JP6892141B2 - Aspergillus that can be used for producing brewed foods and a method for producing brewed foods using the aspergillus that lacks the function of the gene encoding AogdhB. - Google Patents

Description

The present invention relates to a Jiuqu strain suitable for producing a brewed food, which lacks the function of a gene encoding AogdhB, and a method for producing a brewed food using the strain.

The pH of various flavors in the soy sauce brewing process has a great influence on the action of various enzymes and the behavior of fermenting microorganisms, and pH control is very important for controlling the recovery rate and flavor of raw material nitrogen. .. Above all, the pH of soy sauce koji in the koji making process, which is upstream of moromi aging, is complicatedly controlled by various factors such as the medium, koji making conditions, and other microorganisms.

When producing soy sauce jiuqu, the pH of the jiuqu may rise excessively due to the influence of water content, mixing ratio of raw materials, culture temperature, time, etc., which may adversely affect the subsequent fermentation. In addition, if the pH of the soy sauce obtained is high due to the high pH of the jiuqu, the soy sauce may have a darker color or a blurred taste and a bad taste, which may cause a quality problem.

Conventionally, as factors affecting the pH of Jiuqu, it has been reported that the decrease of organic acids such as citric acid in Jiuqu and the production of ammonia cause an increase in pH during Jiuqu production (Non-Patent Document 1). .. In addition, as a method of controlling the pH of the koji making process, a method of blending raw materials, selecting the type of raw material, a koji making method, etc. has been studied (Non-Patent Document 2). It has not been known at all what kind of gene is involved in ammonia production and pH fluctuation.

Various studies have been conducted on the amino acid metabolism pathway by Jiuqu. For example, aspergillus oryzae, which is used in the production of soy sauce and liquor, has increased secretion of acidic proteases as compared with the parent strain, and as a result of comprehensive gene expression analysis of mutant strains with good growth, mutations have occurred. It has been reported that the expression levels of various amino acid dehydrogenases were changed in the strains, and the expression levels of glutamate dehydrogenase AO1008_09334 were significantly increased (Non-Patent Document 3). However, this finding was not an analysis of gene expression during the actual production of soy sauce koji, and it was unclear whether the analysis results reflected the actual conditions in the koji making process.

Japan Salty Sauce Research Institute Magazine Vol. 12, No. 6, pp. 224-228 Summary of Easy Soy Sauce Technology Volume 1 (Japan Soy Sauce Technology Center), pp. 93-99 Biomed Research International, Vol. 2015, Article ID 456802

As described above, the content of ammonia in the production of koji can affect the increase in the pH of the koji and the content of amino acid nitrogen in the soy sauce, and it is important to appropriately control the production of ammonia in the production of the soy sauce. It is very important for proper control of the manufacturing process and the quality of the finished soy sauce.

In addition, ammonia nitrogen is present in 11 to 15% of the total nitrogen in the koji making, which is caused by the decomposition of amino acids. On the other hand, various tasting amino acids such as glutamic acid are components that greatly contribute to the taste, sweetness, and richness of soy sauce, reduce ammonia nitrogen relative to total nitrogen, and increase the content of amino acid nitrogen. This can be expected to be an effective method for increasing the amino acid content in soy sauce and further improving the taste.

Therefore, the subject of the present invention is to identify the enzyme in the aspergillus that is involved in the production of ammonia in the koji making process, thereby facilitating the production of ammonia and the control of pH in the koji making process, and making it easy to obtain high quality soy sauce. To obtain a salty soy sauce strain, and to obtain a method for producing koji and brewed foods using the strain.

The inventor of the present application bred the aspergillus and obtained a mutant strain in which the pH was less likely to increase in the aspergillus production process. Then, as a result of performing the genome sequencing of the strain, the gene consisting of the amino acid sequence shown in SEQ ID NO: 1, which had not been detected at all in the comprehensive analysis as described in Non-Patent Document 3, was mutated and functioned. It was revealed that it was missing.

The gene consisting of the amino acid sequence shown in SEQ ID NO: 1 is referred to as AogdhB because it shows high homology with the gdhB gene encoding glutamate dehydrogenase in Aspergillus nidurance. Although gdhB in Aspergillus nidurance is known to have glutamate dehydrogenase activity, it has not been clarified whether it actually functions in, for example, the koji making process and its contribution.

Therefore, as a further study, the AogdhB gene in the genome that can be used for the production of brewed foods was functionally deficient by genetic recombination, and as a result of koji making using the obtained strain, ammonia production and koji in the koji making process. It was found that the increase in pH was suppressed.

From the above studies, the inventor of the present application found that AogdhB is a gene that is actually involved in pH control of Jiuqu in the Jiuqu manufacturing process of brewed food production and is important for pH control in Jiuqu food production. It was clarified that it is possible to obtain good Jiuqu in which the production of ammonia is small and the increase in the pH of Jiuqu is suppressed by deficient in the function of Jiuqu, and the present invention has been completed.

By obtaining the Jiuqu using the Jiuqu bacterium lacking the function of the AogdhB gene of the present invention, the pH of the Jiuqu in the Jiuqu making process can be kept low, and a stable brewed food can be produced. Further, for example, when soy sauce is produced as a brewed food, the amount of ammonia nitrogen in the soy sauce is reduced, so that a soy sauce having a large amount of free amino acids can be obtained, and it becomes possible to brew a sensually good salty sauce.

FIG. 1 shows the temporal transition of pH when parent strains A and B, which are Yamasa-carrying aspergillus strains, and mutant strains A and B obtained from the respective strains A and B were cultured in a soy sauce medium. FIG. 2 compares the amino acid sequence of the portion of the amino acid sequence of AogdhB in the mutant strains A and B in which the mutation occurs with the sequence of the wild strain. FIG. 3 schematically shows the state of introduction of a mutation into the AogdhB gene of the parent strain (ΔligD strain) and the AogdhB gene structure of the functionally deficient strain (ΔGdhB strain) after the introduction of the mutation. FIG. 4 is an electrophoretogram showing the results of PCR for confirming that the mutation was introduced into the AogdhB gene in the mutagenesis strain. FIG. 5 shows the temporal transition of pH when the ΔGdhB strain and the parent strain were cultured in a soy sauce medium.

In the present invention, the aspergillus that can be used in the production of brewed foods is any aspergillus that can be used in the production of brewed foods such as soy sauce, miso, shochu, sake, and mirin and belongs to the genus Aspergillus, for which safety has been established. There is no particular limitation. Specifically, Aspergillus oryzae, Aspergillus sojae, Aspergillus kawachii, Aspergillus awamori, Aspergillus niger, etc. preferable. Of these, Aspergillus oryzae or Aspergillus sawyer is particularly preferred, and Alpergillus oryzae is even more preferred.

In the present invention, the AogdhB gene refers to a gene whose base sequence is shown in SEQ ID NO: 2 and the amino acid sequence of the gene product is shown in SEQ ID NO: 1. Information on the AodghB gene (AO090001000717 gene) in Aspergillus oryzae can be obtained from, for example, the NCBI website (https://www.ncbi.nlm.nih.gov/gene).

A functionally deficient strain of the AogdhB gene can be obtained by gene disruption by homologous recombination, induction of functional deficiency by mutation introduction, genome editing, or the like.

As a method for disrupting a gene by homologous recombination, a known method can be used. For example, a fragment of the AogdhB gene or a DNA fragment combining the upstream / downstream regions thereof and a marker gene is incorporated into the vector, the vector is incorporated into the aspergillus by the protoplast-PEG method, the electroporation method, etc., and the vector is incorporated into the aspergillus by homologous recombination. Examples thereof include a method of introducing a DNA fragment into the genome of Aspergillus oryzae. Other methods for incorporating the DNA fragment into the Jiuqu cell include a particle gun method, an Agrobacterium method, a microinjection method, and the like.

A known method can be used as a method for confirming that the desired gene has been introduced into Jiuqu by the homologous recombination method. For example, when introducing a gene, an auxotrophic mutant strain is used as a parent strain, and a gene having a function of compensating for the auxotrophy is used as a marker gene, and after transformation, it is normally performed on an auxotrophic medium. Examples include a method of selecting a grown strain. However, it is not possible to confirm whether or not the target locus has been replaced with the introduced marker gene only by such auxotrophy. Therefore, it is necessary to confirm that the target locus is replaced by a marker by appropriately using a PCR method, a Southern hybridization method, or the like according to the auxotrophy.

Further, as the mutation introduction method, a known treatment method can be used, and a physical method of irradiating with ultraviolet rays, an ion beam, radiation, etc., ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, There are chemical methods using mutant agents such as nitrous acid and acrydin dyes. Particularly preferably, a method of irradiating an ion beam or ultraviolet rays can be mentioned.

Furthermore, as a method by genome editing, a site-specific nuclease such as ZFN (Zinc-Finger Nuclease), TALEN (Transcription Activator-Like Effector Nuclease), CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9, etc. Examples thereof include a method in which double-strand breaks in DNA at a genome site are caused, and the DNA repair mechanism induced thereafter is used to cause disruption of the target genome or base substitution.

Whether or not the ammonia-producing ability is actually reduced in a strain in which the function of the AogdhB gene is deleted by the above-mentioned gene disruption method, mutation introduction method, genome editing, etc. should be verified by, for example, the following test method. Is possible.

(Method of verification)
Sauce medium (added water 7ml defatted processed soybean 5g, were mixed well split砕小wheat 5g, 121 ° C. in an autoclave for 40 minutes treatment) and Aspergillus oryzae spores was inoculated so as to be 10 ⁶ cells / g of about a , Cultivate for a predetermined time and verify by quantifying the amount of ammonia in the culture product. To extract ammonia, add 100 ml of water to the culture, stir well, allow to stand at 5 ° C. for 4 hours or more, and then filter to obtain an analytical sample solution. The analysis can be performed using a commercially available amino acid analyzer or F-kit ammonia (JK International).

The aspergillus of the present invention can be used in the production of various brewed foods. Examples of brewed foods include soy sauce, miso, shochu, sake, mirin, etc. Among them, it is preferably used for soy sauce or miso, and particularly preferably for soy sauce.

As a method for producing Jiuqu using the Jiuqu of the present invention and a method for producing a brewed food using the Jiuqu, a known method can be used. As an example, in the production of soy sauce, a mixture of ordinary koji raw materials, for example, a mixture of sprinkled and steamed soy sauce raw materials and roasted and crushed wheat raw materials, is inoculated and mixed with the above-mentioned AogdhB gene-deficient aspergillus. The prepared Jiuqu is charged in a normal preparation tank with an appropriate concentration of salt solution, and fermented and aged for about 3 to 6 months with appropriate stirring to obtain various flavors of soy sauce, which is squeezed, refined, and if necessary. It may be fired to make product soy sauce (raw soy sauce or fired soy sauce).

(Example 1: Acquisition of mutant aspergillus strain with low ammonia production and analysis of mutant site)
Two types of Yamasa-carrying aspergillus strains (Alpergillus oryzae, hereinafter referred to as "parent strain A" and "parent strain B") were irradiated with ultraviolet rays and subjected to mutagen treatment. The method of mutagen treatment was as follows.

(Mutation processing method)
Aspergillus spores were adsorbed on a membrane filter, the filter was irradiated with UV, and then suspended in 0.1 M phosphate buffer (pH 7.0) (survival rate of about 1%). Dilute 1000 times with 0.05% tween solution so that there are about 10 colonies per plate, and the minimum medium containing L-arginine and ornithine (composition: 0.5% sucrose, 0.1% dipotassium phosphate). , Magnesium sulfate 0.05%, Potassium chloride 0.05%, Trace element 0.1%, Bromocresol purple 0.005%, Agar 1.5%, pH 5.5).

In the culture in the medium, the colonies turn blue when the pH rises due to the action of bromocresol purple. After culturing for 3 days, yellow colonies were selected as mutation candidate strains that did not cause a pH increase.

The obtained Jiuqu mutation candidate strain was inoculated into a soy sauce medium (7 ml of water was added to 5 g of defatted soybeans, 5 g of crushed wheat was mixed well, and then treated in an autoclave at 121 ° C. for 40 minutes), and the cells were inoculated at 28 ° C. for 24 minutes. , 48, 72 hours to produce a culture (jiuqu). 100 ml of water was added to each culture, the mixture was stirred well, allowed to stand at 5 ° C. for 4 hours or more, and then filtered through a filter paper. The pH of the filtrate was measured, and strains in which the increase in pH was suppressed were secondarily screened.

As a result, one mutant strain was obtained from each of the mutant candidate strains derived from the parent strain A and the parent strain B (hereinafter, referred to as "mutant strain A" and "mutant strain B", respectively).

The pH measurement results are shown in FIG. As a result, in the parent strains A and B, an increase in pH was confirmed as the culture time became longer, whereas in the obtained mutant strains A and B, an excessive increase in pH was not confirmed.

Therefore, the genome sequences of these mutant strains A and B were decoded, and mutation point analysis was performed. If mutations are found at the same site in different parent strains, it is highly likely that the mutation is a functional gene that suppresses an excessive increase in pH.

The DNeasy Plant Maxi kit (QIAGEN) was used for genome extraction, and the analysis of the obtained genomic DNA was outsourced to Filgen Co., Ltd. For genome analysis, a sequence of 2 Gbp was carried out on PE150 using illumina HiSeq4000 (Illumina Co., Ltd.), and a total of 4 Gbp sequence data was obtained on both sides. The procedure for mutation point analysis was as follows.

(analysis method)
For the sequence data of each of the parent strain and the mutant strain, the entire genome sequence of the Aspergillus oryzae RIB40 strain was mapped as a reference sequence using the Burrows-Wheeler Aligner (BWA). The mutant site specific to the mutant strain was detected by the Haplotype Caller of Genome Analysis Tool Kit (GATK).

As a result of the mutation point analysis, it was confirmed that the mutant strain A had mutations at 27 sites in the gene region of the parent strain A and the mutant strain B had mutations at 48 sites in the gene region of the parent strain B. Among them, the only mutant gene common to both was AO090001000717.

The AO090001000717 gene encodes 1061 amino acids and shows high homology at the amino acid level of 89.6% with gdhB, the glutamate dehydrogenase (GDH) of Aspergillus nidulans. Hereinafter, the gene (AO090001000717 gene) is referred to as an AogdhB gene.

In both mutant strains A and B, a frameshift occurs due to a mutation in the AogdhB gene. Mutant strain A has a frameshift after the 697th (704th Stop codon), and mutant strain B has a 249th or later (298th Stop codon). It was strongly suggested that the function was lost because the amino acid sequence of) was completely different (Fig. 2).

(Example 2: Preparation and evaluation of AogdhB function-deficient strain using donor strain)
(Example 2-1: Preparation of gdhB disrupted strain vector and disrupted strain)
In order to further clarify whether the AogdhB gene in Aspergillus oryzae actually functions during koji production, we attempted to create an AogdhB function-deficient strain using the donor strain as the parent strain.

ORF upstream 1.8 kbp (SEQ ID NO: 3), downstream 1.3 kb region (SEQ ID NO: 4) of the AogdhB gene, sC marker from Aspergillus nidurance (Mol Gen Genet. 1995 May 20; 247 (4): 423-249 The three fragments of.) Were combined using an In-Fusion HD Cloning Kit (TaKaRa) to prepare a disruption vector.

The obtained disrupted fragment was amplified by PCR ^{and transformed using the ΔligD strain (niaD −} , sC ⁻ , ligD :: ptrA) derived from the Aspergillus oryzae RIB40 strain as the parent strain. As a control strain, a strain in which an sC marker derived from Aspergillus nidurance was inserted into the sC gene region of Aspergillus oryzae was used as a control strain. The RIB40 strain is a known donor strain, and ΔligD and a method for producing the same are also known from papers (Mizutani et al., Fungal Genetics and Biology, 45 (2008) pp. 878-889).

Regarding the obtained transformant, it was confirmed by PCR that the DNA fragment derived from the vector was certainly inserted by recombination (FIGS. 3 and 4). In PCR, primer A (SEQ ID NO: 5) was used as the forward primer, and primer B (SEQ ID NO: 6) was used as the reverse primer. The production positions of the primers A and B are as shown in FIG.
By the above method, an Aspergillus oryzae AodghB gene function-deficient strain (hereinafter, may be referred to as "ΔAogdhB strain") was obtained.

(Example 2-2: Growth in raw material medium)
7 ml of water was added to 5 g of degreased soybeans, 5 g of crushed wheat was mixed well, and then the mixture was treated in an autoclave at 121 ° C. for 40 minutes. The obtained raw material, added with a spore suspension of the control strains and ΔAogdhB strain (1.4 × 10 ⁷ cells / ml) by 1 ml, 24 hours, 48 hours, 72 hours, and cultured for 96 hours. 100 ml of water was added to each culture, the mixture was shaken, allowed to stand for 4 hours, and then the pH of the filtrate filtered through filter paper was measured.

[result]
As a result, in the control strain, the pH increased linearly with time, whereas in the ΔAogdhB strain, the pH increase in the late stage of koji making was not confirmed. From this, it was clarified that AogdhB contributed to the increase in pH in the late stage of koji making (Fig. 5).

(Example 2-3: Evaluation of gene-disrupted strain in soy sauce brewing)
260 L of water was added to 200 g of degreased soybeans to absorb water, and the soybeans were steamed according to a conventional method. In addition, 200 g of raw wheat was roasted and crushed according to a conventional method. Spores of ΔAogdhB strain and control strain 1 × 10 ⁶ cfu / g were mixed and koji was prepared according to a conventional method for 48 hours.

700 mL of salt water having a salt concentration of about 25% (w / v) was added to the obtained Jiuqu to prepare a concentrated soy sauce. After stirring well the next day when the jiuqu and salt water were sufficiently familiar, the moromi mash was filtered and the amount of ammonia and pH of each filtrate were measured. The amount of ammonia was measured using an amino acid analyzer (Hitachi High-Tech).

As a result, the ammonia concentration per 1 mL of the filtrate in the control strain was 2.22 (mg / mL), whereas the ammonia concentration in the ΔAogdhB strain was reduced to 1.37 (mg / mL). Table 1 shows the ratio of ammonia content and pH when the measured value in the control strain was 1.00. As described above, in the Jiuqu produced using the ΔAogdhB strain, a significant reduction in the amount of ammonia was confirmed and the pH was also significantly lower than that in the Jiuqu produced using the control strain.

The moromi mash prepared by adding salt water was fermented and aged for 4 months by adding lactic acid bacteria and yeast according to a conventional method. The moromi mash after aging was filtered to obtain raw soy sauce.

The total amount of free amino acids and the amount of ammonia in the raw soy sauce were measured using an amino acid analyzer (Hitachi High-Tech). As a result, the ammonia concentration per 1% (w / v) of the total nitrogen concentration in the raw soy sauce using the control strain was 1.65 (mg / mL), whereas the ammonia concentration in the raw soy sauce using the ΔAogdhB strain was 1.05. It had decreased to (mg / mL). The total free amino acid concentration per 1% (w / v) of the total nitrogen concentration in the raw soy sauce using the control strain was 31.57 (mg / mL), whereas the total free amino acid concentration in the raw soy sauce using the ΔAogdhB strain was 31.57 (mg / mL). Was increased to 35.27 (mg / mL). Table 2 below shows the amount ratio when the measured value in the control strain is 1.00.

As described above, it was confirmed that the dark soy sauce produced using the ΔAogdhB strain significantly increased the amount of free amino acids and decreased the amount of ammonia as compared with the concentrated soy sauce produced using the control strain. ..

(Example 3: Evaluation of practical aspergillus strain in soy sauce brewing)
Soy sauce brewing was carried out using the mutant strain B obtained in Example 1, which is a mutant strain lacking the function of the AogdhB gene.

20 L of water was added to 15 kg of degreased soybeans to absorb water, and the soybeans were steamed according to a conventional method. In addition, 15 kg of raw wheat was roasted and crushed according to a conventional method. Spores of parent strain B and mutant strain B ^{were mixed 1 × 10 6} cfu / g, respectively, and koji was prepared for 48 hours according to a conventional method, and then 50 L of salt water having a salt concentration of about 25% (w / v) was added to the obtained koji. Then, I prepared the dark soy sauce. After stirring well the next day when the jiuqu and salt water were sufficiently familiar, the moromi mash was filtered and the pH was measured. A significant decrease in pH was confirmed in mutant strain B (Table 3).

The moromi mash prepared by adding salt water was fermented and aged for 4 months by adding lactic acid bacteria and yeast according to a conventional method. After aging, the moromi mash was filtered and the amount of free amino acids was measured.

As a result, the ammonia concentration per 1% (w / v) of the total nitrogen concentration in the raw soy sauce using the parent strain B was 1.22 (mg / mL), whereas the ammonia concentration in the raw soy sauce using the mutant strain B was 1. It had decreased to 03 (mg / mL). The total free amino acid concentration per 1% (w / v) of the total nitrogen concentration in the raw soy sauce using the parent strain B was 33.29 (mg / mL), whereas the total free amino acids in the raw soy sauce using the mutant strain B was 33.29 (mg / mL). The concentration had increased to 34.32 (mg / mL). Table 4 below shows the amount ratios when the measured values of the parent strain B are 1.00.

As described above, when the koji was produced using the mutant strain B lacking the function of the AogdhB gene and the concentrated salty soy sauce was produced, the resulting soy sauce was significantly freed as compared with the case where the concentrated salty sauce was produced using the wild strain. An increase in the amount of amino acids and a decrease in the amount of ammonia were confirmed, and it became clear that soy sauce of good quality could be produced.

Claims (5)

Jiuqu that is deficient in the function of the gene encoding AogdhB and can be used in the production of brewed foods. The aspergillus according to claim 1, wherein the aspergillus is Aspergillus oryzae or Aspergillus sojae. A method for producing Jiuqu, which comprises producing Jiuqu using the Jiuqu bacterium according to claim 1 or 2. A method for producing a brewed food, which comprises preparing Jiuqu using the Jiuqu bacterium according to claim 1 or 2, preparing the Jiuqu using the Jiuqu by a conventional method, and fermenting and aging it. The production method of claim 4, wherein the brewed food is soy sauce or miso.

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