Soy sauce production method using npII gene inactivated strain

Abstract

An object of the present invention is to provide a method for producing soy sauce that reduces the generation of pasteurized dregs. The object is to provide a method for producing soy sauce, in which a soy sauce raw material is treated with an Aspergillus sp. strain ( Aspergillus The above-mentioned problems are solved by the above-mentioned method, which comprises a step of subjecting a mutant Aspergillus genus microorganism to a koji-making treatment to obtain soy sauce koji, and by a mutant Aspergillus genus microorganism in which the neutral protease II gene present on the chromosome has been inactivated.

Description

[Technical Field]

[0001] The present invention relates to a method for producing soy sauce using a mutant Aspergillus microorganism as a koji mold. [Background technology]

[0002] Soy sauce by the traditional brewing method is produced by inoculating and cultivating a mixture of a protein raw material such as heat-denatured soybeans and a starchy raw material such as heat-gelatinized wheat with koji starter to produce soy sauce koji, then placing the soy sauce koji thus obtained in salt water for fermentation and aging to produce soy sauce moromi, then subjecting the soy sauce moromi thus obtained to compression and filtration to produce raw soy sauce, and then pasteurizing the raw soy sauce thus obtained.

[0003] When raw soy sauce is pasteurized, residual proteins in the soy sauce become insoluble and precipitate, generating pasteurization dregs. The amount of pasteurization dregs is approximately 5% (v / v) to 15% (v / v) of the amount of soy sauce. For this reason, processes for removing the dregs and removing the dregs are included in the production of pasteurized soy sauce. The mechanism by which pasteurization dregs are generated is presumed to be as follows: (1) heating raw soy sauce causes thermal denaturation of proteins in the soy sauce, (2) the denatured proteins polymerize due to hydrophobic interactions, (3) the resulting fine protein particles become insoluble and form colloidal particles, and (4) the colloidal protein particles aggregate and precipitate, generating pasteurization dregs (see, for example, Non-Patent Document 1).

[0004] Proteases in raw soy sauce include acidic proteases, alkaline proteases, and neutral proteases, which are believed to be involved in the generation of pasteurization dregs (see, for example, Non-Patent Documents 2 to 4). In particular, Non-Patent Document 4 describes that pasteurization dregs were generated by adding alkaline protease, neutral protease I, and neutral protease II to raw soy sauce and incubating the mixture at 60°C, and that the generation time of pasteurization dregs was delayed by adding ethylenediaminetetraacetic acid (EDTA) to the mixture and incubating the mixture at 60°C. Thus, Non-Patent Documents 2 to 4 suggest that proteases in raw soy sauce are involved in the generation of pasteurization dregs, but do not indicate which protease is directly involved in the generation of pasteurization dregs.

[0005] In contrast, Patent Document 1 describes that when raw soy sauce to which alkaline protease was added was subjected to heat treatment at 80°C for 10 minutes, pasteurization dregs were generated, but when raw soy sauce to which neutral protease II was added was subjected to heat treatment at 80°C for 10 minutes, pasteurization dregs were not generated. Similarly, Patent Documents 2 and 3 describe that neutral protease II is hardly involved in the generation of pasteurization dregs, and that in order to suppress pasteurization dregs, it is essential to remove substances that promote the generation of pasteurization dregs. Patent Document 4 describes a mutant neutral protease II characterized by the substitution of cysteine ​​with an amino acid without an SH group in the amino acid sequence of Aspergillus oryzae neutral protease II.

[0006] Non-patent document 5 describes an Aspergillus oryzae RIB40 strain mutant in which the neutral protease II gene is deleted and human lysozyme is heterologously expressed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 58-155057 [Patent Document 2] Japanese Patent Publication No. 60-023822 [Patent Document 3] Japanese Patent Publication No. 60-023823 [Patent Document 4] Japanese Unexamined Patent Application Publication No. 05-168479 [Non-Patent Document]

[0008] [Non-Patent Document 1] H. Hashimoto et al., J. Ferment. Technol., Vol. 52, No. 10, p. 747-758, 1974 [Non-Patent Document 2] H. Hashimoto, Journal of the Japan Society of Brewing, Vol. 71 (1976), No. 7, pp. 496-499 [Non-Patent Document 3] H. Hashimoto et al., J. Ferment. Technol., Vol. 51, No. 9, p. 661-669, 1973 [Non-Patent Document 4] H. Hashimoto et al., J. Ferment. Technol., Vol. 52, No. 5, p. 328-334, 1974 [Non-Patent Document 5] S. Kimura et al., Biosci Biotechnol Biochem, Vol. 72, No. 2, p. 499-505, 2008 [Summary of the Invention] [Problems to be Solved by the Invention]

[0009]

[0010] ​​​​​​​​Even when the information disclosed in Non-Patent Documents 1 to 4 and Patent Documents 1 to 3 are taken together, no effective means for reducing the generation of pasteurization dregs in soy sauce production methods has been clarified. In particular, Patent Document 4 and Non-Patent Document 5 make no mention whatsoever of the relationship between the Aspergillus oryzae RIB40 strain mutant in which the mutant neutral protease II and human lysozyme described therein are heterologously expressed and pasteurization dregs in soy sauce production methods. In particular, Patent Document 4 makes no mention of a yellow Aspergillus oryzae mutant that expresses mutant neutral protease II. In fact, removal of pasteurization dregs is a common practice in soy sauce production methods.

[0010] Therefore, the problem that the present invention aims to solve is to provide a method for producing soy sauce that reduces the generation of pasteurized dregs. [Means for solving the problem]

[0011] In order to solve the above problems, the present inventors have repeatedly conducted trial and error to try to reduce the amount of ignition dregs by taking some measures before, during, and after the generation of ignition dregs.

[0012] As a result, the present inventors prepared a mutant Aspergillus microorganism in which the neutral protease II gene on the chromosome was inactivated, and when soy sauce was produced using the mutant Aspergillus microorganism, they succeeded in reducing the generation of pasteurization dregs after pasteurization of raw soy sauce. This was very surprising in light of the fact that it was known in Patent Documents 1 to 3 that neutral protease II is hardly involved in the generation of pasteurization dregs.

[0013] Based on the above findings and success stories, the present inventors have finally succeeded in creating a soy sauce manufacturing method that reduces the generation of pasteurized dregs, thereby solving the problem of the present invention. The present invention was completed based on the findings and success stories first obtained by the present inventors.

[0014] Therefore, according to each aspect of the present invention, the following embodiments are provided. [I-1] A method for producing soy sauce, The soy sauce raw material was transformed into Aspergillus spp. ( Aspergillus The method further comprises a step of subjecting the mutant microorganism of the genus Bacillus subtilis to a koji-making treatment to obtain soy sauce koji. [I-2] The Aspergillus genus mutant is Aspergillus sojae ( Aspergillus soybeans ), Aspergillus oryzae ( Aspergillus rice ) and Aspergillus tamarii ( Aspergillus childish The method according to item [I-1], wherein the mutant is at least one Aspergillus microorganism selected from the group consisting of: [I-3] The method described in Item [I-1], wherein the neutral protease II gene is a neutral protease II gene having a nucleotide sequence encoding at least one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 5 to 7 or an amino acid sequence having 80% or more sequence identity to the amino acid sequence. [I-4] The method according to item [I-1], wherein the soy sauce is pasteurized soy sauce. [I-5] A mutant Aspergillus spp. in which the neutral protease II gene on the chromosome has been inactivated. [I-6] The Aspergillus genus mutant is Aspergillus sojae ( Aspergillus soybeans ), Aspergillus oryzae ( Aspergillus rice ) and Aspergillus tamarii ( Aspergillus childish The Aspergillus microorganism mutant according to item [I-5], which is a mutant of at least one Aspergillus microorganism selected from the group consisting of: [I-7] The Aspergillus microorganism mutant according to item [I-5], wherein the neutral protease II gene is a neutral protease II gene having a nucleotide sequence encoding at least one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 5 to 7 or an amino acid sequence having 80% or more sequence identity to the amino acid sequence. [II-1] A method for producing soy sauce, The soy sauce raw material was transformed into Aspergillus spp. ( Aspergillus The method further comprises a step of subjecting the mutant microorganism of the genus Bacillus subtilis to a koji-making treatment to obtain soy sauce koji. [II-2] The Aspergillus genus microorganism mutant is Aspergillus sojae ( Aspergillus soybeans ), Aspergillus oryzae ( Aspergillus rice ) and Aspergillus tamarii ( Aspergillus childish The method according to item [II-1], wherein the Aspergillus microorganism is a mutant of at least one Aspergillus microorganism selected from the group consisting of: [II-3] The method according to Item [II-1], wherein the soy sauce is pasteurized soy sauce. [II-4] A mutant Aspergillus microorganism, The neutral protease II gene on the chromosome has been inactivated, and Does not express human lysozyme, The Aspergillus genus microorganism mutant. [II-5] A mutant Aspergillus microorganism, The neutral protease II gene on the chromosome has been inactivated, and The host organism is an Aspergillus microorganism other than Aspergillus oryzae strain RIB40, The Aspergillus genus microorganism mutant. [II-6] The Aspergillus genus mutant is Aspergillus sojae ( Aspergillus soybeans ), Aspergillus oryzae ( Aspergillus rice ) and Aspergillus tamarii ( Aspergillus childish The Aspergillus microorganism mutant according to item [II-4] or [II-5], which is a mutant of at least one Aspergillus microorganism selected from the group consisting of: [II-7] Raw soy sauce having a heat-resistant NpII activity of 0 mU / ml to 0.2 mU / ml. [II-8] Pasteurized soy sauce obtained by pasteurizing the raw soy sauce of item [II-7]. [II-9] Pasteurized soy sauce obtained by pasteurizing raw soy sauce produced by the method described in item [II-1] or [II-2]. [Effects of the Invention]

[0015] According to the present invention, the generation of pasteurization dregs can be reduced in a soy sauce production method, and the process of removing the pasteurization dregs can be simplified or omitted, thereby shortening the process time, reducing the workload, and / or producing soy sauce economically advantageously. DETAILED DESCRIPTION OF THE INVENTION

[0016] Each aspect of the present invention will be described in detail below, but the present invention is not limited to the details of these items and can take various forms as long as the object of the present invention is achieved.

[0017] Unless otherwise specified, each term in this specification is used in the sense commonly used by those skilled in the art of biotechnology, food, and other technical fields, and should not be construed as having an unduly restrictive meaning. Furthermore, the speculations and theories made in this specification are based on the inventors' knowledge and experience to date, and therefore the present invention is not limited solely to such speculations and theories.

[0018] "Comprising" means that elements other than those explicitly stated as being included can be added (same meaning as "comprising at least"), but also encompasses "consisting of" and "consisting essentially of." That is, "comprising" can mean including the explicitly stated elements and any one or more elements, consisting of the explicitly stated elements, or consisting essentially of the explicitly stated elements. Elements include limitations such as ingredients, steps, conditions, and parameters. The term "and / or" means any one or any or all combinations of two or more of the associated listed items. The use of "to" in a numerical range includes both the preceding and following numerical values, and also includes ranges excluding one of the included limits. For example, "0% to 100%" means 0% or more, 100% or less, or 0% or more and 100% or less. "More than" and "less than" refer to the lower and upper limits, respectively, without including the preceding numerical value. For example, "more than 1" means a number greater than 1, and "less than 100" means a number less than 100. "About" refers to an amount within ±10% of the quantity following the term. For example, "about 100" means 100±10%, i.e., 90 to 110. The number of digits in an integer value matches the number of significant digits. For example, 1 has one significant digit, and 10 has two significant digits. Also, the number of digits after the decimal point in a decimal value matches the number of significant digits. For example, 0.1 has one significant digit, and 0.10 has two significant digits.

[0019] "Gene inactivation" refers to partial or complete disruption of the functional expression of a gene, such as when a gene is not transcribed normally or when an enzyme (protein) to be produced by gene expression is not translated to have the function it should have. Gene inactivation can occur, for example, when all or part of the gene is disrupted or deleted, or when functional expression of a gene is disrupted by substitution or introduction of a nucleotide sequence within the gene, resulting in a frameshift mutation, a nonsense mutation, a mutation that causes a splicing abnormality, a mutation in the active center, or a mutation involving an amino acid substitution that reduces thermostability. Gene inactivation can also occur when gene expression is suppressed without altering the sequence of the ORF region encoding the protein, for example, by blocking gene regulation through deletion, insertion, or substitution of the nucleotide sequence in the gene's regulatory region. "Functional expression of a gene" means that an enzyme having an amino acid sequence encoded by the nucleotide sequence of a gene (an enzyme encoded by a gene) is produced with its original structure and activity through transcription, translation, etc. "Gene expression product" means an enzyme produced corresponding to a gene. "Wild strain" refers to a naturally occurring organism that has not been artificially mutated. "Mutant" refers to an organism obtained by artificially mutating a wild strain or mutant. Mutants include microorganisms obtained by artificially inducing mutations in wild strains or mutants, as well as microorganisms mutated using transformation procedures involving the introduction of exogenous nucleic acid fragments into cells or gene editing techniques such as CRISPR / Cas (Cas9, Cas12, etc.), zinc finger nucleases (ZFN), and TALEN. A "wild-type gene" refers to a gene that is naturally present on the chromosome of a wild-type strain. A "wild-type enzyme" refers to an enzyme encoded by a wild-type gene. In this specification, chromosome and genome are synonymous terms. The term "foreign nucleic acid fragment" refers to a nucleic acid fragment consisting of a nucleotide sequence that is not inherently present in the genomic DNA of the microorganism to be introduced. The "foreign nucleic acid fragment" may also be formed by linking a nucleic acid fragment consisting of a nucleotide sequence that is inherently present in the genomic DNA of the microorganism to be introduced.

[0020] [Summary of the Invention] One aspect of the present invention is a method for producing soy sauce. In one aspect of the present invention, a method for producing soy sauce is carried out using Aspergillus ( Aspergillus The method for producing soy sauce according to one embodiment of the present invention is characterized by using a mutant Aspergillus microbial organism. By using a mutant Aspergillus microbial organism, the removal of pasteurized dregs, which is usually performed in the production of soy sauce, can be simplified or omitted.

[0021] [Summary of Aspergillus genus mutants] In one embodiment of the soy sauce production method of the present invention, the mutant Aspergillus microorganism used as the koji starter is a microorganism in which a specific gene on the chromosome of a wild-type strain of the Aspergillus microorganism has been inactivated. The inactivated gene is the neutral protease II gene.

[0022] [Neutral protease II gene] The neutral protease II gene, also called the npII gene, encodes the neutral protease II (NpII) protein. The NpII protein is a metalloprotease secreted and produced by the Aspergillus microorganism known as starter koji (koji mold), and has the ability to degrade proteins (NpII activity) in a near-neutral pH environment. The NpII protein is also called the NpII enzyme.

[0023] The present inventors speculated that by pasteurizing raw soy sauce, the NpII enzyme partially decomposes proteins derived from the soy sauce raw materials and koji mold, and then causes aggregation of the decomposition products due to hydrophobic bonds, resulting in the generation of pasteurization dregs.

[0024] Therefore, we prepared an Aspergillus mutant in which the npII gene of an Aspergillus microorganism having a wild-type npII gene on its chromosome was inactivated, and when raw soy sauce produced using the Aspergillus mutant was pasteurized, the generation of pasteurization dregs was suppressed. Based on this, we identified the npII gene as the gene to be inactivated in the Aspergillus microorganism used to suppress the generation of pasteurization dregs in soy sauce production methods.

[0025] The npII gene is registered in the NCBI GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ) for each Aspergillus microorganism from which it is derived. For example, Aspergillus oryzae ( Aspergillus riceThe npII gene of the RIB40 strain (SEQ ID NO: 1) has been registered as Gene ID AO090010000493. The npII gene is also called the deuterolysin A (deuA) gene, npIIa gene, or nptB gene (see Non-Patent Documents: Biosci Biotechnol Biochem., 80, 1813-1819, 2016; J. Fungi, 7, 658, 2021; Biosci Biotechnol Biochem, 72, 499-505, 2008). The intron-free npII gene of the Aspergillus oryzae RIB40 strain is shown in SEQ ID NO: 20.

[0026] Using the information on the npII gene of Aspergillus oryzae strain RIB40, it is possible to identify the npII genes of other Aspergillus species. One example of an npII gene identified in this way is Aspergillus sojae ( Aspergillus soybeans The npII gene of the NBRC4239 strain (SEQ ID NO: 2) is shown in SEQ ID NO: 21. The intron-free npII gene of the Aspergillus sojae strain (NBRC4239) is shown in SEQ ID NO: 21.

[0027] The NpII enzyme (SEQ ID NO: 5) encoded by the npII gene of the Aspergillus oryzae RIB40 strain has been registered in GenBank under the accession number (version) XP_001827477.1.

[0028] Using the information on the NpII enzyme of Aspergillus oryzae strain RIB40, it is possible to identify the NpII enzymes of other Aspergillus microorganisms. Examples of NpII enzymes identified in this way include the NpII enzyme of Aspergillus sojae strain NBRC4239 (SEQ ID NO: 6) and the NpII enzyme of Aspergillus tamarii ( Aspergillus childish ) NpII enzyme (SEQ ID NO: 7).

[0029] Similarly, Aspergillus tamarii ( Aspergillus childish ) NpII enzyme is KAE8168250.1, and Aspergillus minisclerotiigenes ( Aspergillus minisclerotigens ) NpII enzyme is KAB8274519.1, and Aspergillus steinii ( Aspergillus steyn's The NpII enzyme of the IBT23096 strain has the GenBank accession number (version) XP_024709461.1. The amino acid sequence of the NpII enzyme of the Aspergillus steinii IBT23096 strain is 69.49% identical to that of the Aspergillus oryzae RIB40 strain.

[0030] As described above, the NpII enzyme is well known as an enzyme expressed by microorganisms of the genus Aspergillus. For example, when the amino acid sequence set forth in SEQ ID NO: 5 is searched on BLAST (registered trademark) (URL: https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome), the NpII enzymes (neutral protease 2 or neutral protease II) shown in Table 1 below are found with an E-value of 0.

[0031] [Table 1]

[0032] Among proteins expressed by microorganisms of the genus Aspergillus, proteins that have an amino acid sequence identity of 70% or more, preferably 75% or more, more preferably 80% or more, and even more preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the NpII enzyme of Aspergillus oryzae RIB40 strain may be considered to be NpII enzymes. NpII activity can be measured using a heat-treated enzyme solution and Boc-Arg-Val-Arg-Arg-MCA as a substrate by the method described in the Examples below.

[0033] Even within the same species, Aspergillus microorganisms may have variations due to nucleotide deletions, substitutions, additions, etc. in the nucleotide sequence of the npII gene and / or amino acid deletions, substitutions, additions, etc. in the amino acid sequence of the NpII enzyme, depending on the strain. For example, assuming that there are Aspergillus sojae strain A and Aspergillus sojae strain B, although strains A and B belong to the same species, they are different strains and therefore may differ from each other in the nucleotide sequence of the npII gene and / or the amino acid sequence of the NpII enzyme.

[0034] Therefore, among proteins expressed by Aspergillus microorganisms, those having an amino acid sequence that is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and even more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the amino acid sequence of the NpII enzyme of the same Aspergillus microorganism can be considered to be NpII enzymes. For example, an enzyme from Aspergillus sojae having an amino acid sequence that is 90% or more identical to the amino acid sequence set forth in SEQ ID NO: 6 can be considered to be NpII enzymes.

[0035] Similarly, among genes possessed by Aspergillus microorganisms, genes having a nucleotide sequence that is preferably 80% or more, more preferably 85% or more, and even more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the nucleotide sequence of the npII gene of the same Aspergillus microorganism can be considered to be npII genes. For example, an Aspergillus sojae gene having a nucleotide sequence that is 80% or more identical to the nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 21 can be considered to be an npII gene.

[0036] Furthermore, whether the identified gene and enzyme are the npII gene and NpII enzyme, respectively, can be confirmed by confirming that the expressed protein, using genetic engineering techniques, possesses protease activity at near-neutral pH in the presence of metals such as zinc. Furthermore, NpII enzymes are known to be highly heat-resistant, remaining almost uninactivated even after heat treatment at 100°C (Mol. Gen. Genet., 228, 97-103, 1991). For example, it has been reported that the NpII enzyme of Aspergillus oryzae retains 60% or more of its activity even after heat treatment at 80-100°C for 10 minutes (Biosci Biotechnol Biochem., 80, 1813-1819, 2016). Therefore, NpII enzyme activity can be evaluated by measuring the protease activity of the heat-treated enzyme solution according to the method described in the Examples below.

[0037] The method for determining the sequence identity of nucleotide sequences and amino acid sequences is not particularly limited. For example, it can be determined using a commonly known method by aligning the nucleotide sequences of two types of npII genes or the amino acid sequences of NpII enzymes and using a program to calculate the identity between the two sequences.

[0038] A known program for calculating the percent identity between two nucleotide and amino acid sequences is, for example, the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). A BLAST program using this algorithm was developed by Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Furthermore, Gapped BLAST, a program that determines sequence identity with greater sensitivity than BLAST, is also known (Nucleic Acids Res. 25:3389-3402, 1997). Those skilled in the art can use these programs to search databases for sequences that show high sequence identity to a given sequence. These are available, for example, at the internet website of the US National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0039] [Method for producing mutant Aspergillus microorganisms] For example, a method for inactivating the npII gene on the chromosome of an Aspergillus microorganism can be used, including a step of inactivating the npII gene by modifying the chromosome of a host organism so that the npII gene is deleted or by inserting or substituting a foreign nucleic acid fragment into part or all of the CDS (Coding Sequence) or ORF of the npII gene. Alternatively, the npII gene may be inactivated by modifying the regulatory region (e.g., promoter sequence) of the npII gene instead of the npII gene itself. For example, methods for inactivating the npII gene by modifying the regulatory region of the npII gene include methods for reducing the expression level of the npII gene by introducing a mutation into a cis-element in the promoter sequence or by shortening the promoter sequence. In fact, many cases have been reported in Aspergillus oryzae and Aspergillus sojae where the expression level of a gene encoded by a promoter is reduced due to deletion of a cis-element in the promoter sequence or shortening of the promoter (e.g., Biosci Biotechnol Biochem., 72, 48-53, 2008; Biotechnol Lett., 37, 333-341, 2015; non-patent literature J Gen Appl Microbiol., 67, 77-80, 2021, etc.). Alternatively, the npII gene may be inactivated by modifying the npII gene or its regulatory region (e.g., promoter sequence) without inserting a foreign nucleic acid fragment, using genome editing techniques such as the CRISPR-Cas system, or mutagenesis techniques such as mutagen treatment, UV irradiation, plasma discharge, or radiation exposure.

[0040] The host organism may be any Aspergillus microorganism that possesses the npII gene on its chromosome and can be used as starter koji in soy sauce production. Examples of such Aspergillus microorganisms include Aspergillus sojae, Aspergillus oryzae, and Aspergillus tamarii. Aspergillus microorganisms are used not only in soy sauce brewing but also in traditional Japanese fermented foods such as sake, miso, mirin, amazake, and shochu, and their properties, such as enzyme productivity and secondary metabolite productivity, are diverse. Therefore, Aspergillus microorganisms with properties suitable for each fermented food are used. For example, desirable properties of koji mold for soy sauce brewing include: (1) high productivity of enzymes necessary for soy sauce brewing, (2) vigorous aroma production, resulting in an excellent aroma in the final product, (3) excellent conidial spore adhesion, resulting in easy preparation of starter koji, (4) vigorous growth, resulting in a large amount of fungal body mass in the koji, (5) weak tightness during koji production, resulting in little heat generation and loss of raw materials, (6) low moromi viscosity, resulting in easy pressing, (7) little production of pasteurized lees, (8) genetic stability, and (9) inability to produce mycotoxins such as aflatoxins (Non-Patent Document: Science and Technology of Soy Sauce, edited by Tochikura Tatsurokuro, Brewery Society of Japan, vol. 153, 1988). Therefore, the host organism is preferably an Aspergillus microorganism that possesses any one of these properties or a combination of two to nine of them. Examples include Aspergillus oryzae strain RIB326, isolated from soy sauce koji (Umemura M et al. DNA Res., 19(5), 375-382, 2012), and Aspergillus sojae strain NBRC4239, isolated from a soy sauce brewery (Sato A. DNA Res., 18(3), 165-176, 2011). While the genomes of these koji molds have been sequenced, strains selected for sake and soy sauce production are specialized for enzyme production and are thought to have been domesticated away from the original koji mold. On the other hand, Aspergillus oryzae strain RIB40, the first koji mold whose genome was sequenced, was isolated from a cereal (broad bean) and was selected as a wild-type strain lacking such specialized properties.In other words, this Aspergillus oryzae RIB40 strain is not considered to be an appropriate strain for sake or soy sauce production, and is therefore undesirable (Iwashita Kazuhiro, Chemistry and Biology 47(5), 329-338, 2009).

[0041] The npII gene may be inactivated by introducing a gene mutation using genome editing techniques such as the CRISPR / Cas system, zinc finger nuclease (ZFN), TALEN, homing (mega)nuclease, etc. For example, when modifying a target gene in an Aspergillus microorganism using the CRISPR / Cas9 system, methods known include using an autonomously replicating vector that expresses Cas9 and gRNA, and directly introducing a complex of Cas9 protein and gRNA (J Fungi, 2022, 8(5), 467).

[0042] The target sequence of the gRNA in the CRISPR / Cas9 system can be designed based on the sequence approximately 20 bp upstream of the PAM sequence (NGG) in the target gene sequence. For example, by designing the target sequence of the gRNA to be the CDS or ORF of the npII gene or the sequence of the gene's regulatory region, base substitutions, deletions, or insertions in the nucleotide sequence can cause frameshift mutations, nonsense mutations, mutations that cause splicing abnormalities, mutations in the active center, or mutations involving amino acid substitutions that reduce thermostability, thereby inactivating the npII gene.

[0043] The target sequence of the gRNA can also be designed to the CDS or ORF of the npII gene, or to multiple sequences in the gene's regulatory region. For example, by designing the target sequence of the gRNA to the upstream and downstream sequences of the npII gene on a chromosome, the nucleotide sequence containing the npII gene can be deleted, thereby inactivating the npII gene.

[0044] The npII gene may be inactivated by homologous recombination by introducing a nucleic acid fragment that induces homologous recombination at a desired chromosomal location. For example, a nucleic acid fragment constructed so that a nucleic acid fragment consisting of a sequence other than the npII gene or a nucleic acid fragment consisting of an npII gene sequence with an introduced mutation (deletion, substitution, or insertion) is ligated between homologous recombination regions homologous to the upstream and downstream regions of the npII gene on the chromosome is introduced into a host organism, and the npII gene on the chromosome is replaced with a foreign nucleic acid fragment by homologous recombination. Herein, a nucleic acid fragment prepared for transforming a host organism may be referred to as a "transformation cassette." The foreign nucleic acid fragment preferably contains a selectable marker gene to facilitate selection of the resulting transformant (mutant).For example, selectable marker genes that can be used in the genus Aspergillus include the pyrG gene (orotidine-5'-phosphate decarboxylase gene) and pyrF gene (orotate phosphoribosyltransferase gene) involved in the biosynthesis of uracil, the adeA gene (N-succinyl-5-aminoimidazole-4-carboxyamide ribotide synthase gene) and adeB gene (phosphoribosylaminoimidazole carboxylase gene) involved in the biosynthesis of adenine, the argB gene (ornithine carbamoyltransferase gene) involved in the biosynthesis of arginine, the argC / argE genes (argininosuccinate synthase gene), the argA gene (argininosuccinate lyase gene), and the niaD gene (nitrate reductase gene) involved in the assimilation of nitrate. Examples of selectable marker genes include the sC gene (ATP sulfurylase gene) involved in sulfate assimilation, the amdS gene (acetamidase gene) involved in acetamide assimilation, the trpC gene (glutamine amidotransferase gene / indoleglycerophosphate synthase gene / phosphoribosylanthranilate isomerase gene) involved in tryptophan biosynthesis, the bioDA gene (7,8-diaminopelargonic acid synthase gene / dethiobiotin synthase gene) involved in biotin biosynthesis, the leu2 gene (β-isopropylmalate dehydrogenase gene) involved in leucine biosynthesis, the pyrithiamine resistance marker ptrA gene (thiazole synthase gene), and the benomyl resistance marker benA gene (β-tubulin gene). Furthermore, a method of inactivating genes encoding proteins such as Ku70, Ku80, and LigD involved in non-homologous recombination to increase the frequency of homologous recombination of the target gene may also be combined.

[0045] A transformation cassette can be obtained, for example, by using the chromosomal DNA of an Aspergillus microorganism as a template to obtain nucleic acid fragments of the upstream region of homologous recombination and the downstream region of homologous recombination by polymerase chain reaction (hereinafter referred to as "PCR").Then, the upstream region of homologous recombination, the foreign nucleic acid fragment, and the downstream region of homologous recombination are ligated in that order to the In-Fusion Cloning Site in the multi-cloning site of the plasmid pUC19 to prepare a construct plasmid (DNA), and then amplifying the obtained construct plasmid by PCR using the template DNA.

[0046] The method for extracting chromosomal DNA is not particularly limited, but for example, an Aspergillus microorganism is cultured, water is removed from the resulting mycelia, and the mycelia are physically ground in a mortar or the like while cooled in liquid nitrogen to produce fine powder-like mycelial fragments, from which a chromosomal DNA fraction is extracted by a conventional method. Commercially available chromosomal DNA extraction kits such as the DNeasy Plant Mini Kit (Qiagen) can be used for the chromosomal DNA extraction procedure.

[0047] Methods for transforming Aspergillus microorganisms can be selected from methods known to those skilled in the art. For example, the protoplast-PEG method, which involves preparing protoplasts of a host organism and then using polyethylene glycol and calcium chloride (see, for example, Mol. Gen. Genet. 218, 99-104, 1989; JP 2007-222055 A), can be used. The medium for regenerating Aspergillus microorganisms is appropriate depending on the host organism and the exogenous nucleic acid fragment used. For example, when Aspergillus sojae is used as the host organism and an exogenous nucleic acid fragment containing a drug resistance gene such as the pyrithiamine resistance marker ptrA is used as a selection marker, the transformant can be regenerated using, for example, a minimal agar medium such as Czapeck-Dox medium containing the corresponding drug (pyrithiamine when ptrA is used).

[0048] The creation of an Aspergillus mutant with an inactivated npII gene can be confirmed by performing PCR using a colony or chromosomal DNA of the mutant as a template and sequence-analyzing the PCR product to confirm that the gene mutation has been introduced. When a foreign nucleic acid fragment is introduced using homologous recombination, this can be confirmed by confirming that a PCR product of the foreign nucleic acid fragment is produced, but not that of the npII gene.

[0049] For example, it is preferable to perform PCR using a combination of a forward primer complementary to the upstream region of homologous recombination incorporated into the transformation cassette or a region located further upstream, and a reverse primer complementary to the downstream region of homologous recombination incorporated into the transformation cassette or a region located further downstream, and to confirm that a product of the expected length is produced when homologous recombination occurs.

[0050] Alternatively, the preparation of an Aspergillus microorganism mutant in which the npII gene has been inactivated can be confirmed by measuring the heat-resistant NpII activity in soy sauce koji or soy sauce prepared using the Aspergillus microorganism mutant, as described in the Examples below. The heat-resistant NpII activity in soy sauce koji or soy sauce should be at a lower level than when a wild-type Aspergillus microorganism expressing NpII is used as starter koji.

[0051] Aspergillus mutant microorganisms in which the npII gene on the chromosome has been inactivated can be produced by methods other than introducing mutations using genome editing or using homologous recombination, such as suppressing expression of the npII gene using the antisense RNA method, adding chemical mutagens such as NTG (1-Methyl-3-nitro-1-nitrosoguanidine), 4-NQO (4-Nitroquinoline N-oxide), and EMS (Ethyl ethanesulfonate), or by subjecting the microorganism to mutation treatments such as UV irradiation, plasma discharge (ARTP: Atmospheric Room Temperature Plasma), or radiation (X-rays, gamma rays, heavy particle beams (ion beams), neutron beams).

[0052] A non-limiting embodiment of an Aspergillus microorganism mutant in which the npII gene on the chromosome has been inactivated is an Aspergillus microorganism mutant in which the upstream region of the npII gene on the chromosome and the entire ORF of the npII gene have been deleted, or an Aspergillus microorganism mutant in which a nonsense mutation has been introduced into the ORF of the npII gene, wherein the host organism is Aspergillus sojae or Aspergillus oryzae, as described in the Examples below.

[0053] The Aspergillus mutant microorganism in which the npII gene on the chromosome has been inactivated is used as starter koji for the production of soy sauce, preferably by the honjozo method.

[0054] Another aspect of the present invention is a mutant Aspergillus microorganism. In one embodiment of the present invention, the neutral protease II gene present on the chromosome of the mutant Aspergillus microorganism has been inactivated, and the mutant Aspergillus microorganism does not express human lysozyme. Preferably, the mutant Aspergillus microorganism of one embodiment of the present invention does not contain any foreign genes derived from organisms other than the Aspergillus microorganism, such as the human lysozyme gene.

[0055] In another embodiment of the present invention, the Aspergillus microorganism mutant has the neutral protease II gene on its chromosome inactivated, and the host organism is an Aspergillus microorganism other than the Aspergillus oryzae RIB40 strain.

[0056] In another embodiment of the present invention, the Aspergillus microorganism mutant has the neutral protease II gene on its chromosome inactivated, does not express human lysozyme, and the host organism is an Aspergillus microorganism other than the Aspergillus oryzae RIB40 strain.

[0057] As an indicator of the NpII activity possessed by the Aspergillus microorganism mutant of one embodiment of the present invention, the heat-resistant NpII activity in soy sauce koji or soy sauce obtained using the Aspergillus microorganism mutant of one embodiment of the present invention may be at a lower level than when a wild-type Aspergillus microorganism expressing NpII is used as starter koji. For example, the heat-resistant NpII activity in soy sauce koji is preferably 50% or less, more preferably 20% or less, even more preferably 10% or less, and even more preferably 5% or less, compared to when a wild-type Aspergillus microorganism expressing NpII is used as starter koji. Alternatively, when 1 unit (U) is defined as the amount of enzyme that decomposes 1 μmol of substrate per minute at 50° C., the heat-resistant NpII activity in raw soy sauce is preferably 0.2 mU / mL or less, more preferably 0.1 mU / mL or less, even more preferably 0.05 mU / mL or less, still more preferably 0.025 mU / mL or less, or 0 mU / mL to 0.025 mU / mL. Alternatively, the heat-resistant NpII activity in soy sauce koji is preferably 0.4 mU / g-koji or less, more preferably 0.2 mU / g-koji or less, even more preferably 0.1 mU / g-koji or less, still more preferably 0.05 mU / g-koji or less, or 0 mU / g-koji to 0.05 mU / g-koji.

[0058] [Soy sauce manufacturing method] A soy sauce production method according to one embodiment of the present invention is characterized by comprising a step of obtaining soy sauce koji by subjecting soy sauce raw materials to a koji-making process using an Aspergillus microorganism mutant in which the npII gene present on the chromosome has been inactivated. The soy sauce production method according to one embodiment of the present invention further comprises, after the step of obtaining soy sauce koji, a step of obtaining soy sauce moromi mash and a step of obtaining raw soy sauce, similar to a conventional soy sauce production method. Furthermore, after the step of obtaining raw soy sauce, a step of pasteurizing the raw soy sauce to obtain soy sauce is also included. However, since the soy sauce production method according to one embodiment of the present invention reduces the generation of pasteurization dregs in the soy sauce after pasteurization, the removal of dregs and / or separation of the pasteurization dregs after pasteurization can be omitted or simplified. The soy sauce production method according to one embodiment of the present invention may be a production method described in the Japanese Agricultural Standards (JAS) or a modified version of the production method.

[0059] Soy sauce ingredients may be any ingredients used in conventional soy sauce manufacturing methods, including soybeans (e.g., whole soybeans and defatted soybeans), legumes (e.g., peas, chickpeas, broad beans, adzuki beans, lentil beans, kidney beans, mung beans, and cowpeas), wheat, barley, naked barley, and Job's tears, wheat gluten, rice, foxtail millet, millet, barnyard millet, quinoa, perilla, corn, tomato, yeast extract, soy protein, pea protein, potato protein, rice protein, corn protein, and microbial protein. Sugars such as glucose and starch may also be added as sugar sources. Soy sauce ingredients may be used alone or in combination of two or more. Soybeans are primarily used as protein sources after being heat-denatured, while wheat is primarily used as a sugar source after being heat-gelated. It is preferable that the soy sauce raw material is subjected to pretreatment such as heating, degreasing, pressing, crushing, drying, etc.

[0060] The step of obtaining soy sauce koji can be carried out under the same conditions as those for the conventional soy sauce production method, except that a mutant Aspergillus microorganism in which the npII gene on the chromosome has been inactivated is used as the starter koji. For example, soy sauce koji can be obtained by inoculating a mutant Aspergillus microorganism in which the npII gene on the chromosome has been inactivated as the starter koji into a soy sauce raw material, which is a mixture of steamed and denatured soybeans and roasted and crushed wheat in a predetermined ratio, preferably approximately equal amounts, and then subjecting the mixture to aeration koji production at 10°C to 40°C, preferably at room temperature, for about 12 hours to 4 days.

[0061] By using a mutant Aspergillus microorganism in which the chromosomal npII gene has been inactivated as a seed koji, the heat-resistant NpII activity in soy sauce koji after 10 minutes of treatment at 80°C is reduced. The heat-resistant NpII activity in soy sauce koji may be at a lower level than when a wild-type Aspergillus microorganism is used as a seed koji. For example, the heat-resistant NpII activity in soy sauce koji is preferably 50% or less, more preferably 20% or less, even more preferably 10% or less, and even more preferably 5% or less compared to when a wild-type Aspergillus microorganism is used as a seed koji. Alternatively, when 1 unit (U) is defined as the amount of enzyme required to decompose 1 μmol of substrate per minute at 50°C, the heat-resistant NpII activity in soy sauce koji is, for example, preferably 0.4 mU / g-koji or less, more preferably 0.2 mU / g-koji or less, even more preferably 0.1 mU / g-koji or less, and even more preferably 0.05 mU / g-koji or less. Alternatively, the heat-resistant NpII activity in raw soy sauce is preferably 0.2 mU / mL or less, more preferably 0.1 mU / mL or less, even more preferably 0.05 mU / mL or less, and even more preferably 0.025 mU / mL or less. The heat-resistant NpII activity is measured by the method described in the Examples below.

[0062] The process of obtaining soy sauce moromi mash is carried out by fermenting and aging soy sauce koji. For example, 100 parts by mass of soy sauce koji is added to 50 to 300 parts by mass, preferably 100 to 200 parts by mass, of brine having a salt concentration of 15% (w / v) to 35% (w / v), and optionally soy sauce lactic acid bacteria and / or soy sauce yeast are added to the mixture. The mixture is fermented and aged at 10 to 40°C, preferably 20 to 35°C, with appropriate stirring for 10 to 300 days, preferably 15 to 200 days, to obtain soy sauce moromi mash. When both soy sauce lactic acid bacteria and soy sauce yeast are used, the mixture is preferably subjected to lactic acid fermentation using soy sauce lactic acid bacteria and then to yeast fermentation using soy sauce yeast.

[0063] The soy sauce lactic acid bacteria and soy sauce yeast may be any bacteria that are commonly used in the production of soy sauce. For example, the soy sauce lactic acid bacteria may be Tetragenococcus halophilus ( Tetragenococcus halophilous ), and soy sauce yeast such as Zygosaccharomyces ruxii ( Zygosaccharomyces red-haired ), Candida etquersii ( White Etchell's ), Candida verstilis ( C.versatilis ) and other salt-tolerant yeasts.

[0064] The process of obtaining raw soy sauce is carried out by removing insoluble solids from soy sauce moromi mash. For example, raw soy sauce can be obtained by subjecting the soy sauce moromi mash to solid-liquid separation treatments such as squeezing and filtration. Examples of solid-liquid separation treatments include squeezing using a squeezing device such as a press, filtration using a filter medium such as diatomaceous earth or filter cloth, squeezing filtration using a squeezing device such as a press, and membrane filtration using various permeable membranes such as UF membranes and MF membranes. The solid-liquid separation treatment may be one type of treatment alone or a combination of two or more types, but a combination of squeezing using a squeezing device and filtration using a filter medium is preferred.

[0065] The step of obtaining soy sauce is carried out by heating the raw soy sauce. For example, the raw soy sauce can be obtained by heating the raw soy sauce at 50°C to 100°C, preferably 60°C to 80°C, for 10 to 100 minutes, preferably 10 to 40 minutes.

[0066] In a typical soy sauce manufacturing method, pasteurization of raw soy sauce generates pasteurization dregs. Therefore, the soy sauce and pasteurization dregs are separated by carrying out procedures such as dregs sage, in which the pasteurized soy sauce is allowed to stand and the pasteurization dregs are allowed to settle, and dregs removal, in which the clear portion of soy sauce that does not contain the pasteurization dregs is extracted. However, in a soy sauce manufacturing method according to one embodiment of the present invention, the amount of pasteurization dregs generated can be reduced by using an Aspergillus microbial mutant in which the npII gene on the chromosome has been inactivated. As a result, the separation of soy sauce and pasteurization dregs, which is performed in a typical soy sauce manufacturing method, can be omitted.

[0067] Specific examples of the method for producing soy sauce according to one embodiment of the present invention include, but are not limited to, the following method. A mixture of equal amounts of steamed soybeans and crushed roasted wheat is inoculated with an Aspergillus sojae mutant in which the npII gene on the chromosome has been inactivated as starter koji, and the mixture is aerated at 20°C to 35°C for 30 to 80 hours to obtain soy sauce koji. Next, 100 parts by mass of the soy sauce koji is added to 100 to 200 parts by mass of brine with a salt concentration of 15% (w / v) to 35% (w / v), and the mixture is fermented and aged at 20°C to 35°C for 30 to 200 days with appropriate stirring to obtain soy sauce moromi.

[0068] Next, the obtained soy sauce moromi mash is subjected to a compression treatment using a vertical press type compression device, and then subjected to a filtration treatment using diatomaceous earth to obtain raw soy sauce.

[0069] Next, soy sauce is obtained by subjecting the obtained raw soy sauce to a pasteurization treatment for 10 to 60 minutes at 60 to 90° C. The soy sauce obtained in this manner is clear because the pasteurization residue has been reduced, and has a flavor similar to that of regular soy sauce.

[0070] In the soy sauce production method according to one embodiment of the present invention, various steps or operations can be added before, after, or between the steps described above, as long as the object of the present invention can be achieved.

[0071] The degree of pasteurization dregs in soy sauce obtained by one embodiment of the soy sauce production method of the present invention may be less than the amount of pasteurization dregs in soy sauce (control) obtained by a similar method except for using wild-type microorganisms. For example, when pasteurization is performed for 30 minutes at 80°C, followed by 3 days at 57°C and 8 days at 16°C for dregs removal, the pasteurization dregs volume in the control is preferably 60% or less, preferably 50% or less, more preferably 20% or less, even more preferably 10% or less, and even more preferably 5% or less; and / or the amount of turbidity substances in the control is preferably 50% or less, more preferably 20% or less, even more preferably 10% or less, and even more preferably 5% or less. The degree of pasteurization dregs in soy sauce is evaluated by the method described in the Examples below.

[0072] The soy sauce obtained by the soy sauce production method of one embodiment of the present invention has similar component values ​​to soy sauce obtained using a wild-type strain of an Aspergillus microorganism and has comparable palatability. Therefore, the soy sauce obtained by the soy sauce production method of one embodiment of the present invention can be used to adjust the flavor of food, just like regular soy sauce. The soy sauce obtained by the soy sauce production method of one embodiment of the present invention can be used alone or in combination with other ingredients such as water, vegetable ingredients, yeast extract, meat extract, fruit juice, spices, chemical seasonings, and flavors, as well as dashi, acidulants, amino acids, nucleic acids, organic acids, protein hydrolysates, sugars, sake, mirin, alcohol, thickeners, emulsifiers, and inorganic salts, or in combination with these ingredients, in the cooking and processing of various ingredients. Specifically, the soy sauce obtained by the soy sauce production method according to one embodiment of the present invention can be used in a variety of dishes, including Japanese, Western, and Chinese cuisine. Specifically, the soy sauce can be used in deep-fried foods, grilled meat, udon, soba, ramen, hamburger steaks, meatballs, Chikuzenni, teriyaki, curry, stew, hayashi, and the like, but is not limited to these.

[0073] [Another aspect of the present invention] Aspergillus ( Aspergillus By subjecting raw soy sauce to koji-making treatment using a mutant microorganism of the genus Aspergillus ( ), the amount of pasteurization dregs produced in soy sauce after pasteurization can be reduced. Therefore, another aspect of the present invention is a method for reducing pasteurization dregs in soy sauce production. In one aspect of the method for reducing pasteurization dregs, a soy sauce raw material is treated with a strain of Aspergillus ( ) in which the neutral protease II gene on the chromosome has been inactivated. Aspergillus The present invention also includes a step of subjecting a mutant microorganism of the genus Bacillus subtilis to a koji-making process. Another aspect of the present invention is raw soy sauce and pasteurized soy sauce. The raw soy sauce and pasteurized soy sauce of one aspect of the present invention have a heat-resistant NpII activity of 0 mU / ml to 0.2 mU / ml. The raw soy sauce and pasteurized soy sauce of one aspect of the present invention are preferably produced by the soy sauce production method of one aspect of the present invention.

[0074] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples, and the present invention can take various forms as long as the object of the present invention can be achieved. [Example]

[0075] [Example 1: Creation of the npII gene disruptant As1] Aspergillus oryzae ( Aspergillus rice The nucleotide sequence (SEQ ID NO: 1) of the npII gene (AO090010000493) encoding the thermostable neutral protease on the genome of the RIB40 strain was used to identify the Aspergillus sojae ( Aspergillus soybeans ) The nucleotide sequence of the npII gene on the chromosome of the NBRC4239 strain (SEQ ID NO: 2) was predicted.

[0076] Using the genomic DNA of Aspergillus sojae strain NBRC4239 as a template, the region upstream of the ORF of the npII gene (5' arm sequence for homologous recombination; SEQ ID NO: 3) and the region downstream of the ORF (3' arm sequence for homologous recombination; SEQ ID NO: 4) were amplified by PCR. The amplified DNA fragments were inserted into the pUC19 vector to create a donor DNA plasmid for deleting the npII gene.

[0077] The resulting donor DNA plasmid was used to transform Aspergillus sojae NBRC4241 strain by the protoplast-PEG method. The npII gene region of the resulting transformant was confirmed by colony PCR, and an npII gene disruptant, As1 (ΔnpII strain As1), was isolated, lacking 1 kb upstream of the npII gene and the entire npII ORF.

[0078] [Example 2: Preparation and evaluation of soy sauce koji using the npII gene-disrupted strain As1] A soy sauce starting material, a mixture of equal parts steamed, denatured, defatted soybeans and crushed roasted wheat, was inoculated with ΔnpII strain As1 as a seed koji. The inoculated soy sauce starting material was subjected to a standard koji-making process at 20-35°C for 42 hours to obtain soy sauce koji. As a control, soy sauce koji was obtained in the same manner using a strain of the untransformed Aspergillus sojae NBRC4241 as the seed koji.

[0079] Distilled water was added to the resulting soy sauce koji, and solid-liquid extraction was performed at 20°C to 35°C to extract a crude enzyme solution. NpII activity in the resulting crude enzyme solution was measured using Boc-Arg-Val-Arg-Arg-MCA (Peptide Institute) as a substrate, following Doi et al.'s (Doi Y et al., Biosci Biotechnol Biochem., 2003 Feb; 67(2):264-70). Specifically, 10 μL of the crude enzyme solution, which had been heated at 80°C for 10 minutes, was added to 90 μL of 200 μM Boc-Arg-Val-Arg-Arg-MCA substrate solution. The resulting reaction mixture was incubated at 30°C for 30 minutes for enzymatic reaction. The enzymatic reaction was terminated by adding 200 μL of 0.1 M sodium acetate buffer (pH 4.5). The fluorescence intensity (Ex 360 nm · Em 465 nm) of the resulting solution was measured using a microplate reader to calculate the relative activity of NpII. The results are shown in Table 2.

[0080] [Table 2]

[0081] As shown in Table 2, no NpII activity was detected in the soy sauce koji produced using the ΔnpII strain As1. This confirmed that the ΔnpII strain As1 does not express the NpII enzyme and that the npII gene on its genome was disrupted. Furthermore, no significant differences were observed between the control and the ΔnpII strain As1 in the activity of other proteases, such as alkaline protease (ALP), leucine aminopeptidase I (LapI), and carboxypeptidase (Cpase).

[0082] [Example 3: Production and evaluation of soy sauce using the npII gene disruptant As1] 100 parts by mass of the soy sauce koji prepared in Example 2 was added to 130 parts by mass of brine (salt concentration 26% (w / v)), and the mixture was fermented and aged at 25°C to 30°C for 4 months with appropriate stirring, following standard moromi control. The resulting moromi was pressed and filtered to obtain raw soy sauce.

[0083] The obtained raw soy sauce was pasteurized for 30 minutes at 80° C. 20 mL of the obtained pasteurized soy sauce was placed in a 20 mL measuring cylinder with a stopper and allowed to stand at 57° C. for 3 days and then at 16° C. for 8 days to remove the sediment.

[0084] The volume of the pasteurized dregs that settled after the dregs were removed (dregs volume) was measured using the graduated cylinder's scale. To evaluate the amount of turbidity generated during the pasteurization and dregs removal processes, the turbidity value of the raw soy sauce was subtracted from the turbidity value measured after the graduated cylinder containing the pasteurized soy sauce was thoroughly shaken. The turbidity was measured at a wavelength of 660 nm using a turbidity meter "UT-21" (manufactured by Corona Electric Co., Ltd.). The results (relative values) of the measurement and calculation of the dregs volume and amount of turbidity substances are shown in Table 3.

[0085] [Table 3]

[0086] As shown in Table 3, the soy sauce produced using the ΔnpII strain As1 was clear, with no pasteurization residue after pasteurization and lees removal, and with very little turbidity. There were no significant differences between the control and the ΔnpII strain As1 in the amount of pressed residue and the amount of turbidity in the raw soy sauce.

[0087] Furthermore, when the component values ​​of the control soy sauce and the soy sauce bred by the ΔnpII strain As1 were measured, no significant differences were observed in the total nitrogen content, alcohol content, glutamic acid content, L-lactic acid content, salt content, pH, or amino acid content, with the range of change ranging from 0% to ±5.70%. Similarly, no significant differences were observed in the types and amounts of aroma components or in sensory evaluations such as soy sauce flavor. Therefore, the soy sauce bred by the ΔnpII strain As1 had a flavor comparable to that of conventional pasteurized soy sauce, both in terms of components and sensory evaluation.

[0088] [Example 4: Comparative evaluation of the effect of EDTA in suppressing the generation of slag] Ethylenediaminetetraacetic acid (EDTA) was added to the raw soy sauce prepared in Example 3 to a final concentration of 3 mM. The raw soy sauce after the addition of EDTA was pasteurized and the sediment was removed in the same manner as in Example 3, and the volume of the sediment and the amount of turbidity substances were measured (relative values). The results are shown in Table 4.

[0089] [Table 4]

[0090] As shown in Table 4, in the control soy sauce to which EDTA had been added, the pasteurization dregs no longer settled, but the amount of turbidity substances was only slightly reduced compared to the control without EDTA, resulting in a slightly cloudy soy sauce overall.

[0091] In contrast, soy sauce made with the ΔnpII strain As1 was clear, with no pasteurization dregs and very little turbidity, regardless of whether EDTA was added or not. Therefore, although the addition of EDTA did not produce clear soy sauce with low turbidity, it was found that the use of the ΔnpII strain As1 produced clear soy sauce with low turbidity. Consequently, it was found that the use of starter koji in which NpII activity had been lost resulted in the production of clear, high-quality soy sauce with no pasteurization dregs and low turbidity.

[0092] [Example 5: Construction of npII gene-disrupted strains As2, Ao1, and Ao2 using various Aspergillus oryzae strains as hosts] To confirm that Aspergillus sojae strains and Aspergillus oryzae strains other than the Aspergillus sojae strain NBRC4241 do not produce fire dregs, the npII gene-disrupted ΔnpII strains As2, Ao1, and Ao2 were constructed using the Aspergillus sojae strain NBRC4239, the Aspergillus oryzae strain RIB40, and the Aspergillus oryzae strain RIB326 as hosts, respectively, according to the method described in Example 1.

[0093] The donor DNA plasmid for disrupting the npII gene of Aspergillus sojae NBRC4239 was prepared by PCR amplification of the upstream region of the ORF (5'-arm sequence for homologous recombination; SEQ ID NO: 8), the downstream region of the ORF (3'-arm sequence for homologous recombination; SEQ ID NO: 9), and the pyrG gene (orotidine-5'-phosphate decarboxylase gene) region (SEQ ID NO: 10) of the npII gene nucleotide sequence (SEQ ID NO: 2) using the genomic DNA of Aspergillus sojae NBRC4239 as a template. The amplified DNA fragments were then ligated in the order of the 5'-arm sequence, pyrG gene, and 3'-arm sequence using the In-Fusion HD Cloning Kit (Clontech) and inserted into the pUC19 vector.

[0094] The donor DNA plasmids for the npII gene disruption in Aspergillus oryzae RIB40 and RIB326 strains were prepared by PCR amplification of the upstream region of the npII ORF (5'-arm sequence for homologous recombination; SEQ ID NO: 11), the downstream region of the ORF (3'-arm sequence for homologous recombination; SEQ ID NO: 12), and the pyrG gene (orotidine-5'-phosphate decarboxylase gene) region (SEQ ID NO: 10) of the npII gene using the genomic DNA of Aspergillus oryzae RIB40 as a template. The amplified DNA fragments were then ligated in the order of the 5'-arm sequence, pyrG gene, and 3'-arm sequence using the In-Fusion HD Cloning Kit (Clontech) and inserted into the pUC19 vector.

[0095] Using the resulting donor DNA plasmid for npII gene disruption, the ku70 / pyrG double-disrupted strains of Aspergillus sojae NBRC4239, Aspergillus oryzae RIB40, and Aspergillus oryzae RIB326 were transformed by the protoplast-PEG method. The npII gene was confirmed to be deleted in the resulting transformants by colony PCR, and the ΔnpII strains As2, Ao1, and Ao2, which were npII gene-disrupted strains of Aspergillus sojae NBRC4239, Aspergillus oryzae RIB40, and Aspergillus oryzae RIB326, were isolated.

[0096] [Example 6: Preparation and evaluation of soy sauce koji and soy sauce using npII gene-disrupted strains As2, Ao1, and Ao2] Soy sauce koji was obtained using the ΔnpII strains As2, Ao1, and Ao2 in the same manner as in Example 2. Strains in which pyrG was complemented with the ΔnpII strains As2, Ao1, and Ao2 were used as controls. The koji production process was carried out twice independently for each strain. NpII activity was measured in the same manner as in Example 2. The relative activity values ​​of NpII were calculated and are shown in Table 5.

[0097] [Table 5]

[0098] As shown in Table 5, NpII activity was not detected in soy sauce koji produced using the ΔnpII strains As2, Ao1, and Ao2. This confirmed that the ΔnpII strains As2, Ao1, and Ao2 do not express the NpII enzyme and that the npII gene on their genomes was disrupted. Furthermore, no significant differences were observed between the control strain and the ΔnpII strains As2, Ao1, and Ao2 in the activity of other proteases, such as alkaline protease (ALP), leucine aminopeptidase I (LapI), and carboxypeptidase (Cpase).

[0099] As in Example 3, raw soy sauce was produced using soy sauce koji obtained from the ΔnpII strains As2, Ao1, and Ao2, and then the obtained raw soy sauce was subjected to pasteurization and lees removal, and the lees volume (relative value) was measured. The measurement results are shown in Table 6.

[0100] [Table 6]

[0101] As shown in Table 6, the soy sauces produced using the ΔnpII strains As2, Ao1 and Ao2 did not produce pasteurized dregs after pasteurization and dregs removal.

[0102] [Example 7: Construction of mutant npII gene expression strain] To confirm that a strain expressing the mutant npII gene also produces no dregs, a mutant npII gene expression strain was constructed as follows, which expresses a nonsense mutant npII expression gene in which the 36th glutamine in SEQ ID NO: 6 was changed to a stop codon by substituting the 106th base in SEQ ID NO: 2 from C to T.

[0103] The nonsense mutant npII expression cassette was designed to be inserted in such a way that it disrupted the nucleotide sequence of an ORF that is thought to encode a glucose dehydrogenase (GDH)-like protein (a homologue of AO090103000214).

[0104] Specifically, using the genomic DNA of Aspergillus sojae strain NBRC4239 as a template, the upstream region of the GDH-like protein gene ORF (5' arm sequence for homologous recombination; SEQ ID NO: 13), the downstream region of the ORF (3' arm sequence for homologous recombination; SEQ ID NO: 14), and the npII gene expression cassette (nucleotide sequence consisting of 1127 bp upstream of the npII gene ORF, the npII gene ORF, and 1000 bp downstream of the npII gene ORF; SEQ ID NO: 15) were amplified by PCR. Additionally, using the pPTRII plasmid (Takara Bio Inc.) as a template, the pyrithiamine resistance gene (ptrA; SEQ ID NO: 16), a selectable marker, was amplified by PCR. The amplified DNA fragments were ligated in the order of 5' arm sequence, ptrA gene, npII gene expression cassette, and 3' arm sequence using the In-Fusion HD Cloning Kit (Clontech), and inserted into the pUC19 vector to create a donor DNA plasmid for wild-type npII expression strains. Inverse PCR was performed using the donor DNA plasmid for the wild-type npII expression strain as a template to generate a donor DNA plasmid for the mutant npII expression strain, in which a nonsense mutation had been introduced into the nucleotide sequence of the npII gene.

[0105] The resulting donor DNA plasmid for the mutant npII-expressing strain was used to transform the ΔnpII strain As2 derived from Aspergillus sojae NBRC4239 by the protoplast-PEG method as in Example 5. Colony PCR confirmed that the resulting transformants contained either the mutant or wild-type npII gene expression cassette in the GDH-like gene region, and strains expressing the nonsense mutant npII and wild-type npII were obtained.

[0106] [Example 8: Production and evaluation of soy sauce using mutant npII gene expression strain] Using the obtained nonsense mutant npII-expressing strain and wild-type npII-expressing strain, soy sauce koji was obtained in the same manner as in Example 2, and then NpII activity was measured. The relative activity values ​​of NpII were calculated and the results are shown in Table 7.

[0107] [Table 7]

[0108] As shown in Table 7, no NpII activity was detected in the soy sauce koji produced using the nonsense mutant npII-expressing strain. This confirmed that the nonsense mutant npII-expressing strain does not express the NpII enzyme. Furthermore, no significant differences were observed in the activities of other proteases, such as alkaline protease (ALP), leucine aminopeptidase I (LapI), and carboxypeptidase (Cpase), between the wild-type npII-expressing strain and the nonsense mutant npII-expressing strain.

[0109] Using the soy sauce koji thus prepared, a nonsense mutant npII-expressing strain and a wild-type npII-expressing strain were used to prepare raw soy sauce in the same manner as in Example 3. The resulting raw soy sauce was then pasteurized and the scum was removed, and the scum volume (relative value) was measured. As a result, the scum volume of the pasteurized soy sauce in which the seed koji was a nonsense mutant npII-expressing strain was 0 relative to the scum volume of the pasteurized soy sauce in which the seed koji was a wild-type npII-expressing strain.

[0110] [Example 9: Evaluation of slag formation by adding NpII enzyme solution] The p19-RT-GDH-pyrG3 plasmid, described in International Publication WO 2025 / 089419, contains an expression cassette for the pyrG3 gene (SEQ ID NO: 17) with a shortened promoter length of 56 bp and the GDH gene as a reporter gene. Retrotransposon-like sequences (eight sequences present on the chromosome of Aspergillus sojae NBRC4239) are ligated upstream of Ptef and downstream of the pyrG3 gene. Based on this p19-RT-GDH-pyrG3, we constructed the plasmid p19-RT-npII-pyrG3 (RTup (SEQ ID NO: 18)-Ptef-npII-Talp-pyrG3-RTdown (SEQ ID NO: 19)), in which the GDH gene as a reporter gene was replaced with the npII gene (SEQ ID NO: 2) from Aspergillus sojae NBRC4239.

[0111] A pyrG-deficient strain of Aspergillus sojae NBRC4239 (deleting 48 bp upstream, 896 bp of the coding region, and 240 bp downstream of the pyrG gene) was transformed with p19-RT-npII-pyrG3 by protoplast PEG method to obtain the As-RT-npII-pyrG3 strain, which contained multiple copies of the npII gene expression cassette. The As-RT-npII-pyrG3 strain was inoculated into Czapeck-dox liquid medium (1.5% Czapeck-dox Broth (BD Difco)) containing 0.1% trace element and cultured at 30°C for 7 days with shaking. The culture supernatant was then used as the NpII enzyme solution. SDS-PAGE and CBB staining of the enzyme solution revealed no distinct bands other than those for the NpII enzyme.

[0112] Ten μL of the enzyme solution, heated at 80°C for 10 minutes, was added to 90 μL of 200 μM Boc-Arg-Val-Arg-Arg-MCA substrate solution (50 mM Tris-HCl, pH 7.0). The resulting reaction solution was incubated at 50°C for 2.5, 5, 7.5, and 10 minutes for the enzymatic reaction. The enzymatic reaction was stopped by adding 200 μL of 0.1 M sodium acetate buffer (pH 4.5). The fluorescence intensity (Ex 375 nm · Em 450 nm) of the resulting treated solution and 7-Amino-4-methylcoumarin (AMC, Tokyo Chemical Industry Co., Ltd.) solutions at 0 pmol / well, 50 pmol / well, 100 pmol / well, 150 pmol / well, and 400 pmol / well was measured using a microplate reader (TECAN). A calibration curve was created from the fluorescence intensity of the AMC solution, and 1 unit (U) was defined as the amount of enzyme that produces 1 μmol of AMC per minute at 50° C., and the amount of enzyme was measured from the fluorescence intensity.

[0113] To reduce the amount of NpII enzyme solution added to soy sauce, the NpII enzyme solution was concentrated 10-fold using an AmiconUltra Centrifugal filter (10 kDa cutoff). The enzyme solution was added to soy sauce prepared using the ΔnpII strain As1 prepared in Example 1 to concentrations of 5 mU / mL, 0.5 mU / mL, 0.2 mU / mL, and 0.1 mU / mL. The soy sauce was then pasteurized and the scum was removed as in Example 3, and the scum height was measured. The results are shown in Table 8.

[0114] [Table 8]

[0115] As shown in Table 8, the amount of pasteurized scum was reduced when NpII enzyme solution was added at a concentration of 0.2 mU / mL or less, and no pasteurized scum was formed when NpII enzyme solution was added at a concentration of 0.1 mU / mL or less. This indicates that the formation of scum during pasteurization can be suppressed by creating Aspergillus mutants with reduced NpII activity so that the NpII activity in raw soy sauce during pasteurization is 0.2 mU / mL or less. Furthermore, NpII activity was 0.2 mU / mL or more in raw soy sauce produced with the wild-type Aspergillus sojae NBRC4241 strain, Aspergillus sojae NBRC4239 strain, Aspergillus oryzae RIB40 strain, and Aspergillus oryzae RIB326 strain, as well as in commercially available raw soy sauce. When measuring NpII activity in soy sauce, it is advisable to remove contaminants using an Amicon Ultra Centrifugal filter (10 kDa cutoff) or similar, as the fluorescence intensity due to contaminants will be strong and the blank value will be high. Furthermore, the amount of enzyme in koji (mU / g-koji) can be calculated using the following formula when Y mL of raw soy sauce is obtained from X g of koji. Therefore, the amount of NpII enzyme in soy sauce koji to reduce pasteurized lees is 0.4 mU / g-koji or less, and the amount of NpII enzyme in soy sauce koji to prevent pasteurized lees from being produced is 0.2 mU / g-koji or less. (Amount of enzyme in koji (mU / g-koji)) = (Amount of enzyme in raw soy sauce (mU / mL)) × Y / X When 5 mU / mL of NpII enzyme was added to pasteurized soy sauce produced using the ΔnpII strain As1 produced in Example 1 and pasteurized again, pasteurized scum was generated, similar to the case when NpII enzyme was added to raw soy sauce and pasteurized (Table 8). In other words, pasteurized soy sauce produced using the npII gene-inactivated strain can be distinguished by the fact that the NpII activity is 0.2 mU / mL or less and pasteurized scum is generated when NpII enzyme is added and pasteurized again.

[0116] [Sequence table] The sequences listed in the sequence listing are as shown in Tables 9A to 9C below. The amino acid sequence of the NpII protein encoded by the nucleotide sequences of SEQ ID NOs: 1 and 20 (SEQ ID NO: 1 contains an intron, and SEQ ID NO: 20 does not contain an intron) is shown in SEQ ID NO: 5, and the amino acid sequence of the NpII protein encoded by the nucleotide sequences of SEQ ID NOs: 2 and 21 (SEQ ID NO: 2 contains an intron, and SEQ ID NO: 21 does not contain an intron) is shown in SEQ ID NO: 6. Aspergillus childish The amino acid sequence of the NpII protein of ) is shown in SEQ ID NO:7.

[0117] [Table 9A]

[0118] [Table 9B]

[0119] [Table 9C] [Industrial Applicability]

[0120] By utilizing the present invention, the amount of pasteurization residue generated in soy sauce after pasteurization of raw soy sauce can be reduced. As a result, by utilizing the present invention, soy sauce can be produced on an industrial scale with a shorter process time, a reduced workload, and / or economically advantageous. Cross-reference to related applications

[0121] This application claims priority from Japanese Patent Application No. 2024-102842, filed June 26, 2024, the entire disclosure of which is incorporated herein by reference. Furthermore, the entire disclosures of all documents referenced in the detailed description of the invention of this application, including Patent Documents 1 to 4 and Non-Patent Documents 1 to 5, are incorporated herein by reference.

Claims

1. A method for manufacturing soy sauce, The method comprising the step of subjecting soy sauce raw materials to a koji-making process using a microbial mutant of the genus Aspergillus in which the neutral protease II gene located on the chromosome has been inactivated, thereby obtaining soy sauce koji.

2. The method according to claim 1, wherein the Aspergillus microbial mutant is a mutant of at least one Aspergillus microorganism selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, and Aspergillus tamarii.

3. The method according to claim 1, wherein the soy sauce is pasteurized soy sauce.