KR101146102B1 - method for manufacturing whole soybean milk containing high content of active isoflavon - Google Patents

Abstract

The present invention relates to a method for producing whole soybean milk with high content of active isoflavone, and more specifically, fermented with lactic acid bacteria having high beta-glucosidase activity extracted from kimchi during the production of whole soybean milk, which is made from whole soybeans. The present invention relates to a method of increasing the activity of isoflavones contained in soybean oil.

According to the present invention, it is possible to convert isoflavone glycosides in soy milk into isoflavone non-glycosides with high physiological activity, and by using the bactericidal action of lactic acid bacteria contained in kimchi to kill rot and harmful bacteria, improve the shelf life of soy milk and improve health. Soymilk that is beneficial to the production can be prepared.

Soybean, Beta-Glucosidase, Isoflavones, Fermented, Whole Soymilk

Description

Method for manufacturing whole soybean milk containing high content of active isoflavon}

The present invention relates to a method for producing whole soybean milk with high content of active isoflavone, and more specifically, fermented with lactic acid bacteria having high beta-glucosidase activity extracted from kimchi during the production of whole soybean milk, which is made from whole soybeans. The present invention relates to a method of increasing the activity of isoflavones contained in soybean oil.

Soymilk is a colloidal drink made by grinding soybeans. It contains nutritionally superior ingredients and high functional value. It is low in saturated fatty acids and contains a large amount of unsaturated fatty acids.

It is an optimal nutritional food that can supply dietary fat intake and reduce adult diseases because it has no cholesterol, and can supply high-quality nutrition without side effects to infants and adults with lactose intolerance.

Soybean, a raw material of soy milk, has been one of the important foods that are indispensable in the diet of our nation. Soy protein is a vegetable protein containing abundant essential amino acids. Recently, it has been spotlighted as a substitute for animal protein. As a high functional food containing substances, it is also widely used as snacks and drinks.

Soybean is composed of 20% lipid, 40% protein, 35% carbohydrate, and 5% other ingredients. It contains about 12 ~ 14% water and other ingredients are vitamins, It is composed of minerals.

Soybean contains bioactive substances isoflavones, saponins, lecithin, peptides, amino acids, and the like, and interest in isoflavones, a functional ingredient contained in soybeans, is increasing day by day.

Isoflavones, which are closely related to the efficacy of soybeans, contain 97% glycosides and 3% aglycones, and the glycosides are genistin, didzin, and glycidine ( It is present in soybeans in the form of glycitin, and the nonglycoside is composed of genistein, daidzein, glycidin and other derivatives, and isoflavones present in non-fermented foods. It is known that isoflavone nonglycosides present in fermented foods are more excellent in terms of bioavailability than glycosides.

Glycosides of soybean fermented foods are hydrolyzed by beta-glucosidase of microorganisms and converted into non-glycosides that are easily absorbed into the body. Genistein, a non-glycoside, has an excellent anticancer effect. Among them, prophylactic and breast cancer are known to have high anti-cancer effects. In addition, since it has an effect similar to that of the female hormone estrogen, it is known to have a great effect on the prevention and progression of osteoporosis in postmenopausal women.

However, since most of the isoflavones present in soybean exist as glycosides of genistin, dydazine, and glycidin, there is a problem of low absorption rate in the body. Attempts have been made to convert isoflavones into easy nonglycoside forms.

However, in the case of whole soybean milk, which is prepared by grinding soybeans, the bioactive substances of soybean may be deteriorated during storage, and in particular, sugar or aglycone produced by the decomposition of glycosides by beta-glucosidase There is a problem of increasing the likelihood of corruption of soy milk by acting as a nutrient source of the decaying bacteria present.

In order to solve this, it is usually sterilized at a high temperature to extend the storage period, but high temperature sterilization is not preferable because it destroys not only rot bacteria but also nutrients contained in soymilk.

The problem to be solved by the present invention is to provide a method that can prevent the degradation of the preservation while converting the glycosides contained in soy milk into non-glycosides so as not to harm the human body when manufacturing soymilk.

In order to solve the above problems, the present invention comprises the steps of preparing a soybean powder by grinding the soybeans from which the foreign matter is removed to 550 ~ 650 mesh; Preparing soybean milk by adding 10-12 times water by weight to the soybean powder and then boiling for 50-70 minutes; Cooling the whole soybean milk to room temperature; Inoculating the cooled soybean milk with a strain having high beta-glucosidase enzyme activity extracted from kimchi to 0.05 to 0.15% by weight; And the strain is inoculated whole soybean milk provides a method for producing high soybean milk with high active isoflavone content comprising the step of fermenting at 35 ~ 40 ℃ 15-30 hours.

At this time, the strain extraction method with high beta-glucosidase enzyme activity in the kimchi, the step of filtering the kimchi broth of kimchi to remove solids; Stationary culture of the kimchi broth from which the solids have been removed in an ML liquid medium; Serially diluting the cultured culture medium in saline and then plating the plate in BEA medium and culturing to obtain a single cell colony of black color; Delineating the cell colonies again in BEA medium to obtain a single black cell colony around the cell colonies; And it is preferable to include a step of obtaining a strain by identifying the cell community.

In addition, the kimchi is more preferably aged for 5-6 months at 3-7 ℃ after dipping.

In addition, between the step of inoculating and the fermentation step is preferably added to remove the kimchi broth of the kimchi soup so that the kimchi broth containing 0.5 to 1.5% by weight.

In addition, after the step of fermentation, it is preferable to add the step of preparing the fermented soybean milk in frontal head, powder, granules, capsules, tablet formulation.

According to the present invention, it is possible to convert isoflavone glycosides in soymilk into isoflavone nonglycosides with high bioactivity.

In addition, by using the bactericidal action of lactic acid bacteria contained in kimchi to kill the decayed bacteria and harmful bacteria to improve the shelf life of soy milk can be produced soy milk beneficial to health.

Hereinafter, the present invention will be described in detail.

First, microorganisms producing beta-glucosidase, which converts soybean glycosides into nonglycosides, are isolated from kimchi.

Kimchi, Korea's representative fermented food, is a source of various minerals and vitamins. It has an intestinal action by lactic acid bacteria, and inhibits the action of other harmful bacteria in the intestine to prevent abnormal fermentation and inhibit pathogens. It is effective in suppressing aging and cancer.

Kimchi is a commonly consumed food and there is no side effect to the human body by using microorganisms extracted from kimchi. Kimchi is not limited to cabbage kimchi, radish kimchi, bachelor kimchi, and white kimchi.

After soaking kimchi and aged for 5-6 months at 3 ~ 7 ℃, the kimchi broth of the kimchi is filtered to remove the solids.

When kimchi is aged at 3 ~ 7 ℃ for 5 ~ 6 months, the production of lactic acid bacteria of Kimchi is enriched, especially the production of Lactobacillus plantarum, which is known as a beneficial bacterium for the human body by secreting antibacterial substances and suppressing food poisoning bacteria. Will increase.

The following isolates and identifies microorganisms producing beta-glucosidase from the kimchi broth from which the solids have been removed.

Separation of microorganisms is carried out by a conventional method of separating colonies using a medium, in which a beta-glucosidase produced by the microorganisms in a solid medium containing esculin is contained around the colonies. It is more preferable to use the Bile esculin agar (BEA) method to blacken.

Identification of microorganisms select the highest activity strain by measuring the beta-glucosidase enzyme activity of the isolated microorganisms.

The enzyme activity measurement method may be carried out a general beta-glucosidase enzyme activity measurement method, for example p-nitrophenyl β-D-glucopyranoside (p-nitrophenyl β-D-glucopyranoside: p-NPGlu) Can be measured.

When a strain having high beta-glucosidase enzyme activity is prepared, soybean powder is prepared next.

Soybeans are not limited to peas, green beans, kidney beans, black beans, frosted, seomoktae, chestnuts, cheontae, white tae, small beans, etc., and may be used alone or in combination of two or more.

Soybean powder is prepared by pulverizing soybeans from which foreign substances have been removed with 550-650 mesh.

When the soybean is pulverized to less than 550 mesh, the conversion rate of glycoside to non-glycoside is lowered in the subsequent fermentation process, and if it exceeds 650 mesh, the nutrients contained in soybean are destroyed.

In addition, in the present invention, the raw soybeans are not crushed in water and used as it is pulverized. When soybeans are called in water, the fermentation efficiency is lowered and the vivid texture of the soymilk is reduced because the soybean cells are expanded after the fermentation process. Will be reduced.

Next, water is added to the ground soybean powder and then boiled to prepare soymilk.

The amount of water is preferably added by 10 to 12 times the soybean powder by weight, the boiling time is suitable 50 to 70 minutes.

The soy milk produced as a whole soybean milk (全 豆乳) prepared by grinding the whole soybean contains all the useful components of soybean.

When the preparation of the soybean milk is completed, the whole soybean milk is cooled to room temperature and then fermented after inoculating the chilled soybean milk so that the strain having high beta-glucosidase enzyme activity extracted from the kimchi is 0.05 to 0.15% by weight.

The fermentation is sealed by fermentation for 15 to 30 hours at 35 to 40 ℃.

The fermentation temperature and fermentation time is a range in which the glycosides of isoflavones contained in the soybean milk are converted into non-glycosides at the highest ratio.

In addition, the kimchi broth from which the solid content is removed may be added to the whole soybean oil prepared to separate the microorganisms before the fermentation.

Kimchi broth is rich in minerals such as calcium, phosphorus and iron, and vitamin A and vitamin C. It is rich in antioxidants, and it is effective in inhibiting aging, preventing cancer, and enhancing immunity. Various lactic acid bacteria that have the function of killing other pathogens or rot bacteria.

Therefore, the kimchi broth is added to the soy milk to improve the shelf life of the soy milk by using the sterilizing power of the kimchi soup, so that the useful component of the kimchi soup is contained in soy milk.

The amount of kimchi broth added is preferably 0.5 to 1.5% by weight. If less than 0.5% by weight, the sterilizing power is not sufficient. If the content exceeds 1.5% by weight, the soybean oil is naturally drained and savory. It is undesirable because the texture fades.

During fermentation, the glycosides of isoflavones contained in the soybean milk can be converted into non-glycosides, which are easily absorbed by the body, by the beta-glucosidase enzyme of the strain extracted from kimchi, thereby increasing the human absorption rate of isoflavones of soybean. .

When the fermentation is completed, soybean milk production is completed according to the present invention, in order to ingest the useful components contained in the soybean milk in a more convenient and various ways, food of various formulations, that is, tofu, using the soybean milk, Powders, granules, capsules, tablets and the like can be prepared.

Hereinafter, the present invention will be described in more detail based on the following Examples and Test Examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited by the following examples, and may be changed to other embodiments equivalent to substitutions and equivalents without departing from the technical spirit of the present invention. Will be apparent to those of ordinary skill in the art.

Example 1 Isolation and Identification of Microorganisms Producing Beta-Glucosidase in Kimchi

Chinese cabbage was pickled and seasoned in a general manner, put in an Onggi jar, and aged at 5 ° C. for 6 months to prepare kimchi.

Four kimchi soup samples from which the solids were removed by filtering the kimchi broth after obtaining kimchi broth by homogenizing each of the kimchis on the 135th, 150th, 165th, and 180th day of the kimchi ripening period, and then homogenizing each of them. Got.

1 ml of each of the samples was taken and each sample was placed in 100 ml of a Man-Rogosa-Sharpe (MRS) liquid medium (Difco, USA) and incubated in anaerobic conditions at 37 ° C. for 24 hours.

Each of the cultured samples was serially diluted in 0.85% saline, spread plate on BEA medium, and cultured for 36 hours under anaerobic conditions at 37 ° C to identify a single cell colony. (See Figure 1).

The identified cell colonies were further streaked onto BEA medium to take a single colony of black cells around the cell colonies to separate single species of microorganisms producing beta-glucosidase.

As a result of identification of the isolated microorganism using API 50 CHL kit (product of Biomerieux Co., France), Sample 1 is a major species, Weissella confusa , a kind of lactic acid bacteria, and Sample 2 is lactic acid bacteria. Pediococcus pentosaceus, which is a kind, was determined to be the main species (see FIG. 2). (These strains can be distributed by anyone at the Korea Institute of Bioscience and Biotechnology (http://www.brc.re.kr).)

Sample 3 and sample 4 could not confirm the exact species.

Test Example 1 Measurement of Beta-glucosidase Enzyme Activity of Isolated Microorganisms

1) Extraction of Enzymes

Single cell colonies of each of the samples 1 to 4 of Example 1 were incubated under anaerobic conditions for 24 hours at 37 ° C. in MRS medium (manufactured by Difco, USA).

Centrifuged each of the cultured samples (10000 × g, 10min, 4 ℃) divided into culture supernatant and pellet (pellet), the culture supernatant is stored and the pellet is a buffer (5mM Na ₂ HPO ₄ -citric acid, pH 5.8 , McIlvaine buffer). After washing, cell disruption was performed 10 times with 500 mg glass beads (1 minute per time).

The crushed sample was centrifuged (10000 × g, 10 min, 4 ° C.), and only the supernatant was taken and mixed with the stored culture supernatant, respectively, and 4 times of ethyl alcohol was added thereto, followed by stirring at 4 ° C. for 30 minutes.

After stirring, the supernatant was discarded by centrifugation (10000 × g, 10 minutes, 4 ° C.), and pellets were dissolved in the buffer (McIlvaine buffer) to prepare an enzyme for measuring beta-glucosidase activity.

2) Beta-glucosidase activity measurement

45 μl of each extracted enzyme and 15 μl of 10 mM p-NPGlu were mixed and reacted at 37 ° C. for 15 minutes, and 900 μl of 0.25M sodium carbonate (Na ₂ CO ₃ ) was added to terminate the reaction.

In order to observe the change in absorbance at 400 nm, 135 μl of McIvavaine buffer and 45 μl of 10 mM p-NPGlu were added thereto and reacted at 37 ° C. for 15 minutes. The results are shown in FIG. 3.

As can be seen in Figure 3 of the separated samples showed the highest activity in the enzyme of Sample 2.

Example 2 Preparation of Whole Soymilk Using Microorganisms Extracted from Kimchi

After removing the foreign material 10kg of the selected black beans were ground to 600 mesh to obtain a black bean powder.

110 kg of purified water was mixed with the black soybean powder, and then boiled for 1 hour and cooled to room temperature to prepare 72 kg of soybean oil.

70 g of lactic acid bacteria extracted from Sample 2 of Example 1 having high beta-glucosidase activity was inoculated into the prepared soybean milk.

In Example 1, 700 g of kimchi broth from which solids were removed was added.

The whole soybean milk mixed with the lactic acid bacteria and kimchi broth was sealed at 37 ° C. for 24 hours to ferment to prepare whole soybean milk having high active isoflavone content according to the present invention.

Test Example 2 Determination of Changes in Active Isoflavone Contents

First, Dydjin, Genistin, Dyzedine, and Genistein reagent were prepared using ethanol for high performance liquid chromatography (HPLC), respectively, 10 µg / ml, 50 µg / ml, 250 µg / ml, and 100 µg / ml. After dissolving in ml, 5 µg / ml, 2.5 µg / ml, and 1.25 µg / ml standard solution of each of the reagents was prepared using the solution.

Next, 5 g of the lyophilized sample of the soybean milk prepared in Example 2 was added to a reflux flask, and 50 ml of a 75% ethanol solution was added thereto, followed by extraction under reflux for 3 hours in an 80 ° C. water bath, followed by filtration using a filter paper. Then, the mixture was applied to 75 ml of ethanol solution, 100 ml, filtered through a 0.45 μm syringe filter, and quantified by HPLC. Analysis conditions were set as shown in Table 1 below.

Isoflavone Analysis Conditions Using HPLC Item Condition Detector UV-254 nm Column Agilent Eclipse XDB-C18, 5 μm, 4.6 * 250 mm Column oven temp. 30 ℃ Mobile phase A: 0.1% acetic acid in deionized water B: 0.1% acetic acid in acetonitrile Minutes A (%) B (%) 0 88 12 25 75 25 30 70 30 35 65 35 40 60 40 42 88 12 50 88 12 Flow rate 1.0ml / min Injection volume 10 μl

Using the whole soybean milk before fermentation in Example 2 as a control, the fermented soybean milk of Example 2 was measured by the above method and the results are shown in Table 2 below.

Measurement result of conversion degree of active isoflavone content Glycoside Non-glycoside Sum DIDJIN Jennysteen Dyed Jane Genistein Control 804.0 1075.6 46.3 (2.3) 77.0 (3.8) 2002.9 Example 2 345.1 421.2 370.1 (22.8) 485.2 (29.9) 1621.6

(Unit: ㎍ / g, dry basis (% by weight of total isoflavone content))

As can be seen from Table 2, the active isoflavone conversion content of the non-fermented control was measured at 52.7% in the whole soybean milk of Example 2, compared to 6.1%, which was fermented by lactic acid bacteria extracted from kimchi according to the present invention. It was confirmed that the active isoflavone content of whole soybean milk was high.

<Test Example 3> Determination of active isoflavone content of whole soybean milk with fermentation time

By changing the fermentation time in Example 2, the degree of conversion of the active type isoflavone content according to the fermentation time was calculated based on the following formula and shown in Table 3.

* Conversion degree (%) = non-glycoside / total × 100

Measurement result of conversion of active isoflavone content according to fermentation time Fermentation time
(time) Glycoside Non-glycoside Sum Degree of conversion
(%) DIDJIN Jennysteen Dyed Jane Genistein 0 804.0 1075.6 46.3 77.0 2002.9 6.1 11 422.6 538.1 272.1 396.0 1628.8 41.0 13 384.4 486.2 297.3 427.9 1595.8 45.4 15 344.9 431.2 336.5 461.5 1574.1 50.7 18 353.6 444.5 338.9 465.7 1602.7 50.2 21 302.5 380.7 320.4 438.8 1442.4 52.6 24 339.1 426.6 362.7 497.0 1625.4 52.9 27 309.4 389.1 326.4 447.0 1471.9 52.5 30 320.0 405.4 351.4 480.4 1557.2 53.4 33 335.2 418.1 311.5 431.8 1496.6 49.7 35 342.5 412.7 316.7 417.3 1489.2 49.3

(Unit: ㎍ / g, dry basis)

As shown in Table 3, the conversion to the active isoflavones increased with time, exceeded 50% after 15 hours, and decreased slightly after 30 hours.

Accordingly, it can be seen that the fermentation time of the soybean milk is suitable 15 ~ 30 hours.

Test Example 4 Storage Stability Measurement

In order to determine the shelf life of the soybean milk fermented with kimchi extracted lactic acid bacteria according to the present invention, the pH and the appropriate acidity of the soybean milk of Example 2 were measured at 37 ° C. over time.

As a control, Bifidobacterium animalis ATCC 15707 having beta-glucosidase activity was purchased commercially and inoculated in soybean milk instead of lactic acid bacteria of Example 2, but no kimchi broth was added.

The pH was measured using a pH meter, and the titratable acidity was mixed homogeneously with 10 ml of sample and the same amount of distilled water, followed by neutralization titration with 0.1 N sodium hydroxide (NaOH). It is shown in Table 4 in terms of acidity.

pH and titration acidity changes Storage period
(hr) pH Titration Control Example 2 Control Example 2 0 6.82 6.75 0.24 0.20 12 6.21 6.32 0.32 0.35 24 5.45 6.17 0.68 0.72 36 5.17 5.93 0.81 0.98 48 4.81 5.78 1.03 1.17 60 4.90 5.63 1.10 1.26 72 4.83 5.61 1.23 1.38

As can be seen in Table 4, the pH reduction rate of the soybean milk of Example 2 prepared by the method according to the present invention was lower than that of the control, and the shelf life was improved compared to the lactic acid bacteria having the normal beta-glucosidase activity.

Hereinafter, various exemplary formulations using whole soybean milk having high active isoflavone content according to the present invention are illustrated, but the present invention is not limited thereto.

<Formulation Example 1> Preparation of frontal tofu

After heating the soybean milk prepared in Example 2 to 65 ° C., 0.3 wt% of a mixture of transglutaminase, magnesium chloride, and glucono-delta-lactone was pressed and coagulated. To make the whole head.

The front head is cut and cooled after cooling at 10 ° C. for 10 minutes, and then packaged. The packaged whole head is sterilized at 85 ° C. for 30 minutes and then cooled to 10 ° C. or less.

Formulation Example 2 Preparation of Powder

The whole soybean milk prepared in Example 2 is dried using a vacuum freeze dryer or a spray dryer, and then pulverized and packaged to prepare a high soybean powder containing active isoflavones.

Formulation Example 3 Preparation of Granules

Into a 300 kg fluidized bed granulator, 80 kg of the whole soybean milk powder prepared in Formulation Example 2 was added and suspended. The whole soymilk granules are prepared by spraying 15% hydroxypropylmethylcellulose (HPMC), which is a thickener, on the powdered soybean powder.

<Formulation Example 4> Preparation of Capsule

To the soybean milk granules prepared in Formulation Example 3, after adding 5% by weight of magnesium stearate to prepare a soybean milk capsule using a capsule filling machine.

Formulation Example 5 Tablet Preparation and Coating

After adding 10% by weight of magnesium stearate (Mg-Sterate) or silicon dioxide (SiO ₂ ) to the soybean milk prepared in Example 2, tablets are prepared using a tableting machine.

After purification, hydroxypropylmethylcellulose, glycerin (glycerine), titanium oxide (TiO ₂ ), talc (Talc) mixture is added by 5% by weight of the coating.

As described above, according to the method for preparing whole soymilk with high content of active isoflavone according to the present invention, it is possible to convert isoflavone glycosides in soymilk into isoflavone nonglycosides with high bioactivity by using lactic acid bacteria of kimchi, In addition, by using the bactericidal action of lactic acid bacteria contained in kimchi to kill the decayed bacteria and harmful bacteria to improve the shelf life of soy milk can be produced soy milk beneficial to health.

In addition, by preparing soymilk processed foods of various formulations using the soy milk, it is possible to easily contact the beneficial soy milk for health, which can help to improve the health of consumers and expect the development of soy milk-related industries.

1 is a photograph showing a microbial extracted from kimchi broth to form a cell colony.

Figure 2 is a photograph showing the identification of microorganisms separated from kimchi broth using the API 50 CHL kit.

3 is a graph comparing the relative beta-glucosidase activity of isolated microorganisms.

Claims (5)

Preparing soybean powder by pulverizing soybeans from which foreign substances have been removed with 550-650 mesh; Preparing soybean milk by adding 10-12 times water by weight to the soybean powder and then boiling for 50-70 minutes; Cooling the whole soybean milk to room temperature; Weissella confusa ( Weissella confusa ) extracted from kimchi to the cooled soybean milk Or Pediococcus pentosaceus Strains Inoculating to 0.05 to 0.15% by weight; And Method for producing high soybean milk, characterized in that the strain is inoculated whole soybean milk inoculated at 35 ~ 40 ℃ 15-30 hours. The method of claim 1, Weissella confusa Or Pediococcus pentosaceus strain extraction method, Filtering kimchi broth of kimchi to remove solids; Stationary culture of the kimchi broth from which the solids have been removed in an ML liquid medium; Serially diluting the cultured culture medium in saline and then plating the plate in BEA medium and culturing to obtain a single cell colony of black color; Delineating the cell colonies again in BEA medium to obtain a single black cell colony around the cell colonies; And The method for producing whole soybean milk having high active isoflavone content, characterized in that it comprises the step of obtaining a strain by identifying the cell community. 3. The method of claim 2, The kimchi soybean milk production method with high active isoflavone content, characterized in that aged for 5-6 months at 3 ~ 7 ℃ after dipping. The method of claim 1, Between the step of inoculating and the fermentation step, removing the solids from the kimchi broth of the kimchi and adding so that the kimchi broth containing 0.5 to 1.5% by weight is added to the active isoflavones high content of soybean production Way. The method of claim 1, After the fermentation step, the step of preparing the fermented whole soybean milk in front of tofu, powder, granules, capsules, tablet formulation is added, characterized in that the active isoflavone high content of soybean milk.

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