Cheese manufacturing methods

Abstract

The objective is to provide a method for producing cheese with a high methyl ketone content using filamentous fungi that produce large amounts of methyl ketone during cheese maturation. [Solution] A method for producing cheese, comprising the step of inoculating cheese with a filamentous fungal mutant in which the transcription factor activity of FarB protein is deficient or reduced, and then ripening the cheese.

Description

[Technical Field]

[0001] This invention relates, for example, to a method for producing cheese using filamentous fungi that produce high levels of methyl ketone. [Background technology]

[0002] In recent years, the demand for cheese has been increasing in Japan. However, the microbial starters used in current cheese production, as well as the cheese-making methods themselves, are of foreign origin, making it difficult to differentiate domestic cheeses from imported ones. Amidst these challenges, interest in new, uniquely Japanese cheeses is also growing, leading to the development of a cheese using koji mold, which has long been used in the production of traditional fermented foods in Japan.

[0003] Cheese aged with Aspergillus oryzae exhibits a unique flavor and texture due to the action of Aspergillus oryzae-derived proteolytic and fat-degrading enzymes, which produce amino acids / peptides, fatty acids, and other breakdown products. Aspergillus oryzae has a wide variety of strains with distinctive enzyme activity and metabolites for different uses such as miso, sake, and soy sauce, there is potential to produce a wide range of cheeses by utilizing many strains that are not yet used in cheese production.

[0004] In blue cheeses such as Roquefort, Gorgonzola, and Stilton, which are aged with a mixture of blue molds (Penicillium roqueforti, etc.), methyl ketones, which are lipid metabolites produced by the mold, are a major component in shaping the flavor of high-quality blue cheeses. It is believed that when producing cheese with a flavor similar to blue cheese through aging with Aspergillus oryzae, increasing the methyl ketone content can also lead to the production of high-quality cheese. Therefore, a technology for producing large amounts of methyl ketones through Aspergillus oryzae aging is desired.

[0005] As described above, in blue cheese, methyl ketones, which are lipid metabolites produced by the mold, are a major component in the flavor formation of high-quality blue cheese. Therefore, blue cheese manufacturers employ a technique to increase the amount of methyl ketones in their products by adding lipase enzyme preparations to the product, thereby increasing the amount of free fatty acids, which are then converted into methyl ketones through mold metabolism (Non-Patent Document 1). Furthermore, Patent Document 1 discloses a technique for producing cheese with a high methyl ketone content by inoculating milk-derived protein with Aspergillus oryzae, a type of koji mold, via a filter.

[0006] For example, in Non-Patent Document 1, a lipase enzyme preparation is added to the cheese curd during the production of blue cheese to increase the amount of methyl ketones. However, this is an unnecessary cost incurred to compensate for the fact that the methyl ketone production capacity of the blue mold used is insufficient for producing high-quality blue cheese. In principle, if the blue mold used for fermentation can produce enough methyl ketones, the addition of the lipase enzyme preparation should be unnecessary.

[0007] On the other hand, Patent Document 1 describes a method of producing cheese by filtering Aspergillus oryzae on a milk-derived protein substrate, accumulating enzymes and metabolites derived from Aspergillus oryzae within the substrate, and then adding the resulting "mold culture" to cheese curd as if it were an enzyme preparation itself. While such cheese differs from traditional blue cheese, the technical concept is the same as Non-Patent Document 1 in that it increases the amount of methyl ketones in the cheese by separately adding mold enzymes. Patent Document 1 does not mention the use of a specific strain of Aspergillus oryzae. Therefore, it is presumed that a normal strain of Aspergillus oryzae is used, but it requires a cultivation period of 10 to 30 days to obtain the "mold culture." Considering that the koji-making period for typical fermented foods is about 2 to 3 days, a 30-day koji culture is an unusually long period. While this may be possible in a sterile laboratory, performing such a long-term culture in an actual food manufacturing environment carries a high risk of bacterial contamination. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Patent No. 6946008 [Non-patent literature]

[0009] [Non-Patent Document 1] Cao et al. J. Agric. Food Chem. 62, 5726-5733(2014) [Overview of the project] [Problems that the invention aims to solve]

[0010] In view of the above circumstances, the present invention aims to provide a method for producing cheese with a high methyl ketone content using filamentous fungi that produce a large amount of methyl ketone during cheese ripening. [Means for solving the problem]

[0011] In order to solve the above problems, we conducted intensive research and discovered that filamentous fungi with a functional deficiency mutation in the FarB protein, a transcription factor in lipid metabolism, produce high levels of methyl ketone during cheese ripening, thus completing the present invention.

[0012] In other words, the present invention encompasses the following: [1] A method for producing cheese, comprising the step of inoculating cheese with a filamentous fungal mutant lacking or having reduced transcription factor activity of the FarB protein, and then ripening the cheese. [2] A method for producing methyl ketone, comprising the step of inoculating a filamentous fungal mutant lacking or having reduced transcription factor activity of the FarB protein into a fermentation material and carrying out fermentation. [3] The method according to [2], wherein the methyl ketone is one or more methyl ketones selected from the group consisting of 2-heptanone, 2-pentanone, acetone, 2-hexanone, 2-butanone, and 2-undecanone. [4]The filamentous fungal mutant with defective or reduced transcriptional factor activity of the FarB protein is an Aspergillus oryzae mutant with defective or reduced transcriptional factor activity of the FarB protein, wherein the FarB protein has an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and has the transcriptional factor activity of the FarB protein, and the Aspergillus oryzae mutant according to any one of [1] to [3] of the method described above. [5]The method according to [4], wherein the Aspergillus oryzae mutant increases the amount of methyl ketone as compared with the parent strain in cheese ripening. [6]The method according to [5], wherein the methyl ketone is one or more methyl ketones selected from the group consisting of 2-heptanone, 2-pentanone, acetone, 2-hexanone, 2-butanone, and 2-undecanone. [7]Cheese produced by the method for producing cheese according to [1].

Advantages of the Invention

[0013] According to the present invention, it is possible to produce high-quality cheese with good flavor and having a high content of methyl ketone. In addition, in the production of cheese-like foods such as plant-based foods, a cheese-like flavor can be added.

[0014] In addition, according to the present invention, it is possible to produce a high content of methyl ketone.

Brief Description of the Drawings

[0015] [Figure 1] In the examples, by solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC / MS) analysis, the signal intensities of 2-heptanone, 2-pentanone, acetone, 2-hexanone, 2-butanone, and 2-undecanone, among the methyl ketones, for which a significant difference in signal intensity was observed in the cheese cubes after ripening using the FarB function-deficient strain (farB mutant strain), are shown.

Modes for Carrying Out the Invention

[0016] Hereinafter, the present invention will be described in detail.

[0017] The present invention is a method for producing cheese, which includes a step of performing cheese ripening using a filamentous fungus mutant in which the transcriptional factor activity of the FarB protein, which is a transcriptional factor of the lipid metabolism system, is defective or reduced (hereinafter, may be referred to as "the cheese production method according to the present invention"). According to the cheese ripening using the filamentous fungus mutant, high-quality cheese with a good flavor having a high content of methyl ketones can be produced.

[0018] In the present invention, examples of methyl ketones include one or more (for example, two or more, three or more, four or more, five or more, preferably all six) of 2-heptanone, 2-pentanone, acetone, 2-hexanone, 2-butanone, and 2-undecanone.

[0019] In the present invention, examples of filamentous fungi in which the transcriptional factor activity of the FarB protein is defective or reduced include Aspergillus oryzae, Aspergillus sojae, Aspergillus luchuensis, Aspergillus tamarii, Aspergillus glaucus, Eurotium herbariorum, Penicillium roqueforti, Penicillium candidum, Penicillium camemberti, Rhizopus oligosporus, Neurospora intermedia, Neurospora sitophila, Fusarium venenatum, etc.

[0020] Below, as an example of a filamentous fungal mutant in which the transcription factor activity of the FarB protein is deficient or reduced, an Aspergillus oryzae mutant (hereinafter sometimes referred to as "the Aspergillus oryzae mutant according to the present invention") is described. Other filamentous fungal mutants can be produced in the same manner.

[0021] According to the Aspergillus oryzae mutant of the present invention, the amount of methyl ketones in cheese can be increased during cheese ripening compared to the parent strain. Examples of methyl ketones include one or more (for example, two or more, three or more, four or more, five or more, preferably all six) of 2-heptanone, 2-pentanone, acetone, 2-hexanone, 2-butanone, and 2-undecanone.

[0022] Methyl ketones are produced by the metabolism of mold using free fatty acids as raw materials. Therefore, as mentioned above, conventionally, blue cheese manufacturers have used a technique to increase the amount of methyl ketones by adding lipase enzyme preparations to the cheese raw materials to break down milk fat and release fatty acids. Accordingly, if the farB gene, which encodes the transcription factor FarB protein involved in the expression regulation of the lipase gene of Aspergillus oryzae, is destroyed in Aspergillus oryzae, it is expected that in cheese ripening using the farB gene knockout strain, lipase activity will decrease due to the destruction of the transcription factor, the amount of free fatty acids will decrease, and thus the amount of methyl ketones in the cheese will also decrease. However, the inventors unexpectedly found that in cheese ripening using the farB gene knockout strain, the amount of methyl ketones in the cheese increased significantly.

[0023] In the present invention, examples of Aspergillus oryzae include strains such as A. oryzae RIB40, KC41, KC43, and Q.

[0024] The Aspergillus oryzae mutant according to the present invention is an Aspergillus oryzae mutant obtained by subjecting Aspergillus oryzae, as the parent strain, to a method that depletes or reduces the transcription factor activity of the FarB protein.

[0025] In the present invention, the FarB protein of Aspergillus oryzae is a protein having (or consisting of) an amino acid sequence that has at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and that has transcription factor activity of the FarB protein.

[0026] Examples of genes encoding the FarB protein of Aspergillus oryzae (farB genes) include genes that have (or consist of) a base sequence that has at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the base sequence (genomic DNA sequence) shown in Sequence ID No. 1 or the base sequence (cDNA sequence) shown in Sequence ID No. 2, and that encode a protein having transcription factor activity for the FarB protein.

[0027] Here, "FarB protein transcription factor activity" refers to the activity of binding to a specific region in the promoter region of a group of genes related to lipid metabolism, such as lipase enzyme genes, thereby switching the expression of those genes ON / OFF, or regulating their expression level, site, or timing.

[0028] In Aspergillus oryzae, multiple farB genes, such as alleles or synonymous genes, may exist, but in this invention, it refers to at least one or more of these farB genes.

[0029] In this invention, an Aspergillus oryzae mutant can be obtained by subjecting an Aspergillus oryzae having the farB gene to a method that depletes or reduces the transcription factor activity carried out by the FarB protein encoded by the farB gene.

[0030] Methods for deleting or reducing the transcription factor activity carried out by FarB proteins include, for example, (1) Introduce a mutation targeting the farB gene and disrupt the gene; (2) Suppress the transcription of the farB gene and reduce its expression; (3) Suppress the translation of the farB gene and reduce the translation efficiency of the gene; Several methods can be listed.

[0031] (1) A method of introducing mutations targeting the farB gene Methods for introducing mutations targeting the farB gene include, for example, the Latour method described in the examples (deletion of the farB gene from the Aspergillus oryzae genomic DNA by loop-out transformation using a construct for farB gene deletion), or gene knockout methods using genome editing called ZFN, TALEN, or CRISPR / Cas, which produce mutants lacking the farB gene. Alternatively, after inducing mutations by electromagnetic wave or particle beam irradiation or mutagen treatment, strains with mutations in the farB gene may be selected through an appropriate screening process.

[0032] (2) A method for suppressing the transcription of the farB gene and reducing its expression. One method for suppressing the transcription of the farB gene is to introduce a mutation into the promoter region of the gene in the target Aspergillus oryzae. Another method involves introducing mutations into genes involved in the positive expression regulation of the gene in question, thereby reducing their function. Alternatively, a method could be used in which a mutation is introduced into a gene involved in the negative expression regulation of the gene in question, so that the negative expression regulation is always active.

[0033] (3) A method for suppressing the translation of the farB gene and reducing the translation efficiency of the gene. One method for suppressing the translation of the farB gene is so-called RNA interference.

[0034] The conidial fluid of the Aspergillus oryzae mutant obtained as described above is used to culture cheese (cheese maturation), and the amount of methyl ketones is measured using the method described in the examples. If it can be confirmed that the amount of methyl ketones in the cheese is significantly higher than that of the parent strain, then the creation of an Aspergillus oryzae mutant lacking or with reduced transcription factor activity mediated by the FarB protein according to the present invention is complete.

[0035] The cheese production method according to the present invention involves inoculating cheese with a filamentous fungal mutant in which the transcription factor activity of the FarB protein described above is deficient or reduced, and then performing cheese ripening. For example, green cheese produced by a conventional method (cheese before ripening in the cheese production process) is 10 3 ~10 9 pieces / ml (preferably 10) 6 The conidial solution of a filamentous fungal mutant in which the transcription factor activity of FarB protein, adjusted to a concentration of 1 / ml, is deficient or reduced is passed through the solution and allowed to stand. It is then cultured at a relative humidity of 80-99% (preferably 90%) and a temperature of 15-40°C (preferably 35°C) for 4-30 days (preferably 5 days) and aged. In this way, cheese with a high methyl ketone content can be produced.

[0036] Furthermore, the present invention relates to a method for producing methyl ketones, which can be carried out in accordance with the cheese production method according to the present invention, and includes the step of inoculating a filamentous fungal mutant lacking or having reduced transcription factor activity of FarB protein into a fermentation material and carrying out fermentation. This method for producing methyl ketones can produce methyl ketones with a high content. Examples of fermentation materials include cheese such as green cheese, dairy products such as milk and fresh cream, food products containing vegetable oil or animal oil such as oilseed residue and oil cake, or food residues. [Examples]

[0037] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0038] [Creation of FarB-deficient strains] Deletion of the farB gene (genomic DNA sequence: SEQ ID NO: 1, cDNA sequence: SEQ ID NO: 2, amino acid sequence: SEQ ID NO: 3) was performed using the Latour method. Using the A. oryzaeRIB40 strain genomic DNA as a template, PCR was performed to create a construct for farB deletion. The PCR enzyme was used with Phusion hot start according to the reagent manufacturer's instructions. The primer list used for amplification is shown in Table 1 below.

[0039] [Table 1]

[0040] Four DNA fragments were amplified using primer combinations SAT354 and SAT356, SAT357 and SAT358, SAT368 and SAT370, and SAT363 and SAT364. A mixture of these four fragments was used as a template for fusion PCR using primer combinations SAT355 and SAT369. Sixty-four fusion PCRs were performed in a 50 μl reaction system. After the PCR reaction, the samples were purified by ethanol precipitation and dissolved in 10 μl of Tris-EDTA buffer for use in the transformation of Aspergillus oryzae.

[0041] Aspergillus oryzae transformation was performed according to the standard procedure using the protoplast PEG method. The Aspergillus oryzae strain used for transformation was RIB40 ligD. - ,pyrG - A strain was used. RIB40 ligD was cultured overnight in Czapek Dox with 0.1% uracil. - ,pyrG - The bacterial cells of the strain were recovered, and protoplasts were obtained using Yatarase according to a standard procedure. The entire 10 μl of the fusion PCR product was added to the protoplasts, and transformation was carried out by adding PEG solution according to a standard procedure. The entire amount of PEG-treated protoplasts was selected for uracil-requiring complementation on Czapek Dox protoplast regeneration medium. The protoplasts on Czapek Dox protoplast regeneration medium were cultured at 30°C for 4 days to obtain uracil-free colonies.

[0042] A small amount of conidia was collected from the obtained colonies, and confirmation PCR was performed with primers SAT354 and SAT370 by conidia PCR using an FTA card to confirm RIB40 ligD - ,pyrG - It was confirmed that the band size of the strain was increased by about 2 kb compared to that of the RIB40 ligD

[0043] Next, conidia of the uracil non-requiring strain were cultured in Czapek Dox supplemented with 0.1% uracil, 0.025% uridine, and 0.1% 5-fluoroorotic acid (5-FOA) to obtain 5-FOA-resistant colonies.

[0044] A small amount of conidia was collected from the obtained colonies, and confirmation PCR was performed with primers SAT354 and SAT370 by conidia PCR using an FTA card to confirm RIB40 ligD - ,pyrG - It was confirmed that the band size was reduced by about 2.5 kb compared to that of the RIB40 ligD

[0045] Also, gene deletion was performed on the farA gene in the same manner as described above. Furthermore, instead of the RIB40 ligD - ,pyrG - strain, the parental strain was used as the farA gene deletion strain, and the same operation as above was performed to obtain a farAfarB double gene deletion strain.

[0046] [Preparation of conidial suspension of FarB function-deficient strain] The FarB function-deficient strain obtained by deleting the farB gene was inoculated onto PDA supplemented with 0.1% uracil and cultured for 3 days to sufficiently produce conidia. The conidia were collected according to a standard method and dispersed in a conidia dispersion buffer (0.5% NaCl, 0.002% Tween80).

[0047] [Preparation of green cheese] 45g of NaCl was dissolved in 3kg of raw milk, heated to 75°C, and then quickly cooled to 30°C. 30mg of commercially available lactic acid bacteria starter was added and cultured at 30°C for 40 minutes. Next, 0.09g of commercially available rennet and 0.3g of CaCl2 were added and cultured for another 30 minutes. The agglomerated cheese curd was cut to approximately 50cm using a curd knife. 3 The cheese curds were cut and left to stand for 1 hour. Then, 2.6 L of 1.5% saline solution was added and gently stirred. After letting it stand for another 30 minutes, the cheese curds were packed into a mold (Φ62 mm × 90 mm H) and left overnight at 30°C, occasionally turning the mold to remove the whey.

[0048] [Cheese maturation using FarB-deficient bacterial strains] 10 green cheeses 6 The conidial solution of the FarB-deficient bacterial strain, adjusted to a concentration of cells / ml, was passed through the solution, then placed in a plastic tray and incubated at 90% relative humidity and 35°C for 5 days.

[0049] [Cube cheese aging test] Green cheese was cut into 10 mm cubes and soaked overnight in a 1% uracil solution. Similar to the above [Cheese ripening using FarB-deficient strains], the green cheese cubes removed from the uracil solution were 10 mm cubes. 6 The cells were dipped in a conidial solution containing cells / ml, then placed in a plastic tray and cultured at 90% relative humidity and 35°C. Three cells were sampled on day 3 and three cells on day 5.

[0050] [Solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC / MS) analysis of cube cheese] Similar to the above [Cheese ripening using FarB-deficient strains], one ripened cheese cube was placed in each 20 ml glass headspace vial, sealed tightly, and subjected to GC / MS analysis. Volatile components such as methyl ketones were collected for 20 minutes using SPME fiber (Nitinol core CAR / PDMS) after preheating the vial at 50°C for 10 minutes, desorbed at 230°C in the GC instrument, and injected into the analytical column. Then, individual components were separated using an InertCap-Pure WAX ​​heating program from 30°C to 250°C on the analytical column, detected by mass spectrometer, and GC / MS measurement data was collected. The measurement data was processed using AnalyzerPro XD for peak detection and component identification by searching a mass spectrum database, and the peak area of ​​methyl ketones and other components was obtained as signal intensity for comparison between samples.

[0051] Figure 1 shows the signal intensities of 2-heptanone, 2-pentanone, acetone, 2-hexanone, 2-butanone, and 2-undecanone, which showed significant differences in signal intensity among methyl ketones. For all of these methyl ketones, the signal intensity in the farB mutant and the farA·farB double mutant was significantly higher (p < 0.01) than in the RIB40 strain and the farA mutant. This indicates that the deficiency of farB is strongly involved in the increase of methyl ketones.

Claims

1. A method for producing cheese, comprising the step of inoculating cheese with a filamentous fungal mutant in which the transcription factor activity of the FarB protein is deficient or reduced, and then ripening the cheese.

2. A method for producing methyl ketone, comprising the step of inoculating a filamentous fungal mutant lacking or having reduced transcription factor activity of the FarB protein into a fermentation material and carrying out fermentation.

3. The method according to claim 2, wherein the methyl ketone is one or more methyl ketones selected from the group consisting of 2-heptanone, 2-pentanone, acetone, 2-hexanone, 2-butanone, and 2-undecanone.

4. The method according to any one of claims 1 to 3, wherein the filamentous fungal mutant lacking or reduced transcription factor activity of FarB protein is an Aspergillus oryzae mutant lacking or reduced transcription factor activity of FarB protein, wherein the FarB protein has an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and is a protein having transcription factor activity of FarB protein.

5. The method according to claim 4, wherein the Aspergillus oryzae mutant increases the amount of methyl ketones in cheese ripening compared to the parent strain.

6. The method according to claim 5, wherein the methyl ketone is one or more methyl ketones selected from the group consisting of 2-heptanone, 2-pentanone, acetone, 2-hexanone, 2-butanone, and 2-undecanone.

7. Cheese produced by the cheese production method described in claim 1.

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