WO2015119036A1 - 麹発酵組成物及びそれを用いた調味料、抗酸化剤、食品又は飲料 - Google Patents
麹発酵組成物及びそれを用いた調味料、抗酸化剤、食品又は飲料 Download PDFInfo
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- WO2015119036A1 WO2015119036A1 PCT/JP2015/052513 JP2015052513W WO2015119036A1 WO 2015119036 A1 WO2015119036 A1 WO 2015119036A1 JP 2015052513 W JP2015052513 W JP 2015052513W WO 2015119036 A1 WO2015119036 A1 WO 2015119036A1
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- koji
- black
- fermentation composition
- tea
- composition
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
- A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F3/00—Tea; Tea substitutes; Preparations thereof
- A23F3/06—Treating tea before extraction; Preparations produced thereby
- A23F3/08—Oxidation; Fermentation
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
- A23L2/56—Flavouring or bittering agents
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/14—Yeasts or derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- the present invention relates to a koji fermented composition obtained by fermenting tea leaves with black koji mold such as Aspergillus awamori, and a seasoning, antioxidant, food or beverage using this composition.
- Rice bran is a product obtained by steaming rice, wheat, beans, or the like to propagate an Aspergillus oryzae, of which rice is propagated with Aspergillus oryzae. Rice bran is mainly used for the production of fermented foods such as sake, soy sauce, miso and amazake, but it is also used as a seasoning.
- the koji mold contained in this rice bran has the ability to produce many types of enzymes such as protease and amylase.
- black koji molds are characterized by producing a large number of enzymes such as proteases and amylases, and producing a large amount of citric acid, and are mainly used for the production of shochu such as awamori. Yes. Therefore, regarding black koji mold, as described in Patent Document 2, in order to improve the efficiency of shochu production, research mainly focused on enhancing the enzyme activity of amylase that saccharifies starch in raw materials. Has been done.
- black koji molds are characterized by producing a large amount of citric acid, and the acidic protease produced by black koji molds can retain its activity even in a strongly acidic environment. From this, by using black koji mold as koji mold, for example, koji and koji fermented food having the function of maintaining the activity in the stomach and promoting the digestion of food can be obtained. In addition, black koji molds also produce xylanases that break down the cell walls of plants. Therefore, by using koji mold of black koji molds in plant foods such as vegetables and fruits, the umami components inside the cells of plant foods can be eluted, Effects such as softening can also be obtained.
- the present invention has been devised in view of the above points, and its purpose is to reduce the amount of citric acid produced without losing the acidic protease activity and xylanase activity due to Aspergillus oryzae when using Aspergillus oryzae. It is providing the fermented koji fermentation composition and its manufacturing method.
- Another object of the present invention is to find other activities and functions of the above-described koji fermentation composition and provide a new application.
- the koji-fermenting composition of the present invention that solves the above problems is obtained by inoculating and culturing black koji molds on tea leaves. Breeding black koji molds with tea leaves as raw materials yields a koji fermented composition that reduces citric acid production to less than half compared to rice koji with rice as raw material and has enhanced acidic protease activity and xylanase activity . Therefore, when the koji fermented composition of the present invention is used for foods or is further fermented to produce koji fermented foods, it is possible to obtain foods with low acidity and high enzyme activity described above.
- the protease of the koji fermentation composition of the present invention can maintain its activity even under strong acidity, for example, protein degradation can proceed even in an environment having a low pH such as the inside of the stomach.
- the koji fermentation composition of the present invention has high antioxidant activity and its cell growth inhibitory activity is low, it should be used as a highly safe antioxidant in the fields of food, medicine, cosmetics and the like. Can do.
- the above black koji mold is preferably inoculated into the steamed tea leaves.
- various bacteria in the tea leaves are sterilized, the oxidase contained in the tea leaves is inactivated, the black koji mold can be efficiently propagated in the tea leaves, and the koji fermentation composition can be obtained in a short period of time.
- the fermented cocoon composition of the present invention comprises at least one black gonococcus selected from the group consisting of Aspergillus awamori, Aspergillus luchuensis and Aspergillus gilinuii. It is preferable that Thereby, a suitable bacterial species as a black koji mold for obtaining the koji fermentation composition of the present invention is selected.
- the seasoning of this invention contains the above-mentioned koji fermentation composition or the extract of this koji fermentation composition.
- the fermented koji composition of the present invention has a citric acid production amount reduced to half or less, and an acid protease activity and a xylanase activity are enhanced. Therefore, by using the koji fermentation composition having such characteristics or an extract thereof as a seasoning, the ingredients contained in the food are decomposed by the enzymes contained in the koji fermentation composition, and natural sweetness and umami are obtained. Is granted.
- the food itself can be softened by this enzymatic decomposition action. At this time, since the amount of citric acid produced by the koji fermentation composition of the present invention is reduced, the acidity of citric acid has little effect on the flavor of the food.
- the antioxidant of the present invention contains the above-described koji fermentation composition or an extract of this koji fermentation composition.
- the koji fermentation composition of the present invention has an antioxidant activity that suppresses lipid peroxidation and eliminates active oxygen. Therefore, a koji fermentation composition or an extract thereof can be used as an antioxidant.
- this koji fermentation composition has low cell growth inhibitory activity and is a food-derived composition, it can be used as a highly safe antioxidant.
- the food or beverage of the present invention contains the above-described koji fermentation composition or an extract of this koji fermentation composition.
- This koji fermented composition contains a protease that can maintain its activity even under strong acidity, and can promote protein degradation even in a severe pH environment such as the inside of the stomach.
- this koji fermentation composition has an antioxidant activity that suppresses active oxygen reaction and lipid peroxidation related to the mechanism of various diseases, so it is used as an active ingredient in pharmaceuticals or functional foods. be able to.
- the method for enhancing the umami of the food of the present invention includes a step of adding the above-described koji fermentation composition or the extract of this koji fermentation composition to the food.
- the koji fermentation composition of the present invention by applying the extract to the food material, the components contained in the food material are decomposed by the enzyme contained in the koji fermentation composition, and sweetness and umami are imparted.
- the koji fermentation composition of the present invention since the koji fermentation composition of the present invention has enhanced acidic protease activity, the protein in the food is well degraded, and amino acids constituting umami components such as glutamic acid can be obtained.
- the koji fermentation composition of the present invention also has enhanced xylanase activity, the cell wall mainly composed of hemicellulose in the plant food is easily decomposed, and the sweetness and umami components in the cell wall can be eluted. .
- the koji fermented composition of the present invention can easily increase the umami of the ingredients and process the ingredients to a delicious taste by applying or applying the extract to the ingredients.
- the method for producing a koji fermentation composition with reduced citric acid production and enhanced protease activity and xylanase activity of the present invention comprises a step of inoculating black koji mold on tea leaves and a step of culturing tea leaves. Yes.
- tea leaves as the main raw material, inoculating black koji molds on these tea leaves, culturing them and propagating black koji molds, it is possible to obtain koji with a reduced amount of citric acid and improved acidic protease activity and xylanase activity.
- the black koji mold is selected from the group consisting of Aspergillus awamori, Aspergillus luchuensis, and Aspergillus inuii.
- the black koji mold is selected from the group consisting of Aspergillus awamori, Aspergillus luchuensis, and Aspergillus inuii.
- at least one Aspergillus niger is selected from the group consisting of Aspergillus awamori, Aspergillus luchuensis, and Aspergillus inuii.
- a koji fermentation composition having the following excellent effects can be provided.
- (1) Compared with rice bran, it is possible to obtain a koji fermented composition with a reduced amount of citric acid and enhanced acid protease activity and xylanase activity.
- (3) By adding the fermented koji composition to the foodstuff, the enzyme contained in the koji fermented composition can efficiently decompose the components contained in the foodstuff and easily impart natural sweetness and umami to the foodstuff. .
- the food itself can be softened by the enzymatic decomposition action.
- the fermented koji composition has an action of suppressing lipid peroxidation and removing active oxygen, that is, has an antioxidant activity, and therefore is used as an active ingredient in antioxidants, pharmaceuticals or functional foods. be able to. Moreover, since this koji fermentation composition has a low cell growth inhibitory activity and is a food-derived composition, it is highly safe.
- the manufacturing method of the koji ferment composition concerning embodiment of this invention is the process S0 which prepares the tea leaf which is a raw material, the process S1 which adds water to a tea leaf and absorbs water, the process S2 which steams a tea leaf, It is roughly comprised from the process S3 which inoculates a black tea bacterium to the steamed tea leaf, the process S4 which culture
- the tea leaves in the present invention include not only tea leaves but also stems and branches collected from tea trees.
- the tea leaves used as a raw material for the koji fermentation composition of the present invention are not only fresh tea leaves, but also tea leaves that have been heat-treated immediately after collection, oolong tea or black tea in which tea leaves are oxidatively fermented with oxidase contained in the tea leaves after collection Further, post-fermented tea may be used.
- a tea leaf as a raw material of the koji fermentation composition of the present invention a tea koji after decocting these green tea, oolong tea, black tea or the like can also be used.
- green tea that is a heat-treated tea leaf immediately after collection is preferable because it is easy to handle as a raw material and is not affected by the oxidase contained in the tea leaf in a later step. Moreover, in order to increase the subsequent steaming treatment and the culture efficiency of black koji mold, it is preferable that the tea leaves and tea stems are crushed to a certain size so as not to be too large.
- water absorption treatment step S1 Next, the water absorption treatment step S1 will be described.
- water is added to the tea leaves or immersed in water to absorb water.
- the water absorption is adjusted so that the water content of the tea leaves is 20 to 60%, preferably 30 to 50%.
- the amount of water to be absorbed can be determined by the weight ratio of tea leaves.
- the tea leaves absorbed in the above-mentioned process are put in a steamer, covered, and heated by applying steam to the tea leaves by heating the steamer.
- the cooking time is preferably about 30 to 120 minutes, more preferably 45 to 90 minutes, and particularly preferably about 60 minutes.
- germs in the tea leaf are sterilized, and the oxidase contained in the tea leaf is deactivated, and in the subsequent step, black koji mold can be efficiently propagated in the tea leaf.
- the steamed tea leaves are removed from the steamer, spread evenly on a table, and cooled to about 30-40 ° C. Thereby, it is possible to inoculate the tea leaves with Aspergillus.
- step S3 for inoculating black leaves with tea leaves will be described.
- black koji molds are inoculated on the tea leaves cooled through the above-described steaming step.
- black mold is an Aspergillus fungus that forms black or black-brown conidia (a kind of asexual spore) used in the production of distilled liquor such as Awamori in Okinawa and shochu shochu in Kagoshima. A group of people.
- Aspergillus awamori Aspergillus cytoii, Aspergillus ryukyensis, Aspergillus inui, Aspergillus usami, Aspergillus aureus and the like.
- aspergillus awamori Aspergillus luchuensis, or Aspergillus inuii and Aspergillus inuii and Aspergillus awamori, Aspergillus inchuii and Aspergillus inchui
- spray black koji molds uniformly on tea leaves spread on the table and inoculate them.
- step S4 for culturing black koji mold inoculated into tea leaves.
- black koji molds are propagated in tea leaves inoculated with black koji molds.
- the tea leaves sprayed with black koji mold are placed in a culture room maintained at around 30 ° C.
- the fermentation progresses and the temperature of the tea leaves rises.
- the temperature of the tea leaves is lowered by ventilation and the product temperature of the tea leaves is 30 ° C to 42 ° C.
- the air flow rate is adjusted so as to maintain 30 ° C. to 40 ° C.
- the temperature of tea leaves is adjusted to 35 ° C. to 40 ° C. by ventilation for 12 to 30 hours from the start of culture. Thereafter, the temperature of tea leaves is adjusted to be slightly lower so that the temperature of the tea leaves becomes 30 ° C. to 35 ° C. in the same manner, and the total amount is 1 to 4 days, preferably 36 to 72 hours from the start of the culture.
- the time point after culturing for about 40 to 60 hours is the completion of the koji fermentation composition.
- a machine for example, a drum-type automatic koji making apparatus or the like that can perform raw material washing, water absorption, steaming, koji mold inoculation, and koji making in the same apparatus. It is also possible to do this.
- the obtained fermented koji composition is a so-called “Koji” in which black koji molds propagated in tea leaves.
- the koji fermentation composition of the present invention has a citric acid production amount reduced to less than half compared with ordinary rice koji, etc., despite the propagation of black koji molds, and has acidic protease activity and xylanase. The activity is enhanced.
- the fermented koji composition of the present invention has a high antioxidant activity like the tea leaf of the raw material, but has a low cell growth inhibitory activity unlike the tea leaf.
- the koji fermentation composition obtained by the above-described process is in a state containing a certain amount of water, but the water can be removed by natural drying or low-temperature dehumidification drying.
- the koji fermentation composition from which moisture has been removed can be pulverized into powder or granulated. Even after the water has been removed, the function as a koji fermentation composition can be effectively maintained.
- the enhanced protease activity of the koji fermentation composition of the present invention is the acidity measured according to the method of the National Tax Agency specified analysis method comment (4th revised National Tax Agency specified analysis method, Japan Brewing Association, 1993). It is protease activity, and its enzyme titer expressed in terms of dry matter is preferably 10,000 U / g ⁇ ⁇ ⁇ or more, more preferably 20,000 U / g ⁇ or more, and 30,000 U / g It is particularly preferable that it is g ⁇ or more.
- the enhanced xylanase activity of the koji fermentation composition of the present invention is defined as 1 unit of the amount of enzyme that liberates 1 mg of xylose per 60 minutes at 40 ° C. by the Sommoji Nelson method.
- the value is preferably 2 U / g ⁇ or more, more preferably 5 U / g ⁇ or more, and particularly preferably 8 U / g ⁇ or more.
- the xylanase activity is an activity for hydrolyzing the ⁇ 1,4-glycoside bond of xylan, and is not particularly limited, but the xylanase activity in the present invention is measured as follows.
- xylose-containing standard solution 0 to 240 ⁇ L / mL xylose-containing standard solution is prepared, 0.5 mL of the standard solution is developed by the Sommoji Nelson method, and a standard curve is prepared from the absorbance at 500 nm.
- 10 g of a specimen such as a koji fermentation composition of the present invention is extracted with 50 mL of a pH 5 acetate buffer, filtered, and then diluted with a pH 3.7 acetate buffer as necessary.
- 0.4 mL of 1% xylan solution as a substrate is added to 0.1 mL of the extract, and reacted at 40 ° C. for 60 minutes.
- the acidity value is 10 mL or less. Is preferably 8 mL or less, and particularly preferably 6 mL or less.
- the koji fermentation composition or koji fermentation composition extract of the present invention can be used as a seasoning in food processing or food production.
- a seasoning is a food additive used for the purpose of imparting umami to foods and foods.
- the extract of a koji fermentation composition means what isolate
- the koji fermentation composition of the present invention since the koji fermentation composition of the present invention has enhanced acidic protease activity, the protein in the food is well degraded, and amino acids constituting umami components such as glutamic acid can be obtained. Further, since the koji fermentation composition of the present invention also has enhanced xylanase activity, the cell wall mainly composed of hemicellulose in the plant food is easily decomposed, and the sweetness and umami components in the cell wall can be eluted. . Thus, by applying or applying the fermented koji composition of the present invention or this extract to a food material, the umami of the food material can be easily increased and the food material can be processed to a delicious taste.
- the koji fermentation composition of the present invention contains sugars derived from tea leaves, amino acids, peptides, and the like, umami derived from tea leaves is also imparted. Furthermore, the fermented koji composition of the present invention can soften not only protein-containing food materials such as meat and fish but also plant food materials due to its enhanced enzymatic degradation action.
- the koji fermentation composition or this extract is used by mixing with other materials, or the koji fermentation composition or this extract is used as a seasoning. It can also be used as one of the raw materials. For example, by adding water and salt to a koji fermentation composition and fermenting at room temperature for several days, a salt koji seasoning using the koji fermentation composition can be obtained.
- the koji fermentation composition or the koji fermentation composition extract of the present invention can be used as an antioxidant.
- the antioxidant activity of the koji fermentation composition of the present invention refers to the action of suppressing lipid peroxidation and the action of removing reactive oxygen species (superoxide anion, hydroxyl radical, singlet oxygen, etc.).
- the extract of the koji fermentation composition refers to a product obtained by dissolving a useful component having antioxidant activity from a koji fermentation composition dissolved in a solvent such as water or alcohol. Tea leaves are conventionally known to have high antioxidant activity, while having cell growth inhibitory activity.
- the koji fermentation composition of the present invention has a high antioxidant activity like tea leaves, but its cell growth inhibitory activity is reduced unlike tea leaves. Therefore, it can be widely used as a highly safe antioxidant in the fields of food, medicine, cosmetics and the like for the purpose of preventing the oxidation of contained components.
- the koji fermentation composition or koji fermentation composition extract of the present invention can be used as a food or beverage. Since the koji fermentation composition of the present invention contains the above-described enzyme and also has an antioxidant action, it can be suitably used as a medicine or functional food based on these functions.
- the koji fermented composition or this extract may itself be a food or beverage, but can also be used as one of the ingredients of the food or beverage.
- the further fermented fermented food which has a function of the fermented fermented composition of this invention can also be obtained by adding and fermenting the fermented fermented composition to another foodstuff.
- Example 1 Production of koji-fermenting composition (black koji fungus tea bowl) using black koji fungus Water was added to 1 kg of commercially available green tea, and the water content of green tea was adjusted to be in the range of 30% to 40%. Next, after steaming in a steamer for 60 minutes and cooling to 35 ° C., 1 g (2 billion / g) of black koji mold was uniformly sprayed. Aspergillus awamori was used as the black koji mold, and the amount of application was adjusted so that the number of spores was 1 million or more per 1 g of the raw green tea. After inoculation with the spore, incubation was carried out at 35 ° C.
- Comparative Example 2 Manufacture of black koji mold rice bran using black koji mold Koji made in the same manner as in Example 1 except that white rice was used instead of the raw green tea, and the koji fermented composition of Comparative Example 2 (hereinafter, “ It was called “Kuroiso fungi”.
- Example 2 5. Measurement of Enzyme Activity and Citric Acid Amount of Koji Fermented Composition
- the enzyme activity and acidity of acidic protease and xylanase were measured for black koji mold koji produced in Example 1 and each koji produced in Comparative Examples 1 to 3.
- the enzyme activity and acidity of the acid protease were measured according to the method of the National Tax Agency specified analysis method comment (4th revision of the National Tax Agency specified analysis method, Japan Brewing Association, 1993).
- the xylanase activity was measured by the above-described method for measuring xylanase activity, wherein the amount of enzyme that liberates 1 mg of xylose per 60 minutes at 40 ° C. was defined as 1 unit by the above-mentioned Somogy Nelson method.
- the results are shown in FIGS. 2 (a) to (c).
- the enzyme titers of acid protease and xylanase were expressed in terms of dry matter.
- the black koji fungus bowl (tea koji / black koji) of the present invention has five times or more acidic protease activity compared to black koji mold rice koji (rice koji / black koji). I understood it.
- the black koji fungus bowl (tea koji / black koji) of the present invention has more than twice as many acidic protease activities as compared with the yellow koji fungus tea bowl (tea koji / black koji). Further, from the graph of FIG.
- the black koji mold tea bowl (tea koji / black koji) of the present invention has 10 times or more xylanase activity compared to black koji mold rice koji (rice koji / black koji). I found it. In addition, since the koji mold originally did not produce xylanase, the value of the xylanase activity of koji using koji mold was zero. Further, from the graph of FIG. 2 (c), the black koji fungus tea bowl (tea koji / black koji) of the present invention has an acidity of about one-third compared to black koji fungus rice koji (rice koji / black koji).
- Example 3 6). Confirmation of the umami enhancement effect by the fermented koji ferment composition
- each cocoon produced in Example 1 and Comparative Examples 1 to 3 was dehumidified and dried at low temperature to form a powder.
- 30 mg of each powdered rice cake was uniformly sprinkled on 100 g of pork, and allowed to stand in a refrigerator at 4 ° C. for 12 hours. The pork was taken out of the refrigerator, and the free glutamic acid content of the pork was quantified. The content of free glutamic acid was also quantified in pork that was not sprinkled with spear and treated with nothing. The results are shown in FIG.
- the protein component contained in the meat was decomposed and the amount of free glutamic acid was increased by sprinkling the meat on the meat.
- the pork treated with black koji fungus bowl (tea koji / black koji) of the present invention showed the highest amount of free glutamic acid compared to pork treated with koji from other test areas.
- the umami can be reliably enhanced by applying the black koji fungus tea bowl of the present invention to the foodstuff, and the black koji fungus tea bowl of the present invention is a seasoning that can impart a natural umami taste by ingredients derived from the foodstuff. I understood it.
- Example 4 Measurement of the antioxidant activity of koji fermented composition 1 Water was added to 1 kg of commercially available green tea, and the water content of green tea was adjusted to about 50%. This green tea was put into a drum-type koji making apparatus and steamed at 95 ° C. for 60 minutes. After cooling the tea leaves, 1 g (2 billion pieces / g) of Aspergillus awamori spores were added and mixed, and the mixture was made with a drum-type koji making machine for 3 days, and then the koji fermented composition (hereinafter referred to as “tea koji”). "). The anti-oxidant activity of this tea bowl was evaluated.
- the antioxidant activity of garlic and white rice was evaluated as green tea (used as a raw material for tea bowls) and other food materials.
- four types of tests were conducted: (1) DPPH radical scavenging activity, (2) superoxide anion radical scavenging activity, (3) hydroxyl radical scavenging activity, and (4) singlet oxygen scavenging activity. . Each test was conducted as follows.
- DPPH radical scavenging activity Each sample (tea bowl, green tea, garlic and white rice) was extracted with 50% ethanol to obtain an ethanol extract. About the extract of each sample, DPPH radical (DPPH.) Scavenging activity was measured. A 1.2 M 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution was added to the extract, and the absorbance at 520 nm was measured by a 96-well microplate reader method. DPPH radical scavenging activity was expressed in terms of Trolox equivalent (mgTE / 100 g) per 100 g of sample using Trolox as a standard substance.
- the signal of the obtained spin adduct was detected by an electron spin resonance apparatus (JES-FA100, manufactured by JEOL Ltd.).
- the superoxide scavenging activity was expressed in terms of SOD-like activity (units SOD / g) per 1 g sample from a calibration curve prepared using a superoxide dismutase (SOD) sample.
- DMSO-like activity ( ⁇ mol DMSO / g) per 1 g of a sample from a calibration curve prepared using dimethyl sulfoxide (DMSO) which is a scavenger of hydroxyl radical.
- JES-FA100 electron spin resonance apparatus
- the singlet oxygen scavenging activity was expressed in terms of histidine-like activity ( ⁇ mol histidine / g) per 1 g of a sample from a calibration curve prepared using histidine, which is a scavenger for singlet oxygen.
- FIGS. The results of each antioxidant activity test are shown in FIGS. It has been found that the tea cake of the koji fermented composition of the present invention has a high antioxidant effect compared to other foods such as garlic and white rice. Further, as is conventionally known, green tea contains catechins and vitamins and is known to have a high antioxidant effect.
- the koji fermentation composition (tea koji) of the present invention exhibits a very high antioxidant activity with DPPH radical scavenging activity 1.35 times that of green tea (FIG. 4), and the superoxide scavenging activity is about half that of green tea. (FIG. 5), hydroxyl radical scavenging activity (FIG. 6) and singlet oxygen scavenging activity (FIG. 7) were shown to have more than 60% of the activity of green tea.
- Example 5 8).
- Measurement of antioxidant activity of fermented cocoon composition 2 Thiobarbituric acid reactivity for quantifying the lipid peroxidation of cells with respect to three types of teacups (boiled fermented composition) produced in Example 4, green tea used as a raw material for teacups and bovine serum albumin (BSA) Substance (TBARS) tests were performed to evaluate antioxidant activity. The test was conducted as follows.
- Mouse myoblast cell line C2C12 cells were inoculated into a petri dish and allowed to grow and differentiate into myotube cells.
- the teacup extract, green tea extract and bovine serum albumin described above were each added to the medium at a concentration of 0.1% and cultured for 48 hours.
- Each culture solution was subjected to a TBARS test, and the amount of TBARS was measured.
- the total protein content of the cells in each culture was measured by the Bradford method. Specifically, 500 ⁇ L of the culture solution after 48 hours was taken in a microtube, and 1000 ⁇ L of a 5-fold diluted protein assay staining solution (Bio-Rad Laboratories product) was added and mixed.
- the absorbance at 595 nm was measured, and the total protein content was calculated from a standard curve with bovine serum albumin. From the TBARS value and the total protein content value, the TBARS concentration (TBARS MDAnmol / mg) per 1 mg of cell-derived protein in each culture solution was determined.
- a test was conducted in which oxidative stress was induced with iron nitrilotriacetate (Fe-NTA) to measure the antioxidant activity of the test sample.
- Fe-NTA iron nitrilotriacetate
- a test sample such as teacup extract
- iron nitrilotriacetate that induces oxidative stress is also added and cultured for 48 hours. went.
- the TBARS test and total protein content were measured to determine the TBARS concentration.
- the results are shown in the graph of FIG.
- the vertical axis is the TBARS concentration (TBARS MDAnmol / mg) per 1 mg of cell-derived protein in each culture solution, and no addition (white) indicates the value of the system to which no iron nitrilotriacetate is added, Fe -NTA addition (black) indicates the value of the system to which iron nitrilotriacetate that induces oxidative stress was added.
- TBARS MDAnmol / mg the TBARS concentration per 1 mg of cell-derived protein in each culture solution
- no addition indicates the value of the system to which no iron nitrilotriacetate is added
- Fe -NTA addition black indicates the value of the system to which iron nitrilotriacetate that induces oxidative stress was added.
- the total protein content of the cells in each culture solution (Fe-NTA added) measured in the test of this example is shown in the graph of FIG.
- the vertical axis represents the total protein content of the cells in the petri dish containing the culture solution.
- the koji fermentation composition of the present invention can be used as a seasoning or an antioxidant, and can also be used in the food field, food processing, food manufacturing field, and pharmaceutical field.
Abstract
Description
(1)米麹と比べて、クエン酸生成量が少なく、酸性プロテアーゼ活性及びキシラナーゼ活性が増強された麹発酵組成物を得ることができる。
(2)麹発酵組成物に含まれるプロテアーゼは強酸性下でも活性を維持するため、胃内部における食物の消化促進に寄与する。
(3)麹発酵組成物を食材に添加することにより、麹発酵組成物に含まれる酵素が食材に含まれる成分を効率よく分解し、自然な甘味や旨味を容易に食材に付与することができる。また、酵素分解作用により食材自体を軟らかくすることができる。
(4)麹発酵組成物は、脂質過酸化反応を抑制し、活性酸素を除去する作用、すなわち、抗酸化活性を有しているため、酸化防止剤、医薬又は機能性食品の有効成分として用いることができる。また、この麹発酵組成物は細胞増殖阻害活性が低く、食品由来の組成物であることから、安全性が高い。
まず、図1に示す茶葉を準備する工程S0について説明する。本発明における茶葉には、茶の葉だけでなく、茶樹から採取される茎や枝等も含まれる。本発明の麹発酵組成物の原料として使用される茶葉は、生茶葉だけでなく、採取後すぐに加熱処理された茶葉、採取後茶葉に含まれる酸化酵素で茶葉を酸化発酵させたウーロン茶又は紅茶、さらに後発酵茶であってもよい。また、本発明の麹発酵組成物の原料の茶葉として、これら緑茶、ウーロン茶又は紅茶等を煎じた後の茶滓を使用することもできる。本発明においては、原料として取り扱いがしやすく、後の工程において茶葉に含まれる酸化酵素による影響を受けないことから、採取後すぐに加熱処理された茶葉である緑茶が好ましい。また、後の蒸煮処理や黒麹菌の培養効率を高めるために、茶葉や茶の茎は大き過ぎないよう、ある程度の大きさに破砕されていることが好ましい。
次に、吸水処理工程S1について説明する。本工程では、茶葉に水を加えるか、水に浸漬させて吸水させる処理が行われる。吸水量は、茶葉の水分が20~60%、好ましくは30~50%となるように調整する。具体的には、たとえば、乾燥した緑茶を茶葉原料として使用する際には、茶葉の重量比により吸水させる水の量を決めることができる。
次に、蒸煮処理工程S2について説明する。上述の工程で吸水させた茶葉を蒸し器に入れて蓋をし、蒸し器を加熱することにより水蒸気を茶葉に当てて加熱する。蒸煮処理の時間は、30分~120分程度が好ましく、45分~90分がより好ましく、60分程度が特に好ましい。本工程を行うことにより、茶葉中の雑菌が殺菌されると共に、茶葉に含まれる酸化酵素が失活し、後の工程において、茶葉に黒麹菌を効率よく繁殖させることができる。蒸煮が終了した茶葉は、蒸し器から取り出し、台の上に均等に広げ、30~40℃程度にまで冷却させる。これにより、麹菌を茶葉に接種することが可能となる。
次に、黒麹菌を茶葉に接種する工程S3について説明する。本工程では、上述の蒸煮工程を経て冷却された茶葉に対し、黒麹菌を接種する。本発明において黒麹菌とは、沖縄での泡盛や鹿児島での芋焼酎等の蒸留酒の製造に用いられている黒色又は黒褐色の分生子(無性胞子の一種)を形成するアスペルギルス属のカビの一群のことをいう。具体的には、特に限定されないが、アスペルギルス・アワモリ、アスペルギルス・サイトイ、アスペルギルス・リュウキュウエンシス、アスペルギルス・イヌイ、アスペルギルス・ウサミ、アスペルギルス・アウレス等が挙げられる。本発明においては、酵素活性が向上し、クエン酸生成量が低減する効果が著しい観点から、アスペルギルス・アワモリ(Aspergillus awamori)、アスペルギルス・リュウキュウエンシス(Aspergillus luchuensis)又はアスペルギルス・イヌイ(Aspergillus inuii)及びこれらの組み合わせが好ましい。接種にあたっては、台上に広げた茶葉に黒麹菌を均一に散布し、植菌する。このとき、原料の茶葉1gに対して、黒麹菌の胞子が100万個以上になるように植菌することが好ましい。たとえば、種麹1gに含まれる胞子数が20億個の場合には、茶葉1kgに対して、種麹1g(0.1%)程度を添加すればよい。黒麹菌を茶葉に接種したのち、茶葉をよく撹拌して黒麹菌を茶葉全体に分散させることが好ましい。
ここで、茶葉に接種した黒麹菌を培養する工程S4について説明する。本工程では、黒麹菌を接種した茶葉に黒麹菌を繁殖させる。まず、黒麹菌を散布した茶葉を30℃前後に保った培養室に入れる。時間が経過するにしたがって発酵がすすみ、茶葉の温度が上昇するところ、麹菌は40℃を超えると増殖し難くなるため、通風して茶葉の温度を下げ、茶葉の品温が30℃~42℃、好ましくは30℃~40℃を維持するように通風量を調整する。具体的には、特に限定されないが、培養開始から12~30時間は通風により茶葉の温度が35℃~40℃になるように調整する。その後、同様に通風量を調整するなどして、茶葉の温度を30℃~35℃になるようにやや低めに調整し、培養開始から合計で1~4日間、好ましくは36~72時間、さらに好ましくは40~60時間程度培養した時点を麹発酵組成物の完成とする。
得られた麹発酵組成物は、茶葉に黒麹菌が繁殖した、いわゆる「こうじ」である。ところが、本発明の麹発酵組成物は、黒麹菌を繁殖させているにもかかわらず、通常の米麹等と比較してクエン酸生成量は半分以下に低減しており、酸性プロテアーゼ活性及びキシラナーゼ活性は増強されている。また、本発明の麹発酵組成物は、原料の茶葉同様に高い抗酸化活性を有しているが、細胞増殖阻害活性は茶葉とは異なり低くなっている。上述の工程により得られた麹発酵組成物は、一定の水分を含んだ状態であるが、自然乾燥させたり、低温除湿乾燥させることにより、水分を除去することができる。また、水分を除去した麹発酵組成物を粉砕して粉末状としたり、顆粒状とすることも可能である。いったん水分を除去したものにおいても、麹発酵組成物としての機能は有効に保持され得る。
1.黒麹菌を用いた麹発酵組成物(黒麹菌茶麹)の製造
市販の緑茶1kgに水を加え、緑茶の水分含有量が30%~40%の範囲となるように調整した。次に蒸し器で60分蒸煮し、35℃に冷却したのち、黒麹菌の胞子1g(20億個/g)を均一に散布した。黒麹菌はアスペルギルス・アワモリを用い、散布量は原料の緑茶1gあたり胞子が100万個以上となるように調整した。胞子を接種した後、35℃で保温し培養を行った。発酵が進むにつれて発酵物の温度が上昇したため、培養開始24時間までは、発酵物の温度が40℃以下になるように通風して調整した。その後同じく通風により発酵物の温度を34~35℃程度に下げ、さらに16時間培養を行って本発明の麹発酵組成物(以下、「黒麹菌茶麹」という。)を得た。
2.黄麹菌を用いた麹発酵組成物の製造
黒麹菌であるアスペルギルス・アワモリに代えて、黄麹菌であるアスペルギルス・オリゼーを使用した以外は、上述の実施例1と同様にして、比較例1の麹発酵組成物(以下、「黄麹菌茶麹」という。)を得た。
3.黒麹菌を用いた黒麹菌米麹の製造
原料の緑茶に代えて、白米を使用した以外は、上述の実施例1と同様にして製麹し、比較例2の麹発酵組成物(以下、「黒麹菌米麹」という。)を得た。
4.黄麹菌を用いた黄麹菌米麹の製造
原料の緑茶に代えて白米を使用し、黒麹菌であるアスペルギルス・アワモリに代えて、黄麹菌であるアスペルギルス・オリゼーを使用した以外は、上述の実施例1と同様にして製麹し、比較例3の麹発酵組成物(以下、「黄麹菌米麹」という。)を得た。
5.麹発酵組成物の酵素活性及びクエン酸量の測定
実施例1で製造した黒麹菌茶麹及び比較例1~3で製造した各麹について、酸性プロテアーゼ及びキシラナーゼの酵素活性並びに酸度を測定した。酸性プロテアーゼの酵素活性及び酸度の測定は、国税庁所定分析法注解(第四回改正国税庁所定分析法注解、財国法人日本醸造協会、1993年)の方法に従って行った。他方、キシラナーゼ活性は、上述したソモジー・ネルソン法にて40℃で60分間あたり、1mgのキシロースを遊離する酵素量を1ユニットと定義し、前述したキシラナーゼ活性測定方法にて測定を行った。結果を図2(a)~(c)に示す。酸性プロテアーゼ及びキシラナーゼの酵素力価は乾物換算値で表わした。
6.麹発酵組成物による旨味増強効果の確認
実施例1で製造した本発明の黒麹菌茶麹及び比較例1~3で製造した各麹が、食材にどのような影響を与えるかを確認するため、以下の試験を行った。まず、実施例1及び比較例1~3で製造した各麹をそれぞれ低温除湿乾燥させ、粉末状とした。次に、粉末状の各麹30mgを豚肉100gに均一にそれぞれふりかけ、4℃の冷蔵庫にて12時間静置した。冷蔵庫から豚肉を取り出し、豚肉の遊離グルタミン酸含量を定量した。麹をふりかけず、何も処理しなかった豚肉についても遊離グルタミン酸含量を定量した。結果を図3に示す。
7.麹発酵組成物の抗酸化活性の測定1
市販の緑茶1kgに水を加え、緑茶の水分含有量が約50%となるように調整した。この緑茶をドラム式製麹装置に入れ、95℃条件下で60分蒸煮処理した。茶葉を冷却したのち、黒麹菌(アスペルギルス・アワモリ)の胞子1g(20億個/g)を添加混合し、ドラム式製麹装置で3日間製麹して麹発酵組成物(以下、「茶麹」という。)を得た。この茶麹の抗酸化活性を評価した。対照区として、緑茶(茶麹の原料として用いたもの)、他の食品材料として、にんにく及び白米の抗酸化活性を評価した。抗酸化活性の評価にあたっては、(1)DPPHラジカル消去活性、(2)スーパーオキシドアニオンラジカル消去活性、(3)ヒドロキシルラジカル消去活性及び(4)一重項酸素消去活性の4種類の試験を行った。各試験は次のようにして行った。
各試料(茶麹、緑茶、にんにく及び白米)を50%エタノールで抽出し、エタノール抽出液を得た。この各試料の抽出液について、DPPHラジカル(DPPH・)消去活性を測定した。抽出液に1.2Mの1,1-ジフェニル-2-ピクリルヒドラジル(DPPH)溶液を添加し、520nmにおける吸光度を96穴マイクロプレートリーダー法にて測定した。DPPHラジカル消去活性はTroloxを標準物質とし、試料100gあたりのTrolox相当量(mgTE/100g)に換算して表わした。
各試料(茶麹、緑茶、にんにく及び白米)を90℃のイオン交換水に加え、90℃条件下で5分間抽出して水抽出液を得た。この各試料の水抽出液について、ESRスピントラップ法を用いてスーパーオキシドアニオンラジカル(O2・-)の消去活性を測定した。スーパーオキシドアニオンラジカル(O2・-)はヒポキサンチン/キサンチンオキシダーゼ系で発生させ、スピントラップ剤にはDMPO(5,5-ジメチル-1-ピロリン-N-オキシド)を用いた。得られたスピンアダクトのシグナルを電子スピン共鳴装置(JES-FA100、日本電子株式会社製品)により検出した。スーパーオキシド消去活性は、スーパーオキシドディスムターゼ(SOD)標品を用いて作成した検量線から、試料1gあたりのSOD様活性(units SOD/g)に換算して表わした。
各試料(茶麹、緑茶、にんにく及び白米)を90℃のイオン交換水に加え、90℃条件下で5分間抽出して水抽出液を得た。この各試料の水抽出液について、ESRスピントラップ法を用いてヒドロキシルラジカル(・OH)消去活性を測定した。抽出液50μL、0.55mMのジエチレントリアミン五酢酸(DTPA)を含む0.1mM硫酸第1鉄溶液75μL、1mMの過酸化水素溶液75μL及び8.8mMのDMPO溶液20μLを添加混合した後、直ちに電子スピン共鳴装置(JES-FA100、日本電子株式会社製品)によりシグナルを検出した。ヒドロキシルラジカル消去活性は、ヒドロキシルラジカルのスカベンジャーであるジメチルスルホキシド(DMSO)を用いて作成した検量線から、試料1gあたりのDMSO様活性(μmol DMSO/g)に換算して表わした。
各試料(茶麹、緑茶、にんにく及び白米)を90℃のイオン交換水に加え、90℃条件下で5分間抽出して水抽出液を得た。この各試料の抽出液について、ESRスピントラップ法を用いて一重項酸素(1O2)消去活性を測定した。抽出液40μL、1mMの2-アミノ-4(1H)-プテリジノン(プテリン)40μL、100mMの2,2,6,6-テトラメチル-4-ピペリドン(TMPD)40μL、15mMのジエチレントリアミン五酢酸(DTPA)20μL及び100mMのリン酸バッファー(pH7.4)60μLを添加混合し、40秒間紫外線を照射した後、電子スピン共鳴装置(JES-FA100、日本電子株式会社製品)を用いてシグナルを測定した。一重項酸素消去活性は、一重項酸素のスカベンジャーであるヒスチジンを用いて作成した検量線から、試料1gあたりのヒスチジン様活性(μmol Histidine/g)に換算して表わした。
8.麹発酵組成物の抗酸化活性の測定2
実施例4で製造した茶麹(麹発酵組成物)、茶麹の原料として用いた緑茶及び牛血清アルブミン(BSA)の3種に関し、細胞の脂質過酸化反応を定量するチオバルビツール酸反応性物質(TBARS)検査を行い、抗酸化活性を評価した。試験は次のようにして行った。
Claims (9)
- 茶葉に黒麹菌を接種し、培養することにより得られる麹発酵組成物。
- 前記黒麹菌は、蒸煮処理された茶葉に接種されることを特徴とする請求項1に記載の麹発酵組成物。
- 前記黒麹菌がアスペルギルス・アワモリ(Aspergillus awamori)、アスペルギルス・リュウキュウエンシス(Aspergillus luchuensis)及びアスペルギルス・イヌイ(Aspergillus inuii)からなる群より選ばれる少なくとも1つの黒麹菌であることを特徴とする請求項1又は2に記載の麹発酵組成物。
- 請求項1~3のいずれか1項に記載の麹発酵組成物又は該麹発酵組成物の抽出物を含有する調味料。
- 請求項1~3のいずれか1項に記載の麹発酵組成物又は該麹発酵組成物の抽出物を含有する抗酸化剤。
- 請求項1~3のいずれか1項に記載の麹発酵組成物又は該麹発酵組成物の抽出物を含有する食品又は飲料。
- 請求項1~3のいずれか1項に記載の麹発酵組成物又は該麹発酵組成物の抽出物を食材に添加することを特徴とする食材の旨味増強方法。
- 茶葉に黒麹菌を接種する工程と、前記茶葉を培養する工程とを備えることを特徴とするクエン酸生成量が低減し、プロテアーゼ活性及びキシラナーゼ活性が増強された麹発酵組成物の製造方法。
- 前記黒麹菌がアスペルギルス・アワモリ(Aspergillus awamori)、アスペルギルス・リュウキュウエンシス(Aspergillus luchuensis)及びアスペルギルス・イヌイ(Aspergillus inuii)からなる群より選ばれる少なくとも1つの黒麹菌であることを特徴とする請求項8に記載の麹発酵組成物の製造方法。
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US9961930B2 (en) | 2014-02-10 | 2018-05-08 | Kirishima Highland Beer Co., Ltd. | Koji fermented composition, seasoning using the same, antioxidant, and food or beverage |
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- 2015-01-29 WO PCT/JP2015/052513 patent/WO2015119036A1/ja active Application Filing
- 2015-01-29 JP JP2015560955A patent/JP6392253B2/ja active Active
- 2015-01-29 EP EP15745759.9A patent/EP3109316A4/en not_active Withdrawn
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US9961930B2 (en) | 2014-02-10 | 2018-05-08 | Kirishima Highland Beer Co., Ltd. | Koji fermented composition, seasoning using the same, antioxidant, and food or beverage |
JP2017176175A (ja) * | 2016-03-24 | 2017-10-05 | 二洋商事有限会社 | 醗酵原料として醗酵茶を含む茶味噌及びその製造方法 |
WO2019131274A1 (ja) * | 2017-12-26 | 2019-07-04 | 国立大学法人 筑波大学 | 緑茶抽出物由来の発酵物の製造方法及び緑茶抽出物由来の麹発酵物 |
WO2020008821A1 (ja) | 2018-07-02 | 2020-01-09 | 一般社団法人夢源 | 男性不妊症の治療、予防又は改善用組成物 |
WO2020095590A1 (ja) * | 2018-11-08 | 2020-05-14 | 一般社団法人夢源 | 容器詰飲料及び麹抽出液の保存方法 |
JP2020074749A (ja) * | 2018-11-08 | 2020-05-21 | 一般社団法人夢源 | 容器詰飲料及び麹抽出液の保存方法 |
JP7333089B2 (ja) | 2018-11-08 | 2023-08-24 | 一般社団法人夢源 | 容器詰飲料の製造方法 |
JPWO2020226022A1 (ja) * | 2019-05-07 | 2020-11-12 | ||
JP7170357B2 (ja) | 2019-05-07 | 2022-11-14 | 一般社団法人夢源 | 育毛・発毛用外用剤 |
JP2022151047A (ja) * | 2021-03-26 | 2022-10-07 | 鹿児島県 | 後発酵茶並びにそれを利用した飲料及び食品 |
Also Published As
Publication number | Publication date |
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EP3109316A1 (en) | 2016-12-28 |
US20160309760A1 (en) | 2016-10-27 |
EP3369806A1 (en) | 2018-09-05 |
SG11201605419TA (en) | 2016-08-30 |
JPWO2015119036A1 (ja) | 2017-03-23 |
JP6392253B2 (ja) | 2018-09-19 |
US9961930B2 (en) | 2018-05-08 |
EP3109316A4 (en) | 2017-10-25 |
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