JP4260597B2 - Fermentation processed product and production method thereof - Google Patents

Description

  The present invention ferments the leaves of Momotamana used for street trees and the like with lactic acid bacteria, yeast, Bacillus subtilis, etc., so that it has excellent antioxidant activity, blood glucose level increase inhibitory effect, obesity suppressive effect, in vivo NO The present invention relates to a fermented product having production inhibitory activity, a method for producing the same, a food material containing the same, and a food.

  Momotamana (Terminalia catappa) belongs to the family Combretaceae and is a vegetation that grows from the tropics to the subtropics. Seeds are scattered by ocean currents and are widely used as roadside trees in Okinawa. Momotamana seeds have a flavor similar to almonds and are edible, but portions other than the seeds, for example, leaves of about 25 cm, are leathery and are not used for edible purposes.

  Further, an extract obtained by ashing Momotamana and extracting it with water or seawater is used as a shelf life improving agent for food sterilization and antibacterial use (for example, see Patent Document 1), or a plant mineral salt as a substitute for salt. (For example, refer to Patent Document 2). However, these food disinfectants and the like are applied to food after ashing, and it has not been considered at all to utilize raw or dried leaves of leather-like Momotamana as food or food materials. .

  On the other hand, nitric oxide (NO) has been found to have an important effect in mammalian cells. L-citrulline and NO are produced from L-arginine by the catalytic action of NO synthase (NOS). Here, NOS is classified into a constitutive type (constitutive NOS, cNOS) and an inductive type (inducible NOS, iNOS), and cNOS constantly produces a low concentration of NO in vascular endothelial cells and gastric mucosal cells. On the other hand, iNOS is activated by stimulation of lipopolysaccharide (LPS), cytokine or pathogen in cells involved in inflammation such as macrophages and neutrophils, hepatocytes and vascular smooth muscle. It has an inductive catalytic action that produces a large amount of NO non-constantly. For example, it has been clarified that when macrophages in a living body are stimulated by a pathogen, the expression level of iNOS increases and NO production is promoted from L-arginine. The produced NO acts on an electron transport enzyme in the mitochondrial system of tumor cells to inhibit its activity (immune response regulating action) and inhibit infection. Other effective actions of NO in vivo include relaxing blood vessels and lowering blood pressure, or preventing adhesion of multinucleated leukocytes and platelets to prevent platelet aggregation.

However, when high concentrations of NO are produced from macrophages due to inflammatory diseases such as nephritis, liver damage, ulcerative colitis, rheumatoid arthritis, and allergic diseases, the surrounding tissues are damaged by NO and self- Symptoms similar to the immune phenomenon may occur. For example, when suffering from sepsis, the production of a large amount of NO causes shock symptoms due to myocardial contractility (septic shock). It can also trigger onset. In order to prevent tissue or cell damage due to excessive production of NO, or the development of induced diseases, it may be necessary to suppress the production of NO so that NO is present in the living body at an effective concentration. For this reason, the development of NOS inhibitors that inhibit the action of NOS involved in NO production and NO production inhibitors are being promoted. As typical NOS inhibitors, NG-monomethyl-L-arginine (L -NMMA) and L-arginine derivatives such as NG-nitro-L-arginine methyl ester (L-NAME) (where NG represents that a nitro group is attached to the N atom of the guanidino group). And a compound having a structural formula similar to that of L-arginine as a substrate. Examples of iNOS induction inhibitors include corticosteroids, serine, cysteine protease inhibitors and the like, or laurel derived sesquiterpene costunolide and dehydro having a NO production inhibitory action based on iNOS induction inhibition derived from natural products. Dehydrocostus lactone (see, for example, Non-Patent Document 1), rhubarb-derived stilbene, rhapontigenin, piceatannol, resveratrol (see, for example, Non-Patent Document 2), taxa A derived triterpene, alisol F (for example, see Non-Patent Document 3) and the like have also been reported.
Japanese Unexamined Patent Publication No. 2000-135074 JP 2000-4823 A Matsuda H., Kageura T., Toguchida I., UedaH., Morikawa T., Yoshikawa M. Life Sciences, 66, p. 2151-2157, 2000 Matsuda H., Kageura T., Morikawa T., Toguchida I., Harima S., Yoshikawa M. Bioorganic & Medicinal Chemistry Letters, 10, 323-327, 2000 Matsuda H., Kageura T., Toguchida I., Murakami T., Kishi A., Yoshikawa M., Bioorganic & Medicinal Chemistry Letters, 9, 3081-3086, 1999

  An object of the present invention is to develop an excellent natural resource containing a component having an antioxidant activity and a blood glucose level increase inhibitory activity, and due to its hardness, it is not considered to be used as a food or a food material. Fermented with peach molybana as a food or food material, fermented with plants to improve texture and taste, and have excellent antioxidant activity, blood glucose level increase inhibitory effect, and obesity inhibitory effect The object is to provide a processed food, a production method thereof, and a food material or food containing a fermented product.

  In order to develop excellent natural resources containing active ingredients having an antioxidant activity and the like, the present inventors have conducted research aimed at effective use of plants that are not used as food materials for plants that naturally grow in Okinawa, For guava containing amylase inhibitory activity and antioxidant activity in the leaf, it was found that the medicinal effect of the fermented leaf was enhanced and the astringency and gummy taste were suppressed and the taste was improved and fermented. When fermented foods containing guava leaves have already been developed (Japanese Patent Application No. 2001-63142), and leaves of Asteraceae plants such as Japanese algae and pericarp of citrus plants such as Shikuwasa are fermented with lactic acid bacteria etc. It is improved and easy to take, and it is found that the antioxidant effect increases with the increase in the content of quercetin, which is an antioxidant, and the antihypertensive effect is enhanced. And have developed a lactic acid fermentation process or the like (Japanese Patent Application No. 2002-236155). The present inventors have conducted extensive research on plants that contain a large amount of minerals but have not been fully utilized, and have selected Momotamana, which is often found as a roadside tree in Okinawa, as a subject of research, and leaves of Momotamana, etc. It has been found that the fermented product obtained by fermenting with keratin contains quercetin, which is not contained before fermentation, and has excellent antioxidant activity, thereby completing the present invention.

That is, the present invention is to dry and pulverize the leaves of Momotamana (Terminalia catappa), add water, carbohydrates and proteins to the pulverized product, L. plantarum (L. plantarum), Streptococcus thermophilus (S thermophilus) and Bacillus subtilis (B. subtilis) fermented product obtained by fermentation, characterized in that amylase inhibitory activity is retained and quercetin is contained (claim 1) , vivo NO production inhibitory activity, relates to a fermentation process of claim 1, wherein that the elevated compared to leaves of unfermented processing Terminalia catappa (claim 2).

The present invention also relates to a food material or food (claim 3 ), characterized by containing the fermented product according to claim 1 or 2 .

In addition, the present invention also includes drying and crushing leaves of Momotamana (Terminalia catappa), adding water, carbohydrates and proteins to the pulverized product, and adding Lactobacillus plantarum, Streptococcus thermophilus (S Fermentation using a mixed bacterium of thermophilus and B. subtilis, including a fermentation process that retains amylase inhibitory activity and produces quercetin, and a drying process that dries the processed product after fermentation. method for producing a fermented product according to claim (claim 4) and, in vivo NO production inhibitory activity, fermentation treatment according to claim 4, wherein the raised compared to leaves of unfermented processing Terminalia catappa manufacturing method (claim 5) or of the object, proteins, a process for producing according to claim 4 or 5 fermentation treated according characterized in that it is a rice bran and / or wheat bran (claim 6)

  According to the fermented product of the present invention and the method for producing the same, it is possible to improve the texture and taste by fermentation for the peach tamana plant containing the active ingredient, and can be used as a mineral-rich food, increasing the content of quercetin. Since it has excellent antioxidant activity and retains amylase inhibitory activity, it has an effect of suppressing the increase in blood glucose level and the effect of suppressing obesity, and has great utility as a food or food material.

  The present invention is not particularly limited as long as it is a lactic acid fermentation processed product of leaves of Momotamana (Terminalia catappa). Momotamana, which is applied to the present invention, belongs to the family Sequimidae, is a vegetation that grows from the tropics to the subtropics, has a height of 20 m or more, and is a plant that turns red. The portion to be subjected to the fermentation process of peach tamana may be any of leaves, seeds, fruit peels, etc., but the leaves are suitable, and may be a living body or a dried body.

The fermented product of the present invention is characterized in that the quercetin content is 3.00 × 10 ⁻³ wt% or more. Quercetin is not contained at all in the unfermented peach tamanana, but is produced in the fermented product by hydrolysis of the quercetin glycoside contained in the unfermented plant by the fermenting bacteria. Since quercetin has an antioxidant activity, which is an aglycon, the antioxidant activity caused by quercetin is increased in the fermented product of Momotamana. It is preferable that the content of quercetin is increased by 3.00 × 10 ⁻³ wt% or more, particularly 5.78 × 10 ⁻³ wt% or more by fermentation. The quercetin content was measured by extraction with 100% methanol and measured by high performance liquid chromatography. The quercetin content before and after fermentation is shown in FIG.

  The fermented product of the present invention has excellent antioxidant activity. The strength of the antioxidant activity can be measured by β-carotene method, DPPH method, rhodan iron method and the like. Here, the β-carotene method is a measurement method using β-carotene, which is easily oxidized and fades yellow and becomes transparent, and naturally oxidizes a mixture of β-carotene, linoleic acid, and a specimen. Since the decrease in absorbance over a certain period of time corresponds to the amount of oxidized β-carotene, this is a method for measuring the change in absorbance and determining the oxidation rate of β-carotene. The lower the value of β-carotene oxidation rate, that is, the smaller the β-carotene oxidation amount, the faster the oxidation reaction of the specimen, and the smaller the value, the lower the oxidation rate of the specimen. Represents high activity. Further, the DPPH method is a method based on measurement of radical scavenging ability using 1,1-diphenyl-2-picrylhydrazyl (DPPH), and DPPH whose black purple color fades by capturing radicals easily. When DPPH is added to the liquid to which the analyte has been added, it is immediately oxidized, and the change in absorbance corresponds to the amount of DPPH that has captured radicals. Therefore, the method of measuring the absorbance change and determining the radical consumption rate by DPPH It is. This radical consumption rate indicates that the smaller the value, that is, the smaller the DPPH consumption, the faster the radical capture reaction (oxidation reaction) of the specimen progressed, and the higher the antioxidant activity of the specimen. Represents that.

  The rhodan iron method is a method for measuring the degree of auto-oxidation of linoleic acid with a spectrophotometer (500 nm).

  The antioxidant activity of the fermented product of the present invention shows a high value even when measured by either the β-carotene method or the DPPH method. The measurement of the antioxidant activity of the fermented processed product of the present invention by the β-carotene method described above is performed by using a sample consisting of an 80% ethanol extract of the fermented processed product or a mixture of hot water extract and β-carotene for several hours, for example, After 4 hours of natural oxidation, the absorbance before and after the natural oxidation was measured. From this difference in absorbance, the rate of decrease in absorbance in terms of the decrease in absorbance in the control without addition of the extract of the fermented product was calculated as 100, that is, the oxidation rate. Can be sought. As shown in FIG. 2, the oxidation rate of the fermented product of the present invention thus obtained is lower than that of the control in both 80% ethanol extraction and hot water extraction. The product does not impair the antioxidant activity by fermentation, and in particular, by hot water extraction, it is superior to t-butyl-4-oxyanisole (BHA) or vitamin E (α-Toc) used as an antioxidant. Antioxidant activity. In applying the β-carotene method, 80% ethanol extraction of a sample is extraction with 1 mL of 80% ethanol per 1 mg of sample, and hot water extraction is extraction with hot water at 80 ° C. that is 50 times the weight of the sample. is there.

  Moreover, the measurement of the antioxidant activity of the fermented product of the present invention by the DPPH method is performed by adding DPPH to the 80% ethanol extract or hot water extract of the fermented product, measuring the absorbance after 30 seconds, and adding DPPH. From the difference from the previous absorbance, the rate of decrease in absorbance, that is, the radical consumption rate, can be determined by converting the decrease in absorbance in the control without addition of the extract of the fermented processed product to 100. As shown in FIG. 3, the radical consumption rate of the fermented product of the present invention thus obtained is lower than that of the control in both 80% ethanol extraction and hot water extraction, and the fermentation of the present invention. Even in the measurement by the DPPH method, the treated product does not impair the antioxidant activity by fermentation, and exhibits excellent antioxidant activity comparable to AsA or α-Toc. In applying the DPPH method, 80% ethanol extraction and hot water extraction of the sample are performed by the same method as the extraction in the β-carotene method.

  The fermented product of the present invention has amylase inhibitory activity. The amylase inhibitory activity of the fermented product of the present invention is not reduced by the fermentation treatment, and particularly has excellent α-amylase inhibitory activity. The α-amylase inhibitory activity of the fermented product of the present invention measured by the microplate method is as shown in FIG. The microplate method is a method for detecting the amylase inhibitory activity by measuring the amount of starch decomposed by amylase by utilizing the fact that the starch concentration in the solution is proportional to the turbidity, and measuring the absorbance of the solution. The apparent amylase concentration of the subject is determined using a calibration curve prepared using the amylase standard solution as the absorbance value, and the amylase activity value is determined from the ratio to the actual amylase concentration. The smaller the amylase activity value, the higher the amylase inhibitory activity of the subject. The fermented product of the present invention retains amylase inhibitory activity even by fermentation, and can be applied as a blood sugar level increase inhibitor or an obesity inhibitor based on the amylase inhibitory activity.

  The fermented product of the present invention has NO production suppressing activity in vivo. The fermented product of the present invention has NO production inhibitory activity by suppressing the expression of iNOS which is excessively expressed in vivo and produces excessive NO. The NO production inhibitory activity of the fermented product of the present invention is increased compared to unfermented peach tamana by fermentation treatment, and has an excellent excessive NO production inhibitory activity in vivo, particularly in macrophages. Diseases resulting from, for example, toxic shock, systemic blood pressure decrease by treatment with cytokines such as LPS and IFN-γ, blood pressure response decrease, autoimmune disease, inflammation, arthritis, rheumatoid arthritis, diabetes, inflammatory bowel disease, It is useful for vascular dysfunction, pathologic vasodilation, tissue damage, cardiovascular ischemia, hyperalgesia, cerebral ischemia, angiogenesis-related diseases, cancer, etc. can do.

Such NO production inhibitory action can be measured by a chemiluminescence method, an electrode method, an electron spin resonance (ESR) method, a Griess method, or the like. Griess method is a metabolite of NO NO ₂ ^- and Griess reagent the amount by measuring the absorbance for (sulfanilamide and N-(1-naphthyl) ethylenediamine) and red azo compound produced by diazotization coupling reaction detects, NO ₂ from the amount of the azo compound ^- to calculate the amount, NO ₂ in the control ^- by converting the calculated value of the amount as 100 NO ₂ ^- production of, i.e., obtains the NO generation amount, the now The NO production suppression rate of the specimen is calculated and used as an index for evaluating the NO production inhibitory activity.

  Further, in combination with this, by detecting the number of viable cells by MTT method or the like, the amount of NO produced can be detected, and the degree of NO production inhibitory activity can be determined. In this MTT method, 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium bromide (MTT) is a substrate for intracellular mitochondrial dehydrase, and has high viability. It is a method that utilizes the fact that the amount of MTT that is reduced to a large extent and the resulting yellow to red formazan amount corresponds well to the number of viable cells, and can measure only living cells, see 630 nm The absorbance of the sample at a wavelength of 570 nm is measured as the wavelength, and the cell viability of the sample can be determined by the following calculation formula.

Cell viability = 100 × B / A
In the formula, A represents a value obtained by dividing the absorbance at 570 nm from the absorbance at 570 nm for the control, and B represents a value obtained by dividing the absorbance at 570 nm for the sample from the absorbance at 630 nm. The cell viability detected by the MTT method for the fermented product of the present invention is higher than that of the control, and a larger amount of cells are grown than the control. Therefore, it is clear that the fermented product of the present invention has NO production inhibitory activity. Has been.

  The method for producing a fermented processed product of the present invention is not particularly limited as long as it is a method for fermenting a peach tamana plant, but examples of microorganisms used for fermenting include lactic acid bacteria, yeasts, and Bacillus subtilis. These can be used alone or in combination of two or more, and lactic acid bacteria are preferably used among them, lactic acid bacteria alone, lactic acid bacteria and yeast, lactic acid bacteria and Bacillus subtilis, or lactic acid bacteria and yeast and Bacillus subtilis, etc. Can be used as a combination.

Examples of lactic acid bacteria used in the method for producing a fermented product of the present invention include Streptococcus, Lactobacillus, Leuconostoc, Pediococcus, Bifidobacterium ( Bifidobacterium) or bacteria belonging to the genus Tetragenococcus are preferred, and the genus Lactobacillus is particularly preferred. The bacterium belonging to the genus Streptococcus is preferably Streptococcus thermophilus (S. thermophilus), and specific examples include Streptococcus thermophilus IFO13957 strain. In addition, as a bacterium belonging to the genus Lactobacillus, any of Lactobacillus plantarim (L. plantrum), Lactobacillus delbruckii (L. delbruckii), Lactobacillus pentosus (L. pentosus) or Lactobacillus casei (L. casei) Among these bacteria, Lactobacillus plantarim is particularly preferable. Specific examples of such Lactobacillus plantarim include IFO14712 and IFO14713 strains, Lactobacillus delbriqui as IFO13953 strain, Lactobacillus pentosus as IFO12011 strain, and Lactobacillus casei as IFO15883 strain. Moreover, as a microbe which belongs to Tetragenococcus genus, it is preferable that it is Tetrageno halophyllus (T. halophilus), and tetrageno halophyllus IFO12172 strain can be illustrated concretely. These lactic acid bacteria are preferably used in an amount of usually 10 ³ to 10 ⁷ , particularly 10 ⁶ to 10 ⁷ , per 1 g of dry matter of leaves of Momotamana.

The yeast used in the method for producing a fermented product of the present invention is added mainly for the improvement of aroma, and as such yeast, a bacterium belonging to the genus Candida or Saccharomyces is preferable. As such a bacterium belonging to the genus Candida, Candida versatilis is preferable, and IFO10038 strain can be specifically exemplified as Candida versatilis. The bacterium belonging to the genus Saccharomyces is preferably S. cerevisiae, and IFO0555 strain can be specifically exemplified as Saccharomyces cerevisiae. It is preferable to use 10 ³ to 10 ⁷ , especially 10 ⁶ to 10 ⁷ , of these yeasts per 1 g of dry matter of Momotamana leaves.

Furthermore, as a Bacillus subtilis used in the manufacturing method of the fermented material of this invention, a Bacillus subtilis (B. subtilis) IFO3013 strain can be illustrated specifically. It is preferable to use 10 ³ to 10 ⁷ , especially 10 ⁶ to 10 ^{7 of} these Bacillus subtilis per gram of dry matter of Momotamana leaves.

  In the method for producing a fermented product of the present invention, the microorganism group preferably used is a microorganism group including lactic acid bacteria, yeast and Bacillus subtilis, and among these microorganism groups, Lactobacillus plantarim, Streptococcus thermophilus, Bacillus. It is preferable that they are mixed bacteria of subtilis, and it is preferable to use the same number as the number of bacteria for the dry matter of the leaves of Momotamana. By using the bacteria in such a combination of the number of bacteria, the fermentation time can be shortened and consequently the propagation of various bacteria can be suppressed.

In the method for producing a fermented product of the present invention, the dried leaf of the above-mentioned Momotamana is pulverized to a particle size of 3 mm or less, preferably 0.5 to 1.0 mm. By setting the particle size to 3 mm or less, a sufficient contact area with the fermenting bacteria can be secured, fermentation can proceed effectively, and the particle size in the range of 0.5 to 1.0 mm. This effect can be obtained more remarkably. In order to promote the progress of fermentation, it is preferable to add 1 to 10 parts by weight, particularly about 4 to 6 parts by weight, of water to 1 part by weight of dry matter. To the pulverized plant, the above-mentioned fungus or fungus group is added. It is preferable to add 1 to 10% by weight of each fungus group after culturing each fungus before mixing to the medium and adding to the medium in advance. Fermentation is preferably performed at a temperature of 20 to 50 ° C., preferably 40 ° C., and the fermentation time can be appropriately selected depending on the progress of fermentation under conditions such as pH and the number of bacteria, and preference. If the pH is 4 to 5 and the number of bacteria is 10 ⁶ or more, it is preferably about 72 hours. At the time of fermentation treatment, aeration and deoxygenation treatment can be performed as necessary, but after the deoxygenation treatment, fermentation can be performed in stationary culture. The fermentation format is preferably solid culture rather than liquid culture.

  In such a fermentation treatment, carbohydrates and proteins can be added as an agent for fermenting bacteria. The carbohydrate as the assimilating agent is preferably a commercially available sugar such as glucose, sucrose, molasses, etc., and the amount of these added is preferably 1 to 10% by weight per medium, particularly around 3% by weight. The protein as an assimilating agent is preferably rice bran, bran or the like, and the addition amount thereof is preferably 1 to 5% by weight per medium. These assimilating agents may be used alone or in combination of two or more.

  After completion of fermentation, it is preferable to dry with a dryer so that the moisture value is 10% by weight or less. As a drying method, heat drying or freeze drying can be used. In the case of heat drying, the product temperature is 100 ° C. It is preferable that the following is performed because the deactivation of the physiologically active ingredient can be prevented. After drying, if necessary, sterilization is performed by a known method such as heating to obtain a food material or a fermented product used as an extract raw material.

  The fermented processed product of the present invention includes, as food materials, fermented processed products themselves, tablets, granules, capsules, etc. prepared from extracts extracted into drinking water, tea bags, plastic bottles, cans, tea leaves for drinks be able to. In addition, the fermented product itself or granules produced from the extracted extract can be used as a food material such as sprinkles, or as a health food. In addition, beverages in which such extracts and granules are dissolved in drinking water or juice, baked confectionery such as bread, cakes, rice crackers, Japanese confectionery such as sheep confectionery, bread and confectionery such as frozen confectionery, chewing gum, jelly, udon, soba Noodles such as kamaboko, ham and fish sausage, seasonings such as miso, soy sauce, dressing, mayonnaise, sweeteners, dairy products such as cheese, butter, yogurt, ice cream, pudding, and tofu It can be used as a food blended with various side dishes such as konjac and other boiled salmon.

  In addition, the extract of the fermented product of the present invention can also be used as a pharmaceutical agent such as an antioxidant active composition, an obesity inhibitor, or a therapeutic / preventive agent for diseases, inflammation, cancer, etc. caused by macrophage NO production. In that case, various pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, solubilizers, solubilizers, isotonic agents, etc. Formulation ingredients can be added. These preventive or therapeutic agents can be administered orally or parenterally. That is, it can be administered orally in commonly used dosage forms, such as powders, granules, capsules, syrups, suspensions, etc., or, for example, in dosage forms such as solutions, emulsions, suspensions, etc. These can be administered parenterally in the form of injections, or can be administered intranasally in the form of sprays.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Manufacture of fermented products]
30 g of dried Momotamana leaves were pulverized to a particle size of 0.1 to 3 mm and placed in a container. 150 g of water, 0.9 g of molasses, and 0.9 g of rice bran were added to a container containing Momotamana. After culturing each of the bacteria of Lactobacillus plantarim, Streptococcus thermophilus, and Bacillus subtilis in a container containing the pulverized peach tamamana, the bacteria were mixed at a ratio of 1: 1: 1. Weight% was added, the container was sealed, and fermentation was performed by static culture. The fermentation temperature was 40 ° C. and the fermentation time was 72 hours. Then, after drying at 60 degreeC until the moisture value became 10 weight% or less with a dryer, the sterilization process (130 degreeC vapor | steam, 5 to 15 second) was performed, and 30 g of fermented peach tamana was obtained.

[Measurement of component content of fermented product]
About various minerals, content before fermentation and after fermentation was measured. The measurement was performed by atomic absorption method.

  0.5 g of the sample was put in a uni-seal, 10 mL of nitric acid was added, and the mixture was reacted at 150 ° C. for 90 minutes. After cooling, the mixture was transferred to a 100 mL tall beaker, 10 mL of hydrochloric acid: hydrogen peroxide (1: 1) solution was added and evaporated to dryness on a sand bath, and then 10 mL of diluted hydrochloric acid was added and heated. After standing to cool, the volume was adjusted to 50 mL to obtain a measurement sample. The prepared sample was appropriately diluted, and the contents of various minerals were measured using an atomic absorption meter (manufactured by SHIMADZU: AA-660). The results are shown in Table 1.

[Measurement of antioxidant activity by β-carotene method]
(1) Preparation of sample About the fermented peach tamana obtained in Example 1, the sample extracted with 1 mL of 80% ethanol with respect to 1 mg of the fermented peach tamamana, and each sample extracted with 50 parts by weight of the fermented peach tamamana at 80 ° C hot water. Prepared.
(2) Preparation of Reagents 0.2 g of linoleic acid solution (manufactured by Tokyo Chemical Industry Co., Ltd.) 1 g dissolved in chloroform to 10 mL and 10 mg of β-carotene (manufactured by WAKO Co., Ltd.) in chloroform 0.25 mL of β-carotene solution dissolved to 10 mL and 2 mL of Tween 40 (manufactured by WAKO Co., Ltd.) 2 mL in chloroform were mixed with 0.5 mL of Tween 40 solution, and nitrogen gas was sprayed. After completely removing chloroform, 45 mL of distilled water and 5 mL of 0.2 M phosphate buffer were added and stirred to prepare a reagent.
(3) Measurement 0.1 mL of each sample and 4.9 mL of the above reagent were mixed and incubated at 50 ° C. for 4 hours. Before and after incubation, the absorbance (470 nm) of the sample was measured using a spectrophotometer (manufactured by Shimadzu Corporation, UV-1200 V). Using water as a control, the difference in absorbance between before and after incubation of the sample when the difference in absorbance between before and after incubation of the control was taken as 100 was determined as the oxidation rate. The results are shown in Table 2.

  As a comparative example, the same procedure was performed for Momotamana, BHA, and α-Toc before fermentation. The results are shown in Table 2.

[Measurement of antioxidant activity by DPPH method]
To 0.05 mL of the sample prepared in the same manner as in Example 3, 0.95 mL of 0.05 M Tris-HCl buffer (pH 7.4) (manufactured by WAKO) and 0.1 mM DPPH-ethanol solution (WAKO ( A reagent obtained by mixing 1.0 mL of 100% ethanol and 1.0 mL of 100% ethanol was added. Absorbance (517 nm) was measured before the addition of the reagent and 30 seconds after the addition. Water was used as a control, and the difference in absorbance between before and after the addition of the reagent to the sample, when the difference in absorbance between before and after the addition of the control reagent as 100, was determined as the radical consumption rate. The results are shown in Table 3.

  As a comparative example, the same procedure was performed for Momotamana, AsA, and α-Toc before fermentation. The results are shown in Table 3.

[Measurement of Quercetin Content]
About content of the quercetin of fermented peach tamana obtained in Example 1, when a sample was extracted with 100% methanol and measured by high performance liquid chromatography (HPLC) (made by SHIMADZU Co., Ltd.), it was 5.78 mg%. It was. On the other hand, the content of quercetin was not recognized in Momotamana before fermentation. From the results, it is clear that quercetin is produced by fermentation.

[Measurement of α-amylase inhibitory activity]
Prepared by mixing 1% by weight citrate buffer of starch (manufactured by SIGMA) and 3.2% by weight of citrate buffer of agar (manufactured by WAKO) at a ratio of 1: 1. The reagent was dispensed at 200 μL each on a measurement microplate and incubated at 37 ° C. for 10 minutes. On the incubated microplate, an extract extracted with 20 mL of a 50% ethanol solution with respect to 1 g of fermented Momota mana obtained in Example 1, 0.1 mL of a solution diluted twice with water and α-α having a concentration of 10.8 units. 25 μL of a sample obtained by mixing 0.9 mL of an amylase solution (manufactured by SIGMA) was dispensed onto a microplate, incubated at 37 ° C. for 5 minutes, and the absorbance (655 nm) was measured with a microplate reader ( Japan Bio-Rad Laboratory Co., Ltd. Model 550). Furthermore, incubation was performed at 37 ° C. for 1 hour, and the absorbance was measured in the same manner. As a control, an apparent α-in the case where a sample is added from a calibration curve prepared from the absorbance after addition of an α-amylase solution having a concentration of 0.108, 1.08, and 10.8 units to the above-mentioned reagent and incubation. The amylase concentration was determined, and the α-amylase activity was determined as the ratio of the apparent α-amylase concentration to the actual α-amylase concentration of 10.8 units. The results are shown in Table 4.

  From the results, remarkable α-amylase inhibitory activity was observed for fermented Momotamana.

[Evaluation of NO production inhibitory activity]
(1) Preparation of sample About 10% of fermented peach tamana obtained in Example 1, 10 mL of 80% ethanol aqueous solution was added to 1 gram of fermented peach tamamana, followed by standing extraction for 12 hours, and then separated into a filtrate and a filtration residue. 10 mL of 80% ethanol aqueous solution was added to the filtration residue, extracted again in the same manner, and filtered to obtain a filtrate. The obtained filtrates were combined, and a sample was prepared by adjusting the concentration so that an extract derived from 10 mg of fermented Momota mana dissolved in 1 mL.
(2) NO production Mouse macrophage-derived RAW264.7 cells (purchased from ATCC) were added to a 24-well microplate at 2 × 10 ⁵ cells / well and pre-cultured at 37 ° C. for 12 hours. Thereafter, the plate was washed twice with 1 mL of PBS, 0.5 mL of DMEM medium was added, 5 μL of sample was added, and the final concentration of the sample was 4 μg / mL. For control, 5 μL of DMSO was used. To this, 10 μL of L-arginine (final concentration was 2 mM), 10 μL of LPS (manufactured by SIGMA) (final concentration was 100 ng / mL), and 10 μL of IFN-γ (manufactured by Genzyme) 100 U / mL) and 0.5 mL of DMEM medium were added, the plate was shaken well, and cultured at 37 ° C. for 24 hours.
(3) NO ₂ ^- was added 500 [mu] L of Griess reagent of the following composition in measuring the culture supernatant 500 [mu] L, after stirring, the spectrophotometer absorbance at 543 nm: was measured by (UV-1200 V manufactured by Shimadzu Corporation). The absorbance of the sample was measured with the absorbance in the case of the control as 100. The measured values are shown in Table 5. From the measured absorbance, the reduction rate of NO ₂ ⁻ production amount was calculated as the NO production inhibition rate by the following formula and used as an index for evaluating the NO production inhibition activity in the cells. The measurement was performed 5 times, and the average value was used as the NO production inhibitory activity value. The results are shown in Table 5 and FIG. The p value in FIG. 5 was calculated using Student's t-test (*: p <0.05).

NO production inhibition rate = 100 × (A−B) / A
In the formula, A represents the absorbance of the control, and B represents the absorbance of the sample.

Griess reagent 1% by weight sulfanilamide dissolved in 5% by weight phosphoric acid aqueous solution and 0.1% by weight N- (1-naphthyl) -ethylenediamide dihydrochloride solution are mixed at the same volume ratio. It was.

  From the results, it is clear that fermented peach tamanana significantly increases the NO production inhibitory activity in the cells as compared to motamo mana that is not subjected to fermentation treatment.

[Evaluation of effects on cell viability]
MTT method To each well of the above plate from which the supernatant was removed, 500 μL of DMEM medium and 50 μL of MTT reagent were added, and cultured at 37 ° C. for 1.5 hours. Thereafter, 1 mL of DMSO was added to each well, and the cells were broken with ultrasonic waves (ultrasonic cleaning device: manufactured by Tokyo Science Instrument Co., Ltd.), and then 500 μL of hydrochloric acid / 2-propanol was added to each well and stirred well, at 630 nm. As a reference wavelength, the absorbance at a wavelength of 570 nm was measured with a spectrophotometer (UV-1200 V: manufactured by Shimadzu Corporation). The measured values are shown in Table 6. The cell viability was determined from the measured absorbance by the following formula. The results are shown in Table 6.

Cell viability = 100 × B / A
In the formula, A represents the absorbance of the control, and B represents the absorbance of the sample. The absorbance was a value obtained by dividing the absorbance at a wavelength of 630 nm from the absorbance at a wavelength of 570 nm.

  From the results, it is clear that fermented peach tamanana increases cell viability compared to motamo mana not fermented.

[Inspection of taste]
About fermented peach tamana, the palatability test was done.

  2 g of fermented Momota mana obtained in Example 1 was boiled in 500 mL of boiling water for 5 minutes, and 10 panelists sampled it. The same was done for dry leaf momotamana. Nine panelists felt fermented Momotamana deliciously, and one felt the dry leaf Momotamana delicious.

  From the results, it is clear that the taste of fermented peach tamana was improved and the palatability was improved.

It is a figure which shows the content of the quercetin of the fermentation processed material of this invention. It is a figure which shows the antioxidant activity detected by (beta) carotene method of the fermented material of this invention. It is a figure which shows the antioxidant activity detected by DPPH method of the fermentation processed material of this invention. It is a figure which shows the alpha-amylase activity of the fermented material of this invention. It is a figure which shows NO production suppression activity in the mouse | mouth macrophage of the fermented material of this invention.

Claims (6)

The leaves of peach tamana (Terminalia catappa) are dried and pulverized, and water, carbohydrates and proteins are added to the pulverized product, and then L. plantarum, Streptococcus thermophilus (S. thermophilus) and Bacillus are added. A fermented product obtained by fermenting with a mixed bacterium of B. subtilis and having amylase inhibitory activity and containing quercetin. 2. The fermented processed product according to claim 1, wherein the in vivo NO production inhibitory activity is higher than that of an unfermented treated Momotamana leaf. A food material or food comprising the fermented product according to claim 1 or 2 . The leaves of peach tamana (Terminalia catappa) are dried and pulverized, and water, carbohydrates and proteins are added to the pulverized product, and then L. plantarum, Streptococcus thermophilus (S. thermophilus) and Bacillus are added. Fermentation using a mixed bacterium of B. subtilis, including a fermentation step that retains amylase inhibitory activity and generates quercetin, and a drying step that dries the processed product after fermentation. Processed product manufacturing method. The method for producing a fermented processed product according to claim 4, wherein the in vivo NO production inhibitory activity is increased as compared to the leaves of unfermented treated peach tamanana. The method according to claim 4 or 5, wherein the fermentation process thereof wherein the protein is a rice bran and / or wheat bran.

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