JP4838628B2 - Method for producing composition with high content of γ-aminobutyric acid - Google Patents

Description

  The present invention relates to a method for producing a γ-aminobutyric acid-rich composition, which comprises inoculating and culturing a microorganism having an ability to produce γ-aminobutyric acid in a medium containing a composition obtained from asparagus, and containing the same It relates to food and drink and feed.

  γ-Aminobutyric acid (hereinafter abbreviated as GABA) is a non-protein constituent amino acid that exists widely in the living world but is known to act as a neurotransmitter in the brain. Yes. GABA as a food material has effective physiological effects for modern people with a high awareness of health maintenance, such as blood pressure lowering action, tranquilizing action, brain function improving action, climacteric disorder symptom reducing action, neutral fat increase suppressing action, etc. . In addition, GABA has no side effects even when taken in large quantities by humans, so it is advantageous in terms of safety, and development that is added to food as an ingredient to prevent lifestyle-related diseases, particularly hypertension, where diet is effective. There have been many.

  As such, technologies for producing GABA-enriched grains using the action of enzymes originally contained in rice germ, rice bran, wheat germ, etc. (see, for example, Patent Documents 1 and 2), tomatoes, pumpkins Technology for producing a GABA-enriched composition using the action of enzymes contained in vegetables such as vegetables (see, for example, Patent Documents 3 to 5), producing tea leaves with high GABA content by anaerobic treatment of tea leaves The technique (for example, refer patent document 6) etc. to report are reported.

Moreover, it is known that some microorganisms are excellent in the ability to produce GABA from glutamic acid, and a technique for producing GABA with lactic acid bacteria (for example, Patent Documents 7 and 8) and a technique for producing GABA with koji molds (for example, (See Patent Document 9). Among these, lactic acid bacteria have a high production efficiency of GABA. L. plantarum (for example, see Patent Document 11), Lactobacillus lactis (for example, see Patent Document 12), and the like are disclosed. On the other hand, a method of adding a soybean extract to a culture medium as a GABA production ability promoting factor of lactic acid bacteria has been disclosed (for example, see Patent Document 13).
Japanese Patent No. 2590423 JP 2004-159617 A Japanese Examined Patent Publication No. 7-12296 Japanese Patent Publication No. 7-14333 JP 2001-252091 A Japanese Patent No. 3038373 JP 2001-352940 A JP 2003-70462 A JP-A-11-103825 JP 2004-215529 A Japanese Patent No. 2704493 JP 2001-120179 A JP 2003-157713 A

  However, the conventionally disclosed methods have low GABA production efficiency. For example, Lactobacillus plantarum is finally 2% by mass or less of the culture solution (Patent Documents 7 and 10), and Lactobacillus brevis is about 1.1% by mass ( In Patent Document 10) and Lactobacillus lactis, it was 1% by mass or less (Patent Document 12), and in Lactobacillus hilgardi, it was 4.8% by mass (Patent Document 8) after 48 hours. In addition, Lactobacillus brevis UAS-4 strain isolated by the present inventors is 10.0% in mass of the culture solution after 48 hours, and Lactobacillus brevis UAS-6 strain is 10.2 mass in the culture solution after 48 hours. %, It has been found that it is possible to produce GABA fairly efficiently, so a patent application has already been filed (Japanese Patent Application No. 2005-330200), but it is still not satisfactory, and a large amount of GABA is produced in a shorter time. There was a need for a way to be.

  On the other hand, in the method of adding a soybean extract to a medium as a factor for promoting GABA production ability of lactic acid bacteria (Patent Document 13), even if culturing for 7 days in a medium containing 90% soybean broth, 5% by mass of the culture liquid The glutamic acid of 100% was not converted, and it was never satisfactory.

  An object of the present invention is to provide a method capable of easily producing a composition containing a high concentration of GABA in a short time.

  As a result of earnestly studying the material containing the GABA production ability promoting factor of lactic acid bacteria, the present inventors have increased the GABA production ability of lactic acid bacteria by including a composition obtained from asparagus in the medium. The present inventors have found that GABA can be produced efficiently and have completed the present invention.

  That is, the first of the present invention is characterized in that GABA is obtained from a culture obtained by inoculating and culturing a microorganism having the ability to produce γ-aminobutyric acid in a medium containing a composition obtained from asparagus. The method for producing a GABA high-content composition is as follows. Preferably, the microorganism having γ-aminobutyric acid is a lactic acid bacterium, more preferably a lactic acid bacterium belonging to the genus Lactobacillus, most preferably. Is Lactobacillus brevis (L. brevis) UAS-4 strain (FERM-P20710) or Lactobacillus brevis (L. brevis) UAS-6 strain (FERM-P20711). In the above-described method for producing a GABA high-content composition, preferably, glutamic acid and / or glutamate and / or their contents are added to the medium, and preferably yeast extract is added to the medium. Is the method. In the above-described method for producing a high GABA-containing composition, preferably, the sugar concentration (converted to Brix) of the medium is 0.1 to 40%, and preferably the pH of the medium during the culture is 3 A method of adjusting the pH of the medium during culture to 3.5 to 8.0, preferably glutamic acid and / or glutamate and / or their contents. It is a manufacturing method of the said GABA high content composition which is a method of adding.

  The second aspect of the present invention is a high-GABA composition characterized by being obtained by any one of the production methods described above.

  The third aspect of the present invention is a food or drink or feed characterized by containing the second GABA high-content composition of the present invention.

  According to the present invention, a high-concentration GABA-containing composition can be easily obtained in a short time. Moreover, since the GABA containing composition of this invention contains high concentration GABA, even when mix | blending with food-drinks, an effect can be anticipated with a small amount, and it does not have a bad influence on the taste of food-drinks. In addition, GABA can reduce blood pressure, relax, relieve stress, improve menopause symptoms, improve insomnia, diuresis, kidney function, liver function, etc. It can be expected to synergistically increase the action of GABA, improve gastrointestinal disorders, and have anti-fatigue effects.

  Hereinafter, the present invention will be described in detail.

  First, a method for producing a composition obtained from asparagus in the present invention will be described.

  The composition obtained from asparagus in the present invention is a known technique such as pulverization, crushing, grinding, shredding, extraction, pressing, concentration, solid-liquid separation, heat sterilization, filter sterilization, enzyme treatment, drying, etc. Can be obtained alone or in combination of two or more.

  The asparagus used in the present invention may be any as long as it does not impair the effects of the present invention. Asparagus includes green asparagus that is cultivated in the sun, white asparagus that is cultivated while shading with soil, etc., and mini asparagus that is trimmed while it is thin and short. Green asparagus rich in nutritional components such as is preferable. The production area is not particularly limited, and it may be domestically produced or imported from overseas. The part to be used is not particularly limited and may be a stem part or a tip part, but it is preferable to use a stem part. Among them, the lowest cut when aligning the length of asparagus as a product is most preferable because it can be obtained at low cost.

  Crushing, crushing, crushing, and shredding are methods of physically crushing plants, crushing and crushing by impact, grinding by friction, and shredding by cutting. Grinding, crushing, grinding and shredding may be done manually using a hammer, mortar, kitchen knife, cutter knife, scissors, etc. use. Examples of such an apparatus include a mill, a hammer type pulverizer, a stone mill type pulverizer, a mixer, a blender, and an ultrasonic crusher, and a vegetable shredder may be used. The size of the pulverized, crushed, ground, and shredded plant body is not particularly limited, but is preferably 2 cm or less, and more preferably 5 mm or less.

  Extraction is an operation of adding a solvent to transfer plant components to the plant. As a solvent to be added, water is most preferable, but an organic solvent may be used in combination. The organic solvent may be used in combination with water at the time of extraction, or the extraction may be performed with the organic solvent alone and then mixed with water. In addition, extraction may be performed with an organic solvent alone, and then water may be added after removing the organic solvent by an operation such as concentration to dryness. Although the kind of organic solvent is not specifically limited, Ethanol is preferable at the point which can be used for a foodstuff. In the case of water, the temperature of the solvent at the time of extraction is preferably 0 ° C to 100 ° C, more preferably 10 ° C to 80 ° C, and in the case of an organic solvent, -20 ° C to 200 ° C is preferable, and 0 ° C to 120 ° C. ° C is more preferred.

  Squeezing is a method in which a physical pressure is applied to a plant body to squeeze the liquid and transfer the components to the juice. The pressure may be applied only in one direction, may be applied from two or more directions, and may be accompanied by a shearing force. Although the operation of pressing is easy if a commercially available pressing machine is used, it may be performed by a method that does not use a machine such as hand pressing or stepping. At this time, water or hot water may be added to the plant body and squeezed.

  Concentration is an operation of reducing the amount of water and solvent without reducing other components, and may be performed by any method such as concentration under reduced pressure, concentration by heating, concentration using a filtration membrane, but 20 ° C to 60 ° C. It is preferable to carry out vacuum concentration in the range.

  Solid-liquid separation is a method of separating the solvent and the components dissolved therein from the insoluble solid content. As the separation method, for example, various methods such as filter filtration, squeeze filtration, centrifugation, and decantation are used. it can. When obtaining a clear plant extract, it is preferable to perform filter filtration using a filter aid such as diatomaceous earth.

  Heat sterilization is sterilization by applying heat, and the temperature is preferably 60 ° C to 121 ° C, more preferably 70 ° C to 110 ° C. If it is lower than this temperature range, there is a problem that the effect of sterilization becomes insufficient, and if it is higher than this temperature range, there is a problem that the active ingredient is decomposed.

  Filtration sterilization is an operation for sterilization by passing a membrane of a size through which microorganisms cannot pass under pressure or reduced pressure. The pore diameter of the membrane is preferably from 0.1 to 1.0 μm, more preferably from 0.2 to 0.45 μm.

  Enzyme treatment is treatment with a biocatalyst that promotes the degradation of the components of the plant, and the type of enzyme is not particularly limited, but cellulase, pectinase, arabanase, protease, glutaminase and the like are preferably exemplified. Enzyme treatment conditions may be performed at a temperature, pH, and addition amount suitable for each enzyme. The reaction product after the enzyme treatment may be used for subsequent fermentation as it is, but is preferably deactivated. The deactivation method is not particularly limited, but heat treatment is preferable.

  Drying means removing a solvent such as moisture in the sample, and any known technique such as natural evaporation, heat drying, vacuum drying, spray drying, freeze drying, etc. may be used.

  A preferable example of a combination of these techniques is that asparagus is chopped and extracted with water, then heat sterilized and filtered, and the filtrate is concentrated and then sterilized by filtration.

  In the present invention, a medium is prepared using the composition obtained as described above. The composition derived from asparagus may be used alone for culturing lactic acid bacteria, and even in that case, the GABA production ability of lactic acid bacteria is promoted, but it can also be used by mixing with a known medium for lactic acid bacteria. . As such a medium, GYP medium (D-glucose 1%, peptone 0.5%, yeast extract 1%, sodium acetate trihydrate 0.2%, twin 80 0.05%, magnesium sulfate heptahydrate 0 0.02%, manganese sulfate tetrahydrate 10 ppm, iron sulfate heptahydrate 10 ppm, sodium chloride 10 ppm), commercially available GAM medium (Nissui Pharmaceutical), MRS medium (Difco) and the like. All these components may be added to these media, or only one or more arbitrary components may be added. In particular, when yeast extract is mixed, the GABA producing ability of lactic acid bacteria is the highest. When mixing medium components other than the composition derived from asparagus, the proportion of them in the whole medium is preferably 0 to 90% by mass, more preferably 0 to 50% by mass as the solid content, and 0 to 30% by mass is most preferred.

  Yeast extract is a water-soluble extract that is extracted by treating the useful components of yeast cells with self-digestion, enzymes, hot water, etc., but it contains water-insoluble components such as cells. It doesn't matter. Any yeast cell species may be used as long as it has food experience. Saccharomyces cerevisiae used as beer yeast or baker's yeast, and Candida utilis used as torula yeast. Etc. are examples. The yeast extract may be a commercially available product (Difco, Kirin Brewery, Asahi Breweries, etc.) or may be self-adjusted.

  Although the addition amount of a yeast extract is not specifically limited, 0.01-10 mass% should just be added with respect to a culture medium, Preferably it is 0.1-5 mass%, More preferably, it is 0.5-3 mass%. If the yeast extract concentration falls below this range, the effect is hardly obtained, and if it exceeds this range, the effect cannot be expected to increase, resulting in high costs, adverse effects such as flocking when heated, and a significant impact on taste quality. Occurs.

  In the present invention, the sugar concentration (converted to Brix) of the medium prepared as described above is preferably 0.1 to 40%, more preferably 0.1 to 20%, and 0 Most preferably, it is 5 to 5%. If the sugar concentration (in terms of Brix) is outside this range, the growth of lactic acid bacteria becomes extremely poor. When the sugar concentration (converted to Brix) is less than 0.1%, the sugar concentration can be increased. When the sugar concentration (in terms of Brix) exceeds 40%, it is sufficient to add water to reduce it.

  In the present invention, it is preferable to further add glutamic acid or / and a salt thereof and / or a content thereof to the medium prepared as described above. Of these, any glutamate can be used, but sodium glutamate which is a food additive and has excellent solubility in water is preferable. In addition, any material can be used as the glutamic acid and / or salt thereof, but seasonings such as yeast extract that can be added to foods are preferred. Of these, sodium glutamate, which is excellent in solubility in water, and glutamic acid are desirable in terms of suppressing an increase in pH associated with GABA production, and more preferably a combination thereof.

  Further, in the present invention, instead of adding glutamic acid or / and its salt or / and their contents, or in addition to adding glutamic acid or glutamine in the composition obtained from asparagus by enzymatic treatment, It is also possible to increase the amount. Examples of the enzyme used here include protease, peptidase, and glutaminase that are food additives. The amount of the enzyme used for carrying out the enzymatic decomposition is preferably 0.0001% by mass to 10% by mass, preferably 0.001% by mass to 5% by mass, based on the solid content, although it depends on the type of enzyme and the titer. Is more preferable. If it is less than this range, there is a problem that sufficient enzymatic degradation cannot be expected, and if it is more than this range, an increase in enzymatic degradation can no longer be expected, and there is a problem that changes the taste quality due to enzymes in the product. is there. The temperature during the enzymatic decomposition is preferably 0 ° C to 80 ° C, more preferably 10 ° C to 60 ° C. If it is lower than this range, the progress of the enzymatic reaction tends to be slow and it takes a long time to degrade the protein. If it is higher than this range, the enzyme may be deactivated. Moreover, it is preferable to perform enzyme deactivation treatment before adding lactic acid bacteria. The temperature of the enzyme deactivation treatment is preferably 50 ° C to 120 ° C, more preferably 70 ° C to 100 ° C. The enzyme deactivation treatment time varies depending on the temperature, but is preferably 5 to 30 minutes, more preferably 5 to 15 minutes.

  The amount of glutamic acid or / and its salt or / and content thereof increased by the above method is preferably 0.1 to 40% by mass, more preferably 0.2 to 25% by mass as the final concentration of glutamic acid. 0.5 to 20% is most preferable. If the amount is less than this range, the amount of GABA obtained is small, and if it is more than this range, there is a problem that glutamic acid and / or glutamate remains, or it takes a long time even if the whole amount is converted to GABA. In addition, when adding glutamic acid and / or a salt thereof and / or their contents, a predetermined amount may be added once before culturing, or added in several times during culturing and the concentration described above. It is good. When adding during culture | cultivation, the addition time is not specifically limited. From the viewpoint of workability, it is preferable to add the whole amount at a time, but it is preferable to add a small amount in order to cause rapid pH fluctuations. During the production of GABA, it is preferable to add glutamic acid multiple times because the pH rises.

  The microorganism having the ability to produce GABA used in the present invention is safe even when used in foods and needs to have the ability to produce GABA. Examples of such microorganisms include lactic acid bacteria, yeasts, and koji molds. Of these, lactic acid bacteria are preferred because of their high ability to produce GABA. Lactic acid bacteria include Lactobacillus brevis, L. et al. hilgardi, L.H. plantaram, L.M. casei, L .; paracasei, L .; helveticus, L.H. bulgaricus, L .; acidophilus, L. et al. sp. Streptococcus lactis, S. et al. Examples include lactic acid bacteria belonging to thermophilus, Enterococcus caseiflavus and the like. Among the lactic acid strains belonging to these, a composition obtained from asparagus is highly effective in promoting GABA production. Lactic acid bacteria belonging to brevis are preferred. Among these, L. brevis UAS-4 (FERM P-20710), L.M. brevis UAS-6 (FERM P-20711), L.M. brevis IFO 3345, L.M. brevis IFO12005 is more preferable because of its high ability to produce GABA.

  L. brevis UAS-4 (FERM P-20710), L.M. brevis UAS-6 (FERM P-20711) is a bacterium that the present inventors have isolated asparagus as a separation source, and has the properties shown in Table 1 below.

In addition, as a result of specifying about 500 bases on the 5′-end side of 16S rRNA of UAS-4 and UAS-6 by using a DNA sequencer, both were obtained from the National Center for Biotechnology Information (NCBI). It had a homology of 99% or more with the 16S rRNA sequence of Lactobacillus brevis possessed.

  For the UAS-6 strain, L-arabinose was not assimilated, and the general L. Considering that brevis favorably assimilate L-arabinose, this is a characteristic property of this strain. Moreover, since both UAS-4 strain and UAS-6 strain have significantly higher ability to produce γ-aminobutyric acid from glutamic acid or glutamic acid as described later, The UAS-4 strain is received as the receipt number FERM P-20710, and the UAS-6 strain is the receipt number FERM. It was entrusted on November 11, 2005 as P-20711.

  In the present invention, next, a culture medium containing a composition obtained from asparagus is inoculated with a microorganism capable of producing GABA and cultured, and the conditions will be described.

  The method of adding microorganisms can be propagated by inoculating a small amount of cells directly on a composition obtained from asparagus prepared as described above or a medium containing the same, but the concentration of cells in a short period of time. In order to raise the amount, it is preferable to inoculate a pre-cultured bacterial solution. As the pre-culture solution, a composition obtained from the same asparagus as in the main culture or a medium containing the same may be used, and any of the above-mentioned known mediums may be used. The amount inoculated with the pre-cultured bacterial solution is 1 / 100,000 of the amount of the main culture medium, preferably 1/1000 to 1/10, and preferably 1/200 to 30 minutes. 1 is more preferable. If the inoculation amount is less than this range, there is a problem that it takes time to increase the bacterial cell concentration, and if it is more than this range, it is already a large scale at the time of pre-culture and there is no need to perform main culture. It is.

  Although it depends on the strain used, the culture temperature is 5 ° C to 45 ° C, preferably 15 ° C to 40 ° C, and more preferably 20 ° C to 35 ° C. There is a problem that the growth rate is remarkably inferior even if the culture temperature is higher or lower than this temperature range.

  Although the pH of the culture solution during culture depends on the strain used, it is preferably adjusted to 3.5 to 8.0, more preferably 4.0 to 6.5, and 4.5 to 5. It is most preferable to adjust to 5. When the pH is out of this range, there is a problem that the activity of glutamate decarboxylase is lowered and the production rate of GABA is lowered. Any chemical can be used for pH adjustment, such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, glutamic acid, acetic acid, butyric acid, lactic acid, formic acid, succinic acid, maleic acid, malic acid, oxalic acid, citric acid, sodium hydroxide Potassium hydroxide, calcium hydroxide, aqueous ammonia, sodium glutamate and the like. In the present invention, the pH tends to increase according to the production of GABA, and adjustment is mainly performed by adding an acid. Among these, hydrochloric acid, phosphoric acid, glutamic acid, acetic acid, lactic acid, succinic acid, apple Acid and citric acid are more preferable, and hydrochloric acid, glutamic acid, lactic acid and acetic acid are more preferable, and glutamic acid is most preferable because it is also used as a substrate for GABA.

  Although the oxygen condition at the time of culture depends on the strain used, it can be grown under anaerobic or aerobic conditions. However, since there are strains that can grow under aerobic conditions but the GABA production ability decreases, it is preferable to use anaerobic conditions such as anaerobic conditions or moderate stirring, for example, bubbling or rapid stirring is required. There is no.

  The culture time is not particularly limited, but is preferably 2 hours to 10 days, more preferably 5 hours to 5 days, and most preferably 8 hours to -3 days. If the fermentation time is below this range, the conversion from glutamic acid to GABA becomes insufficient, and if it exceeds this range, no further effect can be expected, and the possibility of contamination and growth of bacteria increases.

  By culturing as described above, GABA accumulates in the culture, and a GABA-rich composition can be obtained by performing the following treatment as it is or if necessary. . As the treatment, operations such as sterilization, sterilization, solid-liquid separation, and drying can be performed to form a clarified liquid or powder. Further, after adding an appropriate carrier, it may be formed into a edible form such as granules, granules, tablets, capsules, gels, pastes, milks, suspensions, liquids, beverages and the like by conventional methods.

  When preparing in a solid form, it is sufficient to use those used in conventional methods such as excipients, binders, disintegrants, lubricants, flavoring agents, colorants, and examples of such carriers. As an excipient, lactose, sucrose, glucose, mannitol, sorbit, dextrin, starch, crystalline cellulose, carboxymethylcellulose, hydroxypropylcellulose, dextran, pullulan, anhydrous silicic acid, calcium phosphate, calcium carbonate, calcium sulfate, etc. Binders include crystalline cellulose, sucrose, mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, gum arabic, dextran, pullulan, water, ethanol, etc., and disintegrants include starch, carboxymethylcellulose, hydroxypropyl Pyrcellulose, dextrin, crystalline cellulose, etc., as a lubricant, stearic acid and its metal salts, talc, boric acid, fatty acid sodium salt, sodium lauryl sulfate, magnesium lauryl sulfate, silicic acid anhydride, etc. Examples include sucrose, orange peel, citric acid, tartaric acid and the like.

  When preparing in a liquid state, it is sufficient to use an emulsifier, a solubilizer, a dispersant, a suspending agent, a thickener, a buffer, a stabilizer, a flavoring agent, etc. As the emulsifier and solubilizer, lecithin, sucrose fatty acid ester, sorbitan fatty acid ester, sodium lauryl sulfate and the like, and as the dispersant and suspending agent, lecithin, sucrose fatty acid ester, methylcellulose, gum arabic, gelatin and the like, As a thickener, methylcellulose, carboxymethylcellulose, gum arabic, gelatin, etc., as a buffer, citrate, succinate, etc., as a stabilizer, lecithin, gum arabic, gelatin, methylcellulose, as a flavoring agent What was mentioned above can be illustrated.

  In addition, sugars, sugar alcohols, salts, oils and fats, amino acids, organic acids, fruit juices, vegetable juices, fragrances, spices, alcohols, glycerin, and the like can be added to improve the taste. In addition, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, butyric acid, lactic acid, succinic acid, malic acid, oxalic acid, citric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, aqueous ammonia, etc. are added for pH adjustment. can do.

  Next, the third food and drink and feed of the present invention will be described.

  The food-drinks and feed of this invention are characterized by containing the GABA high content composition manufactured as mentioned above. The content of the GABA high-content composition in the food and drink and feed is not particularly limited, and the GABA high-content composition itself can be used as the food and drink and feed, but generally converted to GABA per day It is preferable to mix | blend so that the quantity to ingest may be 10-500 mg. If it is less than this range, the effect may not be expected, and if it is more than this range, the increase in the effect may no longer be expected.

  When a GABA high content composition is included in existing food and drink and feed, it is not particularly limited as a base food and drink and feed. For example, processed noodles such as udon and pasta, processed meat products such as ham and sausage Processed foods such as kamaboko and chikuwa, processed milk products such as butter, milk powder and fermented milk, desserts such as jelly and ice cream, processed foods such as breads, confectionery and seasonings, soft drinks, alcohol Beverages such as wines, fruit juice drinks, vegetable juice drinks, milk drinks, carbonated drinks, coffee drinks, and alcohols are preferred.

  The form of the GABA high-content composition of the present invention to be included in foods and drinks and feeds is not particularly limited, and liquids are used for beverages, gummi, candy, etc., and powders are used for tablets, granules, capsules, feeds, etc. You can use things.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. In the examples, the quantitative analysis of amino acids was performed by the following method. That is, it measured on the following conditions by the high performance liquid chromatography method (HPLC method), and detected using the fluorescence detector.
HPLC: Shimadzu Corporation LC-10A
Column: Shim-pack Amino-Li (100 mm L. × 6.0 mm ID)
Mobile phase: Amino acid mobile phase kit Li form Gradient elution flow rate: 0.6 ml / min Column temperature: 39 ° C
Reaction solution: ortho-phthalaldehyde (post column)
Reaction liquid rate: 0.3 ml / min Reaction temperature: 39 ° C.
Detection wavelength: excitation wavelength 350 nm, fluorescence wavelength 450 nm

Reference Example 1 [Selection of material for promoting GABA production ability of lactic acid bacteria]
All 15 kinds of foods listed in Table 2 were pulverized and extracted with a domestic mixer after adding 1 L of water to 1 kg. After sterilization by heating at 85 ° C. for 30 minutes, diatomaceous earth was added and suction filtration was performed using a filter paper (ADVANTEC Toyo No. 5C) to obtain a clear filtrate. Each filtrate was concentrated or diluted with water to adjust Brix to 1.0%. All components of GYP medium and 1% by mass of sodium glutamate monohydrate were dissolved in 1 L of each raw material filtrate, and autoclaved to prepare a medium. This medium was pre-cultured with normal GYP medium. Brevis UAS-4 strain (FERM P-20710) was inoculated in 10 ml, and cultured at 30 ° C. Amino acid analysis was performed by sampling every hour. Since the glutamic acid concentration and the GABA concentration at the start of the culture are different, the GABA production ability was determined by the time required for glutamic acid to decrease by 1% by mass in terms of sodium glutamate monohydrate. The results are shown in Table 1.

From this result, the GABA production ability promotion effect excellent in asparagus was confirmed. The GABA content increased almost the same as the theoretical value from the decrease in glutamic acid, and glutamic acid was not used except for conversion to GABA.

Example 1 [Difference in effect by lactic acid bacteria species]
Since the GABA production ability promoting effect was confirmed in the composition obtained from asparagus in Reference Example 1, The effect on lactic acid bacteria other than brebis UAS-4 was examined.

  10 ml of the culture solution of 4 strains of lactic acid bacteria in Table 2 pre-cultured in normal GYP medium was added to 1 L of medium prepared in the same manner as in Example 1 or 1 L of normal GYP medium, and static culture was performed at 30 ° C. went. After 24 hours, the GABA content in the medium was measured. Table 3 shows the results of GABA increase from the beginning of the culture.

From this result, GABA production ability was promoted in all the strains in the medium supplemented with the composition obtained from asparagus. In particular, the effect was high against the genus Lactobacillus.

Example 2 [Effect of yeast extract]
1 L of water was added to 1 kg of asparagus and pulverized and extracted with a home-use mixer. After sterilization by heating at 85 ° C. for 30 minutes, diatomaceous earth was added and suction filtration was performed using a filter paper (ADVANTEC Toyo No. 5C) to obtain a clear filtrate. The filtrate was concentrated to adjust Brix to 3.0%, and autoclaved. Using this asparagus extract, GYP medium, MRS medium, and yeast extract (1% by mass solution), seven types of media shown in Table 3 were prepared. Note that 1% by mass of sodium glutamate monohydrate was dissolved in all the media. To this medium, 10 ml of a culture solution of Lactobacillus brevis UAS-4 strain (FERM P-20710) pre-cultured in a normal GYP medium was added, and static culture was performed at 30 ° C. Sampling was performed every hour, and the glutamic acid concentration was measured. Since the glutamic acid concentration and the GABA concentration at the start of the culture are different, the GABA production ability was determined by the time required for glutamic acid to decrease by 1% by mass in terms of sodium glutamate monohydrate. The results are shown in Table 4.

From this result, an excellent GABA production ability was obtained with the asparagus extract alone, but was further accelerated by the addition of yeast extract.

Example 3 [Addition of glutamic acid and the like]
20 kg of asparagus was pulverized with a meat chopper, and extracted by adding 20 L of water. After sterilization by heating at 85 ° C. for 30 minutes, diatomaceous earth was added and suction filtration was performed using a filter paper (ADVANTEC Toyo No. 5C) to obtain a clear filtrate. The filtrate was concentrated with an evaporator to adjust Brix to 3.0% and sterilized at 121 ° C. for 20 minutes. To 10 L of the concentrate, 100 ml of a culture solution of Lactobacillus brevis UAS-4 strain (FERM P-20710) pre-cultured in GYP medium and 2% by mass of sodium glutamate monohydrate were added, and static culture was started at 30 ° C. did. After 16 hours, 10 L of asparagus extract concentrate was added, 3% by mass of sodium glutamate monohydrate and 6.5% by mass of glutamic acid were added, and the culture was continued with gentle stirring. After 30 hours and 36 hours from the start of culture, 3% by mass of glutamic acid was further added. When glutamic acid and GABA concentrations were measured after sampling 48 and 48 hours after the start of culture, glutamic acid was converted at a conversion rate of 82.8% at 42 hours and 95.3% at 48 hours.

Example 4 [Addition of yeast extract + glutamic acid, etc.]
20 kg of asparagus was pulverized with a meat chopper, and extracted by adding 20 L of water. After sterilization by heating at 85 ° C. for 30 minutes, diatomaceous earth was added and suction filtration was performed using a filter paper (ADVANTEC Toyo No. 5C) to obtain a clear filtrate. The filtrate was concentrated with an evaporator to adjust Brix to 3.0%, 1% by mass of yeast extract was added, and sterilized at 121 ° C. for 20 minutes. To 10 L of the concentrate, 100 ml of a culture solution of Lactobacillus brevis UAS-4 strain (FERM P-20710) pre-cultured in GYP medium and 2% by mass of sodium glutamate monohydrate were added, and static culture was started at 30 ° C. did. After 16 hours, 10 L of asparagus extract concentrate with yeast extract was added, 3% by mass of sodium glutamate monohydrate and 6.5% by mass of glutamic acid were added, and the culture was continued with gentle stirring. 3% by mass of glutamic acid was added 30 hours and 36 hours after the start of the culture. When glutamic acid and GABA concentrations were measured after sampling 48 and 48 hours after the start of culture, glutamic acid was converted at a conversion rate of 93.2% at 42 hours and 98.9% at 48 hours.

Example 5 [Addition of yeast extract + glutamic acid, etc.]
20 kg of asparagus was pulverized with a meat chopper, and extracted by adding 20 L of water. After sterilization by heating at 85 ° C. for 30 minutes, diatomaceous earth was added and suction filtration was performed using a filter paper (ADVANTEC Toyo No. 5C) to obtain a clear filtrate. The filtrate was concentrated with an evaporator to adjust Brix to 3.0%, 1% by mass of yeast extract was added, and sterilized at 121 ° C. for 20 minutes. To 10 L of the concentrate, 100 ml of a culture solution of Lactobacillus brevis UAS-6 strain (FERM P-20711) pre-cultured in GYP medium and 2% by mass of sodium glutamate monohydrate were added, and static culture was started at 30 ° C. did. After 16 hours, 10 L of asparagus extract concentrate with yeast extract was added, 3% by mass of sodium glutamate monohydrate and 6.5% by mass of glutamic acid were added, and the culture was continued with gentle stirring. 3% by mass of glutamic acid was added 30 hours and 36 hours after the start of the culture. After 42 hours of sampling, glutamic acid and GABA concentrations were measured, and glutamic acid was converted at a conversion rate of 89.0%.

Example 6 [GABA high content powder]
After the culture solution obtained in Example 4 was treated at 100 ° C. for 5 minutes, diatomaceous earth was added and suction filtration was performed using a filter paper (ADVANTEC Toyo No. 5C) to obtain a clear filtrate. When 3 kg of dextrin was dissolved in this filtrate and subjected to spray drying, 5.7 kg of a light brown powder was obtained. The GABA content of this powder was measured and found to be 36.6%.

Example 7 [GABA-containing oolong tea]
50 mg of the GABA high content powder obtained in Example 6 was dissolved in 200 ml of oolong tea to obtain oolong tea containing GABA. The powder dissolved rapidly and there was almost no change in taste and odor.

Example 8 [GABA-containing udon]
The high GABA content powder obtained in Example 6 was mixed with medium strength flour so as to be 0.5 w / w% to prepare udon noodles. When the udon noodles were cooked, they were good noodles with a slight sweetness and a refreshing scent.

Claims (11)

Method for producing a medium containing composition obtained from asparagus, inoculated with lactic acid bacteria, cultured and characterized to obtain a γ- aminobutyric acid from the resulting culture γ- aminobutyric acid-rich compositions. The method for producing a γ-aminobutyric acid-rich composition according to claim 1, wherein the lactic acid bacterium is a lactic acid bacterium belonging to the genus Lactobacillus. Lactobacillus belonging to the genus Lactobacillus is Lactobacillus brevis (L. brevis) UAS-4 strain (FERM-P20710) or Lactobacillus brevis (L. brevis) UAS-6 strain (FERM-P20711) 2. A method for producing a γ-aminobutyric acid-rich composition according to 2. The γ-aminobutyric acid-rich composition according to any one of claims 1 to 3, wherein glutamic acid and / or glutamate and / or a content thereof is further added to a medium containing a composition obtained from asparagus. Production method. The method for producing a γ-aminobutyric acid-rich composition according to any one of claims 1 to 4, wherein a yeast extract is further added to a medium containing a composition obtained from asparagus. The method for producing a γ-aminobutyric acid-rich composition according to any one of claims 1 to 5, wherein the sugar concentration (converted to Brix) of the medium is 0.1 to 40%. The method for producing a high γ-aminobutyric acid-containing composition according to any one of claims 1 to 6, wherein the pH of the medium during culture is adjusted to 3.5 to 8.0. The γ-aminobutyric acid according to claim 7, wherein the method of adjusting the pH of the medium during culture to 3.5 to 8.0 is a method of adding glutamic acid and / or glutamate and / or their contents to the medium. A method for producing a high content composition. A composition containing a high content of γ-aminobutyric acid, which is produced by the method according to claim 1. A food / beverage product comprising the high γ-aminobutyric acid-containing composition according to claim 9. A feed comprising the γ-aminobutyric acid-rich composition according to claim 9.