Microbial growth promoter and method for producing the same

Abstract

The present invention aims to provide a yeast extract composition that can efficiently exhibit a microbial growth-promoting effect, and a method for producing the same. [Solution] The present invention provides a microbial growth promoter comprising (A) an enzyme extract of a yeast cell component having a ribonucleic acid content of 1 to 6% by weight, and (B) a yeast extract composition containing a nucleic acid component having a ribonucleic acid content of 50% by weight or more, and a method for producing a microbial growth promoter comprising (1) preparing an enzyme extract by acting an enzyme on a yeast cell component having a ribonucleic acid content of 1 to 6% by weight, (2) preparing a yeast nucleic acid extract having a ribonucleic acid content of 50% by weight or more by extracting ribonucleic acid from yeast or its culture, and (3) preparing a yeast extract composition containing the enzyme extract and the yeast nucleic acid extract.

Description

Technical Field

[0001] The present invention relates to a microbial growth promoter and a method for producing the same.

Background Art

[0002] In recent years, due to the depletion of fossil resources, rising prices, environmental problems, etc., the development of industrial raw materials derived from biological resources (biomass) has been actively carried out. One of the main biomass-based products is biodegradable plastic. As a method for producing biodegradable plastic, generally, microorganisms are used, and methods such as polymerizing monomers produced by culturing microorganisms and extracting and isolating polymers accumulated in the microorganisms are taken. Representative examples include, for example, the production of polylactic acid using lactic acid produced by using lactic acid bacteria, and the production by extracting and isolating polyhydroxyalkanoic acid (PHA) accumulated in microorganisms such as the genus Bacillus. However, in any case, efficient culturing of microorganisms is essential.

[0003] Yeast extract is widely used as a nitrogen source for culturing microorganisms. However, yeast extract is expensive, and there is a problem that the production cost becomes high. For example, in Patent Document 1, it is described that when culturing lactic acid bacteria, yeast extract is not used as a nutrient source, and the production amount of lactic acid is increased by adjusting the pH.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, methods involving pH adjustment, such as those described in Patent Document 1, do not result in satisfactory bacterial growth, especially for bacterial species with high nutritional requirements, indicating room for improvement. Furthermore, existing yeast extracts sometimes do not demonstrate a sufficient bacterial growth effect commensurate with the amount added. Therefore, there is a need for the development of a yeast extract that can more effectively promote bacterial growth at the same amount added. The present invention aims to provide a yeast extract composition that can efficiently exhibit a microbial growth-promoting effect. [Means for solving the problem]

[0006] In light of the above issues, the inventors conducted diligent research and found that a yeast extract composition combining a predetermined yeast cell component and a nucleic acid component can exhibit a microbial growth promoting effect superior to that of conventional yeast extracts, thus leading to the present invention.

[0007] The present invention provides the following: [1] (A) Enzyme extract of yeast cell components having a ribonucleic acid content of 1-6% by weight, (B) Nucleic acid components having a ribonucleic acid content of 50% by weight or more A microbial growth promoter containing a yeast extract composition containing [the specified substance]. [2] The agent described in [1], wherein the yeast cell component of component (A) is Torula yeast. [3] The agent according to [1] or [2], wherein the content of ribonucleic acid derived from component (A) in the yeast extract composition is 1.0 to 10.0% by weight. [4] The agent according to any one of items [1] to [3], wherein the nucleic acid component of component (B) is yeast nucleic acid extract. [5] The agent according to any one of items [1] to [4], wherein the content of ribonucleic acid derived from component (B) in the yeast extract composition is 1.0 to 30.0% by weight. [6] The agent according to any one of items [1] to [5], wherein the enzyme extract is a protease extract. [7] A solid preparation, as described in any one of items [1] to [6]. [8] (1) Prepare an enzyme extract by reacting an enzyme with a yeast cell component having a ribonucleic acid content of 1-6% by weight. (2) Extract ribonucleic acid from yeast or its culture to prepare a yeast nucleic acid extract having a ribonucleic acid content of 50% by weight or more, and (3) Prepare a yeast extract composition containing the enzyme extract and the yeast nucleic acid extract. A method for producing a microbial growth promoter, including [the specified ingredient]. The method for producing yeast residue having a ribonucleic acid content of 1 to 6% by weight by aqueous extraction of yeast or its culture in (9) (1), and using this as a yeast cell component, as described in (8). The manufacturing method described in [8] or [9], wherein two or more enzymes are used in

[10] (1).

[11] The manufacturing method according to any one of items [8] to

[10] , wherein step (3) is carried out by adding the yeast nucleic acid extract obtained in step (2) to the enzyme extract obtained in step (1), or by adding the yeast nucleic acid extract obtained in step (2) when the enzyme is applied to the yeast cell components in step (1).

[12] The manufacturing method according to [8], wherein in step (1), an enzyme is applied to yeast cell components, and the pH of the resulting stock solution is adjusted to a range of 5.0 to 8.0 to prepare an enzyme extract.

[13] (A) Enzyme extract of yeast cell components having a ribonucleic acid content of 1-6% by weight, (B) Nucleic acid components having a ribonucleic acid content of 50% by weight or more A yeast extract composition containing the following:

[14] (1) Prepare an enzyme extract by reacting an enzyme with a yeast cell component having a ribonucleic acid content of 1-6% by weight. (2) Extract ribonucleic acid from yeast or its culture to prepare a yeast nucleic acid extract having a ribonucleic acid content of 50% by weight or more, and (3) Prepare a yeast extract composition containing the enzyme extract and the yeast nucleic acid extract. A method for producing a yeast extract composition containing [the specified ingredient]. [Effects of the Invention]

[0008] According to the present invention, a yeast extract composition having the effect of promoting the growth of microorganisms can be provided. [Modes for carrying out the invention]

[0009] The details of the present invention will be described below. Unless otherwise specified, "AA~BB%" represents "AA% or more and BB% or less".

[0010] [1. Yeast extract composition] The yeast extract composition contains (A) and (B) below. This allows it to exhibit a growth-promoting effect on microorganisms.

[0011] [1.1 (A) Enzyme Extract] (A) is an enzyme extract of yeast cell components. This enhances the microbial growth-promoting effect of (B).

[0012] -yeast- Examples of yeast used as raw materials for yeast cell components include non-spore-forming yeast and spore-forming yeast, with edible yeast being preferred.

[0013] Examples of non-spore-forming yeasts include yeasts of the genus Torulopsis, such as Torulopsis versatilis; yeasts of the genus Candida, such as Candida utilis, Candida tropicalis, and Candida lipolytica; and yeasts of the genus Rhodotorula, such as Rhodotorula glutinis. Note that Candida utilis is sometimes referred to as torula yeast and is sometimes classified as a yeast of the genus Cyberlindnera (Cyberlindnera jadinii).

[0014] Spore-forming yeasts include, for example, yeasts of the genus Shizosaccharomyces such as Shizosaccharomyces pombe and Shizosaccharomyces octosporus; yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, Saccharomyces uvarum, and Saccharomyces rouxii; and Kluyveromyces fragilis, Kluyveromyces lactis, and Kluyveromyces marxianus. Examples include yeasts of the genus Kluyveromyces (such as marxianus); yeasts of the genus Hansenula (such as Hansenula anomala); yeasts of the genus Pichia (such as Pichia membranaefaciens and Pichia stipitis); yeasts of the genus Debaryomyces (such as Debaryomyces hansenii); yeasts of the genus Zygosaccharomyces (such as Zygosaccharomyces rouxii); and yeasts of the genus Lipomyces (such as Lipomyces starkeyi).The yeast may be yeast generally used in the food industry, such as baker's yeast, brewer's yeast, sake yeast, Torula yeast, etc. (for example, Saccharomyces yeasts such as Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces rouxii; Kluyveromyces yeasts such as Kluyveromyces fragilis; Torulopsis yeasts such as Torulopsis versatilis; Candida yeasts such as Candida tropicalis, Candida lipolytica, Candida utilis; Rhodotorula yeasts such as Rhodotorula glutinis), regardless of whether or not it overlaps with the above examples of yeast. Among these, baker's yeast, brewer's yeast, Torula yeast, or asporogenous yeasts are preferred, and Torula yeast is more preferred. These yeasts have a high cell yield and are easy to culture, so industrial production is easy and they can exhibit a better microorganism growth promoting effect.

[0015] -Yeast cell components- In the present specification, the yeast cell components preferably contain a small amount of ribonucleic acid (RNA). The content is usually 1% by weight or more, preferably 1.2% by weight or more, more preferably 1.4% by weight or more, and still more preferably 1.5% by weight or more. Thereby, the microorganism growth promoting effect can be exerted. The reason is presumed to be that a small amount of ribonucleic acid interacts with (B). The upper limit is usually 6% by weight or less, preferably 5% by weight or less, more preferably 4% by weight or less, and still more preferably 3% by weight or less. Therefore, the content of ribonucleic acid in the yeast cell components is usually 1 to 6% by weight, preferably 1.2 to 5% by weight, more preferably 1.4 to 4% by weight, and still more preferably 1.5 to 3% by weight per solid content.

[0016] Yeast cell components with ribonucleic acid content within the above range may be the yeast cells themselves (for example, yeast cultures recovered at a relatively early culture stage in yeast culture (which may be cultured according to a conventional method)), or components obtained by adjusting the ribonucleic acid content in yeast cells (for example, yeast residues), with yeast residues being preferred. In this specification, yeast residues refer to residues obtained by water extraction (preferably hot water extraction) of yeast, meaning the residues after extraction of flavor components for food additives. By using yeast residues, it becomes easier to adjust the ribonucleic acid content, which also leads to stabilization of the quality and performance of the product. In addition, the residues that would originally be discarded can be reused, reducing the environmental burden, and it is possible to achieve inexpensive production of yeast extract compositions. Yeast residues may be the residues themselves obtained after water extraction, or dried products (for example, those that have undergone heat treatment after water extraction). By water extraction, most of the flavor components such as amino acids and nucleic acids, and most of the soluble components such as vitamins and minerals among the components of yeast are extracted. Therefore, the main components of yeast residues are usually yeast cell walls and proteins, but may also contain a small amount of soluble components such as nucleic acids. The definition and specific examples of nucleic acids will be described later.

[0017] -Enzyme extract- In this specification, "enzyme extract" refers to an extract obtained by adding an enzyme to yeast cell components. The enzyme used for enzyme extraction is preferably an enzyme capable of extracting useful components from yeast cell components (for example, an enzyme that has the ability to break down yeast cell walls and proteins (e.g., hydrolase)). Examples include glycosidic hydrolases such as chitinase, glucanase, cellulase, and mannanase, and proteases. Proteases are preferred from the viewpoint of improving the microbial growth promoting effect of the yeast extract composition. The protease may be either a peptidase (exopeptidase) or a proteinase (endopeptidase), or both. The origin of the enzyme is not particularly limited, and it may be a natural enzyme or an artificial enzyme (a natural enzyme or a biomolecule that has been artificially modified). Proteases originate from, for example, bacteria of the genus Aspergillus (e.g., Aspergillus oryzae, Aspergillus niger, Aspergillus sp.), bacteria of the genus Bacillus (e.g., Bacillus sp., Bacillus licheniformis, Bacillus subtilis, Bacillus polymyxa, Bacillus amyloliquefaciens), and bacteria of the genus Geobacillus (e.g., Geobacillus stearothermophilus). Examples include microorganisms such as stearothermophilus, bacteria of the genus Streptomyces, bacteria of the genus Rhizomucor (e.g., Rhizomucor miehei, Rhizomucor pusillus Lindt, Rhizopus, bacteria of the genus Kluyveromyces (e.g., Kluyveromyces lactis)), plants such as papaya and pineapple, and animals such as pigs (pancreas). Combinations of two or more of these species are also acceptable.Of these, microorganisms are preferred, with Bacillus, Geobacillus, and Aspergillus bacteria being more preferred. One enzyme may be used, but it is preferable to use two or more, and more preferable to use three or more.

[0018] Enzymatic extraction of yeast residue can be performed by a method in which an enzyme is applied to the yeast residue. For example, an enzyme can be added to a suspension of yeast residue and allowed to act, followed by solid-liquid separation (e.g., centrifugation) to obtain the liquid phase, and then the enzyme can be deactivated (and further concentrated and sterilized as necessary). The suspension of yeast residue can be prepared by suspending the yeast residue in water. The pH during enzyme extraction is preferably at or near the optimal pH of the enzyme used, for example, around 5.0 to 10.0. The temperature during enzyme extraction should be at or near the optimal temperature of the enzyme and below the deactivation temperature, for example, around 30 to 90°C or around 70°C. The time for enzyme extraction is not particularly limited, but for example, it is about 2 to 20 hours. When using two or more enzymes, the first enzyme, the second enzyme, and so on can be added sequentially and allowed to act under the optimal conditions of each enzyme or near those conditions and below the deactivation temperature.

[0019] After treating the yeast residue with an enzyme, the resulting stock solution may be used directly as an enzyme extract, but further purification is preferable. The purification is preferably carried out by removing impurities (solid phase) by solid-liquid separation, or by suction filtration of the liquid phase after solid-liquid separation. It is preferable to adjust the pH beforehand when performing solid-liquid separation. This allows for efficient removal of impurities and further increases purity. The pH after preparation is preferably 5.0 to 8.0, more preferably 5.5 to 6.5, and even more preferably 5.7 to 6.0. pH adjustment can be carried out by adding a pH adjusting agent, for example, by adding an acid such as hydrochloric acid or an alkali such as sodium hydroxide.

[0020] The enzyme extract may be a single type, or a combination of two or more types of yeast, yeast cell components, or yeast extraction methods.

[0021] [1.2 (B) Nucleic acid component] (B) is a nucleic acid component. A nucleic acid component refers to a nucleic acid composition whose main component is nucleic acid.

[0022] -Nucleic acid- In this specification, nucleic acids mean molecules containing one or more units consisting of a sugar (deoxyribose or ribose) and a nucleotide. When nucleic acids contain two or more units, each nucleotide is linked by a phosphodiester bond. Nucleic acids are classified into deoxyribonucleic acid (DNA), where the sugar is deoxyribose, and ribonucleic acid (RNA), where the sugar is ribose, and either is acceptable. Examples of nucleotides include adenosine, guanosine, cytidine, thymidine, uridine, or modified versions thereof (for example, nucleic acids containing substitution with methylated bases, deamination, substitution with other atoms such as sulfur atoms, mismatches, and loop structures).

[0023] Nucleic acids may be in the form of salts, at least a portion of which may be salts. Examples of salts include alkali metal salts such as sodium and potassium, and alkaline earth metal salts such as calcium and magnesium.

[0024] The ribonucleic acid content in the nucleic acid component is usually 50% by weight or more, preferably 55% by weight or more, and more preferably 60% by weight or more. This can further promote the growth of microorganisms.

[0025] The molecular weight of the nucleic acid components (total amount as (B)) is preferably 7,000 or more, more preferably 8,000 or more, even more preferably 9,000 or more, and even more preferably 10,000 or more. This creates a difference from the peptide components, which are linked in units of several units of sugar and nucleotides, allowing the nucleic acid molecules to exhibit a good effect in promoting microbial growth. The upper limit is preferably 150,000 or less, more preferably 130,000 or less, even more preferably 100,000 or less, and even more preferably 80,000 or less. This is thought to allow microorganisms to take nucleic acids into their bodies, resulting in a good growth-promoting effect. The molecular weight of the yeast nucleic acid extract is the weight-average molecular weight (Mw), which can be determined by a method of converting to polyethylene glycol using GPC (gel permeation chromatography), and the values ​​in the examples below were also determined by this method.

[0026] The nucleic acid component may be derived from natural products or synthetic products, but it is preferable to include nucleic acids derived from natural products, more preferable to include nucleic acids derived from microorganisms, and even more preferable to include nucleic acid extracts or purified products thereof from microorganisms. Examples of microorganisms include yeast. Specific and preferred examples of yeast are described in the explanation in (A). The nucleic acid extract or product thereof from microorganisms may be derived from one type of microorganism or from two or more types of microorganisms, and it is preferable that it be derived from yeast of the same genus as the yeast from which the yeast cell component in (A) is derived, and more preferably from yeast of the same species.

[0027] -Methods for extracting nucleic acids from natural products- One method for extracting nucleic acids from natural products (e.g., yeast) is denucleation. Taking yeast as an example, denucleation can be any process that allows the separation of the yeast cell wall from other cellular components (for example, a process that breaks or dissolves part or all of the cell wall and elutes the contents containing nucleic acids into a culture medium), and processes including water extraction are preferred. Water extraction can be any extraction method that uses water as the extraction solvent, such as hot water extraction, acidic water extraction, alkaline water extraction, or alkaline ionized water extraction, with hot water extraction being more preferred. In hot water extraction, the extraction temperature (temperature of the hot water) is preferably 80°C or higher, more preferably 90°C or higher. The extraction time (time to maintain the above temperature) is preferably 5 minutes to 2 hours, more preferably 15 minutes to 45 minutes. When separating nucleic acids after water extraction, it is more preferable to further perform cell wall dissolution treatment. Cell wall lysis treatments include treatments using sodium chloride, treatments using cell wall lysis enzymes, separation treatments (e.g., separation treatments using ion exchange resins or separation gels), filtration treatments (e.g., ultrafiltration treatments), and combinations thereof, with treatments using sodium chloride being preferred. Sodium chloride treatment is, for example, a treatment in which sodium chloride is added to the extract, followed by heating and stirring as necessary to adjust the pH to 3 or less (e.g., by adding hydrochloric acid), and the resulting precipitate contains nucleic acids.

[0028] The precipitate or other sediment obtained after cell wall lysis treatment may be used directly as the nucleic acid component, or it may be further purified by re-dissolution treatment and the resulting product may be used as the nucleic acid component. This allows for adjustment, such as increasing the molecular weight of the obtained nucleic acid component, making it a form that is easily utilized by microorganisms, thereby suppressing the depletion of nucleic acid components and ultimately efficiently promoting the growth of microorganisms. Examples of re-dissolution treatments include alkaline treatment (for example, dissolving in an alkaline chemical such as sodium hydroxide, magnesium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, or sodium carbonate, and adjusting the pH to 6.5 or lower, 5 or lower, 3 or higher, or 4 or higher). By adjusting the pH during the re-dissolution treatment, the molecular weight of the obtained nucleic acid can be adjusted. For example, the molecular weight of the obtained nucleic acid tends to be higher as the pH during re-dissolution is higher (preferably 5 or higher, more preferably 5.5 or higher) and lower as the pH is lower. In addition, the obtained nucleic acid may take the form of a salt depending on the type of chemical used in the nucleic acid extraction treatment. For example, when sodium hydroxide is used as the alkaline chemical, the sodium salt of the nucleic acid is obtained, and when magnesium hydroxide is used, the magnesium salt of the nucleic acid is obtained. Furthermore, after nucleic acid extraction, further purification (e.g., contact with peroxides such as hydrogen peroxide, or porous materials such as activated carbon) can remove salts and macromolecules. During the redissolution process, heating and stirring may be performed as needed. Finally, if necessary, dehydration treatment such as spray drying can be performed to obtain the nucleic acid components.

[0029] By appropriately fine-tuning the above purification conditions, it is also possible to adjust the nucleic acid content in the resulting nucleic acid-containing material. For example, this can be done by increasing the pH value during the sodium chloride treatment as a nucleation treatment, or by adjusting the pH to near the upper limit during redissolution in the alkaline agent as a nucleic acid extraction treatment. Through these adjustments, it is possible to adjust the ribonucleic acid content in the nucleic acid component (approximately 50-90% by weight).

[0030] [1.3 Mixing ratio of components (A) and (B)] The weight ratio of components (A) and (B) contained in the yeast extract composition is not particularly limited, but the ratio of ribonucleic acid content derived from (A) to ribonucleic acid content derived from (B) (A / B) is preferably 0.15 or more, more preferably 0.18 or more, and even more preferably 0.2 or more. The upper limit is preferably 0.5 or less, more preferably 0.45 or less, and even more preferably 0.4 or less.

[0031] [1.4 Ribonucleic Acid Content] The ribonucleic acid content in the yeast extract composition is preferably 12% by weight or more, more preferably 12.5% ​​by weight, based on 100% by weight of the composition. The upper limit is preferably 30% by weight or less, more preferably 25% by weight or less, and even more preferably 20% by weight or less.

[0032] [1.5 Optional components] The yeast extract composition may contain other optional components besides components (A) and (B). Examples include antioxidants, flavorings, sweeteners, colorants, thickeners / stabilizers, color fixatives, gum bases, bittering agents, seasonings, enzymes, glazing agents, acidulants, emulsifiers, binders, isotonic agents, buffers, solubilizers, stabilizers, coagulants, excipients, and combinations of two or more selected from these.

[0033] [1.5 Actions of Yeast Extract Composition] The yeast extract composition, by containing a combination of components (A) and (B), can exert a growth-promoting effect on microorganisms and may exhibit a higher effect than a composition obtained by adding purified nucleic acid to a yeast culture (yeast extract). One reason for this is presumed to be as follows: The nucleic acid components in the yeast culture (yeast extract) form complexes with other components such as proteins, making them difficult for microorganisms to utilize. Furthermore, the nucleic acid-other component complexes remaining in the yeast culture also complex with the purified nucleic acid. It is presumed that these forms of existence hinder the utilization of purified nucleic acid by microorganisms. In contrast, by using the enzyme extract (A), the formation of such complexes becomes less likely, and therefore, even if the nucleic acid content is the same, it is presumed that a more significant growth-promoting effect on microorganisms can be exerted.

[0034] There are no particular restrictions on how it can be used as a microbial growth promoter. For example, it can be pre-mixed into the culture medium for culturing microorganisms, or added as needed to a culture medium containing microorganisms during cultivation.

[0035] There are no particular restrictions on the target microorganisms, as long as they can utilize nucleic acids; for example, heterotrophic organisms are included. Heterotrophic organisms include both aerobic and anaerobic microorganisms. Examples of aerobic microorganisms include, but are not limited to, yeast, filamentous fungi (e.g., koji (Aspergillus), Corynebacterium), acetic acid bacteria (Acetobacter, Gluconobacter, Gluconacetobacter, etc.), and some lactic acid bacteria. Examples of anaerobic microorganisms include, but are not limited to, obligate anaerobic bacteria (e.g., Bifidobacterium, Eubacterium, Clostridium, Bacteroides, Fusobacterium, Porphyromonas, Prevotella), facultative anaerobic bacteria (e.g., Escherichia (E. coli), Enterococcus and other enterococci, Lactobacillus and other lactobacillus, Staphylococcus and other staphylococci).

[0036] Microbial growth promoters can be used to promote microbial growth in the manufacturing and development of food and pharmaceuticals, as well as in life science research fields including biotechnology.

[0037] [2. Method for producing yeast extract composition] Yeast extract compositions can be manufactured through the following process.

[0038] [2.1 Step (1): Preparation of enzyme extract] In step (1), an enzyme extract is prepared by reacting an enzyme with a yeast cell component having a ribonucleic acid content of 1-6% by weight. Step (1) is the step for preparing component (A) described above. The yeast cell component, the type of enzyme, and the enzyme extraction conditions are as already described in the description of component (A) above.

[0039] [2.2 Step (2): Preparation of Yeast Nucleic Acid Extract] In step (2), ribonucleic acid is extracted from yeast or its culture to prepare a yeast nucleic acid extract having a ribonucleic acid content of 50% by weight or more. Step (2) is the step of preparing component (A) above. The extraction conditions for ribonucleic acid are as described above for component (B).

[0040] [2.3 Step (3): Preparation of Yeast Extract Composition] In step (3), a yeast extract composition is prepared from the enzyme extract and yeast nucleic acid extract prepared in steps (1) and (2), respectively.

[0041] In step (3), the enzyme extract and yeast nucleic acid extract prepared in advance according to each step may be added and mixed, or the yeast nucleic acid extract (e.g., powder, aqueous solution) prepared in step (2) may be added to the system when the enzyme is applied to the yeast cell components in step (1). Specifically, examples include adding the yeast nucleic acid extract (e.g., powder, aqueous solution) to the yeast residue slurry or powder after water extraction and subjecting it to enzyme extraction, or adding the yeast nucleic acid extract (e.g., powder, aqueous solution) to the reaction solution obtained by adding the enzyme to the yeast residue after water extraction and mixing it. The enzyme extract and yeast nucleic acid extract may be subjected to a drying treatment before or after mixing them. This makes it possible to obtain a yeast extract composition as a solid (powder), which improves convenience. Examples of drying treatments include spray drying, drum drying, cyclone-type hot air treatment, freeze-drying, and airflow drying. Furthermore, it is preferable to adjust the pH of the enzyme extract and yeast nucleic acid extract before drying. The pH after preparation is preferably 5.0 to 8.0, more preferably 5.5 to 6.5, and even more preferably 5.7 to 6.0. Having the pH within this range suppresses the formation of aggregates between nucleic acids and other components, allowing microorganisms to take up nucleic acids and potentially promoting their growth. It also prevents the aggregation of other components that serve as nutrients for microorganisms, such as phosphates, which can further promote microbial growth. [Examples]

[0042] The present invention will be explained with reference to examples. The following examples are merely illustrative and do not limit the present invention.

[0043] Manufacturing Example 1 (Manufacturing of nucleic acid-containing material with high RNA content) A culture solution was prepared by diluting 1 liter of pulverized extract (15% w / v solids) generated from wood chips mainly composed of domestically produced broadleaf trees using sulfurous acid pulverization (150°C, 9 hours). This extract was then diluted with water to a sugar concentration of 3.0%, and a nitrogen source and a phosphorus source (0.1% ammonia, 0.1% monoammonium phosphate) were added.

[0044] Candida utilis was cultured as yeast in the above culture medium for 24 hours. The resulting yeast culture was heated and stirred with 2% by weight sodium chloride aqueous solution (95°C, 30 minutes) to isolate the nucleic acid component. Hydrochloric acid was added to adjust the pH to 1.5 and stirred for 2 hours, and the resulting precipitate was collected. As a redissolution procedure, sodium hydroxide aqueous solution was added until the pH became 4.8, and the mixture was heated and stirred. The resulting solution was spray-dried using a spray dryer to obtain a nucleic acid-containing substance (component (B)). The ribonucleic acid (RNA) content in the above nucleic acid-containing substance was 85% (Table 1). Manufacturing Example 2 (Manufacturing of nucleic acid-containing material with RNA content)

[0045] In Production Example 1, after isolating the nucleic acid component from the obtained yeast culture solution, hydrochloric acid was added to bring the pH to 2.0 and stirred for 1 hour. The purified precipitate was collected, and as a redissolution step, an aqueous sodium hydroxide solution was added until the pH reached 5.8. Otherwise, the nucleic acid-containing substance (component (B)) was obtained in the same manner as in Production Example 1. The RNA content in the above nucleic acid-containing substance was 65%. The following manufacturing of component (A) and mixing with component (B) were carried out under the conditions shown in Tables 1 and 2.

[0046] Example 1 Enzyme reaction (stage 1) 100g of KR yeast (manufactured by Mitsubishi Corporation Life Sciences, RNA content 2.0%: Table 1), which is the yeast residue obtained by hot water extraction of nucleic acid components from Torula yeast, was suspended in 733g of deionized water, heated to an internal temperature of 60°C in a water bath, and the pH was adjusted to 9 with sodium hydroxide aqueous solution. Then, 0.5g of protease (Protin SD-AY10, manufactured by Amanoenzyme Co., Ltd.) was added as an enzyme, and the mixture was stirred and held at 150rpm for 5 hours.

[0047] Enzyme reaction (second stage) Next, the internal temperature was raised to 65°C, the pH was adjusted to 7.3 with hydrochloric acid, and then 0.5 g of protease (Samoase PC10, manufactured by Amanoenzyme Co., Ltd.) was added as an enzyme. The mixture was then stirred and maintained at 150 rpm for 5 hours.

[0048] Enzyme reaction (third stage) Next, the internal temperature was lowered to 55°C, and after checking the pH, if there was any fluctuation, the pH was adjusted to 7.3 with hydrochloric acid. Then, 0.5g of protease (Protamex, manufactured by Novozymes) was added as the enzyme, and the mixture was stirred at 150 rpm for 15 hours to obtain the yeast extract reaction solution.

[0049] pH adjustment / solidification The obtained yeast extract reaction solution was adjusted to pH 5.3 with hydrochloric acid, centrifuged at 8000 rpm for 15 minutes, and the resulting supernatant was filtered by suction to remove impurities and prepare the stock yeast extract (component (A)). To the obtained stock yeast extract, 10.0 g of the nucleic acid-containing material prepared in Production Example 1 was added as the purified nucleic acid component (component (B)), and the mixture was solidified into a powder using a spray dryer (outlet temperature 175°C) to obtain a microbial growth promoter.

[0050] The RNA content in the final product obtained was 14.0% (Table 3), and the molecular weight was approximately 20,000.

[0051] Example 2 Enzyme reaction (stage 1) 100g of KR yeast (manufactured by Mitsubishi Corporation Life Sciences, RNA content 2.0 wt%: Table 1), which is the yeast residue obtained by hot water extraction of nucleic acid components from Torula yeast, and 10.0g of the nucleic acid-containing material produced in Production Example 1 as the purified nucleic acid component were suspended in 733g of ion-exchanged water, heated to an internal temperature of 60°C in a water bath, adjusted to pH 9 with sodium hydroxide aqueous solution, and then 0.5g of protease (Protin SD-AY10, manufactured by Amano Enzyme Co., Ltd.) was added as the enzyme, and the mixture was stirred and held at 150 rpm for 5 hours. Enzyme reaction (second stage)

[0052] Next, the internal temperature was raised to 65°C, the pH was adjusted to 7 with hydrochloric acid, and then 0.5 g of protease (samoase PC10, manufactured by Amano Enzyme Co., Ltd.) was added as an enzyme. The mixture was then stirred and held at 150 rpm for 5 hours.

[0053] Enzyme reaction (third stage) Next, the internal temperature was lowered to 55°C, the pH was measured, and the pH was adjusted to 7.3 with hydrochloric acid or sodium hydroxide solution. Then, 0.5 g of protease (Protamex, manufactured by Novozymes) was added as an enzyme, and the mixture was stirred at 150 rpm for 15 hours to obtain the yeast extract reaction solution.

[0054] pH adjustment / solidification The obtained yeast extract reaction solution was adjusted to pH 5.3 with hydrochloric acid, centrifuged at 8000 rpm for 15 minutes, and the resulting supernatant was filtered by suction to remove impurities and prepare the stock yeast extract (components (A) + (B)). This was then solidified into a powder using a spray dryer (outlet temperature 175°C) to obtain a microbial growth promoter. The RNA content in the final product obtained was 18.0% (Table 3), and the molecular weight was approximately 20,000.

[0055] Example 3 In the procedure "pH adjustment and solidification," the obtained yeast extract reaction solution was adjusted to pH 7.5 with hydrochloric acid or sodium hydroxide aqueous solution. Otherwise, a microbial growth promoter was obtained in the same manner as in Example 1.

[0056] The RNA content in the final product obtained was 13.0% (Table 3), and the molecular weight was approximately 20,000.

[0057] Example 4 A microbial growth promoter was obtained in the same manner as in Example 1, except that peptidase (Sumizyme FP-G, manufactured by Shin Nippon Chemical Industries, Ltd.) was used as the enzyme in the "Enzyme Reaction (Third Stage)" procedure.

[0058] The RNA content in the final product obtained was 13.5% (Table 3), and the molecular weight was approximately 20,000.

[0059] Example 5 A microbial growth promoter was obtained in the same manner as in Example 1, except that the pH was adjusted to 12.2 with an aqueous sodium hydroxide solution in the "Enzyme reaction (third stage)" procedure.

[0060] The RNA content in the final product obtained was 12.8% (Table 3), and the molecular weight was approximately 20,000.

[0061] Example 6 In the "Enzyme Reaction (Stage 1)" procedure, a microbial growth promoter was obtained in the same manner as in Example 1, except that the nucleic acid-containing material produced in Production Example 2 was used as the purified nucleic acid component.

[0062] The RNA content in the final product obtained was 10.0% (Table 3), and the molecular weight was approximately 20,000.

[0063] Comparative Example 1 In the "Enzyme Reaction (Stage 1)" procedure, a microbial growth promoter was obtained in the same manner as in Example 1, except that only Torula yeast was used instead of purified nucleic acid components. The RNA content in the final product obtained was 4.0% (Table 3).

[0064] Comparative Example 2 As the purified nucleic acid-containing material, the nucleic acid-containing material produced in Production Example 1 was used directly as a microbial growth promoter. The RNA content in the final product obtained was 11.5% (Table 3).

[0065] Comparative Example 3 In the "Enzyme Reaction (Stage 1)" procedure, 100 g of dried Torula yeast cells (cultured by Nippon Paper Industries, Inc.) with an RNA content of 11.0% were used, and nucleic acid-containing materials were not used. Otherwise, a microbial growth promoter was obtained in the same manner as in Example 1.

[0066] The RNA content in the final product obtained was 14.0% (Table 3).

[0067] Culture test method ○ Strains used in culture tests Torula yeast: Candida utilis was used. Escherichia coli was used. Lactic acid bacteria: Lactococcus lactis was used.

[0068] ○ Evaluation of culture efficiency Approximately 1.0 mL of bacterial culture solution was collected, diluted 50-fold using a 50 mL volumetric flask and deionized water, and the absorbance at a wavelength of 660 nm was measured (OD660) using a spectrophotometer (JASCO Corporation). The above value was used as an indicator of bacterial culture efficiency (Table 3).

[0069] Evaluation Criteria An OD660 value of 23.0 or higher indicates particularly excellent performance. If the OD660 value is between 18.0 and 23.0, it can be used without any problems. If the OD660 value is between 15.0 and 18.0, the amount added during culture will be large, posing challenges for practical use. An OD660 value below 15.0 makes it impractical.

[0070] ○ Preparation of culture medium 3.0 g of glucose, 0.5 g of yeast extract prepared in each example, and 1.0 g of peptone (manufactured by Fujifilm Corporation) were dissolved in 100 mL of deionized water and stirred until homogeneous. 50 mL of the above culture medium solution was placed in a 200 mL Erlenmeyer flask, sealed with a silicone stopper, and sterilized in an autoclave at 121 °C for 15 minutes to prepare the liquid culture medium for the culture test.

[0071] The liquid culture medium used for pre-culture was prepared using the same composition and procedure as described above, except that the yeast extract used was the test chemical (Difco's "Bacto® Yeast EXTRACT").

[0072] ○ Culture test (pre-culture) Pre-culture was performed in two stages. First, for the first stage of pre-culture, colonies were scraped from the subculturing slant of the refrigerated strain using a platinum loop, placed in a liquid medium for inoculation, and incubated overnight at 30°C at 180 rpm to complete the first stage of pre-culture. Next, after confirming that the OD660 value of the first stage pre-culture solution was within the range of 15.0 to 30.0, 2.5 mL of the pre-culture solution was inoculated into the same liquid medium as in the first stage, and incubated overnight with shaking at 30°C at 180 rpm to complete the second stage of pre-culture.

[0073] Next, we confirmed that the OD660 value of the pre-culture solution from the second stage described above was within the range of 15.0 to 30.0. Then, 2.5 mL of the pre-culture solution was inoculated into a liquid medium prepared for the culture test, and cultured at 30°C at 180 rpm for 24 hours to evaluate the culture efficiency.

[0074] [Table 1]

[0075] [Table 2]

[0076] [Footnote to Table 2] A: Add the nucleic acid from manufacturing example 1 to the enzyme extract. B: When extracting the raw yeast with water, mix in the nucleic acid from Production Example 1.

[0077] [Table 3]

[0078] [Table 4]

[0079] [Footnote to Table 4] Extraction rate: Extraction rate (weight %) of extract (solids) relative to yeast residue (solids). Nitrogen, crude protein, peptides: weight % of total extract

[0080] The test results show that in a culture medium using yeast extract prepared by adding purified nucleic acid to yeast extract obtained by water extraction of nucleic acid components, microbial growth was better compared to yeast extract alone (Comparative Example 1), conventional yeast extract alone (Comparative Example 3), or a culture medium with only purified nucleic acid added (Comparative Example 2). Although there is no clear evidence, the reason for the poor efficiency of the culture medium with only purified nucleic acid added (Comparative Example 2) is presumed to be that the absorption efficiency of the added nucleic acid is consumed faster than the nucleic acid contained in the raw material, leading to faster depletion of the nucleic acid. Furthermore, the analytical values ​​of the final product (Table 4) were no different from the comparative examples and were within the acceptable range as a microbial growth promoter. From this, it can be seen that yeast extract components obtained by enzymatic extraction of yeast residue obtained by water extraction of nucleic acid components from yeast cells, and yeast extracts containing nucleic acid components extracted and purified from yeast cells, respectively, have a significant microbial growth promoting effect that surpasses that of conventional yeast extracts.

Claims

1. (A) Enzyme extract of yeast cell components having a ribonucleic acid content of 1 to 6% by weight, (B) Nucleic acid components having a ribonucleic acid content of 50% by weight or more A microbial growth promoter containing a yeast extract composition containing [the specified substance].

2. The agent according to claim 1, wherein the yeast of the yeast cell component of component (A) is Torula yeast.

3. The agent according to claim 1 or 2, wherein the content of ribonucleic acid derived from component (A) in the yeast extract composition is 1.0 to 10.0% by weight.

4. The agent according to claim 1 or 2, wherein the nucleic acid component of component (B) is a yeast nucleic acid extract.

5. The agent according to claim 1 or 2, wherein the content of ribonucleic acid derived from component (B) in the yeast extract composition is 1.0 to 30.0% by weight.

6. The agent according to claim 1 or 2, wherein the enzyme extract is a protease extract.

7. The agent according to claim 1 or 2, which is a solid preparation.

8. (1) Prepare an enzyme extract by reacting an enzyme with a yeast cell component having a ribonucleic acid content of 1 to 6% by weight. (2) Extract ribonucleic acid from yeast or its culture to prepare a yeast nucleic acid extract having a ribonucleic acid content of 50% by weight or more, and (3) Prepare a yeast extract composition containing the enzyme extract and the yeast nucleic acid extract. A method for producing a microbial growth promoter, including [the specified ingredient].

9. The method for producing a yeast residue having a ribonucleic acid content of 1 to 6% by weight is obtained by aqueous extraction of yeast or its culture in (1), and this is used as a yeast cell component, as described in claim 8.

10. The method for producing the product according to claim 8 or 9, wherein two or more enzymes are used in (1).

11. The manufacturing method according to claim 8 or 9, wherein step (3) is carried out by adding the yeast nucleic acid extract obtained in step (2) to the enzyme extract obtained in step (1), or by adding the yeast nucleic acid extract obtained in step (2) when the enzyme is applied to the yeast cell components in step (1).

12. The manufacturing method according to claim 8, wherein in step (1), an enzyme is applied to yeast cell components, and the pH of the resulting stock solution is adjusted to a range of 5.0 to 8.0 to prepare an enzyme extract.

13. (A) Enzyme extract of yeast cell components having a ribonucleic acid content of 1 to 6% by weight, (B) Nucleic acid components having a ribonucleic acid content of 50% by weight or more A yeast extract composition containing the following:

14. (1) Prepare an enzyme extract by reacting an enzyme with a yeast cell component having a ribonucleic acid content of 1 to 6% by weight. (2) Extract ribonucleic acid from yeast or its culture to prepare a yeast nucleic acid extract having a ribonucleic acid content of 50% by weight or more, and (3) Prepare a yeast extract composition containing the enzyme extract and the yeast nucleic acid extract. A method for producing a yeast extract composition containing [the specified ingredient].

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