TWI377254B - Process for preparation of aglucone isoflavones - Google Patents

Description

1377254 6. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a method of preparing a composition having high phytoestrogenic activity. In particular, the present invention relates to a process for preparing a composition having a high concentration of deglucoside soy isoflavones. The invention is also directed to a composition having a concentration of deglucose soy isoflavones. [Prior Art]

Soy isoflavones such as genistein and daidzein are structurally similar to estrogens and are therefore referred to as "phytoestrogens". Soy isoflavones are known to be involved in certain health-promoting processes, such as the prevention of cancer (Cappe Uetti et al., 2000; Miura et al., 2002; Ravindranath et al., 2004), and the reduction in cardiovascular disease risk (Anth〇ny et al. , 1996;

Goodman-Gruen and Kritz-Silverstein 2001), improvement of bone health (Cotter and Cashman 2003; Weaver and Cheong 2005) and relief of postmenopausal syndrome. In detail, soy isoflavones have been clinically proven to have a therapeutic effect on osteoporosis. 'Including inhibition of bone loss and bone resorption in women after menopause, and can promote bone formation, and no adverse effects on the breast and uterus were observed. The soy isoflavone content in plants is extremely low. For example, soybeans contain only about 0.1/〇 (w/w) soy isoflavones. In addition, the soy isoflavones in plants are in the form of glucoside soy isoflavones which are hydrolyzed to deglucoside soy isoflavones and then absorbed by the intestine (Setche et al, The J〇urnal 〇f Nutrition 2001, Vol. 131 , 1362S 1375S). Studies have shown that the concentration of soy isoflavones in the blood after the administration of sucrose sucrose (4) is 2 to 5 times higher than the concentration of soy isoflavones in the blood after the glucoside soy isoflavones are added to 113300.doc 1377254 ( Izumi et al. 'The J〇urnal Nutriti〇 I1 2000, Vol. 130 '1695·1699). Despite this, the ability of the human intestine to hydrolyze soy isoflavone glucosides decreases with age', causing health problems in the older population. One way to address these problems is to provide de-glucose soy isoflavones directly in food or food supplements. U.S. Patent Nos. 5,320,949, 5,352,384, 5,637,561, 5,637,562, 5,726,034, and 5,821,361 disclose the use of plant- or microbial-based β-glucosidase to convert a substance containing glucoside soy isoflavone A method of containing a substance derived from glucoside soy isoflavones. There are also other patent documents which disclose the use of microbial glucosidase to hydrolyze glucoside soy isoflavones to deglucoside soy isoflavones. For example, U.S. Patent No. 5,554,519 teaches a method for the acidification of soybeans to produce genistein using Saccharopolyspora erythraea; PCT Patent Application No. PCT/JP94/02103 teaches a self-use fungus (k0ji) a method for preparing a glucoside soy isoflavone by a yeast bean crop; and the patent application 2003070439 teaches the addition of purified β-glucosidase produced by Penicii Hum multicolor and other cockroaches to In the preparation of natto, to enhance the deglycosylation of soy isoflavones. A method in which black bean acid is fermented by Bacillus subtilis natto to convert glucoside soy isoflavones in black beans into deglucoside soy yellow is also disclosed (Kuo et al., 2006). H3300.doc 1377254 However, the above method can only produce a product having a low content of deglucosidin soy oxime, and cannot be applied to a large-scale preparation in the field of medicine or health food. Therefore, there is still a need to develop a method for preparing a high concentration of glucoside glycoside. SUMMARY OF THE INVENTION The present invention provides a method for preparing a composition comprising a high concentration of deglucosidin soy isoflavones, which comprises adding glucoside soy isoflavones to a soy matrix which is fermented by microorganisms, which is generally considered safe And can express or produce β-glucosidase. The present invention further provides a method for preparing deglucoside soy isoflavones, wherein glucoside soy isoflavones in soybean-based substances are deglycosylated by using glucosidase, and the improvement is to add glucoside soy isoflavone . The present invention also provides a composition comprising a high concentration of deglucoside soy isoflavones, which is prepared according to the method of the present invention. [Embodiment] The present invention provides a method for preparing a composition containing a high concentration of deglucosidin soy isoflavones, which comprises adding glucoside soy isoflavones to a soybean matrix in a microorganism which is generally considered safe and Express or produce beta-glucosidase. As used herein, the term "glucosinolate isoflavone" refers to plant glucoside soy isoflavones such as daizin and genistin, and A "deglucoside soy isoflavones" means, for example, soy yellow鲷 and genistein plants to glucoside soy isoflavones. 113300.doc 1377254 As used herein, the term "soybean substrate" refers to a substrate containing soy or derived from any form of soybean. Soy-based matrices suitable for use in accordance with the present invention include, but are not limited to, legume crops, soybean meal, soy milk, soy flour, black soybean meal, black soy milk, black soy flour, and the like. Soybean matrix fermentation can be carried out in accordance with methods known in the art, such as those taught in the references mentioned above, in a microorganism that is generally considered safe and can exhibit or produce a terrain. The soy substrate can be sterilized by conventional methods prior to inoculation of the microorganism, for example, autoclaving 0 inoculation of the microorganism to a concentration of 1〇2 cfu/ml at the start of fermentation, and 1〇5 cfu/mi at the end of the fermentation to The range of i〇i2cfu/mi. Preferably, the concentration of the microorganism during the fermentation is 103 efu/n^1() 6 efu/mk range at the start of the fermentation and (10) cfu/ml to 1010 cfu/ml at the end of the acid fermentation. Optimally, the concentration of the microorganism during the fermentation is 1〇5 cfu/m b at the start of the fermentation and cfu/ml at the end of the fermentation. The fermentation of the present invention can be generally considered safe and manifests or produced. A microorganism of glycosidase. Such microorganisms include, but are not limited to, Actinomucor elegans, Taiwan's Phytophthora (A to ~ (10) neem), Aspergillus awamori, A 〇ryzae, soy sauce 麴Bacterium (A. Sojae), Bacillus subtilis, Bacillus subtilis (natto), Bifidobacterium animalis, B breve, B. infantis, Bifidobacterium longum (B"〇ngum), B. thermophilum, Candida spp, 113300.doc 1377254 Debaryomyces spp, Ganoderma lucidum, Lactobacillus acidophilus (Lactobacillus acidophilus), L. casei, L. delbrueckii, L. paracase, Lactobacillus (L. piantarum), Lactobacillus lactis ( Lactococcus lactis), Monascus spp, Mucor spp, rhiz〇pus azyg〇Sporus, Sacchar〇myces spp, red sugar Hobby Ball key letter (Streptococcus thermophilus) and joining the Saccharomyces genus (Zygosaccharomyces spp). The best is the Bacillus subtilis strain. After inoculating the bacteria, they are cultured for a while under suitable conditions. For example, the culture can be carried out at a temperature of about TC (about TC to about 6 (TC). The culture can also be carried out at a shaking rate of about 2 〇Πμπ to about 2000 rpm. Preferably, the culture is carried out at about 20 C to about 40 C. At a temperature and at an oscillating rate of from about 5 rpm to about 1 rpm, optimally, the culture is incubated at a temperature of from about 10 rpm to about 2 rpm.酦 进 进 亍 亍 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又 又+ The time of this can be determined by those skilled in the art. Generally, the η±ρ日° time can be from about 8 hours to 240 hours, preferably from about 20 hours to 120 hours. β-Glucosidase is known. Using its servant ~ degluconosine glucoside soy isoflavones to prepare a large glucosinolate enzyme will be produced in the reaction, the « ^ ^ sugar inhibition, making it difficult to obtain high concentrations of glucoside Soybean different yellow stuffed ^ and again tongue 113300.doc [S] 1377254

Surprisingly, it has been found that when glucose soy ketone is added during fermentation, &&> is highly concentrated during the use of the microorganism to ferment the soy substrate according to the present invention. It is believed that the glucoside soy isoflavones added according to the present invention are glucosylated when the soybean substrate is fermented by microorganisms, and the glucose produced is used as a carbon source for bacterial proliferation, thereby enhancing bacterial proliferation, which in turn produces high concentrations. Glucosinolate isoflavones. Compared with the conventional method (in the case of a microorganism capable of producing glucosidase required for glucosylation, fermenting a substance containing glucoside soy isoflavone), the method of the present invention significantly enhances the glucose-depleted soybean produced by the method of the present invention. The amount of isoflavones. The present invention further provides a method for preparing glucosinolate isoflavones, which uses deglycosylase to glucosylose isoflavones in soy-based substances, and the improvement is to add glucosides and soy Flavonoids. According to the invention, it can be by batch, batch feed or continuous feed

The glucoside soy isoflavone is added to the soy-based matrix fermentation of the microorganism. As is well known in the art, the fermentation can be carried out in any suitable container including, but not limited to, flasks, decimators, and bioreactors. The method of the present invention produces a product having a high concentration of deglucoside soy isoflavones, which exhibits high phytoestrogen activity, that is, high receptor affinity and low a-receptor affinity, and thus is suitable for use in the medical field or Healthy Food Field 0 113300.doc ί S] 1377254 The present invention further provides a composition comprising a high concentration of deglucose soy isoflavones prepared according to the method of the present invention. Since the composition of the present invention has high phytoestrogenic activity, it can be used in medicines or health foods to maintain bone density, reduce cardiovascular risk, prevent cancer (such as breast cancer and prostate cancer) from occurring, and alleviate postmenopausal syndrome. The product of the method of the present invention can be processed by conventional methods to provide a form suitable for use in a pharmaceutical or health food order. The composition of the present invention may be formulated into a form of a key, a pill, a powder, a solution, a syrup or the like according to various needs. For example, the composition can be freeze dried in a conventional manner. The specific examples that follow are illustrative only and are not intended to limit the remainder of the disclosure. It is believed that those skilled in the art will be able to utilise the use of the present invention to the fullest extent. EXAMPLES Example 1 A 100 m crucible containing 5% (w/v) soy cypress was prepared in a SOOnd flask and was at 121. Autoclaving under the armpit for 15 minutes. The Bacillus subtilis was then inoculated into the medium at 37 in the shaker. (:, 125 rpm culture. At the beginning of the culture, the number of bacteria was 9.6 x 1 〇 3 cfu / m for 12 hours, the number of bacteria was 6 x 108 Cfu / m, and it was maintained until the end of the culture (in hours after inoculation). Nitrophenyl-β-D-glycoside (PNPG) was used as a substrate to determine the enzyme activity of β-glucosidase throughout the culture. Beta-glucosidase activity was detected at 8 hours after inoculation, 15th Activity increased to a maximum (3.3 U/ml) from hour to hour 18, followed by β-glucosidase activity 113300.doc -9- 1377254 = as low as 〇·3 u/mi ^ daidzein and dye after 21 hours of culture The concentration of lignin was 76.5 μM and 82.7 μΜ at the beginning of the culture, and began to decrease after 8 hours of culture. The concentration of daidzin and genistein was 25 9 ^^ and t 5 ^Μ, respectively. The concentrations of flavonoids and genistein increased from 28.5 μΜ to m.6 μΜ in i2 hours and from ι〇ΐ 6 μΜ from 54 2 μΜ (Figure 1). Example 2 In a 5〇〇ml flask Prepare 100 ml of 25 〇/〇 (w/v) soybean meal medium, and add the soy isoflavones containing glucose (containing 1 〇m called soybeans). After autoclaving for 15 minutes at 121 °c, the woody grass was inoculated into the medium, and in the shaker, 37. 〇, (2) 『(4) was raised only. When the culture started, the number of bacteria was 10405 efu/ml. After that, the number of raw i was 6.9×10 cfu/m b and was maintained until the end of the culture (four) 24 hours later. The enzyme activity of glucose (iv) was monitored throughout the culture. The β-glucosidase activity was 8 hours after inoculation. It was detected and gradually increased. Its activity reached its maximum at 12 hours after inoculation, and decreased to h/mI between h7 and (4) hours after culture and time. The whole culture was studied. During the period, the glutathionization of glucosinolates of soybean isoflavones was carried out. The concentration of soybean bitter and dyed wood buds was pulsed at the beginning of the culture for 3 hours and cultured for 8 hours. Soybean answered and dyed the rough female one. ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 2 _ and 116 μΜ, while the concentration of soybean yellow net wood phase is at U hour Internally increased from 35 5 _^^4 II3300.doc 1377254 μΜ and increased from 26.9 μΜ to 168 〇μΜ. At the 21st hour after inoculation, all soybean meal (4) wood was converted to daidzein and dye wood yellow steel, large and xanthine and dye The concentrations of the wood phase were 6 μΜ and 2〇6·3 μΜ (Fig. 2). Example 3 1 〇〇mk 5% (w/v) soybean cedar medium was prepared in 00 ml burned, and the addition contained heat extraction. A substance of glucosinolate soy isoflavones (containing 〇 〇 、 、 、 、 、 、 、 、 、 、 、 、 After 12 r (: autoclave (10) minutes, 1 ml of pre-cultured B. natto was inoculated into the medium, and cultured in an oscillator at 37 ° C, 250 rpm. After 15 hours of incubation, glucoside was fed every 6 hours. Soy isoflavones, a total of 8 feeds and a total of 266 mg of genistein and 1〇36 mg of daidzin. The results showed that the bacteria proliferated between the first hour and the tenth hour, and the number of bacteria at the start of the culture was 8.5x104 efu. /ml 'The first hour is increased to i 7χ1〇9 cfu/inl, then enter 1.4\108. 仏/1111 to 2.7<108.!'11/1111 stable period until the end of culture (63 hours after inoculation) The p_glucosidase activity was detected and gradually increased at 12 hours after inoculation. Its activity reached a maximum of 9 7 u/ml at 27 hours after inoculation, and then decreased to 6,3 after 45 hours of culture. U/m b after 8 feedings, that is, at 63 hours after inoculation, the concentrations of daidzein and genistein in the medium reached 1 1548.0 μΜ and 2636.1 μΜ, respectively (Fig. 3). The pH value varied from 7.5 to 8.0. After lyophilizing the product, each gram of powder contains 66 mg of daidzein and 24 mg of genistein. The content of soybean which is 100 times. Example 4 I13300.doc [S] 1377254

2 L of 5 〇/〇 (w/v) soybean meal medium was prepared in a 5 L·acid starter and autoclaved at 121 C for 15 minutes. The precultured 10 ml of Bacillus subtilis Bacillus was inoculated into the medium, and cultured at 37 ° C, 700 rpm under aeration and agitation. The results showed that the bacteria proliferated between the 1st hour and the 12th hour. 'The number of bacteria at the start of the culture was 7 2xl 〇 5 Cfu/m. The 12th hour increased to 2.3x 109 cfu/m b and entered the stable phase until the end of the culture ( 48 hours after inoculation) (Fig. 4A). A continuous feed of the glucoside soy isoflavone matrix was carried out 8 hours after the inoculation. The glucoside soy isoflavone solution was fed at a rate of i 5 ml/mm from 8 hours to 2 hours after inoculation for 1 hour. At the end of the feed, daidzein and genistein were 2464 μΜ and 541 μΜ, respectively (Fig. 4B). Example 5

A sample of the fermentation medium of Example 3 in which the glucosinolate isoflavone conversion was carried out in a bioreactor by batch feeding was taken out at the second hour, 21 hour, 51 hour, and 63 hours after inoculation. The bed was dried and tested for estrogenic activity. The concentration of estrogen activity in a 50% ethanol solution was 1 ng/ml & 100 ng/m. The ch〇-K1 cells (ATCC, CCL 61 were cultured at a density of 2.5 xi 4 cells/well in %). The well plate was incubated with Ham's F-12 medium containing 10% fetal bovine serum at 37 ° C, 5% (: 〇 2 for 24 hours. Then use the reported vector pBK_CMv_GaM_ 1^ ruler (1 (or 0) and mouth 8 〖-匸1^-(1;8-8)4-11<;-test phosphatase (eight?) Transfect the cells for 5 hours. After that, remove the medium containing the transfection agent and dilute the medium to be tested. Samples. 17β-estradiol (E2, Sigma, E2758) was used as a control. Estrogen activity test samples and controls were subjected to estrogen activity 113300.doc ^2 '[S] 1377254 for 48 hours, followed by 48 hours. From the culture ^ θ ^ ^ ; Take a certain amount of medium to determine the alkaline % acid enzyme (ΑΡ) activity. Take the hexahydrate σ field basic bis-bisaluminate salt (ΡΝΡΡ) as the substrate, and in the 96-well plate The sample was reacted for 15 minutes and then the absorbance was measured at 405 nm.

The data show that the product obtained according to the present invention does not make the product obtained by the de-glucosinolate-yellow-yield conversion according to the present invention, whether it is at 10 ng/ml or at 100 ng/ml T, compared with the clinically-producing (iv) β. estradiol. Increased expression of α·receptors (the product of gastric transformation will not increase the risk of breast cancer-related diseases. At the same time, whether at 10 ng/ml or at 1 ,, the sample will make the β receptors follow The conversion time increased and increased (Fig. 5b). This result indicates that the product has a positive effect on increasing bone calcium absorption and inhibiting cardiovascular disease. [Simplified illustration] Figure 1 Deglycosylation of soybean isoflavone glucoside in flask Figure 2. Deglycosylation of soy isoflavone glucosides in flasks.

Figure 3 shows the preparation of deglucose soy isoflavone in a batch feed in a flask. Figure 4A shows the number of cells in the bioreactor for the continuous preparation of glucosinolate soy isoflavones. Figure 4B shows the concentration of soy isoflavones in the bioreactor for the continuous preparation of glucosinolate. Figure 5A Phytoestrogens activity (alpha-receptor) during biotransformation. Figure 5. Phytoestrogens activity (beta-receptor) during biotransformation. 113300.doc •13··

Claims (1)

Patent Application No. 098103083 Those who can express or produce β-glycosidase, wherein the high concentration of glucosinolate isofonone is 699.6 μΜ and 206.3 μΜ, respectively, daidzein and genjstein. 2. The method of claim 1, wherein the glucoside soy isoflavone is added by batch, batch feed or continuous matrix feed. 3. The method of claim </ RTI> wherein the microorganisms are selected from the group consisting of: 'and the group of 'radial hairy hairs'), Taiwan's Phytophthora taiwanensis, 'Aphis faecalis aw (10) 〇rZ), rice bran Bacteria (child, whole oil fungus (baby kiss), Bacillus subtilis μ such as / (4), Bacillus subtilis (5ac (eight) w Subtilis natto), animal double scorpion (mfid〇bacterium a«z_ma/z\s), short Type Bifidobacterium (and office, infant Bifidobacterium / « / (3 « along), long Bifidobacterium (B. / 〇 „gWW), thermophilic bacillus (_g. i / ^ rwo /? / N7ww), Candida (Ca «heart ί/α spp) 'Debaryomyces spp &gt; $ {Ganoderma lucidum) ' 'f ik % Acid bacillus (Z^cio6acz7/w izci'afop/n '/wj), Lactobacillus casei (Z. ca*sez), Lactobacillus delbrueckis (L. G?ei6rwecA: /z·), Lactobacillus paracasei (Z. paracase), Lactobacillus plantarum (I, Lactobacillus lactis (Laciococcws / ac&quot;··?), Rhodobacter genus (Λ/οπαΜΜί spp), Mucor spp, and Rhizopus genus (and; „·ζ〇尸似I13300-I010113. Doc Rl377254 azjvgosporws), Saccharomyces spp), red sugar sleigh (Saccharopolyspora eryi/7rae&lt;3), Streptococcus thermophilus {Streptococcus i/iermop/n./ws) and Zygosaccharomyces spp ° 4 The method of claim 3, wherein the microorganism is Bacillus subtilis natio. The method of any one of claims 1 to 4, wherein the fermentation is carried out at a temperature of from about 10 ° C to about 60 ° C at an oscillating rate of from about 20 rpm to about 2000 rpm for about 8 hours to about 240 hours. 6. The method of claim 5, wherein the fermentation is between about 2 ° C and about 4 〇. The enthalpy at a temperature of from about 5 rpm to about 1000 rpm is carried out for about 2 hours to about 240 hours. 7. The method of claim 6, wherein the fermentation is at about 35. 〇 to about 4 〇. The enthalpy at a temperature of from about 12 rpm to about 2 rpm is carried out for about 2 hours to about 120 hours. 8. 9. 10. A method for preparing a deglucoside soy isoflavone comprising the deglycosylation of glucose # soy isoflavone in soybean base f with β-glucose (IV), modified by the addition of glucoside soy isoflavones. Two: The method of Item 8 wherein the glucoside soy isoflavones are added in a human, batch feed or continuous matrix feed. A method comprising a high concentration of deglucoside according to any one of items 1 to 8 of the composition of claim 7 &quot; ketones. 113300-10101 i3.d〇,