METHOD FOR PRODUCING SOY SAUCE USING npII-GENE-INACTIVATING STRAIN

Abstract

The purpose of the present invention is to provide a method for producing soy sauce in which the generation of heating residue is reduced. The aforementioned purpose is achieved by: a method for producing soy sauce, the method comprising a step for obtaining soy sauce koji by subjecting a soy sauce raw material to a koji-making treatment using a variant of a microorganism of the genus Aspergillus (Aspergillus) in which a neutral protease II gene on a chromosome is inactivated; and a variant of a microorganism belonging to the genus Aspergillus in which a neutral protease II gene on a chromosome is inactivated.

Description

Soy sauce production method using npII gene inactivated strain

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

[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. Therefore, 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.

[0007] JP-A-58-155057, JP-A-60-023822, JP-A-60-023823, JP-A-05-168479

[0008] H. Hashimoto et al., J. Ferment. Technol., Vol. 52, No. 10, p. 747-758, 1974 H. Hashimoto et al., J. Ferment. Technol., Vol. 51, No. 9, p. 661-669,1973H. Hashimoto et al., J. Ferment. Technol., Vol. 52, No. 5, p. 328-334,1974S. Kimura et al., Biosci Biotechnol Biochem, Vol. 72, No.2, p. 499-505, 2008

[0009] Even when the matters described 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 mold 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.

[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 its 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 Patent Documents 1 to 3 have shown 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, comprising a step of subjecting a soy sauce raw material to a koji-making treatment using a mutant Aspergillus microorganism in which the neutral protease II gene on the chromosome has been inactivated to obtain soy sauce koji. [I-2] The method according to item [I-1], wherein the mutant Aspergillus microorganism is a mutant of at least one Aspergillus microorganism selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, and Aspergillus tamarii. [I-3] The method according to 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 said 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 microorganism in which the neutral protease II gene present on the chromosome has been inactivated. [I-6] The mutant Aspergillus microorganism according to Item [I-5], wherein the mutant Aspergillus microorganism is a mutant of at least one Aspergillus microorganism selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, and Aspergillus tamarii. [I-7] The Aspergillus genus 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 said amino acid sequence. [II-1] A method for producing soy sauce, comprising the step of subjecting a soy sauce raw material to a koji-making treatment using an Aspergillus genus microorganism mutant in which the neutral protease II gene on the chromosome has been inactivated, to obtain soy sauce koji.[II-2] The method according to Item [II-1], wherein the Aspergillus genus mutant is a mutant of at least one Aspergillus microorganism selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, and Aspergillus tamarii. [II-3] The method according to Item [II-1], wherein the soy sauce is pasteurized soy sauce. [II-4] The Aspergillus genus mutant, wherein the neutral protease II gene present on the chromosome has been inactivated, and wherein the Aspergillus genus mutant does not express human lysozyme. [II-5] A mutant of a microorganism of the genus Aspergillus, in which 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. [II-6] The mutant of a microorganism of the genus Aspergillus according to item [II-4] or [II-5], which is a mutant of at least one Aspergillus microorganism selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, and Aspergillus tamarii. [II-7] A 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 the raw soy sauce produced by the method described in item [II-1] or [II-2].

[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.

[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] The term "comprising" means that elements other than those explicitly stated as included may be added (same meaning as "comprising at least"), but also encompasses "consisting of" and "essentially consisting 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 components, steps, conditions, and parameters. The term "and / or" means any one of the associated listed items, or any or all combinations of two or more. The term "to" in a numerical range includes both the preceding and following numerical values, and also includes ranges excluding either of the included limits. For example, "0% to 100%" means 0% or more, 100% or less, or 0% or more and 100% or less. "Greater than" and "less than" refer to lower and upper limits, respectively, exclusive of the preceding numerical value. For example, "greater than 1" means a number greater than 1, and "less than 100" means a number less than 100. "About" means 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, for decimal values, the number of digits after the decimal point 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 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 splicing abnormalities, 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 gene expression" refers to the production of an enzyme having an amino acid sequence encoded by the nucleotide sequence of the gene (an enzyme encoded by the gene) with its original structure and activity through transcription, translation, etc. "Gene expression product" refers to an enzyme produced corresponding to a gene. "Wild-type strain" refers to a naturally occurring organism to which no mutations have been artificially introduced. "Mutant" refers to an organism obtained by artificially introducing a mutation. Mutants include microorganisms obtained by artificially inducing mutations in wild-type strains or mutants, and microorganisms into which mutations have been introduced using transformation procedures involving the introduction of foreign nucleic acid fragments into cells or gene editing techniques such as CRISPR / Cas (Cas9, Cas12, etc.), zinc finger nucleases (ZFNs), and TALENs. "Wild-type gene" refers to a gene that is naturally present on the chromosome of a wild-type strain. "Wild-type enzyme" refers to an enzyme encoded by a wild-type gene. Note that, in this specification, chromosome and genome are synonymous terms. "Foreign nucleic acid fragment" refers to a nucleic acid fragment consisting of a nucleotide sequence that is not naturally present in the genomic DNA of the microorganism to be introduced.The "foreign nucleic acid fragment" may be composed of a nucleic acid fragment consisting of a nucleotide sequence that is originally contained 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. The soy sauce production method of one aspect of the present invention is characterized by using a mutant microorganism belonging to the genus Aspergillus as starter koji. By using a mutant microorganism belonging to the genus Aspergillus, the soy sauce production method of one aspect of the present invention can simplify or omit the removal of pasteurization dregs, which is usually performed in soy sauce production methods.

[0021] [Summary of Aspergillus microorganism mutant] In one embodiment of the soy sauce production method of the present invention, the Aspergillus microorganism mutant used as starter koji is a microorganism in which a specific gene on the chromosome of a wild-type strain of Aspergillus microorganism has been inactivated. The gene to be inactivated 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 Aspergillus microorganisms, such as seed koji (koji mold), and has the activity of degrading proteins (NpII activity) in an environment with a near-neutral pH. 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 through hydrophobic bonds, resulting in the generation of pasteurization dregs.

[0024] Therefore, we prepared an Aspergillus microorganism 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 microorganism 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 NCBI GenBank ([URL]https: / / www.ncbi.nlm.nih.gov / genbank / ) for each Aspergillus microorganism from which it is derived. For example, the npII gene (SEQ ID NO: 1) of Aspergillus oryzae RIB40 strain is registered as Gene ID AO090010000493. The npII gene is also called the deuterolysin A (deuA) gene, npIIa gene, nptB gene, etc. (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] By using information on the npII gene of Aspergillus oryzae strain RIB40, it is possible to identify npII genes of other microorganisms belonging to the genus Aspergillus. One example of an npII gene identified in this manner is the npII gene of Aspergillus sojae strain NBRC4239 (SEQ ID NO: 2). The intron-free npII gene of 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 Aspergillus oryzae RIB40 strain is registered in GenBank under the accession number (version) XP_001827477.1.

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

[0029] Similarly, the NpII enzyme of Aspergillus tamarii has GenBank accession number (version) KAE8168250.1, the NpII enzyme of Aspergillus minisclerotigenes has KAB8274519.1, and the NpII enzyme of Aspergillus steynii IBT23096 strain has XP_024709461.1. The amino acid sequence identity between the NpII enzyme of Aspergillus steynii IBT23096 strain and the NpII enzyme of Aspergillus oryzae RIB40 strain is 69.49%.

[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]

[0032] Among proteins expressed by microorganisms of the genus Aspergillus, proteins that have 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 sequence identity with the amino acid sequence of the NpII enzyme of Aspergillus oryzae RIB40 strain may be considered to be NpII enzymes. NpII activity can be measured using the heat-treated enzyme solution and Boc-Arg-Val-Arg-Arg-MCA as a substrate by the method described in the Examples below.

[0033] Even among Aspergillus microorganisms of the same species, different strains 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. 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 microorganisms of the genus Aspergillus, those having an amino acid sequence that has 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 identity with the amino acid sequence of the NpII enzyme of the same species of microorganism of the genus Aspergillus can be considered to be NpII enzymes. For example, an enzyme from Aspergillus sojae having an amino acid sequence that has 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 6 can be said to be NpII enzymes.

[0035] Similarly, among genes possessed by microorganisms of the genus Aspergillus, genes having a nucleotide sequence that has 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 sequence identity with the nucleotide sequence of the npII gene of the same species of microorganism of the genus Aspergillus can be considered to be npII genes. For example, a gene of Aspergillus sojae having a nucleotide sequence that has 80% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 21 can be said 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, using genetic recombination techniques, that the expressed protein has 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, the sequence identity 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] As a program for calculating the identity between two nucleotide sequences and amino acid sequences, 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) is known, and a BLAST program using this algorithm has been developed by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Furthermore, Gapped BLAST, a program that determines sequence identity more sensitively than BLAST, is also known (Nucleic Acids Res. 25: 3389-3402, 1997). Those skilled in the art can use the above programs to search databases for sequences that show high sequence identity to a given sequence, such as those available on the internet website of the U.S. National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0039] [Method for Producing an Aspergillus Microorganism Mutant] An Aspergillus microorganism mutant in which the npII gene present on the chromosome has been inactivated can be produced, for example, 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, thereby inactivating the npII gene. Alternatively, the npII gene may be inactivated by modifying the control region (e.g., promoter sequence) of the npII gene instead of the npII gene itself. For example, methods for modifying the control region of the npII gene to inactivate 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 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 has been 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 Document 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 has the npII gene on its chromosome and can be used as koji starter 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 and excellent aroma of the final product, (3) excellent conidial spore adhesion and easy preparation of starter koji, (4) vigorous growth and a large amount of fungal body in the koji, (5) weak tightness during koji production and little heat generation and loss of raw materials, (6) low moromi viscosity and easy pressing, (7) little production of pasteurized lees, (8) genetic stability, and (9) no ability to produce mycotoxins such as aflatoxin (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 possessing any one of these properties or a combination of any two to nine of these properties. Examples include the Aspergillus oryzae RIB326 strain isolated from soy sauce koji (Umemura M et al. DNA Res., 19(5), 375-382, 2012) and the Aspergillus sojae NBRC4239 strain isolated from a soy sauce brewery (Sato A., DNA Res., 18(3), 165-176, 2011). The genomes of these koji molds have been sequenced, but strains selected for sake and soy sauce production are specialized in enzyme productivity and are thought to have been domesticated away from the original koji mold. On the other hand, the Aspergillus oryzae RIB40 strain, 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 use in sake or soy sauce production, and is therefore undesirable (Iwashita Kazuhiro, Chemistry and Biology 47(5), 329-338, 2009).

[0041] Inactivation of the npII gene may be carried out 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 using the CRISPR / Cas9 system in an Aspergillus microorganism, methods using an autonomously replicating vector that expresses Cas9 and gRNA, and methods of directly introducing a complex of Cas9 protein and gRNA are known (J Fungi, 2022, 8(5), 467).

[0042] The target sequence of gRNA in the CRISPR / Cas9 system can be designed based on the sequence about 20 bp upstream of the PAM sequence (NGG) in the target gene sequence. For example, by designing the target sequence of gRNA to the CDS or ORF of the npII gene, or the sequence of the gene control region, base substitution, deletion, or insertion in the nucleotide sequence causes a frameshift mutation, a nonsense mutation, or a mutation that causes splicing abnormalities, or a mutation in the active center, or a mutation accompanied by an amino acid substitution that reduces thermal stability, etc., 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 sequences of the upstream and downstream regions of the npII gene on the chromosome, the nucleotide sequence containing the npII gene can be deleted, thereby inactivating the npII gene.

[0044] A nucleic acid fragment for inducing homologous recombination at a desired chromosomal location may be introduced to inactivate the npII gene via homologous recombination. For example, a nucleic acid fragment constructed to link 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 a mutation (deletion, substitution, insertion) introduced 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 via 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 gene (argininosuccinate synthase gene) and 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 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 an In-Fusion Cloning Site in the multi-cloning site of the plasmid pUC19 to prepare a construct plasmid (DNA); and then, using the obtained construct plasmid as template DNA, amplifying it by PCR.

[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 fungal cells, and the cells are physically ground in a mortar or the like while cooled in liquid nitrogen to produce fine powder-like fungal fragments, from which a chromosomal DNA fraction is extracted by a conventional method. A commercially available chromosomal DNA extraction kit such as 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, in which protoplasts of a host organism are prepared and then polyethylene glycol and calcium chloride are used (see, for example, Mol. Gen. Genet. 218, 99-104, 1989; JP 2007-222055 A), can be used. A medium for regenerating Aspergillus microorganisms is selected based 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 the 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 microorganism mutant in which the npII gene has been inactivated 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 has been 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 thereof, and a reverse primer complementary to the downstream region of homologous recombination incorporated into the transformation cassette or a region located further downstream thereof, 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 microbial mutants in which the npII gene on the chromosome has been inactivated can be prepared by methods other than those using genome editing to introduce mutations or homologous recombination, such as suppressing the expression of the npII gene by 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), UV irradiation, plasma discharge (ARTP: Atmospheric Room Temperature Plasma), or radiation (X-rays, gamma rays, heavy particle beams (ion beams), neutron beams).

[0052] One 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 genus 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, a mutant Aspergillus microorganism 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 genus mutant microorganism has the neutral protease II gene on its chromosome inactivated, does not express human lysozyme, and uses an Aspergillus genus microorganism other than the Aspergillus oryzae RIB40 strain as a host organism.

[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 that obtained 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 that obtained 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, even 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, even more preferably 0.05 mU / g-koji or less, or 0 mU / g-koji to 0.05 mU / g-koji.

[0058] [Method for Producing Soy Sauce] 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 microbial 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 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 from the soy sauce 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 (e.g., barley, naked barley, and job's oats), 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. The soy sauce ingredients may be one of these ingredients alone or in combination of two or more. Soybeans are primarily used as protein ingredients after being heat-denatured, while wheat is primarily used as a sugar ingredient 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 present 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 present 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 npII gene present on the chromosome has been inactivated as a seed koji, the heat-stable NpII activity in soy sauce koji after treatment at 80°C for 10 minutes is reduced. The heat-stable 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, compared to when a wild-type strain of Aspergillus microorganism is used as a seed koji, the heat-stable NpII activity is preferably 50% or less, more preferably 20% or less, even more preferably 10% or less, and even more preferably 5% or less. 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-stable 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 for 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 a salt solution 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 used in ordinary soy sauce production methods. For example, examples of soy sauce lactic acid bacteria include salt-tolerant lactic acid bacteria such as Tetragenococcus halophilus, and examples of soy sauce yeast include salt-tolerant yeasts such as Zygosaccharomyces rouxii, Candida etchellsii, and Candida versatilis.

[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 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 of these alone or a combination of two or more, 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 raw soy sauce. For example, raw soy sauce can be obtained by subjecting the raw soy sauce to a heat treatment at 50°C to 100°C, preferably 60°C to 80°C, for 10 to 100 minutes, preferably 10 to 40 minutes.

[0066] In conventional soy sauce manufacturing methods, pasteurization of raw soy sauce generates pasteurization dregs. Therefore, the soy sauce and pasteurization dregs are separated by carrying out procedures such as leaving the pasteurized soy sauce to settle (sease), or removing the clear soy sauce portion that does not contain the pasteurization dregs (seiri). However, in one embodiment of the soy sauce manufacturing method 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 conventional soy sauce manufacturing methods, can be omitted.

[0067] Specific embodiments of the soy sauce production method of one aspect 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 and koji-making is carried out 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 having a salt concentration of 15% (w / v) to 35% (w / v), and the mixture is subjected to a fermentation and aging treatment at 20°C to 35°C with appropriate stirring for 30 to 200 days to obtain soy sauce moromi mash.

[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] The resulting raw soy sauce is then pasteurized at 60° C. to 90° C. for 10 to 60 minutes to obtain soy sauce. 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 then 8 days at 16°C to remove the dregs, the amount of pasteurization dregs 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] By performing a koji-making process using a mutant Aspergillus microorganism in which the neutral protease II gene on the chromosome has been inactivated, the amount of pasteurization dregs generated in soy sauce after pasteurization of raw soy sauce can be reduced. Therefore, another aspect of the present invention is a method for reducing pasteurization dregs in soy sauce production. The method for reducing pasteurization dregs of one aspect of the present invention includes a step of subjecting a soy sauce raw material to a koji-making process using a mutant Aspergillus microorganism in which the neutral protease II gene on the chromosome has been inactivated. 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.

[0075] [Example 1: Construction of npII gene-disrupted strain As1] The nucleotide sequence (SEQ ID NO: 2) of the npII gene on the chromosome of Aspergillus sojae strain NBRC4239 was predicted from the nucleotide sequence (SEQ ID NO: 1) of the npII gene (AO090010000493) encoding a thermostable neutral protease on the genome of the koji mold Aspergillus oryzae RIB40 strain.

[0076] Using the genomic DNA of Aspergillus sojae strain NBRC4239 as a template, a region upstream of the ORF of the npII gene (5'-arm sequence for homologous recombination; SEQ ID NO: 3) and a 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 a pUC19 vector to prepare a donor DNA plasmid for deleting the npII gene.

[0077] The resulting donor DNA plasmid was used to transform a strain of Aspergillus sojae NBRC4241 by the protoplast-PEG method. The npII gene region of the resulting transformant was confirmed by colony PCR to obtain an npII gene-disrupted strain As1 (ΔnpII strain As1) in which 1 kb upstream of the npII gene and the entire ORF of the npII gene were deleted.

[0078] [Example 2: Preparation and evaluation of soy sauce koji using npII gene-disrupted strain As1] A soy sauce raw material, which was a mixture of equal amounts of steamed, denatured, defatted soybeans and crushed roasted wheat, was inoculated with ΔnpII strain As1 as seed koji. The soy sauce raw material inoculated with ΔnpII strain As1 was subjected to a koji-making process at 20°C to 35°C for 42 hours by a conventional method to obtain soy sauce koji. As a control, soy sauce koji was obtained in the same manner using a strain of Aspergillus sojae NBRC4241 before transformation 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 the procedure described in Doi et al. (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 solution was subjected to an enzymatic reaction by incubation at 30°C for 30 minutes. The enzymatic reaction was terminated by adding 200 μL of 0.1 M sodium acetate buffer (pH 4.5) to the resulting reaction solution. The fluorescence intensity (Ex 360 nm, Em 465 nm) of the resulting treated solution was measured using a microplate reader to calculate the relative activity of NpII. The results are shown in Table 2.

[0080]

[0081] As shown in Table 2, NpII activity was not detected in soy sauce koji prepared using the ΔnpII strain As1. This confirmed that the ΔnpII strain As1 does not express the NpII enzyme and is a strain in which the npII gene on its genome has been 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 npII gene-disrupted strain As1 One hundred parts by mass of the soy sauce koji produced in Example 2 was added to 130 parts by mass of brine (salt concentration 26% (w / v)), and 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 the sediment was removed at 57° C. for 3 days and then at 16° C. for 8 days.

[0084] The volume of the pasteurized dregs that settled after the dregs were removed (dregs volume) was measured using the scale on the measuring cylinder. To evaluate the amount of turbidity generated in the pasteurization and dregs removal processes, the turbidity value of the raw soy sauce was subtracted from the turbidity value after the measuring 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 of measuring and calculating the dregs height and the amount of turbidity substances (relative values) are shown in Table 3.

[0085]

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

[0087] Furthermore, when the component values ​​of the control soy sauce and the soy sauce produced 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, and 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 and in sensory evaluations such as soy sauce flavor. Therefore, the soy sauce produced by the ΔnpII strain As1 had a flavor comparable to that of conventional pasteurized soy sauce, both in terms of components and sensory aspects.

[0088] [Example 4: Comparative evaluation of the inhibitory effect of EDTA on the generation of pasteurized scum] 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 scum was removed in the same manner as in Example 3, and the volume of the scum and the amount of turbidity substances were measured (relative values). The results are shown in Table 4.

[0089]

[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 to which EDTA had not been added, and the soy sauce as a whole became slightly cloudy.

[0091] In contrast, the soy sauce produced by the ΔnpII strain As1 was clear, with no generation of pasteurization dregs and very little turbidity, regardless of whether EDTA was added or not. Therefore, although the addition of EDTA does not produce clear soy sauce with little turbidity, it was found that the use of the ΔnpII strain As1 produced clear soy sauce with little turbidity. As a result, it was found that the use of starter koji in which NpII activity had disappeared produced clear, excellent-quality soy sauce with no generation of pasteurization dregs and little turbidity.

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

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

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

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

[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. As control strains, strains in which pyrG was complemented with the ΔnpII strains As2, Ao1, and Ao2 were used. Note that the koji production process was carried out independently twice for each strain. NpII activity was measured in the same manner as in Example 2. The results of calculating the relative activity values ​​of NpII are shown in Table 5.

[0097]

[0098] As shown in Table 5, NpII activity was not detected in soy sauce koji prepared 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 are strains in which the npII gene on their genomes has been 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]

[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: Preparation of mutant npII gene expressing strain] In order to confirm that a strain expressing a mutant npII gene does not produce dregs, a mutant npII gene expressing strain expressing a nonsense mutant npII expression gene in which the glutamine at position 36 of SEQ ID NO: 6 was changed to a stop codon by substituting C at position 106 of SEQ ID NO: 2 with T was prepared as follows.

[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 NBRC4239 strain as a template, the upstream region of the ORF of the GDH-like protein gene (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. Furthermore, using the pPTRII plasmid (Takara Bio Inc.) as a template, the pyrithiamine resistance gene (ptrA; SEQ ID NO: 16), a selection marker, was amplified by PCR. The amplified DNA fragment was ligated using an In-Fusion HD Cloning Kit (Clontech) in the order of the 5'-arm sequence, ptrA gene, npII gene expression cassette, and 3'-arm sequence, and inserted into a pUC19 vector to prepare a donor DNA plasmid for a wild-type npII expression strain. Inverse PCR was performed using the donor DNA plasmid for the wild-type npII expression strain as a template to prepare a donor DNA plasmid for a mutant npII expression strain in which a nonsense mutation was introduced into the nucleotide sequence of the npII gene.

[0105] Using the obtained donor DNA plasmid for the mutant npII-expressing strain, the ΔnpII strain As2 derived from Aspergillus sojae strain NBRC4239 was transformed by the protoplast-PEG method in the same manner as in Example 5. Colony PCR was used to confirm that the obtained transformants had the mutant or wild-type npII gene expression cassette in the GDH-like gene region, and a nonsense mutant npII-expressing strain and a wild-type npII-expressing strain were obtained.

[0106] Example 8: Preparation and evaluation of soy sauce using mutant npII gene-expressing strains 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]

[0108] As shown in Table 7, no NpII activity was detected in the soy sauce koji prepared 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 decanted, 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 addition of NpII enzyme solution] p19-RT-GDH-pyrG3, described in International Publication WO2025 / 089419, has an expression cassette for the pyrG3 gene (SEQ ID NO: 17) whose promoter length has been shortened to 56 bp and the GDH gene as a reporter gene, and is composed of retrotransposon-like sequences derived from the Aspergillus sojae strain NBRC4239 (present at eight locations on the chromosome of Aspergillus sojae) linked upstream of Ptef and downstream of the pyrG3 gene. Based on this p19-RT-GDH-pyrG3, a plasmid p19-RT-npII-pyrG3 (RTup (SEQ ID NO: 18)-Ptef-npII-Talp-pyrG3-RTdown (SEQ ID NO: 19)) was prepared in which the GDH gene as a reporter gene was replaced with the npII gene (SEQ ID NO: 2) derived from Aspergillus sojae NBRC4239 strain.

[0111] A pyrG-deficient strain of Aspergillus sojae NBRC4239 (deleting 48 bp upstream, 896 bp coding region, and 240 bp downstream of the pyrG gene) was transformed with p19-RT-npII-pyrG3 by the protoplast PEG method to obtain the As-RT-npII-pyrG3 strain, into which multiple copies of the npII gene expression cassette had been introduced. 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 with shaking at 30°C for 7 days. The culture supernatant was then isolated as an NpII enzyme solution. When the enzyme solution was subjected to SDS-PAGE and stained with CBB, no clear bands other than that of the NpII enzyme were observed.

[0112] 10 μL of the 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 (50 mM Tris-HCl pH 7.0). The resulting reaction solution was subjected to an enzymatic reaction by incubating at 50°C for 2.5 minutes, 5 minutes, 7.5 minutes, and 10 minutes. The enzymatic reaction was stopped by adding 200 μL of 0.1 M sodium acetate buffer (pH 4.5) to the resulting reaction solution. The fluorescence intensity (Ex 375 nm, Em 450 nm) of the resulting treatment solution and 0 pmol / well, 50 pmol / well, 100 pmol / well, 150 pmol / well, and 400 pmol / well solutions of 7-Amino-4-methylcoumarin (AMC, manufactured by Tokyo Chemical Industry Co., Ltd.) was measured using a microplate reader (manufactured by 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. The amount of enzyme was measured from the fluorescence intensity.

[0113] In order to reduce the amount of NpII enzyme solution added to soy sauce, the NpII enzyme solution was concentrated 10 times using an AmiconUltra Centrifugal filter (10 kDa cutoff). The enzyme solution was added to the soy sauce prepared using the ΔnpII strain As1 prepared in Example 1 so that the concentrations were 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 in the same manner as in Example 3, and the scum height was measured. The results are shown in Table 8.

[0114]

[0115] As shown in Table 8, it was confirmed that the amount of pasteurized scum was reduced when the NpII enzyme solution was added in an amount of 0.2 mU / mL or less, and that pasteurized scum was not formed when the NpII enzyme solution was added in an amount of 0.1 mU / mL or less. In other words, it was found that by creating an Aspergillus mutant in which NpII activity was reduced so that the NpII activity contained in the raw soy sauce during the pasteurization process was 0.2 mU / mL or less, it was possible to suppress the formation of scum during pasteurization. In addition, the raw soy sauce produced with the above-mentioned wild-type Aspergillus sojae NBRC4241 strain, Aspergillus sojae NBRC4239 strain, Aspergillus oryzae RIB40 strain, and Aspergillus oryzae RIB326 strain, as well as commercially available raw soy sauce, all had NpII activity of 0.2 mU / mL or more. When measuring NpII activity in soy sauce, it is desirable to remove the contaminants using an AmiconUltra Centrifugal filter (10 kDa cutoff), etc., because the fluorescence intensity due to contaminants is strong and the blank value is 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 dregs is 0.4 mU / g-koji or less, and the amount of NpII enzyme in soy sauce koji to prevent pasteurized dregs 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 the pasteurized soy sauce produced using the ΔnpII strain As1 produced in Example 1 and pasteurized again, pasteurized scum was generated in the same manner as when NpII enzyme was added to raw soy sauce and pasteurized (Table 8). In other words, pasteurized soy sauce produced using an npII gene-inactivated strain can be distinguished by the NpII activity being 0.2 mU / mL or less and the generation of pasteurized scum when NpII enzyme was added and pasteurized again.

[0116] [Sequence Listing] 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. The amino acid sequence of the NpII protein of Aspergillus tamarii is shown in SEQ ID NO: 7.

[0117]

[0118]

[0119]

[0120] By utilizing the present invention, the amount of pasteurization dregs 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. In addition, 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 producing soy sauce, comprising the step of subjecting soy sauce raw materials to a koji-making treatment using a mutant Aspergillus microorganism in which the neutral protease II gene on the chromosome has been inactivated, to obtain soy sauce koji.

2. The method according to claim 1, wherein the mutant Aspergillus microorganism 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 of claim 1, wherein the soy sauce is pasteurized soy sauce.

4. A mutant Aspergillus microorganism, wherein the neutral protease II gene present on the chromosome has been inactivated, and wherein the mutant Aspergillus microorganism does not express human lysozyme.

5. A mutant of a microorganism of the genus Aspergillus, wherein the neutral protease II gene on the chromosome has been inactivated, and wherein the host organism is an Aspergillus microorganism other than the Aspergillus oryzae RIB40 strain.

6. The Aspergillus microorganism mutant according to claim 4 or 5, wherein the Aspergillus microorganism mutant is a mutant of at least one Aspergillus microorganism selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, and Aspergillus tamarii.

7. Raw soy sauce having a heat-resistant NpII activity of 0 mU / ml to 0.2 mU / ml.

8. Pasteurized soy sauce obtained by pasteurizing the raw soy sauce of claim 7.

9. Pasteurized soy sauce obtained by pasteurizing the raw soy sauce produced by the method according to claim 1 or 2.

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