KR100485267B1 - Fermented soymilk having totally hydrolyzed isoflavones and method for preparation of same - Google Patents

Abstract

The present invention relates to a method for producing soy milk fermented milk and to a soy milk fermented milk prepared by fermenting soy milk using Bifidobacterium sp. Int-57 as a lactobacillus starter, thereby isoflavones in soy milk It is contained in the form of a highly active isoflavone aglycone and decomposes alpha-di-galactosyl oligosaccharide (α-D-galactosyl-oligosaccharide), which causes abdominal bloating of soymilk, which is highly nutritious and digestible. It provides high performance soy milk fermented milk that is easy to absorb.

Description

Soymilk fermented milk isohydrolyzed completely and a method for producing the same {FERMENTED SOYMILK HAVING TOTALLY HYDROLYZED ISOFLAVONES AND METHOD FOR PREPARATION OF SAME}

The present invention relates to a method for producing soy milk fermented milk and to a manufactured soy milk fermented milk, and more particularly, using a Bifidobacterium sp. It relates to a method for producing soymilk fermented milk containing isoflavone aglycone) and to a soybean fermented milk produced.

Soybean is a high protein food that contains essential nutrients. Its composition of essential amino acids is excellent, and most of the fat is composed of unsaturated fatty acids, so it is effective in preventing and treating various diseases including cardiovascular diseases. Its usability is increasing.

Soybean contains a protein hydrolase, an inhibitor of trypsin and a protein called lectin as a hemagglutinin, but since these anti-nutrients are mostly destroyed by heat, there is no problem with food. In addition to proteins, B vitamins, and iron, soybeans contain a phytoestrogen called isoflavone, which is a substitute for estrogen, a female hormone, and is a cancer of breast, colon and prostate cancers. It is a beneficial substance for diseases, cardiovascular diseases and osteoporosis.

These isoflavones exist mainly in the glycoside form of diadzin and genistin, which are hydrolyzed by beta-glucosidase produced by bacteria in the colon and isoflavones. The nonglycoside forms of daidzein and genistein are converted and then absorbed.

Isoflavone glycosides are less physiologically active and have a very low absorption rate compared to their nonglycoside forms.

Soy milk is one of the most easily consumed foods using soybeans. Soymilk is a water extract of soybean and does not contain high protein foods or cholesterol or lactose, so it is deficient in lactase. It has been used as a milk substitute for people who cannot digest lactose.

However, soy milk contains a unique beany flavor and contains indigestible alpha-di-galactosyl oligosaccharides, which can lead to bloating. Ingesting soybeans was not easy, and fermentation methods were introduced to overcome these problems and increase nutritional value, but did not completely overcome them.

In addition, converting the isoflavone glycosides of diaidzin and genistin in soymilk into their non-glycoside forms, diaidzein and genistein, which are highly physiologically active and well digested and absorbed. In the past, acid and alkali hydrolysis methods or purified enzymes were used, but they had disadvantages such as destruction of target substance and high cost, and no method using bioconversion using intestinal resident bacteria was attempted. .

Therefore, the present invention has been invented to improve the problems of the prior art, a method for producing a functional soymilk fermented milk 100% isoflavone glycosides contained in soy milk is converted to a non-glycoside form of high physiological activity and the soy milk fermented milk prepared To provide.

Still another object of the present invention is to provide a method for producing soybean milk fermented milk and soybean milk fermented milk which has been eliminated soybean milk specific symptoms and abdominal bloating symptoms.

In order to achieve this object, the present invention provides a Bifidobacterium sp. Int- with beta-glucosidase activity and alpha-galactosidase activity. 57) provides fermented soymilk with excellent physiological activity in which all of the isoflavones in the soymilk are present in nonglycoside form and relieving abdominal bloating.

Bifidobacterium sp. Int-57 is a organized anaerobic bacterium that lives in the intestinal tract of incubated animals, improves the intestinal flora, reduces serum cholesterol, inhibits the growth of potential pathogens, immune responses and anticancer It is a beneficial bacterium that promotes health by stimulating activity.

According to the present invention, the production of soy milk can be carried out under the conventional general fermentation conditions in which some of the conventional methods are soaked in soybeans and purified, mixed with purified water, and ground.

The fermentation method can be carried out under normal fermentation conditions, and after inoculating Bifidobacterium sp. Int-57 with starter to ferment soy milk, pH 5-7, 30-40 ° C., Preferably fermented soymilk is obtained by incubating at 37 ° C. At this time, the strain should be cultured under anaerobic conditions in consideration of being anaerobic.

Bifidobacterium sp. Int-57 is incubated in BHI (brain heart infusion) medium, but is preferably anaerobic, and therefore cultured in modified BHI medium with cysteine added.

Bifidobacterium sp. Int-57 in soymilk has beta-glucosidase activity, so 100% of isoflavone glycosides in soybean fermented milk in their nonglycoside form In addition to being converted to a conversion rate of, it has alpha-galactosidase activity, and thus alpha-digaractyl oligosaccharide (α-D), a non-digestible sugar that has caused abdominal bloating in soybeans. -Galactosyl oligosacharides) can be decomposed to provide high functional soybean fermented milk with good physiological activity, nutrition, and easy digestion and absorption.

In addition, genuine soybean No. 1 in soybeans lacks two kinds (L-2 and L-3) among three kinds of lipoxigenase isozymes that produce ethyl vinyl ketones that cause soybean-specific soybean odor. Genuine soybean 2 is deficient in all lipoxygenase isozymes (L-1, L-2 and L-3). Do.

In the following Examples will be described the present invention in more detail.

Example 1

Microorganisms and Medium Conditions

Five types of commercial bifidus ( Bifidobacterium animalis ATCC 25527, B. adolescentis ATCC 15703, B.longum ATCC 15707, B. bifidum ATCC 29521 and B. infantis ATCC 15697, purchased from American type Culture Collection) and healthy Korean feces the 10 types of bifidobacteria (Bifidobacterium sp. Int-57, B.bifidum BGN 2, B. bifidum BGN 3, B. bifidum BGN 4, Bifidobacterium sp. RD 13, Bifidobacterium sp. SJ 32, Bifidobacterium sp. RD 54, Bifidobacterium sp. RD 3, Bifidobacterium sp. RD 65 and Bifidobacterium sp. SH5, received by Dr. Geun-keok Ji, Dept. of Food and Nutrition, Seoul National University, among which Bifidobacterium adolescentis Int-57 was deposited by Dr. Geun-keok at Korea Microbiological Conservation Center under the deposit number KCCM 10294 on July 18, 2001).

Cultivation of bifidus bacteria was performed at 37 ° C. using a modified BHI liquid medium in which 5% L-cystein-HCl (w / w) (obtained from Sigma Chemical Co., USA) was added to BHI liquid medium (purchased from Difco, USA). It was used after two passages under anaerobic conditions for 24 hours at. After inoculating 15 kinds of Bifidobacteria in 1% BHI liquid medium, the growth of the strain and β-glucosidase activity were measured at 6 hour intervals.

Example 2

Assay of β-glucosidase Activity

In order to select bifidus bacteria having the highest β-glucosidase activity involved in metabolism of isoflavone glycosides, the activity of this enzyme was analyzed.

The analytical method was analyzed by measuring the degree of hydrolysis of p-nitrophenyl-β-glucopyranoside, that is, the resulting para-nitrophenol.

Cells were recovered by centrifugation of the bifidus culture medium at 11,000 rpm for 15 minutes at 4 ° C, washed twice with 0.2 M sodium phosphate buffer (pH 6.0) and resuspended in the same buffer. Beta-glucosidase activity was determined by adding acetone-toluene solution (volume ratio 9: 1) and 1 mM para-nitrophenyl-beta-glucopyranoside (p-nitrophenyl-β-glucopyranoside). The mixture was reacted at 45 ° C. and then 0.5 M sodium carbonate solution was added to terminate the reaction.

The content of free para-nitrophenol in the supernatant of the reaction solution was measured at 400 nm using a model DU 530 spectrometer (purchased from Beckman, USA). One unit of enzyme was defined as the amount of enzyme that liberated 1 μmol of para-nitrophenol from the substrate per minute. Each experiment was repeated three times, and the value is shown as the average value of these results.

As a result of the experiment, Bifidobacterium sp. Int-57 showed the highest enzymatic activity, so the strain was used for fermenting soy milk of the present invention.

Beta-glucosidase activity in modified BHI medium reached its highest after 18-36 hours of incubation, which corresponds to the growth stop phase of the strain, as shown in FIG. It was.

In addition, as shown in FIG. 1, beta-glucosidase activity of Bifidobacterium sp. Int-57 was 24.40 mU / ml after 6 hours of culture, It suddenly increased during the growth phase and then rapidly declined after reaching its peak of 45.70 mU / ml. However, it can be seen that Bifidobacterium sp. Int-57 shows 15.17 mU / ml of high beta-glucosidase activity even at the end of the incubator.

Example 3

Preparation and Fermentation of Soy Milk

Two types of yellow soybeans, Genuine Bean No. 1 (hereinafter referred to as JP # 1) and Genuine Bean No.2 (hereinafter referred to as JP # 2) and two types of black and black bean No.1 (hereinafter referred to as BL # 1) ) And Black Bean No. 2 (hereinafter referred to as BL # 2) were distributed by the Korea Rural Development Administration.

After washing the soybeans, the soaked soybeans were immersed in purified water at 20 ° C. for 8 hours, followed by crushing the soybeans. After filtering using a cotton cloth, soymilk was prepared by autoclaving at 121 ° C. for 15 minutes.

Thereafter, Bifidobacterium sp. Int-57 was inoculated in soymilk at a concentration of 10 ⁶ cfu / ml and fermented at 37 ° C.

Samples were taken at 6 hour intervals for 24 hours and the viable bacterial count of Bifidobacterium sp. Int-57 was measured using a BL solid medium (purchased from Difco, USA). For anaerobic cultivation, an anaerobic jar (purchased from Difco, USA) and a gas pack system (AnaeroGen ^™ Compact, purchased from OXOID Ltd, UK) were incubated at 37 ° C. for 2-3 days.

Figure 2 shows the viable bacterial count of each soymilk (BL # 1 soymilk, BL # 2 soymilk, JP # 1 soymilk, JP # 2 soymilk). After 12 hours of fermentation, the viable bacterial count of Bifidobacterium sp. Int-57 was more than 10 ⁸ cfu / ml in all soymilk after 12 hours of fermentation. Did not look.

Shulter-Malyoth et al. Pointed out that in order to obtain a health benefit from the consumption of fermented milk, it should contain 10 ⁶ -10 ⁸ cfu of bifidus bacteria per 1 ml of fermented milk, and Bifidobacterium ENT-57 ( Bifidobacterium sp. Int-57) can see this effect when ingesting the soy milk fermented milk of the present invention prepared using the present invention, it can be seen that this strain is a soybean fermented milk starter that can provide a beneficial effect on health.

Example 4

PH and titratable acidity of soymilk

The pH of each sample was measured with a pH meter. Titratable acidity was measured by neutralization titration with 0.1 N sodium hydroxide solution and expressed as% lactic acid. Each experiment was repeated three times, and the results are shown in FIG. 3.

The pH of soymilk began to decrease rapidly after 6 hours of fermentation and did not change significantly after 18 hours. The decrease in pH during soymilk fermentation was the greatest decrease in JP # 1 soymilk with a 2.45 pH unit reduction and 2.38, 2.32 and 2.29 pH units decrease in BL # 2 soymilk, JP # 2 soymilk and BL # 1 soymilk, respectively. Kamaly reported that the pH change by B. longum and B. bifidum decreased by 1.01 and 1.30 pH units, respectively.For fermentation with Bifidobacterium sp. Int-57, It can be seen that the decrease is large.

In addition, Angeles and Marth, soy milk forms a curd (curd) at about pH 5.7, Park and Lee reported that the pH of commercial yogurt ranges from 4.2 to 4.4, in the case of the present invention pH soybean fermentation Bifidobacterium sp. Int-57, which not only reaches the pH range that forms curds after coagulation of soy milk protein after 18 hours of fermentation, but also reaches the pH of commercial yogurt. It can be seen that the soybean fermented milk used has a pH suitable as fermented milk.

In the case of the titratable acidity, BL # 1 soymilk, BL # 2 soymilk, and JP # 1 soymilk increased from 0.18% initially to 1.21, 1.15, and 1.08% at the end of fermentation, respectively. Also, the titratable acidity of JP # 2 soymilk increased to 0.9% after 24 hours fermentation.

Example 5

Analysis of Isoflavones and Beta-glucosidase Activity in Soymilk Fermentation

Analysis of isoflavones was performed using HPLC, and standard of Genistin, Dyzedein and Genistein were purchased from Sigma Chemical Co., USA, and Standard of Dydzin was purchased from Indofine, USA.

Each of the soy milk fermented milk was extracted with 80% ethanol at 50 ° C. for 24 hours, and then the supernatant was filtered through a filter system, and the filtrate was analyzed by HPLC.

The mobile phase used acetonitrile containing 0.1% acetic acid and tertiary distilled water containing 0.1% acetic acid. After injection of 20 μl acetonitrile was increased from 15% to 35% over 50 minutes and held at 35% for 10 minutes. The flow rate of the mobile phase was 1 ml / min, and isoflavones were measured at 254 nm. The isoflavone content and composition change during soymilk fermentation are shown in Table 1 below.

Fermentation time (h) Isoflavone Glycosides (µg / mL)   Isoflavone nonglycoside (μg / ml) DIDJIN Jennysteen Dyed Jane Genistein BL # 1 Soymilk 0 21.25 38.90 9.28 3.33 18 n.d. * n.d. 21.23 19.87 BL # 2 Soymilk 0 47.40 94.08 11.73 8.15 18 n.d. n.d. 42.30 56.26 JP # 1 soy milk 0 98.76 129.61 21.52 15.36 18 n.d. n.d. 57.81 55.15 JP # 2 soy milk 0 59.89 85.43 13.14 8.00 18 n.d. n.d. 48.39 45.94

* n.d .: not found

As can be seen from the above table, the isoflavone glycosides Dydzin and Genistin contained in soy milk were treated by Bifidobacterium sp. Int-57 after a relatively short 18-hour fermentation. By hydrolysis reaction with the resulting beta-glucosidase, it can be seen that 100% is completely hydrolyzed to be converted to dydzein and genistein.

In addition, as shown in Figure 4, beta-glucosidase activity in soymilk fermentation was rapidly increased after 12 hours fermentation and BL # 1 soy milk, BL # 2 soy milk, JP # 2 soy milk after 24 hours fermentation Β-glucosidase activity was increased to 59.528, 40.643, 70.844 and 56.962mU / ml, respectively.

As described above, Bifidobacterium sp. Int-57 is suitable as a starter strain for fermenting soy milk, and fermented soy milk using the same has the following effects.

First, Bifidobacterium sp. Int-57 has high beta-glucosidase activity, so isoflavone glycosides in soybean have excellent physiological activity and absorption rate in the body. 100% conversion to the high isoflavone nonglycoside form provides a functional soymilk fermented milk.

Second, Bifidobacterium sp. Int-57 has alpha-galactosidase activity, so alpha-di, an indigestible sugar that can be a problem when ingesting soy milk. Decomposes galactosyl oligosaccharides (α-D-galactosyl-oligosaccharides) to provide soymilk fermented milk that is easy to digest and absorb.

Third, since Genuine Beans 1 and 2 are deficient in lipoxigenase isozyme, which causes soybean oil-specific soybean odor, soy milk fermented milk prepared using it can also eliminate soybean's peculiar taste. Has the effect.

Therefore, the present invention through the experiment, soybean milk prepared with genuine soybeans lacking the enzyme causing soybeany, and inoculated with Bifidobacterium sp. Int-57 ( Bifidobacterium sp. Int-57) high Soymilk fermented milk is prepared by using beta-glucosidase activity, alpha-galactosidase activity, and suitability as a starter in fermented milk production. It is completely converted into isoflavone non-glycoside and has a high functional soymilk fermented milk with excellent digestive absorption ability.

1 is a graph showing beta-glucosidase activity and cell growth according to incubation time when Bifidobacterium sp. Int-57 was incubated at 37 ° C.

Figure 2 is a graph showing the viable cell number according to the fermentation time when inoculated with soy milk at 37 ℃ Bifidobacterium sp. Int-57 ( Bifidobacterium sp. Int-57).

Figure 3 is a graph showing the pH change with the fermentation time when inoculated with soy milk at 37 ℃ Bifidobacterium sp. Int-57.

Figure 4 is a graph showing the beta-glucosidase (β-glucosidase) activity according to the fermentation time when inoculated with soy milk at 37 ℃ Bifidobacterium sp. Int-57.

Claims (5)

Method for producing soymilk fermented milk comprising the step of fermenting soy milk using Bifidobacterium sp. Int-57 as a lactic acid bacterium starter. The method for producing soymilk fermented milk according to claim 1, wherein the soymilk fermentation time is 18 hours. According to claim 1, Bifidobacterium sp. Int-57 ( Bifidobacterium sp. Int-57) is a method for producing soymilk fermented milk, characterized in that cultured in a medium added cystein (cystein). The method for producing soymilk fermented milk according to claim 1, wherein the soybean is a genuine soybean. Soymilk fermented milk comprising isoflavones prepared by the method of any one of claims 1 to 4 in the form of a non-glycoside.