JP6124404B2 - Method for producing sirtuin activity promoter - Google Patents

Description

The present invention relates to the production how sirtuin activity promoter.

  It is known that there is a link between eating habits and aging. Specifically, a sirtuin gene is activated by a diet mainly consisting of a low-calorie meal, and sirtuin that is an anti-aging protein is expressed. And, by the expression of sirtuin, the telomeric gene encoding the protein that controls the life of the cell is protected without being degraded. For this reason, the life of the cells is likely to be prolonged, and aging is unlikely to proceed. On the other hand, if the diet is mainly a high-calorie diet, the sirtuin gene remains inactivated, and the telomere gene is degraded without being protected. Therefore, cell aging is likely to proceed.

  Resveratrol has recently attracted attention as a component that activates the sirtuin gene. However, resveratrol has geometric isomers, and of these, trans-resveratrol is thought to contribute to activation of the sirtuin gene. However, trans-resveratrol is volatile and easily converted to cis-resveratrol. And since cis-resveratrol does not contribute to activation of a sirtuin gene and is inactive, it is difficult to use resveratrol at present. Therefore, there is a demand for a compound (sirtuin activity promoter) having a sirtuin activity promoting effect that can replace resveratrol.

  In view of such circumstances, for example, Patent Document 1 describes a sirtuin activator containing as an active ingredient at least one selected from the group consisting of an extract of a gnetaceae plant and gnetin C.

JP 2011-079797 A

  However, the Gnetaceae plant described in Patent Document 1 is a plant that grows in the tropics. Therefore, growing in Japan may require large-scale facilities. Gnetin C is contained in Gnetumaceae plants, but as mentioned above, it is difficult to grow Gnetumaceae plants in Japan with simple methods and equipment. Therefore, in the technique described in Patent Document 1, it is difficult to easily obtain a sirtuin activity promoter.

The present invention has been made in view of the above problems and to provide a manufacturing how readily available sirtuin activity promoter.

The present inventors have intensively studied to solve the above problems. As a result, the following findings were found. That is, the gist of the present invention is that a branching process for branching burdock, a fermentation process for fermenting the branched burdock using Aspergillus awamori , and the fermented burdock 50 characterized by comprising an extraction step of extracting a burdock extract, the using volume% ethanol aqueous solution, about the method of manufacturing a sirtuin activity promoter.

According to the present invention, it is possible to provide a manufacturing how readily available sirtuin activity promoter.

DESCRIPTION OF EMBODIMENTS Hereinafter, a mode for carrying out the present invention (this embodiment) will be described. However, this embodiment is not limited to the following contents, and can be arbitrarily changed without departing from the gist of the present invention. Can be implemented.
In addition, although the microbial cell name is normally described in italics, in this specification, it describes with a normal font for convenience.

[1. Method for producing sirtuin activity promoter]
The method for producing a sirtuin activity promoter according to the present embodiment includes a branching step of branching burdock, a fermentation step of fermenting the branched burdock using a koji, and the fermented burdock. On the other hand, an extraction step of extracting a burdock extract using an organic solvent is included. However, although production conditions other than these are not particularly limited, it is preferable that the method for producing a sirtuin activity promoter of the present embodiment further includes a drying step and a sterilization step described later in detail.

  Therefore, the production method of the sirtuin activity promoter of this embodiment is exemplified by the case where the production method of the sirtuin activity promoter of this embodiment includes a blanching process, a drying process, a sterilization process, a fermentation process, and an extraction process in this order. A method will be described. However, in the manufacturing method of the sirtuin activity promoter of this embodiment, as mentioned above, except a blanching process, a fermentation process, and an extraction process, it is arbitrary.

(1) Blanching process First, burdock is prepared as a raw material for the sirtuin activity promoter of the present embodiment. There are no particular restrictions on the origin or type of burdock, and any burdock can be used.

  The prepared burdock is usually unprocessed harvested from the soil. In such an untreated state, there is a problem that it is difficult to eat burdock because of its unique flavor. Therefore, it is preferable to perform branching on the burdock first. By branching, the polysaccharide contained in the burdock can be decomposed to obtain inulin, fructo-oligosaccharide, etc., and the sweetness can be increased.

  The blanching can be performed by steaming, for example, but is not limited thereto. For example, it can be carried out by roasting, hot water bathing, roasting, heating in a warm warehouse or steaming chamber, and the like.

  In addition, the blanching processing temperature and processing time can be arbitrarily set in accordance with consumer preferences, such as sweetness intensity. For example, when the blanching treatment temperature is 35 ° C., the treatment time can be 180 minutes, and when the blanching treatment temperature is 95 ° C., the treatment time can be 5 minutes. Note that when the blanching treatment temperature is low and the treatment time is shortened, the polysaccharide is not degraded much, so the amount of fructose produced is reduced and the sweetness tends to be weakened. On the other hand, when the blanching treatment temperature is high and the treatment time is long, polysaccharides are often decomposed, so that the amount of fructose produced increases and the sweetness tends to increase. Therefore, the processing temperature and the processing time may be determined in consideration of these.

  The blanching treatment temperature varies depending on the amount of processing and equipment, but if it is a large steamer, it is preferably 40 ° C or higher, more preferably 50 ° C or higher, and preferably 90 ° C or lower, more preferably. Is preferably 60 ° C. or lower. Moreover, as processing time, it is normally 10 minutes or more and 150 minutes or less.

  Branched burdock is usually peeled and cut (cut) to facilitate drying and fermentation of the burdock and extraction of the burdock and to facilitate crushing. Peeling and cutting of the burdock can be performed with a commercially available automatic vegetable processing machine, such as a burdock peeler or an electric burdock machine. For burdock peel, it is only necessary to remove the outer shell of the burdock. For burdock cuts, thin cuts, round cuts, half-moon cuts, ginger cuts, strip cuts, diagonal cuts, sasaki, etc. may be used. Of course, these can also be performed by a worker.

(2) Drying process The burdock whose skin (skin) has been peeled off and cut is subjected to a drying process. By the drying step, the decrease in the oxidation of the polyphenol component by polyphenol oxidase in the burdock can be suppressed, and the ORAC (Oxygen Radical Absorbance Capacity) value can be maintained high. Moreover, the sensuality such as color, taste, and flavor can be made excellent. The temperature and time during drying are not particularly limited. However, the drying temperature is preferably 30 ° C or higher, more preferably 40 ° C or higher, and preferably 70 ° C or lower, more preferably 60 ° C or lower. Among them, a drying temperature of about 50 ° C. is particularly preferable because the burdock has a light brown color, the sweetness and aroma of the burdock are high, and the content of the polyphenol component is high.

  By drying in such a temperature range, moisture in the burdock can be removed at an early stage, and as a result, reduction in oxidation of the polyphenol component by polyphenol oxidase can be suppressed. In addition, the burdock sensation such as color, taste and flavor can be made excellent. Furthermore, inactivation of the enzyme can be prevented.

  On the other hand, when the drying temperature is less than 30 ° C., drying may be insufficient, and the oxidation reduction of the polyphenol component by the polyphenol oxidase contained in the burdock may be promoted. In addition, it has a dark brown color, has a strong acidity, and may have a raw odor. On the other hand, when the drying temperature exceeds 70 ° C., the film is dried too much, and the Maillard reaction is promoted to give a dark brown color, which may increase the taste and acidity. Moreover, the reduction | decrease by oxidation of a polyphenol component may be accelerated | stimulated.

  Further, the drying time is preferably 30 minutes or more, more preferably 5 hours or more, and preferably 8 hours or less, more preferably 6 hours or less within the above-described temperature range. However, the drying time is not particularly limited as long as the burdock can be dried so as to achieve a desired effect. In addition, since the oxidation of the burdock progresses and the acidity tends to become stronger as the drying time is longer, it is preferably as short as possible.

  As a specific method of drying, for example, any method such as sun drying or hot air drying can be used.

  It is preferable to dry the burdock until the moisture content is 2% or more and 15% or less. If the moisture content is within this range, it is possible to suppress the decrease in the oxidation of the polyphenol component by polyphenol oxidase, to maintain the ORAC value high, and to have excellent sensory features such as color, taste and flavor. On the other hand, if the moisture content exceeds 15%, drying may be insufficient, and the reduction in oxidation of the polyphenol component by polyphenol oxidase contained in the burdock may be promoted. Moreover, the color of the burdock has a dark brown color, a strong acidity, and the flavor may become a raw odor.

(3) Sterilization process The dried burdock is dispersed in water and sterilized. There is no particular limitation on the amount of dry burd to be dispersed, and normally, burdock should be dispersed in an equal amount of water. Specifically, for example, 30 kg of dried burdock can be dispersed in 30 L of water. By making such a quantitative relationship, the dried burdock is sufficiently absorbed.

  The absorbed burdock is sterilized. The pressure, time and temperature at the time of sterilization are not particularly limited. For example, burdock is sterilized by pressurizing at 1 atm (101 kPa (absolute pressure)) and 121 ° C. for 20 minutes. When the sterilization is completed, the burdock is cooled to the vicinity of the seed cutting temperature (the temperature at the time of ingestion of koji described later). Specifically, it is usually cooled to about 35 ° C. or more and 36 ° C. or less.

(4) Fermentation process A rice cake is inoculated to the burdock radiated to the seed cutting temperature (seed cutting). That is, seed buds are inoculated to burdock at the seed cutting temperature (i.e., koji making). The amount of koji consumed is not particularly limited. For example, 30 g of koji can be inoculated with 30 g of seed koji (ie, 0.1% by mass inoculation). After inoculation of koji, burdock fermentation is performed at the temperature and time at which fermentation occurs.

  The temperature and time of fermentation are not particularly limited, but for example, it is preferably 30 ° C. or more and 40 ° C. or less (preferably about 35 ° C.) for about 40 hours. By setting it as such conditions, fermentation can fully advance and it can prevent functional fall, such as the polymerization of polyphenols in a burdock, and the low molecular weight reduction of lignins. The degree of fermentation can be confirmed by measuring the acidic protease activity of the fermented product.

  Thus, in this step, burdock will be fermented using koji. By performing such treatment on the burdock, it is considered that the polymerization of polyphenols in the burdock and the lowering of the molecular weight of the lignin can be promoted. Furthermore, citric acid can be produced by fermenting burdock using straw.

  The specific kind of soot used in this step is not particularly limited. Examples of soot applicable in this step include Aspergillus awamori (A. awamori), Aspergillus kawachii (A. kawachii), Aspergillus inuii (A. inuii), Aspergillussaitoi (A. saitoi), and the like. One of these may be used alone, or two or more of these may be used in any combination. Among these specific examples, from the viewpoint that citric acid can also be produced, the straw is preferably A. awamori.

  Spider is a filamentous fungus of the genus Aspergillus. Such rice bran is a bacterial species that is also used in the production of awamori using rice as a raw material. Therefore, cocoons are commercially available microorganisms, and cocoons are readily available to those who have ordinary knowledge in the technical field to which this invention belongs (so-called persons skilled in the art). Specifically, for example, A. awamori can be purchased from Yagaki F & S. Therefore, in this application, no deposit is made with the depository institution.

  After the burdock is fully fermented by the straw, the whole is dried. The temperature during drying is not particularly limited, but the temperature during drying is preferably not too high. Specifically, the drying temperature may be about 40 ° C., for example. By drying at such a temperature, inactivation of the enzyme produced by fermentation can be more reliably prevented.

  The dried burdock is in a state containing components (enzymes, citric acid, etc.) produced by fermentation. Therefore, the burdock is crushed and sieved in a state containing these components. The size of the pulverized and final product is not particularly limited. By the pulverization, there is an advantage that the extraction efficiency of the sirtuin activity promoter using a solvent is improved in the extraction step described later.

(5) Extraction process For the fermentation and dried burdock, a burdock extract is extracted using an organic solvent. The obtained burdock extract contains a sirtuin activity promoter having a sirtuin activity promoting effect. That is, in this step, a sirtuin activity promoter is obtained (extracted) from the burdock that has undergone the fermentation step.

  The organic solvent used in this step is not particularly limited as long as the effect of the present invention is not significantly impaired as long as it is a solvent containing an organic substance. However, from the viewpoint of good extraction efficiency, a solvent that is easily miscible with water, such as alcohol or acetone, is preferable, and alcohol is more preferable.

  The organic solvent used for extraction may contain an inorganic solvent such as water as long as the effects of the present invention are not significantly impaired. In particular, the solvent used for extraction is preferably a mixed solvent of water and alcohol, and more preferably a mixed solvent (50% by volume alcohol aqueous solution) in which water and alcohol are mixed at a volume ratio of 1: 1. . By using such a solvent, extraction with higher extraction efficiency can be performed, and a burdock extract that can further promote sirtuin activity can be obtained.

  When a solvent containing an alcohol is used as the solvent, the type of alcohol contained is not particularly limited, but a lower alcohol is preferable, and specifically, methanol, ethanol, propanol, butanol and the like are preferable. In particular, when cereal-derived ethanol is used, for example, since the obtained burdock extract is all naturally derived, there is an advantage that it can be easily added to foods and the like.

  The specific conditions for extraction are not particularly limited. For example, after dispersing the powdered burdock powder in a solvent at room temperature (about 25 ° C.), the supernatant can be obtained as a burdock extract, for example, by standing for several minutes. At this time, in addition to standing, stirring may be performed as appropriate. Further, from the viewpoint of improving the extraction efficiency, for example, the extraction may be performed while appropriately stirring using a solvent heated to about 30 ° C. to 40 ° C.

  Separation of the supernatant can be performed using, for example, filter paper. The pore size of the filter paper may be determined according to the particle size of the burdock powder. A burdock extract is obtained by filtering the burdock powder and solvent mixture using filter paper. The burdock extract thus obtained may be concentrated by removing the solvent, if necessary, or may be diluted using another solvent or the like. As described above, the obtained burdock extract contains a sirtuin activator that promotes sirtuin activity. Therefore, concentration, dilution, or the like may be performed using an appropriate solvent so that a desired acceleration effect can be obtained.

[2. Sirtuin activity promoter]
The sirtuin activity promoter of this embodiment is manufactured by the above-described manufacturing method. That is, the burdock extract obtained through the extraction step serves as the sirtuin activity promoter of this embodiment. In addition, when A. awamori having citric acid-producing ability is used in the fermentation process, the burdock extract may contain citric acid together with a sirtuin activity promoter. Therefore, by taking such burdock extract containing citric acid, further advantages such as anti-fatigue effect and H. pylori growth inhibition action can be obtained.

  The sirtuin activity promoter can be used, for example, in the form of a burdock extract obtained in the extraction step from the viewpoint of ease of use. That is, using the above burdock extract, a food and drink composition, a pharmaceutical composition and a cosmetic composition containing a sirtuin activity promoter can be obtained. In addition, as described above, the sirtuin gene is activated, so that the aging of the cells is difficult to proceed. Therefore, the composition containing a sirtuin activity promoter is also suitable as an anti-aging agent.

  The method of taking these compositions and the like is usually taken orally. Therefore, the composition and the anti-aging agent can be ingested in the form of bread, confectionery, side dish, etc., in addition to ingestion in the form as it is. However, if necessary, it may be used as an external preparation, for example, applied to the skin.

  In addition, a sirtuin activity promoter may be included in the food given to animals other than humans (to dogs, cats, etc.) and given to the animals.

[3. effect]
The sirtuin activity promoter of the present embodiment has an effect of promoting the activity of sirtuin, which is an anti-aging protein, and easily promotes various functions of sirtuin in vivo by eating and has excellent functions. Easy to get.

  Specifically, according to the sirtuin activity promoter of the present embodiment, it is possible to provide various disease therapeutic agents, health foods, cosmetics and the like using the same. In addition, it helps to control aging and oxidation (aging) that involve a decrease in sirtuin activity, as well as treatment and prevention of various pathologies such as metabolic syndrome, diabetes and its complications, arteriosclerosis, gastrointestinal diseases, and skin aging. Also useful for prevention. Furthermore, since a sirtuin activity promoter is obtained from burdock, which has been used as a food material for a long time and is easily available in Japan, these functions can be obtained more easily.

  Hereinafter, the present embodiment will be described more specifically with reference to examples.

[1. Preparation of burdock extract]
First, the burdock was washed with water and then blanched at 50 ° C. for 15 minutes (branching step). Then, the branched burdock was water-cooled and then cut (2 mm × 2 mm × 20 mm). Next, the cut burdock was dried with hot air at 50 ° C. for 4 hours (drying step).

  Then, 30 kg of dried burdock was dispersed in 30 L of water having the same weight and pasteurized at 121 ° C. and 1 atm (101 kPa (absolute pressure)) (sterilization process). After cooling to 35 ° C., 30 g of A. awamori (Yagaki F & S) seed meal was inoculated. Then, it culture | cultivated at 35 degreeC for 41 hours (fermentation process). After fermentation, it was blown and dried at 40 ° C. for 4 hours. After drying, pulverization was performed with an airflow pulverizer (Dalton).

  And the extraction process by 50 volume% ethanol (The organic solvent, the mixed solvent of water of ethanol and the same volume water) was performed with respect to the burdock powder dried and grind | pulverized after fermentation, and the extract of Example 1 was obtained. In addition, an extraction process with water was performed on the same burdock powder to obtain an extract of Comparative Example 1.

  In addition, for the burdock after the drying process (the process after the sterilization process is not performed), an extraction process with 50 vol% ethanol (Comparative Example 2) and an extraction process with water (Comparative Example 3) are performed and compared. Extracts of Examples 2 and 3 were obtained.

  Furthermore, a roasting process at 180 ° C. for 13 minutes was performed on the burdock after the sterilization process (the process after the fermentation process was not performed), and after pulverization, a roasted burdock powder was prepared. And the extraction process (comparative example 4) by 50 volume% ethanol and the extraction process (comparative example 5) by water are performed with respect to the obtained roasted burdock powder, and the extract of comparative examples 4 and 5 is obtained. It was.

[2. Evaluation of sirtuin activity promoting effect]
In order to evaluate the activity promoting effect on sirtuin, measurement was performed using a sirtuin (SIRT1) activity measurement kit “SIRT1 Direct Fluorescent Screening Assay Kit (Cayman Chemical Co.)”. The measurement was performed according to the method described in the instruction manual attached to the measurement kit.

<Determination of measurement conditions>
Since there is a possibility that the fluorescence intensity may decrease due to the interference of the developer due to the interference sample of the fluorescence, the decrease was measured by the following method, and the measurement conditions were determined such that the interference was 10% or less after dilution.

  First, 5 μL of diluted fluorescent solution, 5 μL of ethanol, and 90 μL of buffer solution were placed in a well as a blank. For the sample, 5 μL of the diluted fluorescent solution, 5 μL of the sample (each of the extracts of Example 1 and Comparative Examples 1 to 5) in ethanol solution (the sample was diluted 1 to 10,000 times), and 90 μL Buffer was placed in the well. These were allowed to stand at room temperature for 10 minutes, the excitation wavelength was 350 nm to 360 nm, and the fluorescence with a wavelength of 450 nm to 465 nm was measured. In this measurement, the dilution conditions of the sample at which the fluorescence interference was 10% or less were determined, and the following sirtuin activity measurement was performed under the determined dilution conditions.

<Sirtuin activity measurement>
The sirtuin activity was measured by diluting the fluorescent solution under the above conditions so that the interference was 10% or less.

25 μL of buffer, 5 μL of SIRT1 solution and 5 μL of ethanol were placed in the wells of the microplate (100%). The blank used 5 μL of buffer instead of 5 μL of SIRT1. Thereafter, 5 μL of each diluted sample (extracts of Example 1 and Comparative Examples 1 to 5) was placed in each well, and further 15 μL of the substrate solution (SIRT1 peptide and NAD ⁺ ) was added and allowed to react at room temperature for 45 minutes. .

After 45 minutes, 50 μL of stop developer solution was added to stop the reaction and left at room temperature for 30 minutes. Then, the excitation wavelength was set to 350 nm to 360 nm, fluorescence with a wavelength of 450 nm to 465 nm was measured, and sirtuin activity was calculated based on the measured fluorescence. The substrate solution was prepared by adding a buffer to SIRT1 peptide, NAD ⁺ . The stop developer solution was prepared by mixing nicotinamide and buffer in the developer.

  These results are shown in Table 1.

※ 表１中、「５０％ＥｔＯＨ」は、５０体積％エタノールを表す。

* In Table 1, “50% EtOH” represents 50% ethanol by volume.

<Examination>
As shown in Table 1, it was found that the sirtuin activity was particularly high in Example 1, and the effect of promoting sirtuin activity was obtained in the extract of Example 1 obtained using 50% by volume ethanol. Moreover, although it extracted with respect to the same fermented burdock, in the comparative example 1 which extracted with water, the effect of promoting sirtuin activity was hardly obtained. From this, it is considered that the sirtuin activity promoter hardly dissolves in water and easily dissolves in an organic solvent such as 50% by volume ethanol. Therefore, it is thought that it is important to use an organic solvent for extraction.

  Moreover, the sirtuin activity promotion effect was not seen, even if it used either water or 50 volume% ethanol for the extract from dry burdock (Comparative Examples 2 and 3). Compared with the results of Example 1 and the like that are fermented after drying, drying alone is insufficient to obtain an extract containing a sirtuin activity promoter, and it is considered that fermentation is important.

  Furthermore, the effect of promoting sirtuin activity was hardly observed in the extract from the roasted burdock using either water or 50% by volume ethanol (Comparative Examples 4 and 5). However, in Comparative Example 4 using 50% by volume ethanol as a solvent, a sirtuin activity promoting effect was relatively recognized as compared with Comparative Example 5 using water. From this result, it is considered that the sirtuin activity promoter hardly dissolves in water and easily dissolves in an organic solvent such as 50% by volume ethanol, as in the above-described examination of the results of Example 1 and the like.

  Further, in Comparative Examples 4 and 5, roasting was performed without performing fermentation for burdock, but although the sirtuin activity promoting effect was recognized as compared with Comparative Examples 2 and 3 of only drying, Example 1 was subjected to fermentation. The effect of promoting sirtuin activity was smaller. From this result, it is considered that fermentation is important in order to obtain an extract containing a sirtuin activity promoter.

  As mentioned above, a sirtuin activity promoter can be easily obtained by extracting from fermented burdock using an organic solvent. In particular, burdock has been eaten since ancient times, and is easily available in Japan. A awamori, etc., which is a candy, has been used for a long time in the production of food and liquor, and is easily available in Japan. Therefore, according to this embodiment, the manufacturing method of a sirtuin activity promoter which can be obtained easily, a sirtuin activity promoter, and a composition containing the same can be provided.

Claims (1)

A branching process for branching the burdock;
A fermentation process of fermenting the branched burdock with Aspergillus awamori ;
The relative fermented burdock, characterized in that it comprises a, an extraction step of extracting a burdock extract with 50 vol% aqueous ethanol solution, prepared how sirtuin activity promoter.