Method for producing yeast extract

Abstract

The purpose of the present invention is to provide a method for producing a yeast extract having a high peptide ratio. A yeast extract having a high peptide ratio can be produced by a method for producing a yeast extract comprising a step for causing a protease selected from the group consisting of a protease derived from the genus Bacillus, a protease derived from the genus Geobacillus, and a protease derived from the genus Rhizopus, and / or a glutaminase to act on a yeast.

Description

Technical Field

[0001] The present invention relates to a method for producing a yeast extract, and more specifically, to a method for producing a yeast extract having a high peptide ratio or capable of promoting the production of lactic acid bacteria. Background Art

[0002] Yeast extracts are used in a wide range of applications, such as being added to various foods for the purpose of imparting or enhancing a preferred flavor such as a flavor or a rich flavor, or being added to a culture medium for the purpose of utilizing it as a nitrogen nutrient source when culturing microorganisms. In addition, demand for yeast extracts is on the rise against the backdrop of the recent increase in natural and healthy intentions. Therefore, development is being conducted with the purpose of increasing the added value of yeast extracts.

[0003] For example, in order to increase the content of flavor components in yeast extract, the following method is known: after subjecting a yeast suspension to alkali extraction, the proteases and ribonucleases in the bacteria are inactivated by heating, and then 5′-phosphodiesterase and 5′-adenylate deaminase are allowed to act to obtain a yeast extract containing 8% or more of each of the flavor 5′-nucleotides, i.e., 5′-guanylate and 5′-inosinate, and 16% or more of peptides and free amino acids in total (Patent Document 1).

[0004] In addition, in order to enhance the effects of yeast extract on sustaining exercise capacity and preventing and recovering from fatigue, as well as the effects on chronic fatigue syndrome, there is known a yeast extract-containing composition in which a yeast extract containing a predetermined amount of vitamin B1, a yeast extract containing a predetermined amount of peptides, and a yeast extract containing a predetermined amount of glutathione are combined at a predetermined ratio (Patent Document 2).

[0005] Prior art literature Patent Literature Patent Document 1: Japanese Patent Application Laid-Open No. 6-113789 Patent Document 2: Japanese Patent Application Publication No. 2009-107962 Summary of the invention

[0006] Technical problem to be solved by the invention Peptides contained in yeast extracts not only impart a preferred taste to food, but also include functional peptides that exert useful functions in the body when ingested, and in this respect, they are considered to be elements that should be given special attention in imparting added value to yeast extracts. However, in the methods for producing yeast extracts known to date, the peptide ratio in the obtained yeast extracts is still insufficient.

[0007] Therefore, an object of the present invention is to provide a method for producing a yeast extract having a high peptide ratio.

[0008] Technical solutions for solving technical problems The present inventors have found that a yeast extract having a high peptide ratio can be obtained by allowing a protease selected from a protease derived from Bacillus, a protease derived from Geobacillus, and a protease derived from Rhizopus to act on yeast and / or glutaminase. It has also been found that when lactic acid bacteria are cultured using the yeast extract obtained in this way, an excellent ability to promote the production of lactic acid bacteria is exerted. The present invention has been completed through further research based on this finding.

[0009] That is, the present invention provides the following inventions.

[0010] Item 1. A method for producing a yeast extract, comprising the step of allowing a protease selected from a protease derived from Bacillus, a protease derived from Geobacillus, and a protease derived from Rhizopus and / or glutaminase to act on yeast.

[0011] Item 2. The production method according to Item 1, wherein the protease derived from the genus Bacillus is a protease derived from Bacillus licheniformis.

[0012] Item 3. The production method according to Item 1, wherein the protease derived from the genus Geobacillus is a protease derived from Geobacillus stearothermophilus (including a strain formerly known as Bacillus stearothermophilus).

[0013] Item 4. The production method according to any one of Items 1 to 3, wherein the protease derived from the genus Rhizopus is a protease derived from Rhizopus niveus.

[0014] Item 5. The production method according to any one of Items 1 to 4, wherein the step is performed under the condition of pH 7 to 9 using at least the protease derived from the genus Bacillus and / or the protease derived from the genus Geobacillus.

[0015] Item 6. The production method according to any one of Items 1 to 4, wherein the step is performed under the condition of pH 3 to 6 using at least the protease derived from the genus Rhizopus.

[0016] Item 7. A yeast extract obtained by the production method according to any one of Items 1 to 6.

[0017] Item 8. A peptide ratio-enhancing agent for a yeast extract, comprising a protease selected from a protease derived from Bacillus, a protease derived from Geobacillus, and a protease derived from Rhizopus and / or glutaminase.

[0018] Item 9. An agent for improving lactic acid bacteria production ability of a yeast extract, comprising a protease selected from a protease derived from the genus Bacillus, a protease derived from the genus Geobacillus, and a protease derived from the genus Rhizopus and / or glutaminase.

[0019] Item 10. A method for producing a lactic acid bacteria culture, comprising the step of culturing lactic acid bacteria using a culture medium containing a yeast extract obtained by the production method according to any one of Items 1 to 6.

[0020] Effects of the Invention According to the present invention, a yeast extract having a high peptide ratio can be provided. In addition, according to the present invention, a yeast extract having an excellent ability to promote the production of lactic acid bacteria when used for lactic acid bacteria culture can be provided. DETAILED DESCRIPTION

[0021] 1. Method for producing yeast extract The method for producing a yeast extract of the present invention is characterized by comprising a step of allowing a protease selected from a protease derived from Bacillus, a protease derived from Geobacillus, and a protease derived from Rhizopus and / or glutaminase (hereinafter also referred to as "prescribed enzyme") to act on yeast. In this step, more specifically, a mixed solution containing yeast and a prescribed enzyme, typically in water, is subjected to conditions under which the prescribed enzyme acts. Thus, a yeast extract having a high peptide ratio can be obtained. In addition, a yeast extract having an excellent ability to promote lactic acid bacteria production when used in lactic acid bacteria culture can also be obtained.

[0022] 1-1. Yeast The yeast used in the present invention is not particularly limited, and typically includes yeast suitable for use in foods, specifically, yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae and Saccharomyces pastorianus; yeasts of the genus Candida such as Candida utilis; 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; yeasts of the genus Zygosaccharomyces such as Zygosaccharomyces mellis; and yeasts used or produced in the food industry (baker's yeast, brewer's yeast, yeast recovered after brewing, etc.). These yeasts may be used alone or in combination of a plurality of them.

[0023] Among these yeasts, from the viewpoint of further enhancing the peptide ratio-improving effect of the yeast extract and / or the ability to promote lactic acid bacteria production, preferably, yeast of the genus Saccharomyces cerevisiae is used, and more preferably, Saccharomyces cerevisiae is used.

[0024] The form of the yeast used in the present invention is not particularly limited, and examples thereof include a slurry form (more specifically, a yeast concentrate) and a dry form (more specifically, a dry yeast).

[0025] The content of the yeast in the mixed solution is not particularly limited, and examples thereof include 1 to 25% by weight, preferably 5 to 20% by weight, more preferably 7 to 15% by weight, and further preferably 9 to 12% by weight.

[0026] The method for inactivating the endogenous enzyme of yeast is not particularly limited and can be appropriately determined by a person skilled in the art, but preferably, a method of heating at 85 to 95° C. for 20 to 40 minutes is used.

[0027] 1-2. Specified enzymes In the present invention, as the predetermined enzyme, a protease selected from the group consisting of a protease derived from the genus Bacillus, a protease derived from the genus Geobacillus, and a protease derived from the genus Rhizopus (hereinafter also referred to as "the predetermined protease") and / or glutaminase is used. It should be noted that the protease in the present invention refers to an endo-peptidase.

[0028] The protease derived from the genus Bacillus is not particularly limited, and examples thereof include proteases derived from Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus cereus, Bacillus clausii, Bacillus intermedius, Bacillus lentus, and Bacillus thermoproteolyticus, etc. These proteases derived from the genus Bacillus may be used alone or in combination of two or more.

[0029] The protease derived from the genus Geobacillus is not particularly limited, and examples thereof include protease derived from Geobacillus stearothermophilus [including a strain formerly known as Bacillus stearothermophilus] and the like.

[0030] The protease derived from Rhizopus is not particularly limited, and examples thereof include proteases derived from Rhizopus niveus, Rhizopus chinensis, Rhizopus delemar, and Rhizopus oryzae. These proteases derived from Rhizopus may be used alone or in combination of two or more.

[0031] The glutaminase is not particularly limited, and examples thereof include glutaminase derived from Cryptococcus, Bacillus, and Aspergillus. As a glutaminase derived from Bacillus, glutaminase derived from Bacillus amyloliquefaciens can be mentioned. These glutaminases can be used alone or in combination of two or more.

[0032] Among these prescribed enzymes, from the viewpoint of further improving the peptide ratio-improving effect of the yeast extract and / or the ability to promote lactic acid bacteria production, preferably include a protease derived from the genus Bacillus, a protease derived from the genus Geobacillus, a protease derived from the genus Rhizopus, and a glutaminase derived from the genus Bacillus; more preferably include a protease derived from the genus Bacillus, a protease derived from the genus Geobacillus, a protease derived from the genus Rhizopus, and a glutaminase derived from Bacillus amyloliquefaciens; further preferably include a protease derived from the genus Bacillus, a protease derived from the genus Geobacillus, and a protease derived from the genus Rhizopus; further preferably include a protease derived from Bacillus licheniformis, a protease derived from Geobacillus stearothermophilus (including strains formerly known as Bacillus stearothermophilus), and a protease derived from Rhizopus nivalis.

[0033] In the present invention, from the viewpoint of further improving the peptide ratio-enhancing effect of the yeast extract and / or the ability to promote lactic acid bacteria production, it is particularly preferred to combine a prescribed protease and glutaminase, more preferably to combine a protease selected from the group consisting of a protease derived from Bacillus licheniformis, a protease derived from Geobacillus stearothermophilus (including strains previously known as Bacillus stearothermophilus), and a protease derived from Rhizopus nivalis with a glutaminase derived from the genus Bacillus, and even more preferably to combine a protease selected from the group consisting of a protease derived from Bacillus licheniformis, a protease derived from Geobacillus stearothermophilus (including strains previously known as Bacillus stearothermophilus), and a protease derived from Rhizopus nivalis with a glutaminase derived from Bacillus amyloliquefaciens.

[0034] The enzyme specified above can be prepared by the culture fluid of the microorganism that becomes its source or the heterologous host into which the gene encoding the enzyme specified above is introduced. As a specific preparation method, a method for recovering the target enzyme from the culture fluid or bacterial cell of the above-mentioned microorganism can be cited. For example, in the case of using a microorganism that secretes the target enzyme outside the bacterial cell, the bacterial cell can be recovered from the culture fluid by filtering, centrifugation, etc. in advance as needed, and the enzyme is separated and / or purified. In addition, in the case of using a microorganism that does not secrete the target enzyme outside the bacterial cell, the bacterial cell can be recovered from the culture fluid in advance as needed, and the bacterial cell can be broken by pressure treatment, ultrasonic treatment, etc. to expose the enzyme, and the enzyme is separated and / or purified. As a separation and / or purification method of the enzyme, a known protein separation and / or purification method can be used without particular limitation, and various chromatography methods such as centrifugation, UF concentration method, salting-out method, and ion exchange resin can be cited. The separated and / or purified enzyme can be powdered by drying methods such as freeze drying and reduced pressure drying. In addition, it can also be powdered using appropriate excipients and / or drying aids in the drying method. Alternatively, the isolated and / or purified enzyme may be one that has been liquefied by adding appropriate additives and performing filtration sterilization.

[0035] The enzymes specified above may be enzymes prepared by the above methods or commercially available products. Preferred examples of commercially available products include proteases derived from Bacillus licheniformis, Bacillus stearothermophilus (reclassified as Geobacillus stearothermophilus), and Rhizopus niveus manufactured by Amano Enzyme Inc., and glutaminase derived from Bacillus amyloliquefaciens manufactured by Amano Enzyme Inc.

[0036] The usage amount of the above-mentioned prescribed protease is not particularly limited, and the usage amount per 1 g of yeast may be, for example, 4 U or more, and from the viewpoint of further improving the peptide ratio-enhancing effect of the yeast extract and / or the ability to promote lactic acid bacteria production, preferably 20 U or more, more preferably 40 U or more, further preferably 200 U or more, further preferably 360 U or more, 660 U or more, 760 U or more, or 860 U or more. The upper limit of the usage amount range of the prescribed protease per 1 g of yeast is not particularly limited, and may be, for example, 50000 U or less, 10000 U or less, 5000 U or less, 2000 U or less, 1500 U or less, 1000 U or less, 940 U or less, 840 U or less, 740 U or less, 540 U or less, or 440 U or less.

[0037] The amount of glutaminase used is not particularly limited, and the amount per 1 g of yeast may be, for example, 0.01 U or more. From the viewpoint of further improving the effect of improving the peptide ratio of the yeast extract and / or the ability to promote lactic acid bacteria production, the amount is preferably 0.05 U or more, more preferably 0.1 U or more, further preferably 0.5 U or more, and further preferably 0.9 U or more. The upper limit of the range of the amount of glutaminase used per 1 g of yeast is not particularly limited, and may be, for example, 50 U or less, 10 U or less, 5 U or less, 2 U or less, 1.5 U or less, or 1.1 U or less.

[0038] When the above-specified protease and glutaminase are used in combination, the usage ratio of the specified protease to glutaminase is determined based on the above-mentioned usage amounts of each enzyme, and from the viewpoint of further improving the peptide ratio improvement effect of the yeast extract and / or the ability to promote lactic acid bacteria production, the usage amount of the protease per 1U of glutaminase is preferably 25U or more, more preferably 50U or more, further preferably 200U or more, further preferably 360U or more, 500U or more, or 750U or more, and the upper limit of the usage amount range of the protease per 1U of glutaminase is, for example, 50,000U or less, preferably 10,000U or less, more preferably 5,000U or less, further preferably 2,000U or less, further preferably 1,500U or less, 1,000U or less, or 500U or less.

[0039] The activity of the protease was determined by the Folin method at pH 3 (for acidic proteases) or pH 8 (for neutral proteases and alkaline proteases) using casein as a substrate. That is, the activity of the protease is determined by the amount of protease equivalent to 1 ng / mL in 1 minute when the enzyme reaction is carried out at pH 3 (for acidic proteases) or pH 8 (for neutral proteases and alkaline proteases) using a conventional method using casein as a substrate. μ The amount of enzyme increased by the colorimetric substance of Folin's test solution per g of tyrosine is taken as 1 unit (1U).

[0040] For the activity of glutaminase, L-glutamine is used as substrate and 1 μ The amount of enzyme per mol of L-glutamic acid is defined as 1 unit (1U).

[0041] 1-3. Reaction conditions, etc. The reaction conditions (pH, temperature and time) of the mixed solution supplied to allow the predetermined enzyme to act may be appropriately determined by further preliminary experiments, preferably according to the pH and temperature characteristics of the enzyme used, the yeast feed scale and the like.

[0042] For example, the pH (25° C.) of the mixed solution may be 3 to 11, preferably 4 to 10, more preferably 4 to 9, further preferably 4.5 to 8.5, 4.5 to 7, 4.5 to 6, 6 to 8.5 or 7 to 8.5.

[0043] More specifically, when neutral protease or alkaline protease is used as the prescribed enzyme, preferably when protease derived from Bacillus and / or protease derived from Geobacillus is used, the pH (25°C) of the mixed solution is preferably 7 to 9, preferably 7.5 to 8.5. When acidic protease is used as the prescribed enzyme, the pH (25°C) of the mixed solution is preferably 3 to 6, more preferably 4 to 5.5, and further preferably 4.5 to 5.5.

[0044] In addition, when the yeast extract is prepared under relatively high pH conditions, the peptide ratio is significantly reduced. However, according to the present invention, even when the extract is prepared under relatively high pH conditions, the peptide ratio can be effectively increased. From this point of view, examples of the preferred pH (25° C.) of the mixed solution include 7 to 9, preferably 7.5 to 8.5.

[0045] The reaction temperature is, for example, 30 to 80°C, preferably 40 to 70°C, more preferably 40 to 60°C, further preferably 45 to 55°C.

[0046] The reaction time is, for example, 5 minutes to 12 hours, preferably 10 minutes to 6 hours, and more preferably 30 minutes to 3 hours.

[0047] In addition, in the present invention, when glutaminase and a predetermined protease are used in combination, the order in which glutaminase and the predetermined protease are allowed to act on yeast is not particularly limited, and each enzyme may be allowed to act sequentially in an arbitrary order, or both enzymes may be allowed to act simultaneously. In the above case, it is preferred to allow glutaminase and the predetermined protease to act simultaneously from the viewpoint of further improving the peptide ratio-enhancing effect of the yeast extract and / or the ability to promote lactic acid bacteria production.

[0048] 1-4. Other processes The production method of the present invention may include any other steps in addition to the step of allowing the enzyme to act as described above. Specific examples of other steps include a step of inactivating the endogenous enzyme of yeast as a step performed before the above step, and a step of inactivating the enzyme as described above and a step of recovering a yeast extract (for example, a method of obtaining a supernatant as an extract by solid-liquid separation such as centrifugation), a step of concentrating a yeast extract, and a step of drying a yeast extract (for example, freeze drying, vacuum drying, and spray drying, etc.) as a step performed after the above step. In the production method of the present invention, any one of these other steps may be included alone, or two or more steps may be included in combination.

[0049] 2. Yeast Extract The yeast extract of the present invention is a yeast extract obtained by the above-mentioned "1. Method for producing a yeast extract". The yeast extract of the present invention contains peptides at a high ratio. In the present invention, peptides refer to peptides that produce a peak at a molecular weight of 400 or more and 1000 or less in molecular weight distribution chromatography. In addition, in the present invention, the ratio of peptides in the yeast extract refers to the ratio of the weight of peptides contained in the yeast extract to the weight of free amino acids contained in the yeast extract ([weight of peptides contained in the yeast extract] / [weight of free amino acids contained in the yeast extract]).

[0050] The peptide ratio of the yeast extract of the present invention exceeds 1. The specific peptide ratio may vary depending on the pH conditions during the reaction, and examples thereof include 1.7 or more, preferably 2 or more, more preferably 2.5 or more, further preferably 3 or more, and further preferably 3.5 or more. The upper limit of the range of the peptide ratio is not particularly limited, and examples thereof include 4.5 or less or 4 or less.

[0051] Since the yeast extract of the present invention has a high peptide ratio, it can be used in the following applications: taste improvement, taste masking, flavor correction and seasoning of food and beverages (including both food and beverages for humans and feed for animals other than humans. The same applies hereinafter); nutrition enhancement and nutrition addition of food and beverages; and use as a nutrient source in a culture medium for microorganisms.

[0052] In addition, the yeast extract of the present invention has a high peptide ratio and also exhibits excellent lactic acid bacteria production promoting ability when culturing lactic acid bacteria. In the yeast extract of the present invention, the level of the peptide ratio does not correspond to the level of the lactic acid bacteria production promoting effect, so the specific components or compositions that contribute to the lactic acid bacteria production promotion of the yeast extract of the present invention are not yet clear, but it is believed that at least one of the unique components or compositions constituting the unique various compound group including the components contained in the yeast and the components produced by the above-mentioned enzymes in the components exerts the lactic acid bacteria production promoting effect. Therefore, the yeast extract of the present invention is preferably used for the purpose of being mixed with a nutrient source in a culture medium of lactic acid bacteria (i.e., for lactic acid bacteria cultivation).

[0053] The form of the yeast extract is not particularly limited and may be appropriately determined depending on the intended use. Examples of the form include forms having fluidity such as liquid, slurry, and paste, and solid forms. Specific examples of the solid form include powder, granular, and granular forms.

[0054] 3. Yeast extract peptide ratio enhancer The enzyme (protease and / or glutaminase selected from the group consisting of protease derived from Bacillus, protease derived from Geobacillus, and protease derived from Rhizopus) can increase the peptide ratio of the obtained yeast extract by acting on yeast. Therefore, the present invention also provides a yeast extract peptide ratio improving agent, comprising a protease and / or glutaminase selected from the group consisting of protease derived from Bacillus, protease derived from Geobacillus, and protease derived from Rhizopus.

[0055] The types and amounts of the components used in the peptide ratio-enhancing agent of the yeast extract are as shown in the above-mentioned section “1. Method for producing yeast extract”.

[0056] 4. Yeast extract lactic acid bacteria production ability enhancer The enzyme (protease and / or glutaminase selected from protease derived from Bacillus, protease derived from Geobacillus and protease derived from Rhizopus) can improve the lactic acid bacteria production capacity of the obtained yeast extract by acting on yeast. Therefore, the present invention also provides a lactic acid bacteria production capacity improving agent for yeast extract, which comprises a protease and / or glutaminase selected from protease derived from Bacillus, protease derived from Geobacillus and protease derived from Rhizopus.

[0057] The types and amounts of the components used in the yeast extract for improving the lactic acid bacteria productivity are as shown in the above-mentioned "1. Method for producing yeast extract".

[0058] 5. Method for producing lactic acid bacteria culture As described above, the yeast extract shown in the column "2. Yeast extract" is useful for use as a nutrient source in a culture medium for lactic acid bacteria (i.e., for culturing lactic acid bacteria). Therefore, the present invention also provides a method for producing a lactic acid bacteria culture, comprising the step of culturing lactic acid bacteria using a culture medium containing the yeast extract obtained by the above "1. Method for producing a yeast extract".

[0059] The lactic acid bacteria to be cultured are not particularly limited, and examples thereof include lactic acid bacteria of the genus Lactobacillus and the genus Bifidobacterium. In addition, lactic acid bacteria of the genus Lactobacillus include Lactobacillus acidphilus, Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus gasseri. These lactic acid bacteria may be cultured alone or in combination.

[0060] As the culture medium, any culture medium applicable to lactic acid bacteria culture can be used, and specific examples thereof include MRS medium and GYP medium.

[0061] The content of the yeast extract in the culture medium is not particularly limited, and may be adjusted to have a protein content equivalent to that of a known yeast extract.

[0062] The culture conditions may be appropriately determined by those skilled in the art in consideration of the pH characteristics and temperature characteristics of the lactic acid bacteria.

[0063] Example Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not construed as being limited to the following Examples.

[0064] [Yeast and enzymes used] The following yeasts and enzymes were used.

[0065] [Table 1]

[0066] [Protease activity value determination method] After heating 5 mL of 0.6% (w / v) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0 [in the case of neutral proteases other than protease G and alkaline proteases], 0.05 mol / L sodium hydrogen phosphate, pH 6.0 [in the case of protease G] or 0.7% (v / w) lactic acid, pH 3.0 [in the case of acidic proteases]) at 37°C for 10 minutes, 1 mL of the sample solution containing protease is added and immediately shaken to mix. After the liquid is placed at 37°C for 10 minutes, 5 mL of trichloroacetic acid solution (0.44 mol / L trichloroacetic acid) is added and shaken to mix, and the mixture is placed at 37°C for 30 minutes again and filtered. The first 3 mL of filtrate is removed, and the next 2 mL of filtrate is weighed, and 5 mL of 0.55 mol / L sodium carbonate solution and 1 mL of Folin test solution (1→3) are added, and the mixture is shaken to mix thoroughly and placed at 37°C for 30 minutes. For this liquid (enzyme reaction solution), the absorbance AT at a wavelength of 660 nm was measured using water as a control.

[0067] In addition, 1 mL of the sample solution containing protease was weighed, 5 mL of trichloroacetic acid solution (0.44 mol / L trichloroacetic acid) was added and shaken, and then 5 mL of 0.6% (w / v) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0 [in the case of neutral proteases other than protease G and alkaline proteases], 0.05 mol / L sodium hydrogen phosphate, pH 6.0 [in the case of protease G] or 0.7% (v / w) lactic acid, pH 3.0 [in the case of acidic proteases]) was added, and the mixture was immediately shaken and allowed to stand at 37°C for 30 minutes. The same operation as the above enzyme reaction solution was performed, and the absorbance AB of the obtained liquid (blank) was measured.

[0068] Will cause the equivalent of 1 in 1 minute μ The amount of enzyme that increases by 1 g of tyrosine per colorimetric substance of Folin's test solution is taken as 1 unit (1U).

[0069] Weigh 1mL, 2mL, 3mL and 4mL of 1mg / mL tyrosine standard stock solution (0.2mol / L hydrochloric acid), and add 0.2mol / L hydrochloric acid solution to make 100mL. Weigh 2mL of each liquid, add 5mL of 0.55mol / L sodium carbonate solution and 1mL of Folin test solution (1→3), immediately shake to mix, and place at 37°C for 30 minutes. For these liquids, use the liquid obtained by weighing 2mL of 0.2mol / L hydrochloric acid solution and performing the same operation as above as a control, and measure the absorbance A1, A2, A3 and A4 at a wavelength of 660nm. Take the absorbance A1, A2, A3 and A4 on the vertical axis, and take the amount of tyrosine in 2mL of each liquid on the horizontal axis ( μ g), make a standard curve, and calculate the amount of tyrosine corresponding to the absorbance difference of 1 ( μ g).

[0070] [Number 1] Protease activity (U / g, U / mL) = (AT-AB) × F × 11 / 2 × 1 / 10 × 1 / M AT: absorbance of enzyme reaction solution AB: absorbance of blank F: The amount of tyrosine when the absorbance difference is 1 obtained by using the tyrosine standard curve ( μ g) 11 / 2: Conversion factor of total liquid volume after the reaction stops 1 / 10: Conversion factor for every 1 minute of reaction time M: The amount of sample in 1 mL of sample solution (g or mL) [Glutaminase activity value determination method] An appropriate amount of enzyme is weighed and dissolved or uniformly dispersed in acetic acid buffer (0.01 mol / L, pH 6.0, containing polyoxyethylene (10) octylphenyl ether) to make 50 mL, or further diluted 10-fold or 100-fold with the buffer to prepare a sample solution.

[0071] Weigh 2.0 g of L-glutamine, add 70 mL of water to dissolve it, add 10 mL of pH 6.0 acetic acid buffer (1 mol / L), add water to make 100 mL, and use this as the substrate solution. Weigh 1 mL of the sample solution, heat it in a water bath at 37°C for 5 minutes, add 1 mL of the substrate solution preheated to 37°C, immediately shake and mix, and then heat it at 37°C for 10 minutes, add 1 mL of perchloric acid (83→1000) and shake and mix, and immediately cool it in ice water for more than 1 minute. Here, perchloric acid with a mass fraction of 60% is used. Add 1 mL of sodium hydroxide solution (3→100) to this liquid and shake and mix to prepare a test solution. Separately weigh 1 mL of the sample solution, add 1 mL of perchloric acid (83→1000) and shake and mix, heat it in a water bath at 37°C for 5 minutes, add 1 mL of the substrate solution and shake and mix, and cool it in ice water for more than 1 minute. 1 mL of sodium hydroxide solution (3→100) was added to the liquid and shaken to mix to prepare a comparative solution. When the absorbance at a wavelength of 555 nm was measured using an L-glutamic acid assay kit (manufactured by Yamasa Soy Sauce), the absorbance of the liquid obtained by adding the test liquid was greater than the absorbance of the liquid obtained by adding the comparative solution. It should be noted that when the test liquid and the comparative solution for measuring the absorbance are turbid, centrifugation is performed and the supernatant is measured. μ The amount of enzyme per mol of L-glutamic acid is defined as 1 unit (1U).

[0072] [Number 2] Glutaminase activity (U / g, U / mL) = [(AS-ASB)·(AR-ARB)] × 250 × 4 × 1 / 147 × 1 / 10 × n AS: absorbance of reaction solution ASB: Blank solution absorbance AR: Absorbance of L-glutamic acid standard solution (250 mg / L) ARB: water blank absorbance 250: Concentration of L-glutamic acid standard solution (mg / L) 4: Total reaction liquid volume (mL) 147: molecular weight of L-glutamic acid 10: Reaction time (minutes) n: sample dilution multiple [Test Example 1] Yeast powder was suspended in purified water to prepare a 10% (w / w) yeast suspension, which was treated at 90°C for 30 minutes to inactivate endogenous enzymes. After cooling, the pH was adjusted to 5 or 8 using 1N hydrochloric acid or 1N sodium hydroxide. The resulting yeast solution was dispensed into 50 g portions, pre-warmed at 50°C and 200 rpm for 15 minutes, and then the enzymes in the amounts shown in Tables 2 and 3 were added and further reacted for 2 hours. After the reaction, the enzymes were inactivated at 90°C for 15 minutes, centrifuged (2000×g, 30 minutes), and the supernatant was recovered as a yeast extract.

[0073] The peptide ratio of the obtained yeast extract was calculated based on the following formula.

[0074] [Number 3]

[0075] In addition, the "amino acid content" in the above formula refers to the content of free amino acids, which was quantified using an L-amino acid quantification kit (Sigma-Aldrich). In addition, the "peptide content" in the above formula was calculated based on the following formula.

[0076] [Number 4] Peptide content (mg / mL) = protein content (mg / mL) × peptide area ratio in HPLC It should be noted that the "protein content" in the above formula was quantified using a total protein quantification kit (Sigma-Aldrich) based on the Lowry method. In addition, the "peptide area ratio in HPLC" in the above formula was determined as follows: the obtained yeast extract was applied to an HPLC column connected to TSKgel G2000SWxl and YMC-Pack Diol-60 (0.1M potassium phosphate buffer (pH 7) + 0.2M sodium chloride / acetonitrile = 70 / 30 (volume ratio) was used as the separation solvent, the flow rate was 0.35 mL / min, and the detection wavelength was 205 nm.), a chromatogram showing the molecular weight distribution was obtained, and the ratio of the area occupied by the peptide peak with a molecular weight of 400 or more and 1000 or less (calculated based on the calibration curve obtained from the HPLC peptide mixed standard (Sigma-Aldrich)) to the total area occupied by the peak with a molecular weight of 80 or more and a molecular weight of 60000 or less was determined.

[0077] Furthermore, for enzyme extracts having a peptide ratio exceeding 1, the relative value of the peptide ratio when the peptide ratio in an enzyme extract prepared identically except that no enzyme was used was set to 1 was determined as a peptide ratio improvement index. The results are shown in Tables 2 and 3.

[0078] [Table 2]

[0079] [Table 3]

[0080] As shown in Tables 2 and 3, when the predetermined enzyme (i.e., a predetermined protease selected from the group consisting of a protease derived from Bacillus, a protease derived from Geobacillus, and a protease derived from Rhizopus and / or glutaminase) was used (Examples 1 to 4, 5 to 8), it was confirmed that the peptide ratio in the obtained yeast extract was significantly improved compared to the case where the yeast extract was produced without using an enzyme (Comparative Examples 1 and 5). In addition, when the predetermined enzyme was not used, the peptide ratio in the obtained yeast extract was hardly improved (Comparative Examples 2 to 4), or the peptide ratio did not exceed 1 at all (Comparative Example 6). Therefore, the significant improvement effect of the peptide ratio confirmed in Examples 1 to 4, 5 to 8 is considered to be a unique effect brought about by the use of the predetermined enzyme.

[0081] In Examples 1 to 4 and 5 to 8, when the predetermined protease was combined with glutaminase (Examples 4 and 8), the effect of improving the peptide ratio was particularly remarkable.

[0082] Furthermore, as shown by comparison between Comparative Examples 1 and 5, the peptide ratio was particularly low when the yeast extract was produced at a high pH (Comparative Example 5). In view of this, the effect of improving the peptide ratio confirmed when the yeast extract was produced at a high pH using a predetermined enzyme (Examples 5 to 8) is considered to be particularly significant.

[0083] [Test Example 2] The yeast extract (supernatant) obtained in Test Example 1 was concentrated under reduced pressure (60° C., −1 bar) and then spray-dried (inlet temperature: 140° C., outlet temperature: 100° C.) to obtain a dry yeast extract powder.

[0084] MRS medium having the composition shown in the following table was prepared. The amount of yeast extract added was adjusted to be equivalent to 5 g / L protein (confirmed by absorbance at 280 nm) of yeast extract manufactured by Funakoshi.

[0085] [Table 4]

[0086] (*) The protein content was adjusted to be equivalent to 5 g / L of yeast extract manufactured by Funakoshi Co., Ltd.

[0087] The lactic acid bacteria were selected from the lactic acid bacteria shown in the following table. Each lactic acid bacteria was inoculated from a glycerol stock into an MRS medium (the yeast extract in the MRS medium shown in Table 4 was replaced with 5 g / L of yeast extract manufactured by Funakoshi Co., Ltd.), and statically cultured at 37° C. for 3 days under anaerobic conditions to perform pre-culture.

[0088] [Table 5]

[0089] [Test Example 2-1] Inoculate 10 cells in 4 mL of MRS medium listed in Table 4. μ L pre-culture solution was statically cultured at 37°C for 3 days under anaerobic conditions until the stationary phase was reached, and formal culture was performed (n=7). As the yeast extract mixed with the MRS medium, the dry powder of the yeast extract prepared in Example 4 or the dry powder of the yeast extract prepared in Comparative Example 1 for comparison was used, and as the pre-culture solution, the pre-culture solution of lactic acid bacteria a, b, c, d, and e was used. The turbidity (OD660) at the end of the culture was used as an index to evaluate the proliferation promoting effect using the T test. Specifically, the turbidity in the case of using the yeast extract of Example 4 was converted to a relative value when the turbidity in the case of using the comparative yeast extract was set to 1, and the proliferation promoting effect was evaluated based on the relative value (hereinafter referred to as the proliferation promoting index.). If the proliferation promoting index exceeds 1, it can be evaluated that the lactic acid bacteria production promoting effect is exerted.

[0090] As a result, based on the yeast extract of Example 4, the proliferation promotion index of lactic acid bacteria a was 1.05, the proliferation promotion index of lactic acid bacteria b was 1.13, the proliferation promotion index of lactic acid bacteria c was 1.02, the proliferation promotion index of lactic acid bacteria d was 1.12, and the proliferation promotion index of lactic acid bacteria e was 1.02, and the proliferation promotion effect was confirmed in all of them.

[0091] [Test Example 2-2] The growth promoting effect was evaluated in the same manner as in Test Example 2-1 except that the dry powder of the yeast extract prepared in Example 1 was used as the yeast extract mixed with the MRS medium, and the preculture solutions of the lactic acid bacteria b and c were used as the preculture solutions.

[0092] As a result, the growth promotion index of lactic acid bacteria b and the growth promotion index of lactic acid bacteria c based on the yeast extract of Example 1 were 1.11 and 1.07, respectively, indicating that growth promotion effects were observed in both cases.

[0093] [Test Example 2-3] The growth promoting effect was evaluated in the same manner as in Test Example 2-1 except that the dry powder of the yeast extract prepared in Example 2 was used as the yeast extract mixed with the MRS medium, and the preculture solution of lactic acid bacteria a was used as the preculture solution.

[0094] As a result, the growth promotion index of lactic acid bacteria a based on the yeast extract of Example 2 was 1.05, and a growth promotion effect was confirmed.

[0095] [Test Example 2-4] The growth promoting effect was evaluated in the same manner as in Test Example 2-1, except that the dry powder of the yeast extract prepared in Examples 6, 7, and 8 or the dry powder of the yeast extract prepared in Comparative Example 5 for comparison was used as the yeast extract mixed with the MRS medium, and the preculture solution of lactic acid bacteria a was used as the preculture solution.

[0096] As a result, the proliferation promotion index of lactic acid bacteria a based on the yeast extract of Example 6 was 1.09; the proliferation promotion index of lactic acid bacteria a based on the yeast extract of Example 7 was 1.14; and the proliferation promotion index of lactic acid bacteria a based on the yeast extract of Example 8 was 1.08, and the proliferation promotion effect was confirmed in all of them.

Claims

1. A method for producing a yeast extract, characterized in that: include: A step of allowing a protease selected from a protease derived from Bacillus, a protease derived from Geobacillus and a protease derived from Rhizopus to act on yeast and / or glutaminase.

2. The manufacturing method according to claim 1, wherein: The protease derived from Bacillus is a protease derived from Bacillus licheniformis.

3. The manufacturing method according to claim 1, wherein: The protease derived from the genus Geobacillus is a protease derived from Geobacillus stearothermophilus.

4. The manufacturing method according to claim 1, wherein: The protease derived from Rhizopus is a protease derived from Rhizopus nivalis.

5. The manufacturing method according to claim 1, wherein: The step is performed at least using the protease derived from the genus Bacillus and / or the protease derived from the genus Geobacillus under the condition of pH 7 to 9.

6. The manufacturing method according to claim 1, wherein: The step is performed at least at pH 3 to 6 using the protease derived from the genus Rhizopus.

7. A yeast extract, characterized in that Obtained by the production method according to claim 1.

8. A peptide ratio-enhancing agent for a yeast extract, characterized in that: The protein enzyme comprises a protease and / or a glutaminase selected from a protease derived from Bacillus, a protease derived from Geobacillus and a protease derived from Rhizopus.

9. A yeast extract lactic acid bacteria production ability enhancer, characterized in that The protein enzyme comprises a protease and / or a glutaminase selected from a protease derived from Bacillus, a protease derived from Geobacillus and a protease derived from Rhizopus.

10. A method for producing a lactic acid bacteria culture, characterized in that: include: A step of culturing lactic acid bacteria using a culture medium containing the yeast extract obtained by the production method according to claim 1.

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