JP2011217696A - Method for producing pinitol or pinitol-containing composition, and microorganism used for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing pinitol or a pinitol-containing composition, capable of obtaining the pinitol in a short period of time and also in a high purity by a relatively simple treatment while inhibiting the proliferation of sundry bacteria, and microorganisms used for the same.SOLUTION: This method for producing the pinitol is provided by comprising a process of using a pinitol-containing plant material as a resource and performing the culturing of the microorganisms shown in (A) in a high temperature region: (A) bacteria belonging to the thermophilic genus Geobacillus, and having a carbohydrate-decomposing function except the pinitol.

Description

  The present invention relates to a method for producing pinitol or a pinitol-containing composition and a microorganism used therefor.

  Pinitol (3-O-methyl-D-kilo-inositol), which is a kind of inositol, is a natural component present in many plant bodies such as locust bean, soybean, black soybean, pine, clover, pine, etc., and D-kilo -It has a chemical structure in which the hydroxyl group at the 3-position of inositol is methylated to form a methoxy group.

  Pinitol is known to have a hypoglycemic action by alleviating insulin resistance, like D-kilo-inositol (see Non-Patent Document 1). Pinitol has been reported to improve type 2 diabetes caused by insulin resistance and its complications such as liver damage and cataract, as well as anti-inflammatory action and creatine accumulation effect (non-patented). Reference 2-5).

  In recent years, pinitol has been reported to have an obesity-inhibiting effect without causing side effects, particularly an obesity-inhibiting effect caused by the accumulation of subcutaneous fat, and the development of anti-obesity agents using such pinitol has also been reported. (See Patent Document 1).

  Various methods for extracting pinitol from the plant as described above have also been studied (see Patent Documents 2 and 3).

JP 2009-196931 A JP 2009-67716 A Japanese Patent No. 3806663

Kim JI et al., EJCN. 59, 456-458 (2005) Son Dong-gong, Journal of Pharmaceutical University of Pharmaceutical Sciences (2003) Akira Kinzai, Chuo University School of Medicine research report (2004) Geenwood M et al., J. Exercise Physiology 4 (4), 41-47 (2001) Singh RK et al., Fitoterapia72,168-170 (2001)

  However, it is actually quite difficult to extract pinitol with high purity from a plant body as described above. The reason is that carbohydrates other than pinitol (general carbohydrates in plants, such as glucose and sucrose) and contaminants can be obtained only by treating the extract from the plant by physical and chemical methods. This is because they are extracted at the same time and it is difficult to separate these carbohydrates from pinitol.

  As a technique for solving such a problem, for example, in Patent Documents 2 and 3 described above, a technique for extracting pinitol with high purity by passing through a sugar assimilation process using a microorganism having no ability to assimilate pinitol, such as yeast. Has been proposed.

  However, since sugar assimilation by microorganisms such as yeast as described above is usually performed at room temperature, which is the activation temperature of the microorganisms, there is a concern about the propagation of miscellaneous bacteria. When miscellaneous bacteria propagate, pinitol may also be decomposed and consumed. Therefore, complicated processes such as a sterilization process are required. In addition, the microorganisms conventionally used for the above uses do not have so high performance required to achieve the object purpose of obtaining pinitol with high purity, and there is still room for improvement in this respect. Furthermore, the sugar assimilation step by the microorganism usually requires a long fermentation period and is not preferable in terms of productivity, and improvement is also required in this respect.

  The present invention has been made in view of such circumstances, and a pinitol or pinitol-containing composition capable of obtaining pinitol in a short period of time and with high purity while suppressing the propagation of various bacteria by a relatively simple treatment. The object is to produce a product and to provide a microorganism used therefor.

In order to achieve the above object, the present invention provides a method for producing pinitol or a pinitol-containing composition comprising a step of culturing bacteria as shown in (A) below in a high temperature region using a pinitol-containing plant as a resource. It is set as the summary of 1.
(A) A thermophilic Geobacillus bacterium having a resolution of carbohydrates excluding pinitol.

  The second gist of the present invention is Geobacillus kaustophilus PS8 (FERM P-21938), which has the resolution of carbohydrates excluding pinitol.

  That is, the present inventors have intensively studied to solve the above problems. In the course of the research, the present inventors recalled that the sugar utilization process of the pinitol-containing plant body was performed in a high temperature range in order to suppress the propagation of various bacteria, and sought a thermophilic bacterium that could be adapted to it. In the process, we examined the use of a thermophilic bacterium, Diovacillus sp., And as a result of repeating various experiments, it was found that a variety of bacterium belonging to the genus Geobacillus has a resolution of carbohydrates excluding pinitol ( (Mutant strain) was found. Then, by using the plant body as a resource and culturing the bacterial body in a high temperature range, it is possible to easily obtain high-purity pinitol (or a pinitol-containing composition) while suppressing the propagation of various bacteria. The present inventors have found that pinitol can be obtained in a short period of time and with a high purity as compared with microorganisms such as yeast conventionally used for sugar assimilation treatment.

  As described above, the pinitol (or pinitol-containing composition) production method of the present invention uses a pinitol-containing plant as a resource, cultures thermophilic Diobacillus bacteria having special performance in a high temperature range, and a sugar assimilation treatment This is done through a process such as As a result, pinitol can be obtained in a short period of time and with high purity while suppressing the propagation of various bacteria by a relatively simple treatment. Moreover, since it is advantageous also in terms of manufacturing cost, it is industrially useful. And the pinitol (or pinitol-containing composition) obtained by the production method of the present invention is excellent in the improvement / reduction of symptoms such as abnormal blood glucose levels, diabetes, liver disorders, cataracts, obesity and the like without side effects. The effect can be demonstrated.

  In particular, when the thermophilic Geobacillus genus bacterium is Geobacillus kaustophilus PS8 (FERM P-21938), the purification efficiency of pinitol (or a pinitol-containing composition) becomes more excellent.

  Geobacillus kaustophilus PS8 (FERM P-21938) is a novel microorganism newly discovered by the present applicant. This strain does not have the ability to degrade pinitol, but it does not have the ability to degrade pinitol. The ability to decompose impurities is extremely high. Therefore, as described above, it can be advantageously used in the production method of pinitol (or pinitol-containing composition) of the present invention. In addition to pinitol, this strain has been confirmed to have no resolution for inositol such as myo-inositol, D-kilo-inositol, scyllo-inositol, and therefore, this strain is inositol other than pinitol. It is also possible to adapt to the manufacture of

2 is a chromatogram showing the content of pinitol and other carbohydrates in the concentrate obtained by the production method of Example 1. 2 is a chromatogram showing the content of pinitol and other carbohydrates in the concentrate obtained by the production method of Comparative Example 1.

The method for producing pinitol or a pinitol-containing composition of the present invention comprises a step of culturing bacteria as shown in (A) below in a high temperature region using a pinitol-containing plant as a resource. The “pinitol or pinitol-containing composition” refers to both pure pinitol and one containing components other than pinitol.
(A) A thermophilic Geobacillus bacterium having a resolution of carbohydrates excluding pinitol.

  In addition, “having the resolution of carbohydrates except for pinitol” in the above (A) does not mean “having the resolution of all carbohydrates except for pinitol”. , Sucrose and other common carbohydrates (carbon sources) in plant bodies are efficiently decomposed and consumed. And the thermophilic Geobacillus bacterium which has such special performance can be obtained by collection of microorganisms in a specific habitat, selection by so-called screening, or the like. With regard to the screening process, for example, a mutant selected by treatment with ethyl methanesulfonate is subjected to penicillin screening or the like. As a result, it is possible to select cells that can grow on a medium using sugars such as glucose and sucrose as a carbon source but cannot grow on a medium containing only pinitol as a carbon source (pinitol-free strain). it can.

  The ethyl methanesulfonate treatment is performed to convert guanine / cytosine to adenine / thymine by adding a methyl group to the DNA base. In addition, the penicillin treatment on the cells in the above-described penicillin screening is performed to prevent cell division by inhibiting the action of cell wall synthase when the cells grow. In addition, since the above-mentioned penicillin treatment kills only the cells that have started to divide cells, the cells start to divide when there are available nutrients, but stop when there are only unusable nutrients. Thus, it is possible to select only the non-pinitol-utilizing strain as described above.

  It can be confirmed, for example, by measuring the inositol dehydrogenase activity of the microbial cells that the microbial cells obtained as described above do not have pinitol resolution. That is, if the above microbial cells do not have the resolution of pinitol, inositol dehydrogenase activity is also deleted.

And the inositol dehydrogenase activity in the said microbial cell can be confirmed as follows, for example. That is, first, cells cultured in a medium using inositol (pinitol, myo-inositol, etc.) as a carbon source are collected by centrifugation, then washed repeatedly, and then treated with chemicals, centrifuged. Separation treatment, ultrasonic treatment, etc. are performed to obtain an enzyme solution. Then, the enzyme solution, a buffer for measuring enzyme activity (5 mM β-NAD ⁺ 100 ml, 1 M Tris-HCl [pH 8.0] 100 ml, dH ₂ O 660 ml), and 0.5 M inositol as a substrate were each isothermal. The mixture is warmed to 55 ° C. in a tank, 860 ml of buffer and 40 ml of enzyme solution are mixed in the cell, and 100 ml of 0.5 M inositol is further added to the cell and mixed. Inositol dehydrogenase activity is determined by measuring the increase in absorbance at 340 nm of the reaction solution thus obtained over time and applying it to the following formula (1).

  Among the thermophilic Geobacillus bacteria having the above-mentioned characteristics (A) used in the production method of the present invention, in particular, Geobacillus kaustophilus PS8 (Geobacillus kaustophilus PS8, FERM P-21938) contains pinitol (or pinitol). This is preferable because the purification efficiency of the containing composition is more excellent.

  The above-mentioned strain is a strain deposited by the present applicant at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and is a mutant strain of Geobacillus caustophilus HTA426 (JCM12893) obtained by penicillin screening. This strain is almost completely lacking inositol dehydrogenase activity and cannot grow using inositols as a single carbon source. Have. That is, it has been confirmed that in addition to pinitol, it has no resolution for inositol such as myo-inositol, D-kilo-inositol, and scyllo-inositol. Therefore, the use of this strain can be applied to the production of inositol other than pinitol. The strain can be stored frozen at −80 ° C. after mixing 250 ml of 80% glycerol with 750 ml of the culture solution.

  Examples of the pinitol-containing plant used in the production method of the present invention include locust bean, soybean, black soybean, pine, clover, lotus, carnation, bougainvillea and the like. In these plants, the site containing a large amount of pinitol varies depending on the type of plant. That is, pinitol is contained not only in seeds (beans and the like) but also in pods, stems, and leaves. In particular, in soybean (including black soybean), it has been confirmed that the portion to be discarded (sheath, stem, leaf) after removing the seed has a higher pinitol content than the seed (bean). Therefore, as the above-mentioned pinitol-containing plant body, by using such a soybean disposal target site, the material cost can be suppressed, the target pinitol (or pinitol-containing composition) can be obtained at a low price, preferable.

  The plant body may be used as it is, or may be used after performing treatments such as shredding, crushing, cutting, lyophilization, dehydration and the like in advance. Moreover, it is preferable to use these extracts from the viewpoint of pinitol extraction. This extraction liquid is performed, for example, by immersing the plant in a high-temperature extraction solvent. As the extraction solvent, water, a water-soluble solvent such as methanol, ethanol, isopropanol, n-propanol, and acetone, or a mixed solvent thereof is used. Moreover, it is preferable from the point of extraction efficiency that the extraction temperature by the said extraction solvent is 50-120 degreeC. In particular, extraction with hot water (80 to 120 ° C.) is more preferable from the viewpoint of simultaneous sterilization of the plant body. Furthermore, in the extraction method, the extraction medium may be refluxed for extraction.

  Subsequently, the plant body is used as a resource (the plant body extract or the plant body itself), and water, buffer solution (phosphate buffer solution, acetate buffer solution, citrate buffer solution, succinate buffer solution) is appropriately added thereto. And the like, and the above-mentioned bacteria (A) are inoculated therein, and the bacteria are cultured in a high temperature range. In particular, it is preferable to add a phosphate buffer because the bacteria can be cultured well. In addition, the said "high temperature range" means 42 degreeC or more, Preferably it is 42-74 degreeC, More preferably, it is the range of 55-65 degreeC. Since the bacterium (A) is a thermophilic bacterium, it can be cultured at such a high temperature. By setting the bacterium in such a high temperature range, propagation of various bacteria can be suppressed.

  And it is preferable to culture | cultivate the said bacteria on aerobic conditions for 18 to 72 hours, More preferably, it performs for 24 to 48 hours. That is, by culturing the bacterium (A) in a high temperature range, the carbohydrates except for pinitol are efficiently decomposed and consumed even in such a short time. Purity pinitol (or pinitol-containing composition) can be obtained, and the production efficiency can be improved.

  The culture solution obtained by the above culturing process (sugar utilization process) contains high-purity pinitol and can be used as it is. However, if necessary, it is concentrated and then ethanol is added to culture. The supernatant is collected and used through a step of precipitating insoluble substances in the liquid. The insoluble material includes the microbial cell itself of (A), and through this step, higher-purity pinitol (or pinitol-containing composition) can be obtained.

  In addition, after the culture step (or after the precipitation treatment as described above), the culture solution is centrifuged, filtered, precipitated, liquid-liquid distribution method, adsorption chromatography, distribution chromatography, hydrophobic interaction, By applying treatment methods such as action chromatography, ion exchange chromatography, size exclusion chromatography, and countercurrent liquid-liquid partitioning, the target pinitol (or pinitol-containing composition) can be obtained with higher purity. ,preferable. In addition, the said processing method may be applied independently and may apply 2 or more types of processing methods collectively.

  Among the above processing methods, a more preferable processing method is ion exchange chromatography. That is, since pinitol has no polarity and is not adsorbed to the ion exchange column, “the above-mentioned culture solution is passed through the ion exchange column and the pinitol eluate discharged from the ion exchange column is recovered as it is”. This is because pinitol can be obtained with high purity by a relatively simple treatment. In addition, as an ion exchange resin, both a cation exchange resin and an anion exchange resin can be used. For example, the extract can be passed through a cation exchange resin converted into H form with an acid and an anion exchange resin converted into an OH form with an alkali, followed by desalting to purify to the desired purity.

  And the target pinitol (or pinitol-containing composition) can be obtained by concentrating and drying the extract thus obtained.

  The pinitol (or pinitol-containing composition) thus obtained has a high purity (at least 50% purity, usually 95 to 100% purity). Useful to improve. Moreover, since it is highly pure, it is also useful as a research reagent. The purity of the above-mentioned pinitol is obtained, for example, by high performance liquid chromatography (HPLC) analysis under appropriate conditions, and the area ratio between the peak of the target compound (pinitol) and the peaks of other carbohydrates and contaminants is obtained. Can be confirmed.

  And the said pinitol (or pinitol containing composition) can exhibit the effect excellent in the improvement / alleviation of symptoms, such as abnormality of a blood glucose level, diabetes, a liver disorder, a cataract, obesity, and the prevention without a side effect. .

  When the above-mentioned pinitol (or pinitol-containing composition) is dissolved or dispersed in a suitable liquid carrier or mixed with a suitable powder carrier, as a pharmacologically acceptable carrier, for example, shaping in a solid preparation Agents, lubricants, binders and disintegrants, or solvents, solubilizers, suspending agents, isotonic agents, buffers and soothing agents in liquid preparations are used.

  In addition, for formulation of the above-mentioned pinitol (or pinitol-containing composition), various components usually used for formulation are arbitrarily used. Examples thereof include starch, dextrin, lactose, corn starch, inorganic Examples include salts.

  Examples of dosage forms for providing the above-mentioned pinitol (or pinitol-containing composition) as pharmaceuticals or supplements include ampoules, tablets, capsules, granules, fine granules, powders, infusions, and drinks. can give.

  Furthermore, the said pinitol (or pinitol containing composition) can be provided also as a form related to food-drinks. Examples of the foods and drinks include health foods (tablets, powders, granules, concentrated liquids), soft drinks, foods for specified health use, drinks, tea, milk, pudding, jelly, rice cake, gum, yogurt, chocolate, soup, cookies , Snacks, wine, shochu, sake, dressing, boiled beans, tofu, natto, soy milk, roasted beans, dried beans, miso, etc. And by continuously eating and drinking these foods and drinks in the same way as normal foods and drinks, as described above, the effects of pinitol (such as abnormal blood glucose levels, diabetes, liver disorders, cataracts, obesity, etc.) Symptom improvement / relief effect and its prevention effect). In addition, although the caloric sweetener that can cause obesity and the like such as glucose and sucrose is decomposed and removed by the production method of the present invention, since pinitol has a unique sweetness, the above-mentioned food and drink have obesity and the like. There is also an effect that sweetness can be imparted to the food and drink without substantially containing a caloric sweetener that can be a cause.

  The above-mentioned pinitol (or pinitol-containing composition) can be administered to animals such as pets and livestock. Therefore, it can also be provided as a form associated with pet food or feed.

  Next, examples will be described together with comparative examples. However, the present invention is not limited to these examples.

[Example 1]
100 soybean plants (variety name: Ohtsuru) [including all sheaths, stems and leaves] were boiled for 20 minutes in double the amount of water (120 ° C.) to obtain an extract. And the liquid mixture of 250 ml of the said extract, 650 ml of water, and a 10 mM phosphate buffer was prepared, and the Geobacillus kaustophilus PS8 (Geobacillus kaustophilus PS8, FERM P-21938) was inoculated to this. Subsequently, after culturing the bacteria at 60 ° C. for 42 hours, the culture solution was concentrated to 250 ml, an equal amount of ethanol was added, and the mixture was allowed to stand overnight in a low temperature room (4 ° C.). Thereby, since a precipitate was generated, the supernatant was collected, and the supernatant was further filtered. The filtrate was heated to 80 ° C. or higher to evaporate ethanol, and then passed through an ion exchange column to recover and purify pinitol. The ion exchange chromatography using the ion exchange column was more specifically performed by adding 250 ml of the filtrate after adding the filtrate to a column packed with 250 ml of strong cation exchange resin (DOWEX HCR-S H + type, manufactured by Dow Chemical). Add pure water, collect 500 ml of all eluate flowing out during this period, and then add all of the eluate to a column packed with 250 ml of strong anion exchange resin (Muromac A202 OH-type, manufactured by Chromati Technos) Thereafter, 500 ml of pure water was further added, and 1000 ml of all the eluate that flowed out during this period was collected. The eluate was concentrated by evaporating moisture by heating in a microwave oven.

[Comparative Example 1]
Bacterial inoculation and culture were not performed. Otherwise, the eluate containing pinitol was collected and concentrated in the same manner as in Example 1.

  Next, concentrated liquids obtained by the methods of Examples and Comparative Examples were used as samples, and pinitol content and other carbohydrate contents in the concentrated liquids were analyzed by high performance liquid chromatography (HPLC). Specifically, an HPLC LaChrom Elite (L-2490, 2300, 220, 2130) manufactured by Hitachi High-Tech Co., Ltd., which is an analysis apparatus, was added to a separation column Wakosil5NH2 (4.6 × 250 mm) and a guard column Wakosil5NH2 (4 .6 × 10 mm) and 80% acetonitrile as a solvent, and the solute was detected by a differential refractometer at a flow rate of 2 ml per minute at 25 ° C.

  The analysis result (chromatogram) of the sample of Example 1 by the HPLC is as shown in FIG. Moreover, the analysis result of the sample of the comparative example 1 by the said HPLC is as showing in FIG. In FIG. 2, in addition to pinitol (corresponding to a peak eluting at 7 minutes), peaks of other carbohydrates such as sucrose (corresponding to a peak eluting at 15 minutes) are also confirmed, whereas in FIG. It can be seen that peaks of carbohydrates other than pinitol are hardly confirmed. Therefore, it can be seen that pinitol was obtained with high purity by the production method of Example 1.

  It should be noted that high purity pinitol (or pinitol) can be obtained from plant materials other than soybeans (plants containing pinitol such as locust bean, pine, clover, lotus, carnation and bougainvillea) by applying the production method as shown in the examples. It has been confirmed by experiments that the composition can be easily obtained. Moreover, in the said Example, although the site | part (sheath, stem, leaf) to be discarded after removing soybean seeds (beans) is used, in soybean, there are many pinitol contents in such sites. This has been confirmed by experiments. Therefore, by using such a soybean disposal target site as the above-mentioned pinitol-containing plant body, the material cost can be suppressed, and the target pinitol (or pinitol-containing composition) can be obtained at a low cost.

  The production method of pinitol (or pinitol-containing composition) according to the present invention can obtain pinitol in a short period of time and with high purity while suppressing the growth of various bacteria by a relatively simple treatment, and also in terms of production cost. However, since it is advantageous, it is industrially useful. In addition, pinitol obtained with high purity as described above is useful for improving the functionality of the drug or food or drink when applied as a material for the drug or food or drink. Furthermore, since pinitol can be obtained with high purity as described above, pinitol obtained by the above production method is also useful as a research reagent. In addition, pinitol obtained by the above production method is easily soluble in water, and can be applied to plants other than animals including humans, and is useful for preventing drying and improving salt tolerance. It becomes.

Claims (11)

A method for producing pinitol or a pinitol-containing composition comprising a step of culturing bacteria as shown in (A) below in a high temperature region using a pinitol-containing plant as a resource.
(A) A thermophilic Geobacillus bacterium having a resolution of carbohydrates excluding pinitol.   The method for producing pinitol or a pinitol-containing composition according to claim 1, wherein the culture is performed in a high temperature range of 42 to 74 ° C.   The method for producing pinitol or a pinitol-containing composition according to claim 1 or 2, wherein the bacterium shown in (A) is Geobacillus kaustophilus PS8 (FERM P-21938).   The method for producing pinitol or a pinitol-containing composition according to any one of claims 1 to 3, wherein a hot water extract of a pinitol-containing plant is used as the resource.   The manufacturing method of the pinitol or pinitol containing composition as described in any one of Claims 1-4 which adds a phosphate buffer to the said resource and performs the said culture | cultivation.   6. The pinitol according to claim 1, wherein the pinitol-containing plant is at least one selected from the group consisting of locust bean, soybean, black soybean, pine, clover, lotus, carnation and bougainvillea. Or the manufacturing method of a pinitol containing composition.   The manufacturing method of the pinitol or pinitol containing composition as described in any one of Claims 1-5 which uses the site | part for which the soybean discard object after removing a bean as said pinitol containing plant body.   The method for producing pinitol or a pinitol-containing composition according to any one of claims 1 to 7, wherein the culture is performed under aerobic conditions for 18 to 72 hours.   After the above culture step, the culture solution is subjected to centrifugation, filtration, precipitation, liquid-liquid partition, adsorption chromatography, partition chromatography, hydrophobic interaction chromatography, ion exchange chromatography, size exclusion chromatography. And a method for producing pinitol or a pinitol-containing composition according to any one of claims 1 to 8, further comprising a step of purification using at least one treatment method selected from the group consisting of a countercurrent liquid-liquid distribution method. .   The pinitol or pinitol-containing composition according to claim 9, further comprising a step of concentrating the culture solution obtained in the culture step and adding ethanol thereto to precipitate insoluble substances in the culture solution before the purification step. The manufacturing method.   Geobacillus kaustophilus PS8 (FERM P-21938) characterized by having a resolution of carbohydrates excluding pinitol.

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