Patent Application
20090253643
The present invention relates to an equal concentration regulator, a selective medium for a microorganism having conversion ability to equal and a method of detecting the same.
Isoflavone rich in soybean food is known as a functional component effective in improving menopausal disorders such as malaise, preventing osteoporosis, preventing hyperlipidemia and arteriosclerosis, preventing breast cancer and prostate cancer and so on. Recent studies have revealed that one of the isoflavones called daidzein is metabolized in vivo by intestinal bacteria into equal, which has stronger estrogen action and antioxidation action. Equal has attracted attention as one of the important active ingredients performing the aforementioned actions in vivo.
In-vivo production of equal from daidzein is not equally performed in all humans and the production ability thereof varies between individuals. It has been reported that 30 to 50% of the humans have equal production ability (Non-Patent Document 1). By such the circumstances, research has been enthusiastically conducted for finding intestinal bacteria having equal production ability and substances accelerating production of equal. Microorganisms having equal production ability that have been so far reported are Bacteroides ovatus, Streptococcus intermedius and Streptococcus constellatus (Patent Document 1). It has been reported that an equal containing egg is obtained by giving a feed containing, e.g., daidzein and a soybean oligosaccharide to domestic fowls (Patent Document 2). A fructo-oligosaccharide (Non-patent Document 2) and twintose (R)(Non-Patent Document 3) have been reported to accelerate production of equal. Note that, Patent Document 1 discloses oligosaccharides such as lacto-oligosaccharide, soybean oligosaccharide, lacturose, lactitol, fructo-oligosaccharide and galacto-oligosaccharide as components contributing to survival and growth of microorganisms having equal production ability. However, each of these carbohydrates is mentioned simply as a nutritional component generally known to contribute to survival and growth of microorganisms; however, no mention is made of how these carbohydrates act upon equal production.
A substance exhibiting estrogen action in vivo is generally called an environmental hormone (endocrine disrupting chemical), which may be involved in reduction of sperm cells and reproduction capacity, and an increase in breast cancer occurrence concerning humans. Isoflavone and equal are each known as one of the phytoestrogens. Excessive intake and excessive in-vivo production of them are likely to have an adverse effect upon humans. Particularly when equal, which has estrogen activity several tens fold as high as other isoflavones, is excessively produced in vivo, it is important to suppress the production of equal. However, examples of microorganisms and substances capable of suppressing the production of equal that have been so far reported are Lactobacillus gasseri (Non-Patent Document 4), insulin (Non-Patent Document 5) and fructo-oligosaccharide (Non-Patent Document 6) alone.
Accordingly, it is very important to appropriately regulate the concentration of equal in-vivo in view of not only treating, improving or preventing various diseases as mentioned above but also avoiding adverse effects caused by the environment hormone-like action of equal. It has been therefore desired to develop a substance having a role in regulating the concentration of equal in vivo and capable of being taken for a long time with high safety.
However, microorganisms and substances capable of regulating the equal concentration in viva are only those mentioned above. Choices are extremely limited and their effects are insufficient. In addition, there have been no reports on a selective medium for a microorganism having conversion ability to equal. In the circumstances, it has been strongly desired to develop a selective medium for simply and quickly screening a microorganism having conversion ability to equal and detecting a microorganism having conversion ability to equal in a specimen.
[Patent Document 1] WO99/7392
[Patent Document 2] JP-A-2003-310177
[Non-Patent Document 1] Proc Soc Exp Biol Med, Vol. 217, No. 3, p 335-339 (1998)
[Non-Patent Document 2] J Nutr, Vol. 132, p 2048-2054 (2002)
[Non-Patent Document 3] Summary of lectures of 2005 annual meeting of the Japanese Society for Bioscience Biotechnology and Agrochemistry, p 97 (2005)
[Non-Patent Document 4] Food Research Organization Information, No. 17, p 18-19 (2005)
[Non-Patent Document 5] J Agric Food Chem, Vol. 52, No. 10, p 2827-2831 (2004)
[Non-Patent Document 6] Arch Microbiol, Vol. 183, No. 1, p 45-55 (2005)
Accordingly, an object of the present invention is to provide a medical drug and food and drink having a role in regulating the concentration of equal in vivo and capable of being taken for a long time with high safety, and to provide a selective medium for a microorganism having conversion ability to equal and a detection method thereof.
The present inventors have conducted intensive studies with a view to attaining the aforementioned object. As a result, they found that various types of carbohydrates, which are safe substances widely taken as food for a long time, have a role in raising or reducing the concentration of equal. They further found that a microorganism having conversion ability to equal can be selectively grown in a medium containing a carbohydrate having a role in raising the concentration of equal. Based on the findings, the present invention was accomplished.
More specifically, according to the present invention, there is provided an equal concentration-raising agent comprising, as an active ingredient, at least one element selected from adonitol, arabinose, erythritol, galactose, lactitol, melezitose, trehalose, ribose, sorbose, xylose, inositol and sorbitol; use of at least one element selected from these carbohydrates for producing the equal concentration-raising agent; and a method of raising the concentration of equal characterized by administrating at least one element selected from these carbohydrates in an effective dose.
According to the present invention, there is further provided an equal concentration-reducing agent comprising, as an active ingredient, at least one element selected from glucose, lactose, lacturose, melibiose, raffinose, sucrose and galacto-oligosaccharide; use of at least one element selected from these carbohydrates for producing the equal concentration-reducing agent; and a method of reducing the concentration of equal characterized by administrating at least one element selected from these carbohydrates in an effective dose.
According to the present invention, there is further provided a selective medium for a microorganism having conversion ability to equal, comprising at least one element selected from adonitol, arabinose, erythritol, galactose, lactitol, melezitose, trehalose, ribose, sorbose, xylose, inositol and sorbitol.
According to the present invention, there is further provided a method of detecting a microorganism having conversion ability to equal in a specimen, comprising culturing the microorganism having conversion ability to equal contained in the specimen by using a selective medium containing at least one element selected from adonitol, arabinose, erythritol, galactose, lactitol, melezitose, trehalose, ribose, sorbose, xylose, inositol and sorbitol.
Each carbohydrate to be used in the present invention has an excellent equal concentration-raising action or reducing action and is widely taken as food for a long time with high safety. Therefore, an equal concentration-raising agent or reducing agent according to the present invention can be daily used in safety for appropriately regulating the concentration of equal in vivo. Furthermore, use of a selective medium according to the present invention makes it possible to simply and quickly screen a microorganism having conversion ability to equal and detect the microorganism having conversion ability to equal in a specimen.
Examples of the carbohydrate to be used in the present invention include adonitol, arabinose, erythritol, galactose, lactitol, melezitose, trehalose, ribose, sorbose, xylose, inositol, sorbitol, glucose, lactose, lacturose, melibiose, raffinose, sucrose and galacto-oligosaccharide. The carbohydrate may be D-form or L-form; however, preferably, D-form. Furthermore, an anhydride or a hydrate such as a 5 hydrate may be used.
Trehalose has α,α isomer, α,β isomer and β,β isomer, which differ in the manner of linkage between two glucose molecules. Although any one of these isomers can be used in the present invention, preferably, α,α isomer is used.
Inositol to be used in the present invention has nine stereoisomers: myo-inositol, D(+)-inositol, L-(−)inositol, muco-inositol, scyll-inositol, cis-inositol, epi-inositol, allo-inositol and neo-inositol. Myo-inositol, D(+)-inositol, L-(−)inositol, muco-inositol and scyll-inositol are naturally occurring ones. However, myo-inositol is preferably used in view of availability. Two types or more of inositol stereoisomers may be used in combination.
The galacto-oligosaccharide to be used in the present invention is a general term referring to oligosaccharide having at least one galactose residue in a molecule. For example, a carbohydrate having 2 to 9 mono-carbohydrates linked to each other may be mentioned. Examples of the galacto-oligosaccharide include those galactose having a β1-2 linkage, β1-3 linkage, β1-4 linkage and β1-6 linkage; however, those having a β1-4 linkage and β1-6 linkage are particularly preferable. In the present invention, a mixture of these galacto-oligosaccharides can be used.
In the present invention, a commercially available carbohydrate such as a synthetic carbohydrate and a carbohydrate extracted from natural product may be used. Alternatively, natural occurring material rich in these carbohydrates may be used. More specifically, mention may be made of a material rich in adonitol such as a plant root and a riboflavin-containing material, a material rich in sorbose such as fruit, a material rich in melezitose such as honey or secreting fluid of plant and a material rich in trehalose such as mushroom.
The equal concentration regulating activity of a carbohydrate can be checked by taking feces from a human having ability to produce equal (equal producer), adding daidzein serving as a substrate for equal and a target carbohydrate to the feces thus taken, culturing the feces, determining the concentration of equal in the culture medium and fitting the determined equal concentration to the following equation in comparison with the concentration of equal in a culture medium containing no carbohydrate.
Equal concentration regulating activity (%)=(equal concentration of a culture medium containing a target carbohydrate)/(equal concentration of a culture medium containing no carbohydrate)×100
The feces taken from the equal producer are preferably washed by centrifugation for use. Culture is desirably performed in anaerobic conditions in order to reproduce the state within the human intestinal tract. The concentration of equal can be determined in accordance with a customary method such as liquid chromatography or LC-MS. A carbohydrate having an equal concentration-raising action according to the present invention is one having an equal concentration regulating activity of 200% or more, and particularly preferably, 400% or more. Furthermore, a carbohydrate having an equal concentration-reducing action according to the present invention is one having an equal concentration regulating activity of 50% or less, and particularly preferably, 10% or less.
When the feces taken from an equal producer are anaerobically cultured after adding daidzein and an antibiotic thereto, production of equal from daidzein is inhibited. From this, it is considered that the equal concentration regulating actions of carbohydrates may be mediated by microorganisms present in the feces. More specifically, it is estimated that a carbohydrate having an equal concentration-raising action selectively accelerates growth of a microorganism having conversion ability to equal or enhances the conversion ability to equal of the microorganism; whereas, a carbohydrate having an equal concentration-reducing action selectively inhibits growth of a microorganism having conversion ability to equal or interferes with the conversion ability to equal of the microorganism.
A medium containing a carbohydrate having an equal concentration-raising action can be used as a selective medium for a microorganism having conversion ability to equal. The selective medium contains at least one element selected from adonitol, arabinose, erythritol, galactose, lactitol, melezitose, trehalose, ribose, sorbose, xylose, inositol and sorbitol. Other than these carbohydrates, daidzein serving as a substrate for equal is preferably contained. A medium can be used as long as it contains a carbohydrate, for example, in an amount of 0.3 to 3% by mass and daidzein in an amount of 0.0025 to 0.25% by mass relative to the total amount of the medium in a usable state. Examples of the medium to be used herein include not only a medium that can be immediately used for culturing but also a mixture of medium constituent components except water to be subjected to culture after dissolving in water and sterilizing it. The medium can be used as a liquid medium or a solid medium resulted from adding agar or the like thereto. An antibiotic may be added to the medium in order to increase selectivity of a target microorganism. Colistin and chloramphenicol may be added, for example, in an amount of 1 to 100 μg/ml relative to the total amount of the medium in a usable state. Furthermore, an appropriate component (other than those mentioned above) such as a nitrogen source may be added. However, it is not preferable to use a component that inhibits growth of a microorganism having conversion ability to equal and interferes with the conversion ability to equal of a microorganism. Examples of the component that can be added include peptone, trypticase peptone, yeast extract, hemin, vitamin such as vitamin K1, L-cysteine hydrochloride, KH2PO4, K2HPO4, NaCl, (NH4)2SO4, CaCl2 and MgSO4. A composition of the medium according to the present invention except for a carbohydrate having an equal concentration-raising action and daidzein may be the same as that of PY medium, GAM medium or BHI medium.
A microorganism having equal production ability (daidzein-to-equal conversion) can be screened and obtained by sub-culturing a specimen such as feces in the selective medium while confirming equal conversion ability from daidzein. In addition, a microorganism having conversion ability to equal present in a specimen can be detected by use of the selective medium. A microorganism having conversion ability to equal can be detected, for example, by culturing a specimen in the medium according to the present invention and determining the concentration of equal in the medium. At this time, if the concentration of equal increases, a microorganism having equal production ability is determined to be present in the specimen. In this case, as a control for determining an increase of equal concentration, the medium containing the same components except for a carbohydrate according to the present invention may be used.
A specimen for use in a detection method according to the present invention is not particularly limited. However, when a microorganism having conversion ability to equal is screened, a specimen supposed to contain a microorganism having conversion ability to equal is desirably used. In particular, specimens such as feces and content of the digestive tract are preferably used.
The carbohydrate having an equal concentration-raising action according to the present invention (adonitol, arabinose, erythritol, galactose, lactitol, melezitose, trehalose, ribose, sorbose, xylose, inositol or sorbitol) can be used as an equal concentration-raising agent in the body, blood and intestine such as large intestine. The equal concentration-raising agent containing the carbohydrate as an active ingredient can be used, for example, for treating, improving or preventing various types of diseases in which isoflavone plays a part including menopausal disorders such as malaise, osteoporosis, hyperlipidemia, arteriosclerosis, breast cancer, prostate cancer and premenstrual syndrome. The carbohydrate having an equal concentration-raising action according to the present invention may be used alone or in combination with two or more types.
Furthermore, the carbohydrate according to the present invention is preferably used in combination with daidzein serving as a substrate for equal. A commercially available daidzein such as synthesized daidzein and daidzein extracted from a natural product may be used. Alternatively, naturally occurring material rich in daidzein and a processed product thereof may be used. Specific examples of the material rich in daidzein include soybean, pea, kudzu and crowbar. Examples of the processed product thereof include tofu, soybean milk, fried bean curd, fermented soybeans, soy sauce, soybean paste and tempeh. Furthermore, an isoflavone glycoside is generally converted into an aglycone by the action of intestinal bacterium in vivo. Therefore, glycoside-compounds of daidzein such as daidzin, malonyldaidzin and acetyldaidzin may be used.
The carbohydrate according to the present invention may be used in combination with a microorganism having conversion ability to equal and obtained in the selective medium according to the present invention. When the microorganism is used, the form of the microorganism is not particularly limited. Living bacteria, inactivated bacteria with heat (dead bacteria) or lyophilized bacteria may be used. Alternatively, cultured products containing these bacteria may be used.
The carbohydrate having an equal concentration-reducing action according to the present invention (glucose, lactose, lacturose, melibiose, raffinose, sucrose or galacto-oligosaccharide) can be used as an equal concentration-reducing agent in the body, blood and intestine such as large intestine. The equal concentration-reducing agent containing the carbohydrate as an active ingredient can be used for preventing adverse effects such as reduction of sperm cells and reduction of reproductive capacity caused by an environmental hormone like action of equal. The carbohydrate having an equal concentration-reducing action of the present invention may be used alone or in combination with two or more types.
Appropriate use of the equal concentration-raising agent and the equal concentration-reducing agent according to the present invention enables to appropriately regulate the concentration of equal in vivo. For example, the equal concentration-reducing agent according to the present invention may be applied to infants, young children and pregnant women, who are said to be highly sensitive to environmental hormones, in order to reduce a risk of exposure to equal. Conversely, the equal concentration-raising agent according to the present invention may be applied to the middle aged and advanced aged persons, who have a high risk of menopausal disorders such as malaise, osteoporosis and cancer. It is very important for a person who has no ability to produce equal (non-equal producer) and a person who has less ability to produce equal to use an equal concentration-raising agent on a daily basis in view of prevention of various diseases in which isoflavone plays a part. Whether a target person has ability to produce equal in vivo or not can be checked by measuring the concentration of equal in the urine or blood of the person by a customary method such as HPLC.
The carbohydrate according to the present invention serving as an active ingredient of the equal concentration-raising agent or the equal concentration-reducing agent has been widely used as food for a long time with high safety. Therefore, the dose of the carbohydrate to be used in the equal concentration-raising agent or the equal concentration-reducing agent is not strictly limited. However, it is desirable to specify an appropriate dose since the obtained effect varies depending upon various conditions in use such as persons and diseases to which the agent is applied. The dose thereof is 0.1 mg to 100 g per day and particularly preferably 50 mg to 50 g.
The equal concentration-raising agent and the equal concentration-reducing agent according to the present invention may be administered orally or non-orally; however, oral administration is preferable. The agent can be administered as a common pharmaceutical preparation by blending a carbohydrate serving as an active ingredient with a solid or liquid pharmaceutically nontoxic carrier suitable for administration manner such as oral or intrarectal administration or injection.
Examples of such a preparation include solid agents such as tablets, granules, powder and encapsulated agents; liquid agents such as a solution, suspension and emulsion, and freeze-dried agents. These preparations can be obtained by customary pharmaceutical means. Examples of the pharmaceutically nontoxic carrier include starch, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acids, gelatin, albumin, water and saline. Furthermore, if necessary, customarily used additives such as a stabilization agent, a wetting agent, an emulsifying agent, a binder, an isotonization agent, an excipient may be added.
The carbohydrate according to the present invention may be used not only as a pharmaceutical preparation as described above but also as food and drink. In this case, the carbohydrate according to the present invention may be contained as it is in food and drink or together with various types of nutritional components. A carbohydrate having an equal concentration-raising action can be used as a health food or food material useful for raising the concentration of equal in vivo or improving and preventing menopausal disorders such as malaise, osteoporosis, hyperlipidemia, arteriosclerosis, breast cancer, prostate cancer and premenstrual syndrome. To the food and drink or the containers thereof, a label informing the aforementioned effect of the products may be attached. A carbohydrate having an equal concentration-reducing action can be used as a health food or food material useful for reducing the concentration of equal in vivo or preventing reduction of sperm cells and reproductive capacity caused by an environmental hormone like action of equal. To the food and drink or the containers thereof, a label informing the aforementioned effect of the products may be attached. When a carbohydrate according to the present invention is blended with food and drink, the carbohydrate may be molded into edible form such as granules, particles, tablets, capsules and paste by customary means with the addition of appropriate additives available for food and drink. Alternatively, the carbohydrate can be added to various types of foods such as processed meat products such as ham and sausage, processed fish products such as fish minced and steamed food and tubular fish meat, bread, confection, butter, powdered milk, fermented dairy products and can be added to drinks such as water, fruit juice, milk, soft drinks and tea drinks. Note that animal feed is included in food and drink.
The present invention will be explained more specifically by way of Experimental Examples and Examples. However, the present invention is not limited to these.
Fresh feces of an equal producer were suspended in a dilute solution (10-fold by volume) containing 0.00255% of KH2PO4, 0.00255% of K2HPO4, 0.006% of NaCl, 0.002556 of (NH4)2SO4, 0.000255% of CaCl2, 0.000255% of MgSO4, 0.1% of a 0.1% resazurin solution, 2.2% of an 8% Na2CO3 solution and 0.05% of L-cysteine hydrochloride by use of glass beads (φ 3 mm) in anaerobic conditions and solid matter was removed by use of sterilized gauze. The solution was centrifuged at 8,000 rpm for 10 minutes. Precipitation was suspended in a dilute solution in the same amount thereof. The solution was stored in a freezer at −30° C. as it was.
The feces dilution solution was thawed and centrifuged again. Precipitation was resuspended in PY medium (100-fold by volume) containing 0.5% of peptone, 0.5% of trypticase peptone, 1% of yeast extract, 0.00005% of hemin, 0.0001% of vitamin K1, 0.05% of L-cysteine hydrochloride, 0.0006% of KH2PO4, 0.0006% of K2HPO4, 0.0012% of NaCl, 0.0006% of (NH4)2SO4, 0.00006% of CaCl2 and 0.00006% of MgSO4. At this time, adonitol, arabinose, cellobiose, erythritol, fructo-oligosaccharide, fructose, galactose, glucose, glycogen, inositol, insulin, lactitol, lactose, lactulose, maltose, mannitol, mannose, melezitose, melibiose, raffinose, rhamnose, ribose, sorbitol, sorbose, sucrose, trehalose, xylose, or galacto-oligosaccharide was added so as to obtain a final concentration of 1%. Simultaneously, a series of sample containing no carbohydrate was prepared. To the series of samples, daidzein was added so as to obtain a final concentration of 100 μM. Culture was performed in completely anaerobic conditions at 37° C. for 16 hours. Note that culture was performed independently in four series. From the culture solution obtained, daidzein and equal were extracted and subjected to HPLC analysis.
Note that inositol used herein is myo-inositol manufactured by SIGMA and glycogen was one derived from oyster and manufactured by SIGMA. Trehalose is D(+) trehalose (α,α form) manufactured by SIGMA and galacto-oligosaccharide is TOS-S, which is a mixture of 4′galactosyl-lactose and 6′galactosyl-lactose, and manufactured by Yakult Pharmaceutical Industry Co., Ltd. Fructo-oligosaccharide is a mixture of 1-kestose, nystose and 1-fructosyl-D-nystose manufactured by Wako Pure Chemical Industries, Ltd.
Daidzein and equal were extracted by the following manner. To 500 μL of a culture solution, 250 μL of diethyl ether was added and sufficiently stirred. The solution mixture was centrifuged at 2,000 rpm for 10 minutes to obtain a diethyl ether layer. The remaining aqueous layer was subjected to diethyl ether extraction performed in the same manner. The ether layers obtained in the two extraction operations were combined, concentrated and dried under spray of nitrogen gas flow at 40° C. The dried product was dissolved in 250 μL of 80% methanol and filtrated through a filter to obtain a sample to be subjected to measurement.
Furthermore, HPLC was performed in the following conditions:
Apparatus: LC module 1 (Waters)
Column: YMC-Pack CN (manufactured by Y.M.C.)
Detection: Ultraviolet absorption photometer (determined at a wavelength of 280 nm)
Column temperature: 40° C.
Mobile phase: solution mixture of 0.1% formic acid solution/acetonitrile/methanol (87:3:10)
Flow amount: 2.5 mL/min
Injection amount of sample: 10 μL
Daidzein and equal standard products were poured in the aforementioned conditions to form calibration curves, respectively. In this manner, daidzein and equal concentrations of the samples were determined. Based on the concentrations thus obtained, and in accordance with the equation shown in paragraph [0020], equal concentration regulating activity was calculated.
The results are shown in Table 1 and
On the other hand, in each case of adonitol, arabinose, erythritol, galactose, lactitol, melezitose, trehalose, ribose, sorbose, xylose, inositol and sorbitol, the daidzein-to-equal conversion rate was significantly increased to 200% or more. From this, it was found that these carbohydrates have positive equal concentration regulating activities. In particular, in the cases of lactitol, melezitose, trehalose, sorbose, xylose and inositol, 70% or more of 100 μM daidzein added to the medium were converted into equal. The equal concentration regulating activities were 400% or more.
A feces suspension solution stored in the process according to Experimental Example 1 was added to 500 μL of PY mediums (to which adonitol or sorbose was previously added so as to obtain a final concentration of 1%; and distilled water was added in place of adonitol or sorbose for preparing a control sample) so as to obtain a content of 1/10 and daidzein was added so as to obtain a final concentration of 100 μM. Each medium was cultured at 37° C. for 16 hours in completely anaerobic conditions. After the culturing, an aliquot of 50 μL was taken from the culture solution and transferred to a fresh PY medium (500 μL). The remaining culture solution was subjected to daidzein and equal extraction. Thereafter, the same operation was repeatedly performed. In this manner, sub-culture was performed. At this time, a bacterial solution was appropriately subjected to gram staining and microscopically observed. The extraction of daidzein and equal, and quantification by HPLC were performed in accordance with the method described in Experimental Example 1. Each obtained daidzein and equal concentration was fitted into the following formula to obtain a daidzein-to-equal conversion rate. Note that culture was performed independently in three series to obtain an average conversion rate and standard deviation thereof.
Daidzein-to-equal conversion rate (%)=100×(equal concentration of culture solution)/(addition of equal concentration and daidzein concentration of culture solution)
As a result, as shown in
The following components were mixed in accordance with the following formula, granulated, dried, sized and made into tablets.
The components were mixed by a customary method in accordance with the following formula and homogenized to obtain a soft drink. A brown bottle was charged with the obtained soft drink, sealed with an aluminum cap and subjected to heat treatment.
The components were mixed in accordance with the following formula, adjusted to 1 L with water and sterilized to prepare a selective medium for a microorganism having daidzein-to-equal conversion ability.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-319548 | Nov 2005 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2006/321963 | 11/2/2006 | WO | 00 | 5/1/2008 |
