Method for producing yeast extract

Abstract

The present invention addresses the problem of providing technology capable of improving the ribonucleotide yield in the production of yeast extract.　The present invention provides a method for producing a yeast extract, the method comprising reacting a filamentous-fungi-derived protease, a phosphodiesterase, and AMP-deaminase, simultaneously or in any order, in a suspension of yeast cells.

Description

Yeast extract manufacturing method

[0001] The present invention relates to a method for producing a yeast extract with an improved yield of ribonucleotides.

[0002] In the food market, yeast extract is used as an additive to impart or enhance umami and richness. Yeast extracts are broadly divided into high-nucleic acid types with a high nucleic acid content and high-amino acid types rich in amino acids. The main taste components in the former high-nucleic acid type yeast extract are 5'-guanylic acid (GMP) and 5'-inosinic acid (IMP).

[0003] Various studies have been conducted on methods for producing high nucleic acid yeast extracts. For example, Patent Document 1 discloses that a yeast extract with a high nucleic acid content can be produced by adjusting the pH of a yeast cell suspension to neutral or alkaline and heat-treating it to a predetermined temperature, then adding nuclease and protease to the yeast cell suspension to degrade nucleic acids and proteins, and then performing a deaminase treatment. Furthermore, Patent Document 2 discloses that simultaneous treatment of a yeast lysate with ribonuclease and AMP-deaminase can simplify the process, improve production efficiency, and reduce production costs in the production of high nucleic acid yeast extracts.

[0004] International Publication No. WO 03 / 55333 International Publication No. WO 2021 / 66130

[0005] By utilizing the technology of Patent Document 2, the production efficiency of high nucleic acid yeast extract can be significantly improved. However, further improvement is desired in terms of the yield of ribonucleotides (GMP, IMP, etc.), which are umami components contained in yeast extract. Therefore, an object of the present invention is to provide a technology that can improve the yield of ribonucleotides in the production of yeast extract.

[0006] The present inventors have conducted extensive research to solve the above-mentioned problems and have found that a yeast extract with a significantly improved ribonucleotide yield can be obtained by allowing a filamentous fungal protease, phosphodiesterase, and AMP-deaminase to act simultaneously or in any order on a suspension of yeast cells. The present invention was completed based on this finding and through further research.

[0007] That is, the present invention provides inventions relating to methods for producing yeast extract, etc., in the following embodiments. Item 1. A method for producing yeast extract, in which a protease, phosphodiesterase, and AMP-deaminase derived from a filamentous fungus are allowed to act simultaneously or in any order in a suspension of yeast cells. Item 2. The production method according to Item 1, in which a protease, phosphodiesterase, and AMP-deaminase derived from a filamentous fungus are allowed to act simultaneously. Item 3. The production method according to Item 1 or 2, in which the protease is derived from a filamentous fungus of the genus Aspergillus. Item 4. The production method according to Item 3, in which the filamentous fungus of the genus Aspergillus is Aspergillus oryzae. Item 5. The production method according to any one of Items 1 to 4, in which the protease is an acid protease. Item 6. The production method according to any one of Items 1 to 5, in which the phosphodiesterase is derived from Penicillium citrinum. Item 7. Item 7. A method for producing yeast extract according to any one of Items 1 to 6, wherein the AMP-deaminase is derived from Streptomyces myrinus. Item 8. A method for producing yeast extract according to any one of Items 1 to 7, wherein the ratio of the total mass of IMP and GMP to the total mass of IMP, GMP, UMP, CMP, and AMP is 53% or more relative to 100%. Item 9. A yeast extract obtained by the production method of any one of Items 1 to 8. Item 10. An agent for improving the yield of ribonucleotides in yeast extract production, comprising a protease, phosphodiesterase, and AMP-deaminase derived from a filamentous fungus. Item 11. Use of a protease, phosphodiesterase, and AMP-deaminase derived from a filamentous fungus to improve the yield of ribonucleotides in yeast extract production.

[0008] According to the present invention, in a method for producing a yeast extract in which a protease, a phosphodiesterase, and an AMP-deaminase are allowed to act on yeast cells, by using a protease derived from a filamentous fungus, it is possible to obtain a yeast extract with a significantly improved yield of ribonucleotides.

[0009] 1. Method for Producing Yeast Extract The present invention is a method for producing yeast extract, which is characterized by allowing a protease, a phosphodiesterase, and an AMP-deaminase derived from a filamentous fungus to act simultaneously or in any order in a suspension of yeast cells. The production method of the present invention is described in detail below.

[0010] [Yeast Cells] The type of yeast cells used in the present invention is not particularly limited as long as it is a yeast that can be used for food applications, and examples include yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae and Saccharomyces pastorianus; yeasts of the genus Candida; yeasts of the genus Kluyveromyces such as Kluyveromyces lactis and Kluyveromyces marxianus; yeasts of the genus Pichia such as Pichia pastoris; yeasts of the genus Debaryomyces such as Debaryomyces hansenii; and yeasts of the genus Zygosaccharomyces such as Zygosaccharomyces mellis. Among these, yeasts of the genus Saccharomyces and yeasts of the genus Candida are preferred, yeasts of the genus Saccharomyces are more preferred, and Saccharomyces cerevisiae is even more preferred. Furthermore, yeast cells recovered after brewing beer, sake, etc. may also be used. Furthermore, the yeast cells may have an increased nucleic acid content through pretreatment or the like. In the present invention, one type of yeast cell may be used alone, or two or more types may be used in combination.

[0011] The protein content of the yeast cells is not particularly limited, but may be, for example, 30% by weight or more, calculated as the dry weight of the yeast cells. From the viewpoint of further enhancing the effect, the content is preferably 35% by weight or more, more preferably 40% by weight or more, even more preferably 45% by weight or more, and particularly preferably 50% by weight or more. The upper limit of the content range is not particularly limited, but may be, for example, 90% by weight or less, preferably 80% by weight or less, more preferably 70% by weight or less, and even more preferably 60% by weight or less. More specific examples of the protein content contained in the yeast cells include 30 to 90% by weight, 30 to 80% by weight, 30 to 70% by weight, 35 to 90% by weight, 35 to 80% by weight, 35 to 70% by weight, 40 to 90% by weight, 40 to 80% by weight, 40 to 70% by weight, 45 to 90% by weight, 45 to 80% by weight, 45 to 70% by weight, 50 to 90% by weight, 50 to 80% by weight, or 50 to 70% by weight, calculated as the dry weight of the yeast cells.

[0012] Furthermore, the yeast cells used in the present invention may be in an active or inactivated state. It is desirable that the yeast cells are heat-inactivated to inactivate endogenous enzymes in the yeast cells. The heat-inactivation treatment may be carried out before preparing a yeast cell suspension, or may be carried out on a yeast cell suspension before enzyme treatment. Furthermore, the yeast cells may be in any form, such as liquid, powder, or slurry.

[0013] [Protease derived from filamentous fungi] Proteases are enzymes that hydrolyze proteins. In the present invention, the use of a protease derived from filamentous fungi as the protease that lyses yeast cells can improve the yield of ribonucleotides in the resulting yeast extract.

[0014] The microorganism from which the protease used in the present invention is derived is not particularly limited as long as it is a filamentous fungus, and examples thereof include filamentous fungi of the genus Aspergillus, filamentous fungi of the genus Rhizopus, filamentous fungi of the genus Mucor, filamentous fungi of the genus Neurospora, filamentous fungi of the genus Penicillium, filamentous fungi of the genus Rhizomucor, filamentous fungi of the genus Sclerotinia, etc. Among these filamentous fungi, from the viewpoint of producing a yeast extract with an even higher yield of ribonucleotides, filamentous fungi of the genus Aspergillus and Rhizopus are preferred, and filamentous fungi of the genus Aspergillus are more preferred. Specific examples of filamentous fungi of the genus Aspergillus include Aspergillus oryzae, Aspergillus melleus, Aspergillus niger, Aspergillus sojae, Aspergillus luchuensis, Aspergillus flavus, etc. Of these filamentous fungi of the genus Aspergillus, preferred are Aspergillus oryzae and Aspergillus melleus, and more preferred is Aspergillus oryzae.

[0015] Furthermore, the type of protease derived from a filamentous fungus is not particularly limited, and may be, for example, an acidic protease (aspartic acid protease), a neutral protease, or an alkaline protease. From the viewpoint of producing a yeast extract with an even higher yield of ribonucleotides, preferred are acidic proteases and neutral proteases, and more preferred are acidic proteases.

[0016] Specific examples of the protease derived from a filamentous fungus include an acidic protease derived from Aspergillus oryzae, a neutral protease derived from Aspergillus oryzae, and a neutral protease derived from Aspergillus melleus. Among these, from the viewpoint of producing a yeast extract with a further increased yield of ribonucleotides, an acidic protease derived from Aspergillus oryzae is preferred.

[0017] The protease derived from a filamentous fungus may be either a wild-type or a mutant. In the present invention, the protease derived from a filamentous fungus may be used alone or in combination of two or more types.

[0018] Proteases derived from filamentous fungi can be prepared by known methods. One example of a method for producing a protease derived from a filamentous fungus is a method of recovering the protease from the culture medium or cells of the filamentous fungus. For example, when a protease-secreting filamentous fungus is used, the cells can be recovered from the culture medium in advance by filtration, centrifugation, or the like, as necessary, and the protease can then be separated and / or purified. When a protease-nonsecreting filamentous fungus is used, the cells can be recovered from the culture medium in advance by pressure treatment, ultrasonication, or the like, to expose the protease, and the protease can then be separated and / or purified. Known protein separation and / or purification methods can be used to separate and / or purify the protease, including, for example, centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins. The separated and / or purified protease can be powdered by drying methods such as lyophilization and vacuum drying, or by using an appropriate excipient and / or drying aid in the drying method. The isolated and / or purified protease can also be liquefied by adding an appropriate additive and sterilizing it through filtration.

[0019] In the present invention, a commercially available protease derived from a filamentous fungus may also be used, such as an acidic protease preparation derived from Aspergillus oryzae manufactured by Amano Enzyme Inc.

[0020] [Phosphodiesterase] Phosphodiesterase is an enzyme that hydrolyzes one bond of a phosphodiester bond. In the production method of the present invention, the phosphodiesterase hydrolyzes ribonucleic acid in yeast to produce nucleotides such as GMP, which is a taste-producing substance. The phosphodiesterase used in the present invention is preferably a ribonuclease (an enzyme that hydrolyzes the intramolecular phosphodiester bond of ribonucleic acid (RNA)).

[0021] The origin of the phosphodiesterase used in the present invention is not particularly limited, and may be derived from a microorganism, a plant, etc. Preferred examples of the phosphodiesterase include those derived from a microorganism. Examples of microorganisms from which phosphodiesterases are derived include filamentous fungi of the genus Penicillium such as Penicillium citrinum; filamentous fungi of the genus Leptographium such as Leptographium procerum; filamentous fungi of the genus Aspergillus such as Aspergillus niger; filamentous fungi of the genus Streptomyces such as Streptomyces aureus, Streptomyces avermitilis, Streptomyces cinnamoneus, Streptomyces griseus, Streptomyces thermoviolaceus, Streptomyces violaceus, Streptomyces Among these microorganisms, from the viewpoint of producing a yeast extract with an even higher yield of ribonucleotides, filamentous fungi of the genus Penicillium and Leptographium are preferred, and Penicillium citrinum is more preferred.

[0022] The phosphodiesterase may be either a wild-type or a mutant. In the present invention, one type of phosphodiesterase may be used alone, or two or more types may be used in combination.

[0023] Phosphodiesterase can be prepared by known methods. For example, phosphodiesterase derived from Penicillium fungi can be easily prepared by culturing Penicillium fungi that produce phosphodiesterase and isolating the phosphodiesterase by known means, or by a method using genetic recombination technology.

[0024] In the present invention, commercially available phosphodiesterases can also be used, such as Penicillium citrinum-derived ribonuclease manufactured by Amano Enzyme Inc.

[0025] [AMP-deaminase] AMP-deaminase is an enzyme that hydrolyzes AMP to produce IMP and ammonia. In the production method of the present invention, AMP-deaminase converts the liberated AMP into IMP, a flavor component.

[0026] The origin of the AMP deaminase used in the present invention is not particularly limited, and may be derived from a microorganism, a plant, or the like. The AMP deaminase is preferably derived from a microorganism. The microorganism from which the AMP deaminase is derived is not particularly limited, and examples thereof include filamentous fungi of the genus Aspergillus, such as Aspergillus niger; and actinomycetes of the genus Streptomyces, such as Streptomyces murinus, Streptomyces celluloflavuys, and Streptomyces griseus.

[0027] Suitable examples of AMP deaminases used in the present invention include those with high thermostability and a reaction temperature of about 40 to 70°C. Specific examples of such highly thermostable AMP deaminases include AMP deaminases derived from Streptomyces actinomycetes (specific examples include Streptomyces myrinus, Streptomyces celluloflavus, and Streptomyces griseus) (see, for example, WO 2005 / 105991). AMP deaminase derived from Streptomyces myrinus is particularly excellent in terms of thermostability and is particularly suitable for use in the present invention.

[0028] The AMP deaminase may be either a wild-type or a mutant. In the present invention, one type of AMP deaminase may be used alone, or two or more types may be used in combination.

[0029] AMP-deaminase can be prepared by known methods. For example, AMP-deaminase derived from Streptomyces actinomycetes can be easily prepared by culturing AMP-deaminase-producing Streptomyces actinomycetes and isolating the AMP-deaminase by known means, or by a method using genetic recombination technology.

[0030] In the present invention, commercially available AMP deaminase can also be used. Examples of commercially available AMP deaminase include AMP deaminase derived from Streptomyces myrinus, manufactured by Amano Enzyme Inc.

[0031] [Enzyme Treatment] In the production method of the present invention, a yeast extract is produced by allowing a protease, phosphodiesterase, and AMP-deaminase derived from a filamentous fungus to act simultaneously or in any order in a suspension of yeast cells. Specifically, the three enzymes are added simultaneously or in any order to a suspension of yeast cells, and the enzymatic reaction caused by the three enzymes is allowed to proceed, thereby producing a yeast extract with an increased yield of ribonucleotide nucleic acids.

[0032] The dispersion medium for the yeast cell suspension is not particularly limited as long as it allows the action of the filamentous fungus-derived protease, phosphodiesterase, and AMP-deaminase, and examples thereof include water, a buffer solution, etc. Water is preferred as the dispersion medium.

[0033] The concentration of yeast cells in the suspension is not particularly limited, but may be, for example, 10 to 1000 g / L, preferably 20 to 500 g / L, more preferably 50 to 300 g / L, and even more preferably 100 to 200 g / L, calculated as the dry weight of yeast cells.

[0034] The amount of filamentous fungal protease added to a suspension of yeast cells may be appropriately determined depending on the concentration of yeast cells in the suspension, reaction time, etc., but may be, for example, 0.1 U or more of filamentous fungal protease per 1 g of dry weight of yeast cells contained in the suspension. From the viewpoint of producing a yeast extract with a further increased ribonucleotide yield, the amount of filamentous fungal protease added per 1 g of dry weight of yeast cells is preferably 1 U or more, more preferably 10 U or more, even more preferably 20 U or more, particularly preferably 40 U or more, and even more preferably 80 U or more. The upper limit of the amount of filamentous fungal protease added per 1 g of dry weight of yeast cells is not particularly limited, but may be, for example, 2000 U or less, 1000 U or less, 500 U or less, 200 U or less, or 170 U or less. The amount of filamentous fungal protease added per gram of dry weight of yeast cells contained in the suspension is typically 1 to 2,000 U, preferably 10 to 1,000 U, more preferably 20 to 500 U, even more preferably 40 to 200 U, and particularly preferably 80 to 170 U. Herein, protease activity is defined as the amount of enzyme that liberates 1.5 μg / mL of tyrosine per minute when a 7 w / v % casein solution (100 mM acetic acid / sodium acetate buffer, pH 5.5) is used as a substrate solution and the substrate solution is treated at 60°C, and the amount of protease activity is defined as 1 unit (1 U). Specific conditions for measuring protease activity are described in the Examples section.

[0035] The amount of phosphodiesterase added to a suspension of yeast cells may be appropriately determined depending on the concentration of yeast cells in the suspension, reaction time, etc., but may be, for example, 0.001 U or more of phosphodiesterase per 1 g of dry weight of yeast cells contained in the suspension. From the viewpoint of producing a yeast extract with a further increased ribonucleotide yield, the amount of phosphodiesterase added per 1 g of dry weight of yeast cells is preferably 0.01 U or more, more preferably 0.1 U or more, even more preferably 0.5 U or more, particularly preferably 1 U or more, and even more preferably 1.4 U or more. The upper limit of the amount of phosphodiesterase added per 1 g of dry weight of yeast cells is not particularly limited, but may be, for example, 1000 U or less, 500 U or less, 300 U or less, 200 U or less, or 140 U or less. The amount of phosphodiesterase added per gram of dry weight of yeast cells contained in the suspension is specifically 0.01 to 1000 U, preferably 0.1 to 500 U, more preferably 0.5 to 300 U, even more preferably 1 to 200 U, and particularly preferably 1.4 to 140 U. In this specification, 1 U of phosphodiesterase activity is defined as the amount of enzyme that liberates 1 μmol of phosphate per minute when phosphodiesterase is allowed to act on 3'-AMP as a substrate at the optimal temperature and optimal pH of the enzyme. Specific conditions for measuring phosphodiesterase activity are described in the Examples section.

[0036] The amount of AMP-deaminase added to the yeast cell suspension may be appropriately determined depending on the concentration of yeast cells in the suspension, the reaction time, etc., and may be, for example, 0.1 U or more of AMP-deaminase per 1 g of dry weight of yeast cells contained in the suspension. From the viewpoint of producing a yeast extract with an even higher ribonucleotide yield, the amount of AMP-deaminase added per 1 g of dry weight of yeast cells is preferably 0.2 U or more, more preferably 0.5 U or more, even more preferably 1 U or more, and particularly preferably 2 U or more. The upper limit of the amount of AMP-deaminase added per 1 g of dry weight of yeast cells is not particularly limited, and may be, for example, 1000 U or less, 500 U or less, 250 U or less, or 100 U or less. Specific examples of the amount of AMP-deaminase added per 1 g of dry weight of yeast cells contained in the suspension include 0.2 to 1000 U, preferably 0.5 to 500 U, more preferably 1 to 250 U, and even more preferably 2 to 100 U. In this specification, the AMP-deaminase activity is a value measured using the decrease in optical density at 265 nm (OD265) during the reaction as an index, and specifically, 1 U is defined as the amount of enzyme that reduces the absorbance difference by 0.001 per minute when measured under the conditions described in the Examples section.

[0037] In the production method of the present invention, the three enzymes may be allowed to act simultaneously or in any order in a suspension of yeast cells, but preferred examples include allowing the three enzymes to act in any of the following modes 1 to 4. From the viewpoint of improving work efficiency, the following mode 1 (simultaneous treatment of three enzymes) is preferred. Mode 1: A protease, phosphodiesterase, and AMP-deaminase derived from a filamentous fungus are reacted simultaneously, allowing the three enzymes to react in one step. Mode 2: A protease and phosphodiesterase derived from a filamentous fungus are reacted simultaneously, followed by AMP-deaminase, allowing the three enzymes to react in two separate steps. Mode 3: A protease derived from a filamentous fungus is reacted, followed by phosphodiesterase and AMP-deaminase are reacted simultaneously, allowing the three enzymes to react in two separate steps. Aspect 4: After reacting with a protease derived from a filamentous fungus, phosphodiesterase is reacted, and then AMP-deaminase is reacted, and three types of enzymes are reacted in three separate steps.

[0038] When three types of enzymes are reacted in two or three separate steps as in the above-described embodiments 2 to 4, the enzymes used in the previous enzymatic reaction may be inactivated by carrying out a heat treatment or the like after the completion of the previous enzymatic reaction and before the initiation of the subsequent enzymatic reaction, or the subsequent enzymatic reaction may be started without inactivating the enzymes used in the previous enzymatic reaction.

[0039] In the production method of the present invention, the reaction temperature, pH, time, and other factors used when reacting the three enzymes can be appropriately set depending on the characteristics of the enzymes used, the amount of enzyme added, the concentration of yeast cells, and other factors, and optimal conditions can be determined through preliminary experiments. For example, the reaction temperature can be 0 to 80°C, preferably 10 to 75°C, and more preferably 40 to 70°C. The pH of the suspension during the reaction can be 3 to 9, preferably 3.5 to 8, and more preferably 4 to 7. The reaction time can be 1 minute to 48 hours, preferably 2 minutes to 24 hours, and more preferably 2 hours to 12 hours. When reacting the three enzymes in two or three stages, as in Aspects 2 to 4, the reaction temperature, pH, and time for each stage can be set within the above ranges.

[0040] [Recovery of yeast extract] The reaction solution (suspension after the enzyme reaction) after the three enzyme reactions contains extracted components contained in yeast, including ribonucleotides. The reaction solution after the enzyme reaction is subjected to solid-liquid separation treatment such as centrifugation or filtration to recover the liquid fraction, thereby obtaining a liquid yeast extract. If necessary, the obtained liquid yeast extract may be subjected to concentration treatment to obtain a concentrated liquid, or may be subjected to drying treatment to obtain a dried product (powder, granules, etc.).

[0041] [Uses of Yeast Extract] The yeast extract obtained by the production method of the present invention has an improved yield of ribonucleotides (IMP, GMP, UMP, CMP, and AMP). In particular, the yeast extract obtained by the production method of the present invention is characterized by a high ratio of the total amount of IMP and GMP, which are umami components, to all ribonucleotides. In the yeast extract obtained by the production method of the present invention, the ratio of the total amount of IMP and GMP to all ribonucleotides is not particularly limited, but for example, the ratio of the total mass of IMP and GMP to the total mass of IMP, GMP, UMP, CMP, and AMP (100%) is 53% or more, preferably 53 to 60%.

[0042] The yeast extract obtained by the production method of the present invention is useful as a high nucleic acid yeast extract, and can be used to enhance or adjust the flavor of various foods or beverages. The types of foods and beverages to which the yeast extract obtained by the production method of the present invention can be applied are not particularly limited, and examples include processed seafood products (chikuwa, kamaboko, hanpen, dried squid, dried fish, salted fish, fish sausage, tsukudani, canned goods, etc.), processed meat products (ham, bacon, sausage, jerky, corned beef, formed meat, etc.), processed vegetables (canned and bottled vegetables, processed tomatoes, processed mushrooms, pickled vegetables, dried vegetables, vegetable tsukudani, etc.), noodles and breads (various noodles, bread, sweet bread, etc.), processed grain products (cereals, oatmeal, muesli, processed rice products, wheat gluten, barley tea, etc.), dairy products (milk, processed milk, dairy drinks, concentrated milk, powdered milk, condensed milk, fermented milk, lactic acid bacteria drinks, butter, cheese, ice cream, etc.), and the like. Examples of suitable foodstuffs include: processed fruit products (canned and bottled fruit, jams, marmalade, dried fruit, etc.), sweets and desserts (biscuits, baked goods, rice crackers, fried sweets, fresh Japanese sweets, fresh Western sweets, semi-fresh sweets, dried Japanese sweets, candies, chocolates, chewing gum, snacks, frozen desserts, etc.), beverages (soft drinks, carbonated drinks, fruit juice drinks, coffee drinks, vegetable juice drinks, tea drinks, non-alcoholic drinks, alcoholic drinks, etc.), seasonings (sauces, soups, dressings, etc.), soups, roux (curry roux, stew roux, etc.), nutritional supplements and beverages (protein powders, protein drinks, supplements, energy drinks, etc.), pet food, nutritional supplements for pets, etc.

[0043] When the yeast extract obtained by the production method of the present invention is blended into a food or beverage, the blending amount may be appropriately determined depending on the type of food or beverage, the desired taste, etc., but may be, for example, 0.001 to 5 wt %, preferably 0.01 to 1 wt %, calculated as the dry weight of the yeast extract per total amount of the food or beverage.

[0044] 2. Agent for improving ribonucleotide yield in yeast extract production As described above, yeast extract with an increased ribonucleotide yield can be produced by using a protease, phosphodiesterase, and AMP-deaminase derived from a filamentous fungus. Therefore, in another embodiment of the present invention, there is provided an agent for improving ribonucleotide yield in yeast extract production, comprising a protease, phosphodiesterase, and AMP-deaminase derived from a filamentous fungus. The yield improver is an enzyme preparation used in the method for producing yeast extract, and is used to improve the yield of ribonucleotides.

[0045] As long as the yield enhancer contains a protease, phosphodiesterase, and AMP-deaminase derived from a filamentous fungus, it may be any of a single-agent type consisting of an enzyme preparation containing a mixture of these three enzymes, a two-agent type consisting of an enzyme preparation containing a mixture of two of these three enzymes and an enzyme preparation containing the remaining enzyme, and a three-agent type containing each of these three enzymes as a separate enzyme preparation.

[0046] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0047] 1. Measurement of Enzyme Activity The activity of each enzyme used in the following Examples and Comparative Examples was measured according to the following method.

[0048] 1-1. Protease Activity: 0.2 mL of enzyme sample solution containing protease was added to 2 mL of 100 mM acetic acid / sodium acetate buffer (pH 5.5) containing 7 w / v% casein and allowed to stand at 60°C for 30 minutes. The reaction was stopped by adding 2 mL of TCA test solution (18 g of trichloroacetic acid and 19 g of sodium acetate trihydrate were dissolved in 800 mL of water, followed by 20 mL of glacial acetic acid, and the volume was adjusted to 1000 mL). The reaction mixture was then allowed to stand at 37°C for another 30 minutes to obtain a reaction solution. For a blank, 2 mL of 100 mM acetic acid / sodium acetate buffer (pH 5.5) containing 7 w / v% casein was allowed to stand at 60°C for 30 minutes, after which 2 mL of TCA test solution was added, followed by 0.2 mL of sample solution containing protease, and the mixture was allowed to stand at 37°C for 30 minutes to obtain a blank solution. The resulting solution was centrifuged, and the absorbance at 275 nm of the resulting supernatant was measured. Separately, a calibration curve showing the relationship between tyrosine concentration and absorbance (275 nm) was prepared using a tyrosine standard solution. Using this calibration curve, the tyrosine concentration (μg / mL) in the reaction solution was determined from the absorbance of the reaction solution minus the absorbance of the blank solution. The protease activity was calculated using the following formula, where 1 unit (1 U) is the amount of enzyme required to produce 1.5 μg / mL of tyrosine per minute.

[0049] 1-2. Phosphodiesterase Activity The activity of each phosphodiesterase was measured based on Method 1 of the Phosphodiesterase Activity Test Method in the 9th Edition of the Japanese Standards for Food Additives. A substrate solution was prepared by dissolving 20 mg of adenosine-3'-monophosphate in 10 mL of barbital sodium hydrochloride buffer (adjusted to pH 5.0, the optimal pH for each phosphodiesterase used) and filtering through a membrane filter (0.45 μm). Separately, 0.50 g of 2,4-diaminophenol dihydrochloride and 10.0 g of sodium bisulfite were weighed, dissolved in water to make 50 mL, and filtered to prepare an amideol test solution. 0.4 mL of the freshly prepared substrate solution was left to stand for exactly 5 minutes at 70 ± 0.5°C (the optimal temperature for each phosphodiesterase used), and 0.1 mL of enzyme sample solution was added, followed by leaving the solution to stand for exactly 15 minutes at 70 ± 0.5°C (the optimal temperature for each phosphodiesterase used). Next, 4 mL of 6% perchloric acid test solution, 0.4 mL of amidol test solution, and 0.2 mL of hexaammonium heptamolybdate tetrahydrate solution (8.3 → 100) were mixed to obtain a reaction solution. Separately, a solution containing 0.4 mL of substrate solution, 4 mL of 6% perchloric acid test solution, and 0.1 mL of water was shaken and left at 70 ± 0.5 °C for exactly 15 minutes. Next, 0.4 mL of amidol test solution and 0.2 mL of hexaammonium heptamolybdate tetrahydrate solution (8.3 → 100) were mixed to obtain a blank solution. The prepared reaction solution and blank solution were left in running water for 15 minutes, after which the absorbance at 750 nm was measured. Separately, a calibration curve showing the relationship between phosphate concentration and absorbance (750 nm) was created using a phosphate standard solution. Using this calibration curve, the phosphate concentration (μg / mL) in the reaction solution was calculated by subtracting the absorbance of the blank solution from the absorbance of the reaction solution. The activity of phosphodiesterase was calculated using the following formula, where 1 U is the amount of enzyme that liberates 1 μmol of phosphate per minute.

[0050] 1-3. AMP-deaminase activity First, 1 ml of enzyme sample solution containing AMP-deaminase was added to 3 ml of a solution prepared by mixing 0.017 M 5'AMP-2Na and 1 / 15 M phosphate buffer (pH 5.6) at a volume ratio of 1:2 to prepare a reaction solution, which was then allowed to react at 37°C for 15 minutes. After 15 minutes, 4 mL of 2 v / v% aqueous perchloric acid solution was added to stop the reaction, and 100 μL was then measured. Water was added to the measured 100 μL to make a total of 5 mL, and the OD265 was measured. A blank was prepared by measuring the same except that the reaction time was set to 0 minutes. Under the above conditions, the amount of enzyme required to reduce the absorbance difference by 0.001 per minute of reaction was defined as 1 U.

[0051] 2. Production and Evaluation of Yeast Extract 2-1. Production of Yeast Extract 800 g of water was stirred at speed 2 in a thermomixer (Thermomix TM6, manufactured by Vorwerk). 120 g of yeast powder derived from Saccharomyces cerevisiae (Active Dry Yeast, manufactured by Red Star) was gradually added, and once the entire amount was added, the temperature was raised to 90°C. The mixture was held at 90°C for 10 minutes to inactivate the endogenous enzymes of the yeast. The mixture was then cooled to 35°C and adjusted to pH 5.5 with 1N hydrochloric acid to obtain a yeast suspension.

[0052] Water was added to the resulting yeast suspension to a final volume of 960 mL, and 48 mL aliquots were dispensed into Erlenmeyer flasks. The proteases shown in Table 1, phosphodiesterase (mainly containing ribonuclease) derived from Penicillium citrinum (Amano Enzyme), and AMP deaminase derived from Streptomyces murinus (Amano Enzyme) were added in the amounts shown in Table 2, and an enzymatic reaction was carried out. The total volume of the enzymes added was adjusted to 2 mL so that the total volume of the yeast suspension after enzyme addition was 50 mL. The enzymatic reaction was carried out at 60°C for 6 hours with shaking (200 rpm). After completion of the enzymatic reaction, the yeast suspension was centrifuged at 13,300 rpm (17,000 G) for 10 minutes, and the yeast extract (supernatant) was recovered.

[0053]

[0054] 2-2. Analysis of Ribonucleotide Composition of Yeast Extract The obtained yeast extract was diluted 51-fold with 50 mM potassium dihydrogen phosphate solution (pH 4.6) and passed through a membrane filter (0.22 μm) to prepare an analytical sample. The analytical sample was analyzed for ribonucleotide composition using high-performance liquid chromatography (detector SPD-20A, Shimadzu Corporation) under the following conditions, and quantification was performed using standards for each ribonucleotide (Sigma). <HPLC Analysis Conditions> Column: Shim-pack WAX-1 (4.0 mm x 5 cm, Shimadzu Corporation) Temperature: 40°C Apply: 10 μl Detector: SPD Measurement wavelength: 260 nm Flow rate: 0.5 ml / min Solvent: 50 mM potassium dihydrogen phosphate solution (pH 4.6)

[0055] 2-3. Results The results are shown in Table 2. When yeast extract was produced by treating a yeast suspension with protease, phosphodiesterase (mainly including ribonuclease), and AMP-deaminase, the yield of ribonucleotides in the resulting yeast extract was significantly higher when a protease derived from a filamentous fungus was used (Example 1) than when a protease derived from a bacteria was used (Comparative Examples 1 to 4). Furthermore, the yeast extract obtained using a protease derived from a filamentous fungus (Example 1) also had a higher ratio of the umami components IMP and GMP to total ribonucleotides and a higher total amount of IMP and GMP than when a protease derived from a bacteria was used (Comparative Examples 1 to 4). Furthermore, the yeast extract obtained using a protease derived from a filamentous fungus (Example 1) had an excellent clear appearance and a good taste when consumed.

[0056] Since the amounts (activities) of the enzymes used in Example 1 and Comparative Examples 1 to 4 were all the same, these results revealed that in the production of yeast extract using protease, phosphodiesterase, and AMP-deaminase, using a protease derived from a filamentous fungus contributes to improving the yield of ribonucleotides in the resulting yeast extract.

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Claims

1. A method for producing a yeast extract, which comprises allowing a protease, phosphodiesterase, and AMP-deaminase derived from a filamentous fungus to act simultaneously or in any order on a suspension of yeast cells.

2. The production method according to claim 1, wherein a protease, a phosphodiesterase, and an AMP-deaminase derived from a filamentous fungus are simultaneously reacted.

3. The method according to claim 1 or 2, wherein the protease is derived from a filamentous fungus of the genus Aspergillus.

4. The method according to claim 3, wherein the filamentous fungus of the genus Aspergillus is Aspergillus oryzae.

5. The method according to claim 1 or 2, wherein the protease is an acid protease.

6. The method of claim 1 or 2, wherein the phosphodiesterase is derived from Penicillium citrinum.

7. The method according to claim 1 or 2, wherein the AMP deaminase is derived from Streptomyces murinus.

8. A production method according to claim 1 or 2, wherein in the yeast extract produced, the ratio of the total mass of IMP and GMP to the total mass of IMP, GMP, UMP, CMP, and AMP (100%) is 53% or more.

9. A yeast extract obtained by the production method described in claim 1 or 2.

10. A ribonucleotide yield enhancer in yeast extract production, comprising a protease, a phosphodiesterase, and an AMP-deaminase derived from a filamentous fungus.

11. Use of a protease, a phosphodiesterase, and an AMP-deaminase derived from a filamentous fungus to improve the yield of ribonucleotides in yeast extract production.

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