leuconostoc pseudomesenteroides K-AP3 (Accession No. KFCC 11409P) having high α-galactosidase and β-glucosidase activity. And by converting the functional ingredients of soybean-derived food material into a form of high availability in the body can be used in the manufacture of health functional foods and pharmaceuticals using the same.

Soybean is a high protein and fat-rich agricultural product as well as one of the world's most important edible fats and oils, as well as one of food or processed food raw materials. Soy, a nutritious food, is an important source of protein and fat that is likely to be lacking in a vegetarian diet, making it an essential food for our diet. Korea has developed a unique dietary culture by developing various soybean fermented foods suitable for our constitution with the wisdom and experience of our ancestors. Representatives include doenjang, soy sauce, and cheonggukjang, and non-fermented foods include tofu and soy milk.

In recent years, interest in bioactive substances in soybeans has been rapidly increasing, and researches on the functionalities of soybean foods have been actively conducted internationally. Therefore, interest in soybean processed products and soybean fermented products by westerners in non-soybean cultures is increasing. The trend is increasing.

Soybeans contain bioactive substances such as isoflavones, dietary fibers, oligosaccharides, lecithin, saponins, vegetable sterols and phenolic compounds. Soy isoflavones are called phytoestrogen because they have a structure and function similar to that of the female hormone estrogen. In addition, isoflavones have been reported to lower blood cholesterol and prevent osteoporosis in postmenopausal women, as well as physiological activities of isoflavones such as cardiovascular disease inhibition and anticancer effects by antioxidant activity.

Diisozin, genistin and their aglycones, the main isoflavones in soybean, were first isolated by Walter and Naim et al., Glycyite. Then, Ohta and Kudou et al. Identified and identified acetyl derivatives and malonyl derivatives. Isoflavones were divided into three aglycones, genistein, daidzein, and glycidin, respectively. There are three kinds of glycosides (glycosides, malonylglucosides, acetylglucosides), and a total of 12 kinds of isoflavone compounds have been reported. In general, soy isoflavones contain about 1.5% to 2.5% of dry weight. Among them, isoflavone glycosides (dyzein and genistin) account for the largest portion (60-70%). Digestin and genistein content of various non-glycoside forms of physiological activity is known to be only about 0.01 ~ 0.15%.

Among the reported studies on the bioactive function of genistein, a non-glycoside of soybean, Wang . Et al. Reported that genistein inhibits the binding of 17β-estradiol to the estrogen receptor of breast cancer cells and decreases the estrogen receptor of breast cancer cells. Lamartiniere . Et al. Reported that breast cancer induced with dimethylbenz-anthracene (DMBA) was effectively inhibited by genistein, particularly that pre-pubertal administration of genistein was effective in preventing breast cancer. Prostate cancer is also associated with dihydro-testosterone (DHT) concentrations, Evans . Et al. Reported that genistein had an inhibitory effect of 5-α-reductase, an enzyme that converts testosterone to DHT. In addition, genistein has been reported to have an anticancer effect on bladder cancer, stomach cancer, lung cancer, liver cancer, and the like.

Antioxidative substances contained in soybeans include tocopherols, phenolic acids, and isoflavones. Tocopherols decrease as fermentation progresses, while isoflavones in the glycoside form are not affected by the action of β-glucosidase. It is known to increase glycosides and increase its antioxidant power. In addition, the antioxidant effect of isoflavone is known to protect against intracellular oxidative stress in vitro, and plasma nitrite concentration and red blood cell catalase and glutathione in vitro are known. It is known to increase glutathione peroxidase activity. As such, ingestion of isoflavones in the form of nonglycosides affects vascular disease related factors and the antioxidant enzyme system of red blood cells and liver.

Alternative therapies for the prevention and treatment of osteoporosis by reducing the secretion of estrogen, a female hormone after menopause, include oral administration of phytoestrogen or the consumption of foods containing it. In addition to binding to the estrogen receptor (ER) in the cell has an estrogen effect and does not cause side effects, the content of diystein and genistein of medicinal soybean extract is higher than that of soybean, and the growth of MG-63 osteoblasts It is known to selectively express IGF-I, a growth-related factor.

Ingestion of xenstein, the non-glycoside of isoflavones, and ingestion of the genistein, the glycoside, resulted in higher body recovery in the urine of genistein, which means that the bioavailability of isoflavone when glycosides are converted to nonglycoside It indicates an increase.

Malonyl glycoside-formed isoflavones, which accounted for about 60% of the isoflavone content using β-glucosidase, decreased to 3% and nonglycoside-formed isoflavones increased from 3% to 56%. In some cases, the use of these fortified food ingredients has been reported to be possible.

Β-D-glucoside glucohydrolase (EC 3.2.1.21), which is distributed in plants, fungi, yeasts, bacteria and animal tissues, is a cellulase compound. Endo-glucanase is cleaved, followed by cellobiohydrase and finally β-glucosidase, which is known to produce glucose, the end product.

Let us study the β- glucoside of microbial origin is the Lactobacillus casei sub Spanish ramno suspension system (Lactobacillus casei subsp. Rhamnosus), bifidobacteria spp bacteria (Bifidobacterium sp.), Spokane roteuri kyum pilreom Cellulofine (Sporotrichum cellulophilum), Bacillus subtilis , Aspergillus niger , Aspergillus nidulans , Trichoderma reesei , Thermomonospora fusca , Penicillium Penicillium verruculosum , Hermicola Grzey Bar. Β-glucosidase produced by Humicola grisea var. Thermoidea is reported to be involved in the hydrolysis of isoflavone glycosides.

It is known that soybeans contain various harmful ingredients in addition to physiologically active substances. When processing and using soybeans as foods, a pretreatment process for removing these harmful ingredients is required. Trypsin inhibitors, hemagglutinin (hemagglutinin virus), phytate, which inhibits the absorption of minerals, and gastrointestinal flatulence factors are known. Of these, oligosaccharides, which are gas-generating factors, contain stachyose and raffinose in soybeans, and the human body lacks α-galactosidase in the digestive tract, so these oligosaccharides are not completely digested and absorbed. It is fermented by Clostridium sp. And Bacteroides sp. In the intestine, and the bloating and intestinal flatulence due to gas production of water, carbon dioxide, methane, etc. ), Cramping pains, and borborygmus. Recently, many countries have been trying to develop a method for removing starchios and raffinose, which are known to cause gas generation, before manufacturing processed foods using soybeans and soybeans in various countries.

Α-galactosidase (EC 3.2.1.22), an enzyme that degrades oligosaccharides such as stachyose and raffinose, is widely distributed in animals, plants, and microorganisms, and contains galactose residues. Hydrolyzes α-galactose residues of α-1,6 bonds in oligosaccharides such as galactomannan polysaccharides, melibiose (galactose-α-1,6-glucose), raffinose, and stachyose.

Studies on α-galactosidase of microbial origin include Aspergillus niger , Aspergillus oryzae , Aspergillus nidulans , Aspergillus awamori , Aspergillus ficuum , Aspergillus tamarii, Trichoderma reesei , Penicillium simplicissimum , Saccaromyces cerevisiae , Hansenula polymorpha , Mortierella vinacea, Bifidobacterium adolescentis , Bifidobacterium angulatum , Bifidobacterium longum , Scopulariopsis brevicaulis , Lactobacillus plantarum, Leuconostoc mensenteroides , Bacillus stearothermophilus , Α-galaccosidase enzymes produced by Bacillus licheniformis , Escherichia coli , and the like have been reported.

 The invention of microorganisms with high activity of α-galactosidase and β-glucosidase enzymes having proven their usefulness has been demanded in the food industry, the pharmaceutical industry, and the like.

As a result of continuous efforts by the present inventors to meet the demands of the industry as described above, it is α-galactosine to industrially produce foods or pharmaceuticals with high content of non-glycoside in the form of isoflavones and low content of indigestible oligosaccharides. Recognizing that it is a prior problem to find strains with high activity of the agent and β-glucosidase, the study was to find a new strain with high activity of α-galactosidase and β-glucosidase. .

The present invention provides a new strain, Leuconostoc pseudomesenteroides K-AP3 (Accession No. KFCC 11409P), to solve the above problems.

The present invention also provides a method for preparing an isoflavone concentrate obtained by fermenting soybean or soybean embryo with the Leukonostock Pseudome centroides K-AP3.

The present invention, Leukonostock Pseudomecentroides K-AP3 has a high activity of β-glucosidase, and thus isoflavone (digin, zenithine) in glycoside form, which is contained in foods, especially soybeans, in a non-glycoside form of isoflavone ( Diyzein and Genistein) have a very high conversion rate, and because the activity of α-galactosidase is very high, it is excellent in degrading effect of oligosaccharides such as stachiose and raffinose, which are intestinal gas generating factors. Preparation of Soybean Powder or Embryo Powder Fermented with Healthy Functional Food Ingredients, Soybeans or Embryos with Leukonostock Pseudomecentroides K-AP3 Suitable for

Recently, with the spread of well-being culture, there is increasing interest and demand for health foods and drugs with improved functions. Therefore, in the food industry, the pharmaceutical industry, etc., studies are being actively conducted to improve functions of existing health foods and drugs. As a result of constant research by the present inventors in this atmosphere, the high activity of β-glucosidase and α-galactosidase results in the improvement of the function of the soybean food, the most representative well-being food, which is Ryukonostoke pseudometheroides K-. Leuconostoc pseudomesenteroides K-AP3 (Accession No. KFCC 11409P) was provided. Hereinafter will be described in detail with respect to the present invention Leukonostock Pseudome centroides K-AP3.

Generally, the leukonostock strain is catalase-negative Gram-positive cocci, which is present as a flora in the large intestine and mucosa of humans and animals, and is used to prepare milk, dairy products and various fermented foods. It is known to not cause infection. These strains are Leuconostoc mesenteroides , Leuconostoc lactis , Leuconostoc pseudomesenteroides , Leuconostoc citreum , Leuconostoc citreum Eight species of Leuconostoc fallax , Leuconostoc argentinum , Leuconostoc carnosum , and Leuconostoc gelidum are included. However, these strains are difficult to classify and identify because different heterogeneous species with different biochemical properties exist within the same species. For accurate identification, PCR for each strain or specific 16S or 23S rDNA sequence or There is a difficulty in analyzing cell wall components.

The present inventors have overcome the above-mentioned difficulties through continuous research, and cultured, identified, selected, isolated and β-glucose from microorganisms from dairy products, kimchi, mammal's bowel movement, and cheonggukjang, which are generally known to contain leuconosstock strains. A new strain was provided through the oxidase and α-galaccosidase activity experiments.

The present invention also relates to an isoflavone concentrate enriched in aglycone and reduced indigestible oligosaccharides, in which soybeans or soybean embryos are fermented with Leukonostock Pseudomecentroides K-AP3.

The isoflavone concentrate fermented 42-48 hours with the Leuconosestock Pseudomecentroides K-AP3 of the present invention contains a large amount of non-glycoside isoflavones in the range of 0.7 to 0.8 wt%, which is shown in FIG. You can check it through the graph.

In addition, the isoflavone concentrate fermented 42-48 hours with Leukonostock Pseudomecentroides K-AP3, the isoflavone concentrate will contain less than 4% by weight of stachyose, raffinose (raffinose) 2 It will be contained in a weight percent or less, it can be confirmed through the graph (C) of Figure 4, which is reduced by more than 40% compared to the general soybean embryo.

That is, the present invention can reduce the content of stachyose and raffinose, which are indigestible oligosaccharides present in soybeans or soybean embryos, by about 50% compared to normal embryos, and glycoside iso is present in soybeans or soybean embryos. The conversion of flavones to nonglycoside isoflavones at high conversions can increase the content of nonglycoside isoflavones by at least 20%.

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Detailed description of the method for producing the isoflavone concentrate of the present invention described above,

Mixing soybean or soybean embryo with water in a ratio of 1 to 7 to 10, and then sterilizing; And

Inoculating and fermenting the leuconosstock pseudometh centroides K-AP3 to the sterilized mixture; Isoflavone concentrate is prepared through the above, wherein the leukonostock Pseudomecentroides K-AP3 is preferably inoculated at about 1 × 10 ⁵ to 1 × 10 ⁷ CFU / g, and fermentation is performed at room temperature for 24 to 60 hours. More preferably, the fermentation is carried out for about 36 to 48 hours.

The present invention is described in more detail as follows.

Leuconostock Pseudome centroides K-AP3 of the present invention was selected according to the following method. However, the following manufacturing method is an example for the practice of the present invention, and the present invention is not limited thereto.

1.Method of manufacturing medium

(1) The embryos used in this example were used after being milled to a solid content of 10% by hydrolysis (embryonic: water = 1: 8 weight / volume, w / v).

(2) The microbial separation medium was a soy medium prepared using soybean (from Goesan-gun, Chungbuk, 2005), and soybean and distilled water were 1: 4 w / v (weight / volume) at 121 ° C. for 15 minutes. After the heat treatment, the soybeans were removed using a filter network, the pH of the filtrate was adjusted to 3.5, and the mixture was allowed to stand at 4 ° C. for 6 hours to remove precipitated protein with filter paper (Whatman, Maidstone England). Prepared by readjusting the pH to 6.5.

(3) Solid medium was added to the prepared medium, 1.5% (w / v) agar and 0.005% (w / v) bromocresol purple (manufactured by Yakuri chemicals Co. Osaka, Japan) and 121 It was prepared by sterilization at 15 ℃.

2. Microbial isolation and culture method

Take 1g samples from dairy products, kimchi, pig and cow intestine contents, vegetables, virgin tract, infant feces, and Cheonggukjang and dilute them sequentially with sterile saline to 10 ^-1 ~ 10 ^-3 100 μl of soy plate agar was smeared and incubated at 37 ° C. for 24 hours, and different microorganisms were separated according to the yellowish colonies. In order to isolate anaerobic microorganisms, an anaerobic incubator using an anaerobic system Gaspack (BBL, Becton Dickinson, USA) was incubated at 37 ° C. for 24 hours. Standard strains and isolated strains were subcultured at 37 ° C. for 24 hours using a soy medium to be used for later experiments.

3. Preparation of crude enzyme solution

After culturing the strain in the liquid medium for enzyme production, the supernatant recovered by centrifugation (10,000 × g, 5 minutes) was used as an extracellular coenzyme solution. The cells were recovered and washed twice with phosphate buffered saline (PBS), and the cells were sonicated on ice for 5 minutes using an ultrasonic cell crusher (Sonosmasher, Ulsso Hitech Co., Korea). It was. Then, the supernatant recovered by centrifugation (10,000 × g, 5 minutes) was used as an intracellular coenzyme solution.

4. Enzyme Activity Measurement Method

Enzyme activity is Yeo . It was carried out according to the method. Substrates were prepared in 50 mM sodium phosphate buffer (pH 6.5), ρ-nitrophenyl-α-D-galacto-pyranoside, PNPG, and Sigma Chemical. Co.) and ρ-nitrophenyl-β-D-galacto-pyranoside (P-nitrophenyl-β-D-gluco-pyranoside (PNPG, Sigma Chemical Co.) were respectively prepared to 10 mM, and then 100 100 μl of coenzyme solution was added to the substrate solution in 10 μl and reacted for 10 minutes at 37 ° C. Then, 100 μl of 0.5 M Na ₂ CO ₃ solution was added to stop the reaction to release ρ-nitrophenol (ρ- The amount of nitrophenol, PNP, and Sigma Chemical Co.) was measured by absorbance at 400 nm The enzyme activity unit was defined as 1 unit of enzyme amount necessary to release 1 μmole of PNP in 1 minute.

5. Method for Investigating Growth Characteristics of Selected Microorganisms

The growth characteristics of the microorganisms selected through the enzyme activity measurement and microorganism identification were investigated by the following method. The number of viable microorganisms of the selected microorganisms was taken in 1 g of sample, diluted 10 with 0.85% NaCl, 100 μl of the diluting solution was smeared on MRS agar (Difco, Sparks, MD, USA) and incubated at 30 ° C. for 48 hours to provide colorony. The numbers were counted to be between 50 and 200. pH was measured by a pH meter (ATI Orion, Boston, MA, USA), titratable acidity (titratable acidity) was titrated with 0.1N NaOH solution to express the lactic acid content as a percentage using the following equation (1). 4 ml of distilled water and 50 μl of 1% phenolphthalein were added to 1 ml of the culture, and the growth characteristics were examined by measuring the amount of NaOH consumed at the end point of red color with titration with 0.1 N NaOH solution.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited by the following examples.

Example 1

Primary selection of strains from isolated microorganisms and selection of secondary strains from primary selection strains

By the microbial separation and culture method described above, 1g of each sample was taken from dairy products, kimchi, pig and bovine intestine contents, vegetables, virgin tract, infant feces, and Cheonggukjang, and diluted with sterile physiological saline. 100 μl of agar plate soybean medium supplemented with 0.005% (w / v) bromocresol purple indicator was incubated at 37 ° C. for 24 hours, followed by acid production to yield yellow colored colonies (colony). Depending on the form, 491 strains of different microorganisms were isolated. Of these, 422 strains were anaerobic strains and 69 strains were anaerobic strains.

The supernatant was separated by incubating the isolated strain in a liquid medium for producing α-galactosidase and β-glucosidase at 37 ° C. for 24 hours, followed by centrifugation. The cell was used as an extracellular coenzyme solution, and the supernatant was used as an intracellular coenzyme solution by sonicating the cells and centrifuging again. The α-galactosidase enzyme was measured using 10 mM ρ-nitrophenyl-α-D-galacto-pyranoside as a substrate and β-glucosidase. For enzyme measurement, extracellular coenzyme solution and intracellular coenzyme solution were added using 10 mM ρ-nitrophenyl-β-D-galacto-pyranoside as a substrate. The reaction was carried out at 37 ° C. for 10 minutes. Here, when α-galactosidase and β-glucosidase decompose PNPG, ρ-nitrophenol (ρ-nitrophenol, PNP) is separated and yellow, so the higher the titer, the darker the yellow. Thereafter, the reaction was stopped by adding 0.5M Na ₂ CO ₃ solution to measure absorbance at 400 nm. Enzyme activity of 422 strains of anaerobic strains and 69 strains of anaerobic strains was quantified in the standard curve of ρ-nitrophenol, and 192 strains of high activity of α-galactosidase and β-glucosidase were selected first.

192 strains of the first selected strain were compared to α-galactosidase and β-glucosidase enzyme activities in the same manner, and 48 strains of the strain were selected secondarily.

Example 2

Selection of tertiary strains

① 89 strains isolated from vegetables on the basis of α-galactosidase and β-glucosidase enzyme activity among the second selected 48 weeks ② ② V13-4 strains isolated from virgin tract, ③ K-M1-4 and ④ K-AP3 strains isolated from Kimchi, ⑤ CMB 16 strains and ⑥ S2C5 strains isolated from Cheonggukjang were selected.

The six strains selected were Gram-stained and observed under the microscope. All of them were observed as Gram-positive bacteria. The primary identification was performed by API CHL kit test through sugar availability review. 16S ribosomal DNA analysis of the results are shown in Table 1 below. The isolates selected were Weissella genus (isolate 89, isolate V13-4, isolate K-M1-4), isolate K-AP3, enterococcus genus (isolate CMB 16). , isolate S2C5) was confirmed.

division API kit (CHL 50) 16S rDNA sequencing 89 strains  Lactobacillus fermentum Weissella confusa V13-4 strain Lactobacillus coprophilus Weissella cibaria K-M1-4 strain Lactobacillus coprophilus Weissella cibaria K-AP3 strain Leuconostoc mensenteroides ssp. mensenteroides / dextranicum Leuconostoc pseudomensenteroides BF-BW-31 strain euconostoc mensenteroides ssp. mensenteroides / dextranicum Leuconostoc pseudomensenteroides CMB 16 strain  Lactobacillus plantarum Enterococcus faecium S2C5 strain  Lactobacillus plantarum Enterococcus faecium

Experimental Example 1

Enzyme activity test of 3rd selected strain and selection of 4th strain

Seven strains of the third strain isolated in Example 2 were tested by the enzyme activity test method described above for the α-galactosidase and β-glucosidase enzyme activities, and the strains were liquid medium. ) At 37 ° C. for 24 hours, and the results are shown in Table 2 below.

89 strains, K-M1-4 strains and K-AP3 strains showed higher α-galactosidase and β-glucosidase activity than other strains. By selecting the growth and characteristics of these strains were carried out.

Experimental Example 2

Growth and Characterization of Fourth Selected Strains

The fourth selected strains were cultured and fermented in embryo medium for 30 ° C. for 48 hours, and the changes of pH, acidity and viable cell number were examined, and the results are shown in FIGS. 1, 2, and 3, respectively.

(1) Looking at the pH change experiment 1, the initial pH of the fermentation was about 6.3, but after 12 hours fermentation K-AP3 strain <K-M1-4 strain <89 strains in the order of high pH.

(2) Titratable acidity (hereinafter referred to as "TA") Looking at Figure 2, the experimental result of the change, after 12 hours fermentation, K-M1-4 strain 89 strains <K-AP3 strains in the TA high can confirm.

(3) The viable cell count increased rapidly until 12 hours of fermentation and remained almost constant, similar to the change of pH and acidity, but after K-M1-4 and K-AP3 strains were fermented for 12 hours, 10 The viable cell number of ⁹ was shown. Through experiments of pH, titratable acidity and viable cell number after 12 hours of fermentation through Experimental Example 2, K-AP3 strains showing low pH, high titratable acidity and high viable cell count of average pH 4 showed the best growth. I could confirm it.

Experimental Example 3

Oligosaccharide Resolution Confirmation Experiments of Fourth Screened Strains

In the sugar decomposition experiment, 4 ml of distilled water was added to 0.5 g of the lyophilized sample, shaken for 2 hours in an 80 ° C. constant temperature bath, and 10 ml of ethanol was added to extract sugar in the sample for 30 minutes in an 80 ° C. constant temperature bath. Then, the supernatant was collected by centrifugation at 10,000 rpm for 5 minutes, filtered through a 0.2 μm membrane filter, and subjected to HPLC (High Performance Liquid Chromatograph System) analysis under the conditions shown in Table 3.

HPLC Agilent 1100 series Column Eclipse XDB-C18 (4.6cm x 150mm, 3.6μm) Guard column Eclipse XDB-C18 Mobile phase
(ingredient) A: 0.1% phosphoric acid
B: Acetonitrile Detector 255 nm Flux 0.8 ml / min

89 strains, K-M1-4 strains and K-AP3 strains, 4th selected strains, were inoculated into 1% (v / v) of non-bearing medium and embryo medium, and then cultured at 30 ° C for 48 hours for 6 hours. HPLC was used to analyze the changes in the contents of sugars such as stachyose, raffinose, sucrose, glucose, galactose, and fructose. The analysis results are shown in FIG. 4.

Referring to Figure 4 it can be seen that the indigestible oligosaccharides containing the high proportion of starchios and raffinose, 89 strains and K-M1-4 strains can be confirmed that almost no use of starchios and raffinose, K- It can be seen that the AP3 strain is gradually reduced in the content of starchios and raffinose as the fermentation time passes.

Experimental Example 4

Isoflavone aglycone conversion rate confirmation experiment of 4th selected strain

First, the selection microorganisms were cultured in a liquid medium for 37 hours at 37 ° C. for 48 hours, and then cultured (250 ml) were separated by centrifugation (10,000 × g / 15 min), and then the supernatant was taken and lyophilized.

For isoflavone nonglycoside analysis, 20 g of 70% methanol was added to 0.5 g of the lyophilized sample, sonicated for 10 minutes and shaken for 20 minutes. Thereafter, the supernatant was collected by centrifugation at 10,000 rpm for 5 minutes, filtered through a 0.2 μm membrane filter, and HPLC analysis was performed under the conditions shown in Table 4 below. .

HPLC Agilent 1100 series Column Sugar-Pak TM1, 6.5cm x 300mm (Waters, USA) Column temp. 80 ℃ Mobile phase H ₂ O Detector RID (Waters, USA) Flux 0.5 ml / min

The change of isoflavones was shown by the 4th selection strain, but as the fermentation time passed, glycoside (glycoside), which is a common glycoside, was decreased in all strains, while aglycone, which was a non-glycoside, was gradually increased. It showed the condition. In particular, in the case of the K-AP3 strain, it was confirmed that the content of the aglycone, which is a non-glycoside, increased significantly from 620 mg / 100g to 760 mg / 100g as shown in FIG. 5.

K-AP3 strains have excellent activity characteristics against α-galactosidase and β-glucosidase enzymes through growth and characterization, sugar degradation and isoflavone nonglycoside content analysis of the strains of Experimental Examples 2 to 4 It was found that it has, and accordingly the K-AP3 strain was finally selected and identified.

Experimental Example 5

Identification of K-AP3 Strains

Identification method

Identification method was examined for morphological characteristics by Gram staining and microscopic observation. The API 50 CHL kit (bioMerieux Co., France) investigated the availability of the 49 carbon sources shown in Table 5 below and the results were analyzed using API 50 CHL database V3.0 (http://apiweb.biomerieux.com). It was tentatively identified. For more accurate identification, 16S ribosomal DNA gene sequencing analysis was performed. Isolation of chromosomal DNA of selected microorganisms followed by demineralization at 94 ° C for 1 minute using 27F (5'-AGAGTTTGATCATGGCTCAG-3 ') and 1492R (5'-GGATACCTTGTTACGACTT-3') primers commonly used for 16S rDNA sequencing. (denaturation), annealing at 51 ° C for 1 minute, and polymerization at 72 ° C for 1 minute and 30 seconds were amplified by PCR (Minicycler ^™ , MJ research Inc., Waltham, Mass., USA). The amplified fragment of about 1400 bp was transformed after binding to a T vector (T vector, Invitrogen, Carlsbad, CA, USA). The sequencing was performed using a T vector sequencing primer, and the results were analyzed using the BLAST search (http://www.ncbi.nlm.nih.gov) program and GENBANK (NCBI, Bethesda, MD, USA) was identified as compared to ribosomal DNA gene sequencing.

Microscopic observation of the K-AP3 strain with the highest enzymatic activity was Gram-positive cocci and identified by API 50 CHL KIT TEST to examine glucose availability. Leuconostoc pseudomesenteroides ssp., 99.9% homology with leukonostock mesentroides / dextranicum was shown in Table 5 below.

For more accurate identification, 16S ribosomal DNA analysis of selected microorganisms was performed.

The results of the 16S rDNA gene sequence are shown in SEQ ID NO: 1, and the base sequence was compared with the data of BLASTN to obtain a phylogenetic diagram showing molecular biological correlations, which are shown in FIG. 6.

According to the identification result, the selection microorganism appeared to be the closest flexible relationship with Leukonostock Pseudome centroides ATCC8293. Therefore, the selection microorganisms were identified as Leuconostok pseudomeenteroides and Leuconostoc pseudomesenteroides . It was named K-AP3 (Accession No. KFCC 11409P).

In Table 5, + represents a positive reaction,-represents a negative reaction.

Production Example

Functional embryo powders having increased glycoside isoflavone content and reduced indigestible oligosaccharides were prepared using the final selected Leukonostock Pseudomecentroides K-AP3.

In the manufacturing process, crushed embryos (22 kg) and water (176 kg) were put into a fermenter (300 L), sterilized at 121 ° C. for 10 minutes, cooled to 30 ° C., and then seeded with 1 × 10 ⁶ CFU / g. Incubated at 30 ° C. for 24 hours, and the culture was lyophilized to prepare a product.

The present invention, Leukonostock Pseudomecentroides K-AP3 is very high activity of the α-galactosidase and β-glucosidase enzyme, it can be used in the manufacture of health foods such as pharmaceuticals, fermented foods in the future It is expected.

1 is a pH change test results of the fourth selected strains of Experimental Example 2.

Figure 2 is a test result of the change in titratable acidity (TA) of the fourth selected strains of Experimental Example 2.

Figure 3 is a test result of the change in viable cell number of the fourth selected strains of Experimental Example 2.

Figure 4 shows the results of sugar degradation in the embryo medium of Experimental Example 3, (A) 89 strains, (B) K-M1-4 strains, (C) is the experimental results for the K-AP3 strain.

5 is an isoflavone content analysis test result in the embryo medium of Experimental Example 4, (A) is 89 strains, (B) is the K-M1-4 strain, (C) is the experimental result for the K-AP3 strain. .

Figure 6 is a schematic diagram showing the molecular biological correlation of the nucleotide sequence of the K-AP3 strain identified by Experimental Example 5 with the data of BLASTN.

<110> MAEIL DAIRY INDUSTRY CO., LTD. <120> Leuconostoc pseudomesenteroides K-AP3 having High activity of          alpha-galactosidase and beta-glucosidase and Isoflavon          concentrates using <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 980 <212> DNA <213> Leuconostoc pseudomesenteroides K-AP3 <400> 1 tgcttagacg gctccttcct aaaaggttag gccaccggct ttgggcatta caaactccca 60 tggtgtgacg ggcggtgtgt acaagacccg ggaacgtatt caccgcggcg tgctgatccg 120 cgattactag cgattccgac ttcatgtagt cgagttgcag actacaatcc gaactgagac 180 gtactttaag agattagctc accctcgcgg gttggcaact cgttgtatac gccattgtag 240 cacgtgtgta gcccaggtca taaggggcat gatgatctga cgtcgtcccc gccttcctcc 300 ggtttgtcac cggcagtctc gctagagtgc ccatctgaat gctggcaact aacaataagg 360 gttgcgctcg ttgcgggact taacccaaca tctcacgaca cgagctgacg acgaccatgc 420 accacctgtc actttgtctc cgaagagaac acttctatct ctaaaagctt caaaggatgt 480 caagacctgg taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg 540 cgggtccccg tcaattcctt tgagtttcaa ccttgcggtc gtactcccca ggcggaacac 600 ttaatgcgtt agcttcggca ctaagaggcg gaaacctcct aacacctagt gttcatcgtt 660 tacggtgtgg actaccaggg tatctaatcc tgtttgctac ccacactttc gagcctcaac 720 gtcagttaca gtccagtaag ccgccttcgc cactggtgtt cttccatata tctacgcatt 780 ccaccgctac acatggagtt ccacttacct ctactgcact caagttaacc agtttccaat 840 gccattccgg agttgagctc cgggctttca catcagactt aatcaacgtc tgcgctcgct 900 tacgccaaat caagctggca tacgtatacg tgctggacga tttacgtcct tgaggtacgt 960 caactaaatc tccattagct 980  

Claims (6)

Leuconostoc pseudomesenteroides K-AP3 (Accession No. KFCC 11409P) having high activity of α-galactosidase and β-glucosidase. delete delete delete delete Mixing soybean or soybean embryo with water in a ratio of 1 to 7 to 10, and then sterilizing; And Inoculating and fermenting the sterilized mixture with the leukonostock pseudometh centroides K-AP3 of claim 1; Method for producing an isoflavone concentrate, characterized in that it comprises.

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