Fermented food composition production method

Abstract

To provide a method for effectively using a dried bonito shaving extraction residue which is industrial waste.SOLUTION: A fermented food composition production method includes the steps of: extracting stock from dried fish shaving; separating a dried fish shaving extraction residue from a mixture of the dried fish shaving and the stock; and fermenting the dried fish shaving extraction residue with a filamentous fungus belonging to the genus Aspergillus having a xerophilous nature. The method further may include a step of causing a proteolytic enzyme to act on the dried fish shaving extraction residue. The proteolytic enzyme is preferably at least one of an endo-peptidase or an exo-peptidase derived from the filamentous fungus belonging to the genus Aspergillus having the xerophilous nature.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a fermented food composition. [Background technology]

[0002] Dried fish such as bonito flakes are basic seasonings for traditional Japanese cuisine, along with kelp, dried sardines, and shiitake mushrooms. For example, dashi stock extracted from shaved bonito flakes, a type of dried fish, is used in clear soups, miso soup, simmered dishes, and noodle soups.

[0003] In the process of extracting water-soluble components from dried bonito flakes and producing liquid seasonings and powdered soup stock from the resulting liquid stock, a large amount of dried bonito extract residue is generated as a food manufacturing by-product, which is usually disposed of as industrial waste. In recent years, there has been a push to turn food manufacturing by-products, surplus food, cooking waste, farm waste, etc. into silage or liquefaction in order to improve feed self-sufficiency and recycle food (Ministry of Agriculture, Forestry and Fisheries, Livestock Industry Bureau, Feed Division, September 2022, Situation Surrounding Ecofeed).

[0004] The main components of dried bonito extract residue are protein and water, and if the fattening effect of mixing it with existing feed is clarified, its use as feed will be promoted. For example, as a prior art, the use of the extract residue as self-supplied feed for Aigamo ducks is being considered (Non-Patent Document 1). There have been many attempts to turn food manufacturing by-products, including the residue from the extraction process (e.g., okara, soy sauce and sake cakes, soybean oil cake, rice bran, bran, etc.), into feed, but it would be desirable to develop an effective method of utilizing the components of the residue from the extraction process. For example, it is expected that the extraction residue can be processed to produce multifunctional food ingredients, health functional foods, and fine chemicals that exhibit various physiological activities.

[0005] As a method for utilizing dried bonito extract residue, which is mainly composed of protein, for example, there is a liquefaction method by hydrolysis. Prior art related to this liquefaction method that has been proposed so far includes an acid hydrolysis method using hydrochloric acid (Non-Patent Document 2) and an enzymatic hydrolysis method using a commercially available protease (Patent Document 1), etc. These methods can shorten the processing time, but excessive decomposition can cause deterioration of flavor and coloration. For example, when decomposing eggshell membrane protein, which is mainly composed of protein, it has been pointed out that acid or alkali treatment can cause problems such as brown coloring and the generation of unpleasant odors due to further decomposition of amino acids (Patent Document 2). Therefore, unless an amino acid solution with enhanced umami and rich flavors can be successfully produced, its use may be limited to the above-mentioned livestock feed, etc. On the other hand, if liquefaction were to be achieved solely by enzymatic hydrolysis using commercially available proteases, it would be easy to predict that the production costs would be enormous.

[0006] In addition, a decomposition method using the Aspergillus fungus, which is used in the brewing of alcohol, miso, and soy sauce, as a source of protease is known as a prior art. Examples include a method for producing a seasoning from a mash consisting of dried bonito extract residue, soy sauce koji, and ethanol (Patent Document 3), a method for producing a seasoning ingredient by decomposing dried bonito extract residue using solid koji or koji mold (Patent Documents 4 and 5), and a method for liquefying dried bonito extract residue by combining koji mold with a commercially available enzyme preparation (Non-Patent Document 3). Both methods use koji mold, which has been eaten for a long time, and therefore take safety into consideration. However, it is necessary to produce koji by separately culturing Aspergillus filamentous fungi, and it is not clear which of the proteolytic enzymes secreted and produced by koji mold will work efficiently on the proteins in the residue of dried bonito extract, making it difficult to maintain and optimize the quality of the manufactured products. In addition, since koji is added, it cannot be said to be a composition mainly composed of dried bonito extract residue. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 01-300872 [Patent Document 2] Patent Publication No. 2016-152816 [Patent Document 3] Patent Publication No. 2001-149033 [Patent Document 4] Patent Publication No. 2005-080627 [Patent Document 5] Patent Publication No. 2011-36206 [Non-patent literature]

[0008] [Non-Patent Document 1] Agricultural Technology Management Journal 25(4) 93-97 (2019) [Non-Patent Document 2] Journal of the Japanese Society of Fisheries Science 86(3) 196-203 (2020) [Non-Patent Document 3] Yamaguchi Prefectural Industrial Technology Center Technical Research Report 24 18-20 (2012) Summary of the Invention [Problem to be solved by the invention]

[0009] The object of the present invention is to provide a method for producing a fermented food composition by microbial fermentation of dried fish extract residue, which is a by-product of food production, and thereby enabling the entire amount to be utilized as a food ingredient without waste. Another object of the present invention is to provide a method for producing a fermented food composition with improved taste by microbial fermentation, and further, a method for producing a fermented food composition with added value that also has various physiological activities. [Means for solving the problem]

[0010] As a result of extensive research into the fermentation of dried fish extract residue, the inventors discovered that when fermentation involves the use of xerophilic filamentous fungi belonging to the genus Aspergillus, which secrete high amounts of proteolytic enzymes, it is possible to produce a fermentation product containing soluble amino acids and peptides that exhibit flavors such as umami and various physiological activities, and thus completed the present invention.

[0011] That is, the present invention provides: [1] A process for extracting stock from fish flakes; Separating a fish flakes extraction residue from the mixture of fish flakes and stock; The present invention provides a method for producing a fermented food composition, which comprises a step of fermenting a residue of dried fish extract using a xerophilic filamentous fungus belonging to the genus Aspergillus.

[0012] Another aspect of the present invention is [2] A step of subjecting a fish flakes extraction residue to a proteolytic enzyme, The present invention provides a method for producing a fermented food composition according to the above-mentioned [1], wherein the protease is at least one of an endo-type peptidase and an exo-type peptidase derived from a xerophilic filamentous fungus belonging to the genus Aspergillus.

[0013] Another aspect of the present invention is [3] A step of subjecting a fish flakes extraction residue to a proteolytic enzyme, The protease contains an endopeptidase derived from a xerophilic filamentous fungus belonging to the genus Aspergillus, The present invention provides a method for producing a fermented food composition according to the above item [2], wherein the endopeptidase is two types of serine protease.

[0014] Another aspect of the present invention is [4] A step of subjecting a fish flakes extraction residue to a proteolytic enzyme, the protease contains an exopeptidase derived from a xerophilic filamentous fungus belonging to the genus Aspergillus, The present invention provides a method for producing a fermented food composition according to the above item [2], wherein the exopeptidase is an aminopeptidase.

[0015] Another aspect of the present invention is [5] Provided is a method for producing a fermented food composition according to any one of [1] to [4] above, in which the xerophilic filamentous fungus belonging to the genus Aspergillus has a proteolytic enzyme activity of at least one of serine protease and aminopeptidase of 0.3 units / g of culture or more.

[0016] Another aspect of the present invention is [6] A method for producing a fermented food composition according to any one of [1] to [5] above, wherein the fermentation period using a xerophilic filamentous fungus belonging to the genus Aspergillus is in the range of 1 to 3 weeks.

[0017] Another aspect of the present invention is [7] The present invention provides a method for producing a fermented food composition according to any one of [1] to [6] above, wherein the xerophilic filamentous fungus belonging to the genus Aspergillus is Aspergillus glaucus MA0196, which is deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE AP-02122.

[0018] Another aspect of the present invention is [8] Provided is a method for producing a fermented food composition according to any one of [1] to [7] above, which method includes a production method for a fermented food composition for at least one use selected from the group consisting of fermented food compositions for radical scavenging activity, fermented food compositions for antioxidant activity, fermented food compositions for angiotensin-converting enzyme inhibitory activity, and fermented food compositions for promoting the growth of lactic acid bacteria.

[0019] The present invention also provides [9] A step of extracting stock from the fish flakes; Separating a fish flakes extraction residue from the mixture of fish flakes and stock; The method comprises the step of fermenting a fish extract residue using a xerophilic filamentous fungus belonging to the genus Aspergillus. The present invention provides a method for improving the quality of a fermented food composition, the method comprising at least one method selected from the group consisting of a method for improving the taste of a fermented food composition, a method for improving the radical scavenging activity of a fermented food composition, a method for improving the antioxidant activity of a fermented food composition, a method for improving the angiotensin-converting enzyme inhibitory activity, and a method for improving the growth-promoting activity of lactic acid bacteria. Effect of the Invention

[0020] According to the production method of the present invention, the entire amount of the residue of dried fish extraction, which is a by-product of food production, can be used as a food ingredient without waste. Furthermore, according to the production method of the present invention, it is possible to produce a fermented food composition with improved taste, and furthermore, it is possible to produce a fermented food composition with added value that also has various physiological activities. [Brief description of the drawings]

[0021] [Figure 1] 1 is a graph showing the results of taste measurement using a taste sensor for a fermented food composition obtained by the production method of the present invention. [Diagram 2] 1 is a graph showing the growth-promoting effect of lactic acid bacteria, Lactobacillus acidophilus NBRC13951, when a culture medium for lactic acid bacteria containing a soluble fraction prepared from a fermented food composition was used. [Diagram 3] 1 is a graph showing the growth-promoting effect of lactic acid bacteria, Lactobacillus brevis NBRC107147, when a culture medium for lactic acid bacteria containing a soluble fraction prepared from a fermented food composition is used. [Figure 4] 1 is a graph showing the growth-promoting effect of lactic acid bacteria, Lactobacillus casei NBRC15883, when a culture medium for lactic acid bacteria containing a soluble fraction prepared from a fermented food composition is used. [Diagram 5]1 is a graph showing the growth-promoting effect of lactic acid bacteria, Lactobacillus delbrueckii NBRC3202, when a culture medium for lactic acid bacteria containing a soluble fraction prepared from a fermented food composition is used. [Figure 6] 1 is a graph showing the growth-promoting effect of lactic acid bacteria, Lactobacillus fermentum NBRC15885, when a culture medium for lactic acid bacteria containing a soluble fraction prepared from a fermented food composition was used. [Figure 7] 1 is a graph showing the growth-promoting effect of lactic acid bacteria, Lactobacillus lactis subsp. lactis NBRC 100933, when a culture medium for lactic acid bacteria containing a soluble fraction prepared from a fermented food composition is used. [Figure 8] 1 is a graph showing the growth-promoting effect of lactic acid bacteria, Lactobacillus plantarum NBRC 15891, when a culture medium for lactic acid bacteria containing a soluble fraction prepared from a fermented food composition is used. [Figure 9] 1 is a graph showing the results of ultraviolet absorption analysis (280 nm) illustrating the distribution of polypeptides contained in a soluble fraction prepared from a fermented food composition. [Figure 10] 1 is a graph showing the amino acid and peptide amounts (ninhydrin method, converted into glycine) of the soluble fraction prepared from the fermented food composition. [Figure 11] 1 is a graph showing the results of a radical scavenging test for a soluble fraction prepared from a fermented food composition. [Figure 12] 1 is a graph showing the results of a radical scavenging test for a soluble fraction prepared from a fermented food composition. [Figure 13] 1 is a graph showing the results of an antioxidant power test for a soluble fraction prepared from a fermented food composition. [Figure 14] 1 is a graph showing the results of an angiotensin-converting enzyme inhibitory activity test for soluble fractions prepared from fermented food compositions. [Figure 15]1 is a graph showing the results of size exclusion chromatography of the molecular weight distribution of components separated from a soluble fraction prepared from a fermented food composition. [Figure 16] 1 is a graph showing the results of reverse phase chromatography analysis of components separated from a soluble fraction prepared from a fermented food composition. [Figure 17] 1 is a graph showing the analysis results of fraction number 18 by reverse phase chromatography. [Figure 18] 1 is a graph showing the analysis results of fraction number 25 by reverse phase chromatography. [Figure 19] 1 is a graph showing the analysis results of fraction number 30 by reverse phase chromatography. [Figure 20] 1 is a graph showing the analysis results of fraction number 47 by reverse phase chromatography. [Figure 21] 1 is a graph showing the analysis results of fraction number 83 by reverse phase chromatography. [Figure 22] This is a photograph in place of a drawing showing the results of electrophoresis to investigate whether the crude enzyme solution is involved in the decomposition of proteins derived from bonito meat. [Figure 23] 1 is a graph showing the results of separation of the crude enzyme solution after dialysis by column chromatography under weakly acidic conditions. [Figure 24] 1 is a photograph in place of a drawing showing the results of electrophoresis of purified protease. [Diagram 25] 1 is a graph showing the results of separation of the crude enzyme solution after dialysis by column chromatography under neutral conditions. [Figure 26] 1 is a photograph in place of a drawing showing the results of electrophoresis of purified protease. [Figure 27] 1 is a photograph of electrophoresis showing the results of decomposition of bonito meat-derived protein using purified protease. [Figure 28] 1 is a photograph of electrophoresis showing the results of decomposition of bonito meat-derived protein using purified protease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The method for producing the fermented food composition of the present invention includes a fermentation step using dried fish extract residue and a xerophilic filamentous fungus of the genus Aspergillus.

[0023] The dried fish extract residue used in the present invention refers to the residue obtained by extracting soup stock from dried fish shavings produced from dried fish such as rough fish and dried fish, and refers to a food manufacturing by-product also known as soup stock residue. As the raw material of the dried fish used in the present invention, for example, one or more kinds of fish such as bonito, mackerel, tuna, and sardine can be used. As the raw material of the dried fish used in the present invention, for example, bonito is preferable, and as the dried fish, dried bonito is more preferable.

[0024] The process for extracting soup stock from dried fish shavings is not particularly limited as long as it is within the scope of what is normally carried out in daily life. For example, use 20g to 60g of dried bonito flakes for 1kg of boiling water. When the water boils, stop heating and add the dried bonito flakes. The bonito flakes will sink in about 1 to 10 minutes, then separate them. There is no limitation on the method for separating the dried bonito flakes from the stock. For example, one or more of the following methods may be used: a method for separating by filtration using a filter cloth, filter paper, etc., and a method for separating by centrifugation. By the above operation, a fish flakes extraction residue is obtained.

[0025] It is preferable to dry the residue of the dried fish extract. There is no limitation on the method for drying the residue of dried fish extract, and one or more of the following methods can be used: drying in the sun, placing in a heating oven and heating while circulating air, placing in a container and reducing the pressure.

[0026] In the fermentation step included in the production method of the present invention, it is preferable that the moisture content of the dried fish extract residue at the start of fermentation is low, and more preferably the water activity is in the range of 0.8 or less, since this can reduce the possibility of contamination by other microorganisms.

[0027] The xerophilic Aspergillus filamentous fungi used in the fermentation process are a group of filamentous fungi that can grow under the above-mentioned low water activity conditions. For example, the fungi include one or more of Aspergillus amstelodami, Aspergillus chevalieri, Aspergillus pseudoglaucus, Aspergillus ruber, Aspergillus sydowii, etc., which live on the surface of dried fish such as bonito flakes during fermentation and maturation.

[0028] The xerophilic filamentous fungus of the genus Aspergillus used in the fermentation step is preferably one that is particularly excellent in productivity of proteolytic enzymes. The xerophilic Aspergillus filamentous fungus used in the fermentation process is preferably a filamentous fungus that not only secretes components obtained by decomposing proteins but also secretes at least one of endo-type peptidase and exo-type peptidase, and more preferably a filamentous fungus that secretes both.

[0029] Furthermore, it goes without saying that the xerophilic Aspergillus filamentous fungus used in the present invention is a filamentous fungal species that has been shown not to produce mycotoxins (Takenaka et al. Identification and characterization of extracellular enzymes secreted by Aspergillus spp. involved in lipolysis and lipid-antioxidation during katsuobushi fermentation and ripening. Int. J. Food Microbiol. 353:109299 (2021).).

[0030] The temperature range for the fermentation step is usually 15 to 45°C, preferably 25 to 35°C, and more preferably 28 to 32°C. The fermentation time range is preferably from 1 week to 3 weeks.

[0031] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples in any way. EXAMPLES

[0032] The Aspergillus sydowii MA0196 used in this example is the same as the Aspergillus glaucus MA0196 deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE AP-02122.

[0033] [Production of fermented food composition derived from bonito extract residue] The residue of dried bonito extract generated after making soup stock from dried bonito flakes (shaved bonito) was dried in a constant temperature incubator at 70°C for 48 hours until the moisture content was 2.7%. The dried bonito extract residue may be dried in an incubator without a blower function, or may be dried in the sun without using a dryer, and it is preferable to dry it to a moisture content of 3.0% or less.

[0034] The dried residue was mixed with ion-exchanged water in an Erlenmeyer flask (5 g residue / 50 ml Erlenmeyer flask to 50 g residue / 500 ml Erlenmeyer flask), sterilized in an autoclave at 120°C for 20 minutes, and then cooled to room temperature. To this, a spore suspension of Aspergillus sydowii MA0196 (2.5 × 10 6 The culture was then statically cultured for 2 to 3 weeks in an incubator maintained at a temperature of 28 to 30° C. and a humidity of 75 to 85° C. to obtain a fermented food composition. The amount of ion-exchanged water added to the dried material was in the range of 0.75 volume (g / g). The range is preferably 0.5 to 1 volume (g / g). The concentration of the spore suspension was 1.0 to 5.0 × 10 6 It is preferable to work in the range of spores / ml.

[0035] The fermented food composition obtained by the above method was subjected to component analysis (free amino acid content, soluble polypeptide content, amino acid composition, taste). The sample was prepared based on the bonito stock extraction method: 6 g of the culture and about 200 ml of distilled water heated to 90°C were added to a 500 ml stainless steel mug, sealed, and left to stand for 3 minutes. Next, the mixture was suction filtered using a Buchner funnel equipped with filter paper (Advantec No. 2), and the resulting filtrate (hereinafter referred to as the soluble fraction) was adjusted to a constant volume of 200 ml and used as a sample for component analysis.

[0036] The amounts of free amino acids and soluble polypeptides in the soluble fraction were determined by the ninhydrin method (based on the amount of glycine; Sun et al., Efficiency improvements on ninhydrin method for amino acid quantification. J. Food Comp. Anal., 19(2-3): 112-117 (2006)) and the Lowry method (based on the amount of Bovain serum albumin (BSA); Lowry et al., Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265275 (1951).).

[0037] The composition of free amino acids was determined by HPLC analysis using the ninhydrin post-column derivatization method. In addition, taste measurements were made using a taste sensor (taste recognition device) such as the SA402B manufactured by Intelligent Sensor Technology Co., Ltd. Based on previously reported methods, five types of taste sensors were used - CA0 (sourness), CT0 (saltiness), C00 (bitterness / off-flavors), AE1 (astringent stimulation), and AAE (umami) - to measure a total of eight taste items: the initial tastes of sourness, bitterness / off-flavors, astringent stimulation, umami, and saltiness, and the aftertastes of bitterness, astringency, and umami richness.

[0038] The results obtained by the above methods are shown in Table 1 (total amount of free amino acids and total amount of soluble protein), Table 2 (amino acid composition, etc.) and Figure 1 (taste measurement results). The soluble fraction prepared from the culture obtained by inoculating and culturing Aspergillus sydowii MA0196 showed significantly increased amounts of free amino acids and soluble polypeptides compared to the uninoculated control. In addition, the soluble fraction prepared from the culture obtained by culturing Aspergillus oryzae RIB40 under the same culture conditions showed no significant difference from the control not inoculated with the filamentous fungus (Table 1). Therefore, it was demonstrated that xerophilic filamentous fungi such as Aspergillus sydowii MA0196 are suitable for fermenting dried bonito extract residue. When converted to 1 g of culture, the amount of amino acids was 0.12 mmol / culture (g), and the amount of soluble protein was 1.55 mg / culture (g).

[0039] [Table 1]

[0040] [Table 2] The results in Table 2 confirm that the soluble fraction contains large amounts of aspartic acid and glutamic acid, which are related to umami taste.

[0041] As shown in Figure 1, the change in membrane potential for bitterness, off-flavors, saltiness, and umami tastes was greater than that of the control, and these tastes tended to be enhanced. On the other hand, the change in sourness was smaller.

[0042] The soluble fraction prepared from the culture of Aspergillus sydowii MA0196 was examined for radical scavenging activity, antioxidant potential, angiotensin converting enzyme inhibitory activity, and growth promotion effect on lactic acid bacteria.

[0043] a) ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) scavenging activity The method of Malunga & Beta was followed (Malunga and Beta. Antioxidant capacity of arabinoxylan oligosaccharide fractions prepared from wheat aleurone using Trichoderma viride or Neocallimastix patriciarum xylanase. Food Chem. 167:311―319 (2015).). Briefly, the ABTS radical stock solution was prepared by adding 2.45 mM potassium peroxodisulfate to 7 mM ABTS aqueous solution at a 1:1 (v / v) ratio and leaving it at room temperature in the dark for 12-16 hours. For the measurement of ABS radical scavenging activity, this stock solution was diluted with deionized water to an absorbance of approximately 0.7 at 734 nm. The sample (50 μl) was added to the ABTS radical solution (850 μl), and the mixture was left at room temperature in the dark for 30 minutes, after which the absorbance at 734 nm was measured. Standard Trolox solutions were prepared at 0.05 to 0.4 μM. The sample solution was diluted 2-fold with ethanol to make a 50% ethanol solution. The ABTS radical scavenging activity was calculated as Trolox equivalent (μmol / L) using a calibration curve prepared using Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Tokyo Chemical Industry Co., Ltd., Tokyo) as the standard substance.

[0044] b) DPPH (2,2-Diphenyl―1-picrylhydrazyl) scavenging activity The method of Veenashri & Muralikrishna was followed (Veenashri and Muralikrishna. In vitro anti-oxidant activity of xylo-oligosaccharides derived from cereal and millet brans -A comparative study. Food Chem. 126:1475-1481(2011).). 50 μl of 200 mM MES-NaOH buffer (pH 6.0) and 50 μl of 0.8 mM DPPH solution were added to 100 μl of sample or standard Trolox solution (50% (v / v) ethanol solution), and the mixture was incubated at room temperature for 20 minutes, after which the absorbance at 520 nm was measured. Standard Trolox solutions were prepared at 0.08 to 0.8 μM. Sample solutions were diluted 2-fold with ethanol to make 50% ethanol solutions. Scavenging activity was calculated as Trolox equivalent (μmol / l).

[0045] c) ferric ion reducing antioxidant power (FRAP) The method of Benzie & Strain was followed (Benzie & Strain. The ferric reducing ability of plasma (FRAP) as a measurement “antioxidant power”: The FRAP assay. Anal. Biochem. 239:70-76(1996).). Fe 3+ From Fe 2+ The antioxidant power was measured using a reduction reaction to The FRAP reagent was prepared by mixing 10 mM FRAP, 40 mM HCl solution, 300 mM sodium acetate buffer (pH 3.6), and 20 mM iron (III) chloride aqueous solution in a ratio of 10:1:1 (v / v / v). 50 μl of sample or standard Trolox solution (50% (v / v) ethanol solution) was added to 1.5 ml of FRAP reagent, left at room temperature for 3 minutes, and then the absorbance at 593 nm was measured. Standard Trolox solutions were prepared at 0.08 to 0.8 μM. Sample solutions were diluted 2-fold with ethanol to prepare 50% ethanol solutions. Standard Trolox solutions were prepared at 0.08 to 0.8 μM. Sample solutions were diluted 2-fold with ethanol to prepare 50% ethanol solutions. Scavenging activity was calculated as Trolox equivalent (μmol / l).

[0046] d) Angiotensin Converting Enzyme (ACE) inhibitory activity The procedure was carried out according to the ACE inhibitory activity kit manufactured by Dojin Glocal Co., Ltd. and the accompanying instruction manual, and the ACE inhibitory activity value (inhibition rate %) and the sample amount equivalent to 50% inhibition rate (peptide amount converted into glycine concentration) were calculated.

[0047] Compared with the control, the soluble fraction exhibited significant radical scavenging activity and ferric reducing power activity (Table 3). [Table 3] The angiotensin-converting enzyme inhibitory activity (IC50, converted into glycine concentration (mM)) of the soluble fraction was 0.0421 mM.

[0048] The growth-promoting effect of the soluble fraction on lactic acid bacteria was examined. The following seven strains of lactic acid bacteria were used in the test. These strains were purchased from the National Institute of Technology and Evaluation. Lactobacillus acidophilus NBRC13951 (Figure 2), Lactobacillus brevis NBRC107147 (Figure 3), Lactobacillus casei NBRC15883 (Figure 4), Lactobacillus delbrueckii NBRC3202 (Figure 5), Lactobacillus fermentum NBRC15885 (Figure 6), Lactobacillus lactis subsp. lactis NBRC 100933 (Figure 7) and Lactobacillus plantarum NBRC 15891 (Figure 8). The test medium was based on MRS agar medium (MRSagar: deMan, Rogosa, and Sharpeagar). In other words, a liquid medium consisting of yeast extract (5.5 g / L), peptone (12.5 g / L), glucose (11.0 g / L), dipotassium hydrogen phosphate (0.25 g / L), potassium monohydrogen phosphate (0.25 g / L), sodium acetate (10.0 g / L), magnesium sulfate (0.1 g / L), manganese sulfate (5.0 mg / L), and ferrous sulfate (5.0 mg / L) was used.

[0049] After adding 1 / 10th the amount (v / v) of the soluble fraction to the test medium, the lactic acid bacteria test strain was inoculated and cultured at 30°C in an Anaeropack square jar containing an Anaeropack® Kenki pouch. The growth amount (OD 600 In addition, a medium in which one-tenth the amount (v / v) of distilled water was added to the test medium was also transplanted and cultured in the same manner, and the growth amount was compared with that of the control to examine the effect of the soluble fraction on the growth of lactic acid bacteria.

[0050] Of the seven test strains, the growth-promoting effect of the soluble fraction was observed in Lactobacillus acidophilus NBRC13951 (Figure 2), Lactobacillus casei NBRC15883 (Figure 4), Lactobacillus delbrueckii NBRC3202 (Figure 5), and Lactobacillus lactis subsp. lactis NBRC 100933 (Figure 7).

[0051] The growth-promoting effect on lactic acid bacteria is expected to improve the human intestinal flora. In addition, the soluble fraction obtained from the culture can be used as an additive for promoting lactic acid fermentation and the growth of lactic acid bacteria. Furthermore, the fermented composition can be used as a functional food.

[0052] The soluble fraction (100 ml) was concentrated to 10 ml, and insoluble matter was removed using a centrifugal filter membrane. 2 ml of the concentrate that passed through was separated using a column (2.5 x 50 cm) packed with Toyopearl HW-40 to separate peptides. Soluble peptides were eluted using 10 mM ammonium acetate solution at a flow rate of 20 ml / h. The obtained fractions (5 ml / tube) were examined for polypeptide content (UV 280 nm), amino acid and peptide content (ninhydrin method, converted to glycine), ABTS, DPPH, FRAP, and angiotensin-converting enzyme inhibitory activity.

[0053] Absorption at 280 nm due to the side chains of aromatic amino acids (tyrosine and tryptophan) was observed in fractions 18, 25, 30, 47, and 83 (FIG. 9). On the other hand, the ninhydrin reaction revealed that oligopeptides and amino acids were contained around fraction number 30 (Figure 10). ABTS, DPPH, FRAP, and angiotensin-converting enzyme inhibitory activities were also observed for these fractions (Figures 11 to 14, respectively). 11 and 12 show the antioxidant titer in terms of Trolox amount, and FIG. 13 shows the iron reduction / antioxidant titer in terms of Trolox amount.

[0054] Fractions 18, 25, 30, 47 and 83, which showed biological activity, were examined by size exclusion chromatography for the molecular weight distribution of peptides contained in the fractions. 10 μl of the solution was applied to a YMC-Pack Diol-60 (4.6 × 300 nm) (YMC, Kyoto) equilibrated with an eluent (100 mM KH2PO4-K2HPO4 (pH 7.4)-acetonitrile (80:20, v / v)), and the peptides were eluted at a flow rate of 0.25 ml / min and a detection wavelength of 216 nm. In addition, standard proteins and peptides Myoglobin (molecular weight, 17,000 Da; elution time 8.93 min), Cytochrome C (12,400 Da; 9.64 min), Bacitracin (1,422 Da; 11.83 min), N-Hippuryl His Leu (429 Da; 12.83 min), and Glycylglycylglycine (189 Da; 13.55 min) were also analyzed under the same conditions.

[0055] Based on the elution times of standard proteins, it was found that the soluble fraction contained various polypeptides, oligopeptides and amino acids with molecular weights of 12,400 Da or less (reference number 17 in FIG. 15). On the other hand, the control also contained various peptides and amino acids with molecular weights of 1,422 Da or less, but in small amounts (reference number 18 in FIG. 15). The reference symbols M, C, B, H and G in Figure 15 are abbreviations of standard proteins and peptides for molecular weight indication, and respectively represent myoglobin, cytochrome c, bacitracin, N-hippuryl His-Leu hydrate and glycylglycylglycine. The same applies to the following cases:

[0056] Fraction numbers 18, 25, 30 and 83 were found to contain peptides and amino acids with molecular weights of approximately 12,400 Da, approximately 1,422 Da, approximately 429 Da and less than 189 Da, respectively (Figure 16).

[0057] Fractions 18, 25, 30 and 83 were subjected to peptide mapping using reverse phase chromatography. A C18 column COSMOSIL 5C18-AR-II (4.6×250 mm, manufactured by Nacalai Tesque) was connected to the liquid chromatography apparatus. Mobile phase A was 5 mM TFA / water, mobile phase B was 5 mM TFA / acetonitrile, and the gradient was 1 to 40% mobile phase B concentration per 75 min. The flow rate was 0.5 mL / min, and the change in absorbance at UV 216 nm was examined (Figures 17 to 21). Fraction 18 contained almost no peptides of a particular chain length, while fractions 25 and 30 contained five to six types of peptides. Fraction 83 was predicted to consist of one type of peptide.

[0058] The amounts of free amino acids and soluble polypeptides in the culture obtained by inoculating and culturing Aspergillus sydowii MA0196 were significantly increased, so the activities of exo-type peptidases secreted by this bacterium were examined according to a previously reported method (Blinkovsky et al. A non-specific aminopeptidase from Aspergillus. Biochim. Biophys. Acta. 1480, 171181 (2000)).

[0059] Peptidase activity was measured using leucyl-p-nitroanilide, and the amount of p-nitroaniline released upon hydrolysis was measured as the increase in absorbance at A405.

[0060] On the other hand, to measure the activity of endo-type peptidase, hemoglobin (acidic conditions) and casein (neutral conditions) were used, and the enzyme activity was determined by quantifying peptides containing tyrosine residues released upon hydrolysis using Folin-Ciocalteu reagent, and calculating the amount produced in terms of tyrosine (Takenaka et al. Isolation and characterization of an aspartic protease able to hydrolyze and decolorize heme proteins from Aspergillus glaucus. 99, 2042-2047 (2019)).

[0061] The crude enzyme solution was prepared according to a previously reported method. That is, 30 ml of 20 mM sodium acetate buffer (pH 5.0) was added to 5 g of the culture ( / 50 ml Erlenmeyer flask) and shaken at room temperature for 3 hours. Then, the mixture was suction filtered using a Buchner funnel with filter paper (Advantec No. 2) placed on it, and the resulting filtrate (crude enzyme solution) was used for enzyme activity measurement.

[0062] Using the above method, the activity against leucyl-p-nitroanilide was 0.043±0.002 U / ml, and the activities against hemoglobin and casein were 0.050±0.004 U / ml and 0.080±0.004 U / ml, respectively. Therefore, it is believed that exo- and endo-peptidases that show hydrolytic activity against these substrates are involved in the hydrolysis of proteins derived from bonito meat.

[0063] Next, we investigated whether the crude enzyme solution was involved in the decomposition of proteins derived from bonito meat. First, soluble proteins were extracted from dried bonito powder (arabushi powder) as follows. 5 g of dried bonito powder was placed in a 100 mL beaker, 50 mL of 0.1 M KCl-20 mM Tris-HCl buffer (pH 7.5) was added, and the mixture was stirred at room temperature for 1 hour. The effluent (soluble protein solution) was collected using a Buchner funnel. Enzyme solution (50 microL) was added to the soluble protein solution (500 microL), and the reaction was carried out at 30°C. The degree of degradation was determined by comparing the electrophoretic patterns. The reaction solution (50 microL) was mixed with denaturing buffer for SDS-PAGE (50 microL), and then incubated at 100°C for 3 minutes. 10 microL of the same sample was subjected to SDS-PAGE. Electrophoresis was performed using an AE-6530 Rapidus mini-slab electrophoresis tank manufactured by ATTO Corporation, the ready-made gel e-PAGEL E-T12.5L, and the electrophoresis buffer for SDS-PAGE AE-1410, and proteins were detected using AE-1340 EzStain Aqua CBB staining solution for protein detection.

[0064] The results of electrophoresis performed according to the above method are shown (Figures 22(a) and (b)). The crude enzyme solution was dialyzed against (a) 20 mM Tris-HCl buffer (pH 7.5) or (b) 20 mM sodium acetate buffer (pH 5.0), and the resulting solution was used to degrade soluble proteins derived from bonito meat. The electrophoretic patterns revealed that the crude enzyme solution treated at either pH degrades the soluble proteins. Because 0.1M KCl-20 mM Tris-HCl buffer (pH 7.5), which contains salt, was used to extract proteins derived from bonito meat, it is expected that myofibrillar proteins such as actin and myosin, and sarcoplasmic proteins (muscle-forming proteins) including myoglobin and hemoglobin were extracted, and it is believed that the crude enzyme solution can degrade these proteins derived from bonito meat. In addition, in Fig. 22, the reference symbol BM means broad range protein marker, and the reference symbol LM means low range protein marker. The same applies to the following cases.

[0065] Based on the above results, we decided to identify the protease by purifying the protease contained in the crude enzyme solution and identifying its amino acid sequence. Enzyme purification was performed under conditions of 20 mM sodium acetate buffer (pH 5.0) and 20 mM Tris-HCl buffer (pH 7.5).

[0066] [Purification of protease under weakly acidic conditions] All chromatographic supports used in the purification of the enzyme were manufactured by Tosoh Corporation. 1) DEAE-Toyopearl column chromatography The crude enzyme solution was dialyzed against 20 mM sodium acetate buffer (pH 5.0). The crude enzyme solution after dialysis was designated as fraction 1. The fraction was applied to a DEAE-Toyopearl column (1.5 x 20 cm) equilibrated with the same buffer, and the protein was eluted / fractionated (6 ml / tube) using a linear gradient method (dotted line in Figure 23) with a salt concentration of 0 to 0.3 M NaCl-20 mM sodium acetate buffer (pH 5.0) at a flow rate of 1 ml / min. When the enzyme activity of each fraction was measured using hemoglobin as a substrate, two activity peaks were found (Figure 23). These are designated as fractions 5Pro1-2 and 5Pro2-2 in order of elution.

[0067] 2) Toyopearl GigaCapQ column chromatography Fraction 5Pro1-2 was dialyzed against 1 liter of 20 mM sodium acetate buffer (pH 5.0) for 6 hours in the first round and for 12 hours in the second round. The solution was applied to a Toyopearl GigaCap Q column (1.5 × 20 cm) equilibrated with the above buffer, and the protein was eluted / fractionated (6 ml / tube) using a linear gradient method with a salt concentration ranging from 0 to 0.5 M NaCl-20 mM sodium acetate buffer (pH 5.0) at a flow rate of 1 ml / min. The protease activity of each fraction was measured, and the highly active fraction was pooled and designated as fraction 5Pro1-3.

[0068] 3) Toyopearl HW-55F Fraction 3-1 was concentrated by centrifugal filtration and separated using a column (2.5 x 50 cm) packed with Toyopearl HW-55. Proteins were eluted / fractionated (3.5 ml / tube) with 0.2 M NaCl-20 mM sodium acetate buffer (pH 5.0) at a flow rate of 0.35 ml / min. Protease activity in each fraction was measured, and the highly active fractions were collected. This was named 5Pro1-4.

[0069] 4) Butyl-Toyopearl column chromatography Ammonium sulfate was added to 5Pro2-2 to a final concentration of 2M. The mixture was then applied to a Butyl-Toyopearl column (1.5 x 20 cm) equilibrated with 2M ammonium sulfate-20 mM sodium acetate buffer (pH 5.0), and the protein was eluted / fractionated (6 ml / tube) at a flow rate of 1 ml / min using a linear gradient method in which the ammonium sulfate concentration was changed from 2M to 0M-20 mM sodium acetate buffer (pH 5.0). The protease activity of each fraction was measured, and the highly active fractions were collected. This fraction was named 5Pro2-5. The proteases (5Pro1 and 5Pro2) contained in fractions 5Pro1-2 and 5Pro2-2 could be purified into single proteases by the above column chromatography (Figure 24). The total activity, total protein amount, specific activity, etc. at each purification step are summarized (Table 4).

[0070] [Table 4]

[0071] [Purification of proteolytic enzymes under neutral conditions] 1) DEAE-Toyopearl column chromatography The crude enzyme solution was dialyzed against 20 mM Tris-HCl buffer (pH 7.5). The crude enzyme solution after dialysis was designated as fraction 1. It was applied to a DEAE-Toyopearl column (1.5 x 20 cm) equilibrated with the same buffer, and the protein was eluted / fractionated (6 ml / tube) at a flow rate of 1 ml / min using a linear gradient method with salt concentrations ranging from 0 to 0.3 M NaCl-20 mM Tris-HCl buffer (pH 7.5). Enzyme activity was measured for each fraction using casein as a substrate, and one activity peak was found. This was designated as fraction 2.

[0072] 2) Butyl-Toyopearl column chromatography Ammonium sulfate was added to fraction 2 to a final concentration of 2 M. The mixture was then applied to a Butyl-Toyopearl column (1.5 x 20 cm) equilibrated with 2 M ammonium sulfate-20 mM Tris-HCl buffer (pH 7.5), and the protein was eluted / fractionated (6 ml / tube) using a linear gradient method in which the ammonium sulfate concentration was changed from 2 M to 0 M-20 mM Tris-HCl buffer (pH 7.5) at a flow rate of 1 ml / min. The protease activity of each fraction was measured, and the highly active fractions were collected. When the enzyme activity of each fraction was measured using casein as a substrate, two activity peaks were found (Figure 25). The proteases contained in fractions 7Pro3-1 and 7Pro3-2 in the order of elution were single (7Pro1 and 7Pro2) (Figure 26). The total activity, total protein amount, specific activity, etc. at each purification step are summarized (Table 5).

[0073] [Table 5]

[0074] The activity of the purified proteases (5Pro1, 5Pro2, 7Pro1 and 7Pro2) in decomposing bonito muscle protein was examined. FIG. 27(a) shows a reaction system to which 5Pro1 was added, and FIG. 27(b) shows a reaction system to which 5Pro2 was added. FIG. 28(a) shows a reaction system to which 7Pro1 was added, and FIG. 28(b) shows a reaction system to which 7Pro2 was added.

[0075] 5Pro1 showed almost no degradation (Figure 27(a)), while degradation was confirmed for 5Pro2, 7Pro1 and 7Pro2 (Figure 28(b), Figures 22(a) and (b)). Therefore, it is believed that 5Pro1 cooperates with 5Pro2, 7Pro1 and 7Pro2 to promote the degradation of proteins derived from bonito meat.

[0076] The purified proteases (5Pro1, 5Pro2, 7Pro1 and 7Pro2) were subjected to SDS-PAGE electrophoresis, and each band was excised. The amino acid sequence of each protease was analyzed according to the method of Takahata et al. (Takahata et al. Close proximity of phosphorylation sites to ligand in the phosphoproteome of the extreme thermophile Thermus thermophilus HB8. Proteomics 12, 14141430 (2012).), and peptides were prepared by in-gel digestion with trypsin, and the peptide sequences were identified by LCMSMS-Mascot analysis.

[0077] The identification results and genetic analysis of the MA0196 strain revealed that 5Pro2 and 7Pro1 were identical.

[0078] Using the obtained array information as a reference, the genes 5Pro1 (7Pro1), 5Pro2, and 7Pro2 were cloned with the genomic DNA and cDNA of strain MA0196 as templates. The extraction and purification of genomic DNA from strain MA0196, and the extraction of total RNA and preparation of cDNA were carried out according to the previously reported methods (Takenaka et al. Identification and characterization of extracellular enzymes secreted by Aspergillus spp. involved in lipolysis and lipid―antioxidation during katsuobushi fermentation and ripening. Int. J. Food Microbiol. 353:109299 (2021)). Also, the PCR primers for gene cloning were as shown in Table 6. PCR was performed using TaKaRa PrimeSTAR MAX DNA polymerase, and the reaction solution composition and gene fragment amplification conditions were in accordance with the methods described in the instruction manual.

[0079] Sequence Listing Free Text

[0080]

Table 6

[0081] The sequences of 5Pro1, 5Pro2 (7Pro1), and 7Pro2 are as follows.

[0082] Base and amino acid sequences of 5Pro1 (aminopeptidase) 5Pro_1_from Aspergillus sydowii MA0196

[0083] Sequence Listing Free Text 5Pro_1_from Aspergillus sydowii MA0196 <Notation consisting of Coding region and non-coding region> 10 20 30 40 50 60 ATGTGTGGTTCTCGTCGTGCTGGGGTCTCTGGTTCGACCAACGTTCCTGACCGTGAGGTT M C G S R R A G V S G S T N V P D R E V 70 80 90 100 110 120 CTCCCCACCAACGTTAAGCCAACCCACTATGACTTGACTCTTGAGCCGAACTTTGAGACC L P T N V K P T H Y D L T L E P N F E T 130 140 150 160 170 180 TTTAAATATGACGGGACTGTCGTTATCGAGtatgtagcactacggacgttctgtggccga F K Y D G T V V I D 190 200 210 220 230 240 tcccaaatcggatcttgaagctgatagttccgtgtagcTTACAGGTTGTCGAGGACACGA L Q V V E D T T 250 260 270 280 290 300 CATCCATCTCGCTCAACTCTACTGAAATCAACATCCACACAGCTACCGTGTCCTCTCAAG S I S L N S T E I N I H T A T V S S Q G 310 320 330 340 350 360 GATCAGTAGTCAGCGCGTCACCTGAGATTTCCCTCAACGATGACAAGCAAGAGGCTACAA <h2 style=";text-align:left;direction:ltr">SVVSASPEISLNDDKQEATI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 370 380 390 400 410 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TCAAGTTTCGGATGTTATCCGCACCGGAACATCCGCTCAACTTAAGCTGACCTTCACCG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> KFSDVIRTGTSAQLKLTFTG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 430 440 450 460 470 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GTACTCTCAATGACAACATGGCCGGTTTCTACCGCTCATCTTACAAGACCGTCGGACGGCG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TLNDNMAGFYRSSYKTSDGE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 490 500 510 520 530 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AGACCAAATACATTGCGGTCAACTCAAATGGAGCGACCGATTGCCGGAGAGCTTTCCCTT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TKYIASTQMEPTDCRRAFPC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 550 560 570 580 590 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GCTTCGAACGAGCCTGCGCTCAAGGCTAAATTTACTGTCAAACTGATTGCGGACAAGAGCA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FDEPALKAKFTVKLIADKSM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 610 620 630 640 650 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TGACTTGTTTTGAGCAACATGGACGTTGCATCCGAAGAGGATATTGATGGTGGGAAGAAAA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TCLSNMDVASEEDIDGGKKI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 670 680 690 700 710 720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TTGTCAAGTTCAATGAGTCTCCGGTCATGTTCTACCTACCTCGTGGCTTTCATTGTCGGCC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VKFNESPVMSTYLVAFIVGH<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 730 740 750 760 770 780<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ACCTGAAGTACATTGGACCAAGGACTTCCGTGTCCCTATCCGTGGTCTATGCTACACCAG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LKYIETKDFRVPIRVYATPD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 790 800 810 820 830 840<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ATCAGGACATTGAACATGGCCGTTTCTCCCTGAACTTGGCGGCCAAGACGCTTGCCTTCT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> QDIEHGRFSLNLAAKTLAFY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 850 860 870 880 890 900<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ACGAAAAGGCTTTCGATAACGAATTCCCTCTTCCAAAGATGGACATGGTTGCGGTCCCCG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EKAFDNEFPLPKMDMVAVPD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 910 920 930 940 950 960<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ATTTCAGTGCTGGAGCCATGGAGAACTGGGGTTTGATCACCTACCGTATCCGTTGATGTGC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FSAGAMENWGLITYRIVDVL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 970 980 990 1000 1010 1020<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">TCCTGGATGAGAAGACAAGCAGTGCATCGCGGAAGGAGCGCATTGCTGAAACTGTTCAGC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LDEKTSSASRKERIAETVQH<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1030 1040 1050 1060 1070 1080<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ACGAGCTTGCCCACCAGTGGTTTGGTAACTTGGTTACCATGGATTTCTGGGACGGTCTAT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ELAHQWFGNLVTMDFWDGLW<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1090 1100 1110 1120 1130 1140<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GGCTGAACGAAGGCTTTGCTACTTGGATGTCGTGGTATTCTTGCAACACCTTCTACCCCG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LNEGFATWMSWYSCNTFYPE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1150 1160 1170 1180 1190 1200<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AATGGAAGGTCTGGCAAACATATGTCATCGACAACCTGCAAAGTGCGCTCTCCCTCGATT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> WKVWQTYVIDNLQSALSLDS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1210 1220 1230 1240 1250 1260<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> CTCTCCGCAGCAGTCACCCCATCGAAGTGCCCGTGAAGCGCGCGGACGAGATAAACCAGA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LRSSHPIEVPVKRADEINQI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1270 1280 1290 1300 1310 1320<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">TATTTGATGCCATCTCCTACTCCAAGGGTTCTTCTGTCCTGCGCATGATCTCAAAGTATC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FDAISYSKGSSVLRMISKYL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1330 1340 1350 1360 1370 1380<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TGGGTGAAGACACCTTCCTGCAGGGCGTTCGTAATTACATTAAGAAGCACGCTTACGGCA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GEDTFLQGVRNYIKKHAYGN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1390 1400 1410 1420 1430 1440<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ACACACAAACCGGTGACCTGTGGTCCGCCCTTGCTGGGTGCTAGTGGCAAGCCCGTGGAGG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TQTGDLWSALAGASGKPVEE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1450 1460 1470 1480 1490 1500<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AGGTCATGGATATCTGGACCAAGAATGTTGGATTCCCAGTCGTCTCTGTGTCGGAAAACC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VMDIWTKNVGFPVVSVSENP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1510 1520 1530 1540 1550 1560<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> CAAGCTCCCTCATCCATCAAGGTGAAACAGAATCGATTTCTCCGCACAGGCGACGTTCGAC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SSSSIKVKQNRFLRTGDVRP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1570 1580 1590 1600 1610 1620<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">CCGAGGAAGACACGACTCTCTTCCCTGTGATGCTTGGATTGCGAACAAAGGAGGGGAGTTG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EEDTTLFPVMLGLRTKEGVD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1630 1640 1650 1660 1670 1680<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ACGAAAACACCCTGCTGTCATCGCGCGAGGAAGAGTTCAAGGTTCCCGATCTCGATTTT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ENTLLSSREEEFKVPDLDFY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1690 1700 1710 1720 1730 1740<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ACAAGCTTAATGCCGATCACACCGCAATTTACCGCACATCCTACACCCCTGAGCGGTTAA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> KLNADHTAIYRTSYTPERLT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1750 1760 1770 1780 1790 1800<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> CCAAGCTGGGCAACGCTGCCAAGGAGGGTCTGCTTACTGTTGAGGACCGCGCGGGTATGA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> KLGNAAKEGLLTVEDRAGMI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1810 1820 1830 1840 1850 1860<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TTGCTGATGCTGGTGCACTGGCTGCTTCTGGTTACCAGAAAACGTCTGGTTTGCTATCGC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ADAGALAASGYQKTSGLLSL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1870 1880 1890 1900 1910 1920<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">TGCTTGCAGGATTTGATGGCGAGCCCGAATTTATTGTTTGGAACGAGATCTTGACTCGTA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LAGFDGEPEFIVWNEILTRI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1930 1940 1950 1960 1970 1980<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TTGGTGCCCTACGTGCCGCTTGGATATTCGAGGATTCCCAGTCCAAGGACGCACTGGAGG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GALRAAWIFEDSQSKDALEA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1990 2000 2010 2020 2030 2040<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> CATTCCAGCGTGCCTTGGTCGGCGAGAAAGCGCAGAAACTGGGATGGGAGTTTTCCGACA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FQRALVGEKAQKLGWEFSDK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2050 2060 2070 2080 2090 2100<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AGGACGACCATATTCTACAACAATTCAAGGCTCTCTTGTTCAGTGCCGCTGGCAATGCTC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DDHILQQFKALLFSAAGNAQ<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2110 2120 2130 2140 2150 2160<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AGGAACCAACTGTAGTCAAGGCCGCCCAGGACATGTTCGAGCGCTTCGCTGCTGGTGATA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EPTVVKAAQDMFERFAAGDI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2170 2180 2190 2200 2210 2220<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">TCAGCGCCATCCACCCTAACATTCGCGGCAGCGTTTTCTCCATCGTCCTGAAAAACGGCG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SAIHPNIRGSVFSIVLKNGG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2230 2240 2250 2260 2270 2280<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GTGAAAGGGAGTACAATGTTGTGTATGACCGCTTCCGCAATGCCCCCACCTCTGATGAGA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EREYNVVYDRFRNAPTSDEK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2290 2300 2310 2320 2330 2340<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AGACCACGGCTCTCCGCTGCCTTGGTACGGCCGAAGATCCGGCCCTCATCAAACGCACTC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TTALRCLGTAEDPALIKRTL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2350 2360 2370 2380 2390 2400<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TCGGCTTGGCCCTGGGTGACGAGGTTAAGAACCAGGACATTTACATGCCGCTTGGCGGAC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GLALGDEVKNQDIYMPLGGL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2410 2420 2430 2440 2450 2460<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TCCGCACTCATGCCGCCGGTATTGAAGCGCGATGGACCTGGGTGAAGGATAACTGGGACA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> RTHAAGIEARWTWVKDNWDT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2470 2480 2490 2500 2510 2520<h2 style=";text-align:left;direction:ltr"> CCCTATACCAGCGCCTGCCCCCCGGATTGGGCATGCTCGGAACTGTCCTACAAATCTGCA L Y Q R L P P G L G M L G T V L Q I C T 2530 2540 2550 2560 2570 2580 CTGCCTCTTTCTGTACGGAGGCGCAGCTGAAGGATGTTGAGGGCTTCTTCGCCAACAAAG A S F C T E A Q L K D V E G F F A N K D 2590 2600 2610 2620 2630 2640 ATACCAAGgtaagtttcttgttatctgcgtccaggcggcgtataactaacactttcttag T K 2650 2660 2670 2680 2690 2700 GGCTATGACCGTGCTATTGAGCAAAGCCTCGACGCAATCCGCGCCAAGGTCAGCTGGGTT G Y D R A I E Q S L D A I R A K V S W V 2710 2720 2730 2740 2750 2760 CACCGTGACCGCGCGGATGTTACAGACTGGTTGAAGTCGAAGGGGTACTTCAAAGAAGGT H R D R A D V T D W L K S K G Y F K E G 2770 GGCAAGCTTTAA G K L *

[0084] Sequence Listing Free Text 5Pro_1 from Aspergillus sydowii MA0196 <CDS notation of DNA consisting of coding region>

[0085] Sequence Listing Free Text 5Pro_1_from Aspergillus sydowii MA0196 <CDS notation of amino acid sequence consisting of Coding region> MCGSRRAGVSGSTNVPDREVLPTNVKPTHYDLTLEPNFETFKYDGTVVIDLQVVEDTTSISLNSTEINIHTATVSSQGSVVSASPEISLNDDKQEATIKFSDVIRTGTSAQLKLTFTGTLNDNMAGFYRSSYKTSDGETKYIASTQMEPTDCRRAFPCFDEPALKAKFTVKLIADKSMTCLSNMDVASEEDIDGGKKIVKFNESPVMSTYLVAFIVGHLKYIETKDFRVPIRVYATPDQDIEHGRFSLNLAAKTLAFYEKAFDNEFPLPKMDMVAVPDFSAGAMENWGLITYRIVDVLLDEKTSSASRKERIAETVQHELAHQWFGNLVTMDFWDGLWLNEGFATWMSWYSCNTFYPEWKVWQTYVIDNLQSALSLDSLRSSHPIEVPVKRADEINQIFDAISYSKGSSVLRMISKYLGEDTFLQGVRNYIKKHAYGNTQTGDLWSALAGASGKPVEEVMDIWTKNVGFPVVSVSENPSSSSIKVKQNRFLRTGDVRPEEDTTLFPVMLGLRTKEGVDENTLLSSREEEFKVPDLDFYKLNADHTAIYRTSYTPERLTKLGNAAKEGLLTVEDRAGMIADAGALAASGYQKTSGLLSLLAGFDGEPEFIVWNEILTRIGALRAAWIFEDSQSKDALEAFQRALVGEKAQKLGWEFSDKDDHILQQFKALLFSAAGNAQEPTVVKAAQDMFERFAAGDISAIHPNIRGSVFSIVLKNGGEREYNVVYDRFRNAPTSDEKTTALRCLGTAEDPALIKRTLGLALGDEVKNQDIYMPLGGLRTHAAGIEARWTWVKDNWDTLYQRLPPGLGMLGTVLQICTASFCTEAQLKDVEGFFANKDTKGYDRAIEQSLDAIRAKVSWVHRDRADVTDWLKSKGYFKEGGKL*

[0086] Sequence Listing Free Text 5Pro_1_from Aspergillus sydowii MA0196 <Notation consisting only of the coding region> 10 20 30 40 50 60 ATGTGTGGTTCTCGTCGTGCTGGGGTCTCTGGTTCGACCAACGTTCCTGACCGTGAGGTT M C G S R R A G V S G S T N V P D R E V 70 80 90 100 110 120 CTCCCCACCAACGTTAAGCCAACCCACTATGACTTGACTCTTGAGCCGAACTTTGAGACC L P T N V K P T H Y D L T L E P N F E T 130 140 150 160 170 180 TTTAAATATGACGGGACTGTCGTTATCGAGTTACAGGTTGTCGAGGACACGACATCCATC F K Y D G T V V I E L Q V V E D T T S I 190 200 210 220 230 240 TCGCTCAACTCTACTGAAATCAACATCCACACAGCTACCGTGTCCTCTCAAGGATCAGTA S L N S T E I N I H T A T V S S Q G S V 250 260 270 280 290 300 GTCAGCGCGTCACCTGAGATTTCCCTCAACGATGACAAGCAAGAGGCTACAATCAAGTTT V S A S P E I S L N D D K Q E A T I K F <h2 style=";text-align:left;direction:ltr">310 320 330 340 350 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TCGGATGTTATCCGCACCGGAACATCCGCTCAACTTAAGCTGACCTTCACCGGTACTCTC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SDVIRTGTSAQLKLTFTGTL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 370 380 390 400 410 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AATGACAACATGGCCGGTTTCTACCGCTCATCTTACAAGACGTCGGACGGCGAGACCAAA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> NDNMAGFYRSSYKTSDGETK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 430 440 450 460 470 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TACATTGCGGTCAACTCAAATGGAGCCGACCGATTGCCGGAGAGCTTTCCCTTGCTTCGAC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YIASTQMEPTDCRRAFPCFD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 490 500 510 520 530 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GAGCCTGCGCTCAAGGCTAAATTTACTGTCAAACTGATTGCGGACAAGAGCATGACTTGT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EPALKAKFTVKLIADKSMTC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 550 560 570 580 590 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TTGAGCAACATGGACGTTGCATCCGAAAGAGGATATTGATGGTGGGAAGAAAATTGTCAAG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LSNMDVASEEDIDGGKKIVK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 610 620 630 640 650 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">TTCAATGAGTCTCCGGTCATGTTCTACCTACCTCGTGGCTTTCATTGTCGGCCACCTGAAG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FNESPVMSTYLVAFIVGHLK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 670 680 690 700 710 720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TACATTGAGACCAGGACTTCCGTGTCCCTATCCGTGTCTATGCTACACCAGATCAGGAC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YIETKDFRVPIRVYATPDQD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 730 740 750 760 770 780<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ATTGAACATGGCCGTTTCTCCCTGAACTTGGCGGCCAAAGACGCTTGCCTTCTACGAAAAG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> IEHGRFSLNLAAKTLAFYEK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 790 800 810 820 830 840<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GCTTTCGATAACGAATTCCCTCTTCCAAAGATGGACATGGTTGCGGTCCCCGATTTCAGT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AFDNEFPLPKMDMVAVPDFS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 850 860 870 880 890 900<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GCTGAGCCATGGAGAACTGGGGTTTGATCACCTACCGTATCCGTTGATGTGCTCCTGGAT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AGAMENWGLITYRIVDVLLD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 910 920 930 940 950 960<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GAGAAGACAAGCAGTGCATCGCGGAAGGAGCGCATTGCTGAAACTGTTCAGCAGAGCTT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EKTSSASRKERIAETVQHEL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 970 980 990 1000 1010 1020<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GCCCACCAGTGGTTTGGTAACTTGGTTACCATGGATTTCTGGGACGGTCTATGGCTGAAC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AHQWFGNLVTMDFWDGLWLN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1030 1040 1050 1060 1070 1080<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GAAGGCTTTGCTACTTGGATGTCGTGGTATTCTTGCAACACCTTCTACCCCGAATGGAAG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EGFATWMSWYSCNTFYPEWK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1090 1100 1110 1120 1130 1140<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GTCTGGCAAACATATGTCATCGACAACCTGCAAAGTGCGCTCTCCCTCGATTCTCTCCGC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VWQTYVIDNLQSALSLDSLR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1150 1160 1170 1180 1190 1200<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AGCAGTCACCCCATCGAAGTGCCCGTGAAGCGCGCGGACGAGATAAACCAGATATTTGAT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SSHPIEVPVKRADEINQIFD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1210 1220 1230 1240 1250 1260<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GCCATCTCCTACTCCAAGGGTTCTTCTGTCCTGCGCATGATCTCAAAGTATCTGGGTGAA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">AISYSKGSSVLRMISKYLGE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1270 1280 1290 1300 1310 1320<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GACACCTTCCTGCAGGGCGTTCGTAATTACATTAAGAAGCACGCTTACGGCAACACACAA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DTFLQGVRNYIKKHAYGNTQ<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1330 1340 1350 1360 1370 1380<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ACCGGTGACCTGTGGTCCGCCCTTGCTGGTGCTAGTGGCAAGCCCGTGGAGGAGGTCATG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TGDLWSALAGASGKPVEEVM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1390 1400 1410 1420 1430 1440<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GATATCTGGACCAAGAATGTTGGATTCCCAGTCGTCTCTGTGTCGGAAAACCCAAGCTCC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DIWTKNVGFPVVSVSENPSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1450 1460 1470 1480 1490 1500<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TCATCCATCAAGGTGAAACAGAATCGATTTCTCCGCACAGGCGACGTTCGACCCGAGGAA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SSIKVKQNRFLRTGDVRPEE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1510 1520 1530 1540 1550 1560<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GACACGACTCTCTTCCCTGTGATGCTTGGGATTGCGAACAAAGGAGGGAGTTGACGAAAAC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">DTTLFPVMLGLRTKEGVDEN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1570 1580 1590 1600 1610 1620<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ACCCTGCTGTCATCGCGCGAGGAAGAGTTCAAGGTTCCCGATCTCGATTTTTACAAGCTT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TLLSSREEEFKVPDLDFYKL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1630 1640 1650 1660 1670 1680<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AATGCCGATCACACCGCAATTTACCGCACATCCTACACCCCTGAGCGTCTAACCAAGCTG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> NADHTAIYRTSYTPERLTKL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1690 1700 1710 1720 1730 1740<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GGCAACGCTGCCAAGGAGGGTCTGCTTACTGTTGAGGACCGCGCGGGTATGATTGCTGAT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GNAAKEGLLTVEDRAGMIAD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1750 1760 1770 1780 1790 1800<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GCTGGGTGCACTGGCTGCTTCTGGTTACCAGAAAACGTCTGGTTTGCTATCGCTGCTTGCA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AGALAASGYQKTSGLLSLLA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1810 1820 1830 1840 1850 1860<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GGATTTGATGGCGAGCCCGAATTTATTGTTTGGAACGAGATCTTGACTCGTATTGGTGCC<h2 style=";text-align:left;direction:ltr"> G F D G E P E F I V W N E I L T R I G A 1870 1880 1890 1900 1910 1920 CTACGTGCCGCTTGGATATTCGAGGATTCCCAGTCCAAGGACGCACTGGAGGCATTCCAG L R A A W I F E D S Q S K D A L E A F Q 1930 1940 1950 1960 1970 1980 CGTGCCTTGGTCGGCGAGAAAGCGCAGAAACTGGGATGGGAGTTTTCCGACAAGGACGAC R A L V G E K A Q K L G W E F S D K D D 1990 2000 2010 2020 2030 2040 CATATTCTACAACAATTCAAGGCTCTCTTGTTCAGTGCCGCTGGCAATGCTCAGGAACCA H I L Q Q F K A L L F S A A G N A Q E P 2050 2060 2070 2080 2090 2100 ACTGTAGTCAAGGCCGCCCAGGACATGTTCGAGCGCTTCGCTGCTGGTGATATCAGCGCC T V V K A A Q D M F E R F A A G D I S A 2110 2120 2130 2140 2150 2160 ATCCACCCTAACATTCGCGGCAGCGTTTTCTCCATCGTCCTGAAAAACGGCGGTGAAAGG <h2 style=";text-align:left;direction:ltr">IHPNIRGSVFSIVLKNGGER<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2170 2180 2190 2200 2210 2220<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GAGTACAATTGTTGTGTATGACCGCTTCCGCAATGCCCCCACCTCTGATGAGAAGACCACG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EYNVVYDRFRNAPTSDEKTT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2230 2240 2250 2260 2270 2280<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GCTCTCCGCTGCCTTGGTACGGCCGAAGATCCGGCCCTCATCAAACGCACTCTCGGCTTG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ALRCLGTAEDPALIKRTLGL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2290 2300 2310 2320 2330 2340<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GCCCTGGGTGACGAGGTTAAGAACCAGGACATTTACATGCCGCTTGGCGGACTCCGCACT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ALGDEVKNQDIYMPLGGLRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2350 2360 2370 2380 2390 2400<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> CATGCCGCCGGTATTGAAGCGCGATGGACCTGGGTGAAGGATAACTGGGACACCCTATAC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> HAAGIEARWTWVKDNWDTLY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2410 2420 2430 2440 2450 2460<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> CAGCGCCTGCCCCCGGATTGGGCATGCTCGGAACTGTCCTACAAATCTGCACTGCCTCT<h2 style=";text-align:left;direction:ltr"> QRLPPGLGMLGTVLQICTAS 2470 2480 2490 2500 2510 2520 TTCTGTACGGAGGCGCAGCTGAAGGATGTTGAGGGCTTCTTCGCCAACAAAGATACCAAG FCTEAQLKDVEGFFANKDTK 2530 2540 2550 2560 2570 2580 GGCTATGACCGTGCTATTGAGCAAAGCCTCGACGCAATCCGCGCCAAGGTCAGCTGGGTT GYDRAIEQSLDAIRAKVSWV 2590 2600 2610 2620 2630 2640 CACCGTGACCGCGCGGATGTTACAGACTGGTTGAAGTCGAAGGGGTACTTCAAAGAAGGT HRDRADVTDWLKSKGYFKEG 2650 GGCAAGCTTTAA GKL*

[0087] A Protein BLAST search for the amino acid sequence deduced from the 5Pro1 base sequence revealed that it shared 55% identity with an aminopeptidase derived from Candida albicans (accession number EAK91143), which is classified as an arginine / alanine / leucine-specific metallo-aminopeptidase based on substrate specificity tests. Therefore, 5Pro1 is classified as an exo-type peptidase and is expected to hydrolyze the protein derived from skipjack tuna from the terminal. As described above, 5Pro1 alone cannot proceed with the degradation of the protein, but 5Pro2 (7Pro1) and 7Pro2 further degrade the fragmented peptides into amino acids and short-chain peptides, which is thought to contribute to the taste and physiological activity of the culture.

[0088] Sequence Listing Free Text Base / Amino Acid Sequence of 5Pro2 / 7Pro1 (Serine Protease) 5Pro_2 / 7Pro_1_from Aspergillus sydowii MA0196 (Trypsin-like serine protease) <Consisting only of the Coding region notation> 10 20 30 40 50 60 ATGAAGACCACCCTCGCCTTCCTCTCCGTCCTCTCCAGCGTCCTCGCCAAGGAGAACATC M K T T L A F L S V L S S V L A K E N I 70 80 90 100 110 120 GTCGGCGGCTCCGAGGCCTCCATCGAGGAGTTCCCCTACCAGATCGGCCTGCTGCAGGGC V G G S E A S I E E F P Y Q I G L L Q G 130 140 150 160 170 180 GGCTCGCTCATCTGCGGTGGCAGCATCATCGACAACAAGTACATTGTCACCGCCGGCCAC G S L I C G G S I I D N K Y I V T A G H 190 200 210 220 230 240 TGCGCCGAGGGCCAGTCCGCCAGCGACCTCAGCATCCGCGCCGGCTCCGACTCCTCGTCC C A E G Q S A S D L S I R A G S D S S S 250 260 270 280 290 300 TCCGGCGGCACCACCGTGGGCGTCTCCTCCATCGCCGTCCACCCGGACTACGACGCCCAG S G G T T V G V S S I A V H P D Y D A Q 310 320 330 340 350 360 ACCGTCAACAACGACATTGCCATTCTCACCCTCGACCAGGACCTCAAGTACGGCCCCGGC T V N N D I A I L T L D Q D L K Y G P G 370 380 390 400 410 420 ATCGAGGCCGTCAAGCTCCCTGCCAAGAACGCCTCGCTCCCCTCCGCCGGCACCGTCGGC I E A V K L P A K N A S L P S A G T V G 430 440 450 460 470 480 ACCATCACCGGCTGGGGCGCCCTCCAGGAGGGCGGCAACGTCTCGCCCACCCTGCAGTCC T I T G W G A L Q E G G N V S P T L Q S 490 500 510 520 530 540 GTCGAGGTCCCTGTTGTCGACAAGAAGACCTGTGCCCAGGACTACCAGGGCTTCAACGAG V E V P V V D K K T C A Q D Y Q G F N E 550 560 570 580 590 600 ATCACCGCGAGCATGCTGTGCGCTGGTGAGGAGGAGGGTGGCAAGGACGGCTGCCAGGGT I T A S M L C A G E E E G G K D G C Q G 610 620 630 640 650 660 GACTCCGGTGGTCCCTTCGTCTCCAACGGTGTCCTTATTGGTATCACCTCCTGGGGCAAT D S G G P F V S N G V L I G I T S W G N 670 680 690 700 710 720 GGCTGTGCGCGCGCTGGATACCCCGGTGTCTACTCGAGCCCGGCTTACTTCCGTGACTTT G C A R A G Y P G V Y S S P A Y F R D F 730 740 ATCGAGTCGGTGACTGGTATTTAA I E S V T G I *

[0089] Sequence Listing Free Text 5Pro_2 / 7Pro_1_from Aspergillus sydowii MA0196 (Trypsin-like serine protease) <CDS notation of DNA consisting of coding region> ATGAAGACCACCCTCGCCTTCCTCTCCGTCCTCTCCAGCGTCCTCGCCAAGGAGAACATCGTCGGCGGCTCCGAGGCCTCCATCGAGGAGTTCCCCTACCAGATCGGCCTGCTGCAGGGCGGCTCGCTCATCTGCGGTGGCAGCATCATCGACAACAAGTACATTGTCACCGCCGGCCACTGCGCCGAGGGCCAGTCCGCCAGCGACCTCAGCATCCGCGCCGGCTCCGACTCCTCGTCCTCCGGCGGCACCACCGTGGGCGTCTCCTCCATCGCCGTCCACCCGGACTACGACGCCCAGACCGTCAACAACGACATTGCCATTCTCACCCTCGACCAGGACCTCAAGTACGGCCCCGGCATCGAGGCCGTCAAGCTCCCTGCCAAGAACGCCTCGCTCCCCTCCGCCGGCACCGTCGGCACCATCACCGGCTGGGGCGCCCTCCAGGAGGGCGGCAACGTCTCGCCCACCCTGCAGTCCGTCGAGGTCCCTGTTGTCGACAAGAAGACCTGTGCCCAGGACTACCAGGGCTTCAACGAGATCACCGCGAGCATGCTGTGCGCTGGTGAGGAGGAGGGTGGCAAGGACGGCTGCCAGGGTGACTCCGGTGGTCCCTTCGTCTCCAACGGTGTCCTTATTGGTATCACCTCCTGGGGCAATGGCTGTGCGCGCGCTGGATACCCCGGTGTCTACTCGAGCCCGGCTTACTTCCGTGACTTTATCGAGTCGGTGACTGGTATTTAA

[0090] Sequence Listing Free Text 5Pro_2 / 7Pro_1_from Aspergillus sydowii MA0196 (Trypsin-like serine protease) <CDS notation of the amino acid sequence consisting of the Coding region> MKTTLAFLSVLSSVLAKENIVGGSEASIEEFPYQIGLLQGGSLICGGSIIDNKYIVTAGHCAEGQSASDLSIRAGSDSSSSGGTTVGVSSIAVHPDYDAQTVNNDIAILTLDQDLKYGPGIEAVKLPAKNASLPSAGTVGTITGWGALQEGGNVSPTLQSVEVPVVDKKTCAQDYQGFNEITASMLCAGEEEGGKDGCQGDSGGPFVSNGVLIGITSWGNGCARAGYPGVYSSPAYFRDFIESVTGI

[0091] When the amino acid sequence deduced from the nucleotide sequence of 5Pro2(7Pro1) was searched by Protein BLAST for homology, it had 41% identity with a trypsin-like serine protease derived from Anopheles gambiae (accession number, P35035). Therefore, 5Pro2(7Pro1) was classified as an endo-type peptidase and was expected to hydrolyze the interior of the sequence of the protein derived from skipjack tuna, and it is considered that the degradation of the protein was also observed electrophoretically (Figs. 12(b) and 13(a)).

[0092] Nucleotide / amino acid sequence of 7Pro2 (serine protease) 7Pro_2_from A. sydowii MA0196 (elastinolytic serine protease)

[0093] Sequence Listing free text 7Pro_2_from A. sydowii MA0196 (elastinolytic serine protease) <Notation consisting of Coding region and non-coding region>

[0094] Sequence Listing free text 10 20 30 40 50 60 <h2 style=";text-align:left;direction:ltr">ATGTATTCAGTCAAGCGCACCATCCTCCTGCTCCGGAGCTCTTCTTCCTGCGGTCTTTGGT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> MYSVKRTILLLGALLPAVFG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 70 80 90 100 110 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GCTCCTATCCTTGAATCTCGCCGGCCCACTGAAAAGGTCCCCCGGGAAGTACATCGTGACG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> APILESRRPTEKVPGKYIVT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 130 140 150 160 170 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TTCAAATCTGGCCTGCAGGCCGAGCAGATCGACGCTCACACAACCTGGGCCTCGAATGTC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FKSGLQAEQIDAHTTWASNV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 190 200 210 220 230 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> CACAAGCGCAACCTAGAGCGCCGTGATTTGGCTGACCGGATCTGTACCGGGTATTGAG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> HKRNLERRDLADRDLYPGIE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 250 260 270 280 290 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAGAACTTCAAGATCCATAAGTTTGCGGCGTACGCCGGTTCCTTTGATGATGCCACCATC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> KNFKIHKFAAYAGSFDDATI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 310 320 330 340 350 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GAAGAGATTCGAAACAGCAAAGATgtttgtgcccgtcttgaatccccagttgtgctataa E E I R N S K D 370 380 390 400 410 420 ttgttgacgaccccccagGTGGCTTACGTGGAGGAGGACCAAGTCTGGTACATTGATGCC V A Y V E E D Q V W Y I D A 430 440 450 460 470 480 GTAACCACCCAGAGCGATGCTCCCTGGGGTCTTGGAGCTATCTCTCACCAAGGAGATGCG V T T Q S D A P W G L G A I S H Q G D A 490 500 510 520 530 540 AGCACCGACTATATTTACGACACAAGCGCGGGAGCCGACACTTACGCATATGTGGTGGAC S T D Y I Y D T S A G A D T Y A Y V V D 550 560 570 580 590 600 ACGGGTATCAACGTTGATCACTCGGAATTCGACGGACGCGCCAGTCTTGCCTACAACGCT T G I N V D H S E F D G R A S L A Y N A 610 620 630 640 650 660 GCTGGTGGCCAGCACGTTGACAGCGTCGGCCACGGTACCCATGTTGCCGGCACTATCGGT A G G Q H V D S V G H G T H V A G T I G 670,680,690,700,710,720 GGCAAAACCTACGGTGTTTCCAAGAAGGCCAATCTCTCTCTGTCAAGGTCTTCCAAGGGC GKTYGVSKKANLLSVKVFQG 730,740,750,760,770,780 GAATCTAGCAGCACCTCCATTATCCTCGATGGCTACAACTGGGCTGCTAACGACATTGTA ESSSTSILDGYNWAANDIV 790,800,810,820,830,840 AGCAAGGGCCGCACCGGCAAGTCGGCAATCAATCTGAGTTTGGGTaagtattcgctactg SKGRTGKSAINLSLG 850,860,870,880,890,900 cctttccgaaagagtaacattgctgactggacattgtaggcGGTGGATACTCTTATGCT GGYSYA 910,920,930,940,950,960 TTCAACAATGCAGTCGAAAGCGCATTTGATGAGGGGTTCTCTCTGTTGTAGCAGCCGGC FNNAVESAFDEGVLSVVAAG 970 980 990 1000 1010 1020 AACGAAAACGTGagtctgttcgatattatctgcccaaaagccgatgtctaacaagcatgc N E N V 1030 1040 1050 1060 1070 1080 aacctcaggttGATGCATCGAACACCAGTCCCGCTTCTGCACCTAACGCTCTTACTGTTG D A S N T S P A S A P N A L T V A 1090 1100 1110 1120 1130 1140 CGGCTAGCACAGAAAGAAATGCTCGCGCCTCGTTCTCCAACTATGGTAGTGTGGTCGACA A S T E R N A R A S F S N Y G S V V D I 1150 1160 1170 1180 1190 1200 TCTTTGCCCCCGGTGAAGACATCCTCTCGGCCTGGATTGGCGGAACCACTGCCACTAACA F A P G E D I L S A W I G G T T A T N T 1210 1220 1230 1240 1250 1260 CCATTTCTGGCACATCTATGGCCACTCCCCATATTGTTGGTTTGTCCCTCTACTTGATCG I S G T S M A T P H I V G L S L Y L I A 1270 1280 1290 1300 1310 1320 CTCTTGAGGGGCTCTCCACTCCCGCCGATGTGACTTCCCGAATCAAGGAGCTGGCTACCC L E G L S T P A D V T S R I K E L A T Q 1330 1340 1350 1360 1370 1380 AGGACGTTCTCTCAAACGTGTCTGGCAGCCCCAACGCGCTTGCCTACAACGGCGCTGAAT D V L S N V S G S P N A L A Y N G A E * AA

[0095] Sequence Listing Free Text 7Pro_2_from A. sydowii MA0196 (elastinolytic serine protease) <CDS notation of DNA consisting of coding region>

[0096] Sequence Listing Free Text 7Pro_2_from A. sydowii MA0196 (elastinolytic serine protease) <CDS notation of the amino acid sequence consisting of the Coding region> MYSVKRTILLLGALLPAVFGAPILESRRPTEKVPGKYIVTFKSGLQAEQIDAHTTWASNVHKRNLERRDLADRDLYPGIEKNFKIHKFAAYAGSFDDATIEEIRNSKDVAYVEEDQVWYIDAVTTQSDAPWGLGAISHQGDASTDYIYDTSAGADTYAYVVDTGINVDHSEFDGRASLAYNAAGGQHVDSVGHGTHVAGTIGGKTYGVSKKANLLSVKVFQGESSSTSIILDGYNWAANDIVSKGRTGKSAINLSLGGGYSYAFNNAVESAFDEGVLSVVAAGNENVDASNTSPASAPNALTVAASTERNARASFSNYGSVVDIFAPGEDILSAWIGGTTATNTISGTSMATPHIVGLSLYLIALEGLSTPADVTSRIKELATQDVLSNVSGSPNALAYNGAE

[0097] Sequence Listing Free Text 7Pro_2_from A. sydowii MA0196 (elastinolytic serine protease) <Notation consisting only of the Coding region> 10 20 30 40 50 60 ATGTATTCAGTCAAGCGCACCATCCTCCTGCTCGGAGCTCTTCTTCCTGCGGTCTTTGGT M Y S V K R T I L L L G A L L P A V F G 70 80 90 100 110 120 <h2 style=";text-align:left;direction:ltr">GCTCCTATCCTTGAATCTCGCCGGCCCACTGAAAAGGTCCCCCGGGAAGTACATCGTGACG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> APILESRRPTEKVPGKYIVT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 130 140 150 160 170 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TTCAAATCTGGCCTGCAGGCCGAGCAGATCGACGCTCACACAACCTGGGCCTCGAATGTC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FKSGLQAEQIDAHTTWASNV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 190 200 210 220 230 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> CACAAGCGCAACCTAGAGCGCCGTGATTTGGCTGACCGGATCTGTACCGGGTATTGAG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> HKRNLERRDLADRDLYPGIE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 250 260 270 280 290 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAGAACTTCAAGATCCATAAGTTTGCGGCGTACGCCGGTTCCTTTGATGATGCCACCATC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> KNFKIHKFAAYAGSFDDATI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 310 320 330 340 350 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GAAGAGATTCGAAACAGCAAAGATGTGGGCTTACGTGGAGGAGGACCAAGTCTGGTACATT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EEIRNSKDVAYVEEDQVWYI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 370 380 390 400 410 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GATGCCGTAACCACCCAGAGCGATGCTCCCTGGGGTCTTGGAGCTATTCTCTCACCAAGGA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DAVTTQSDAPWGLGAISHQG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 430 440 450 460 470 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GATGCGAGCACCGACTATATTTACGACACAAGCGCGGGAGCCGACACTTACGCATATGTG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DASTDYIYDTSAGADTYAYV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 490 500 510 520 530 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GTGGACACGGGTATCAACGTTGATCACTCGGAATTCGACGGACGCGCCAGTCTTGCCTAC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDTGINVDHSEFDGRASLAY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 550 560 570 580 590 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AACGCTGCTGGTGGCCAGCACGTTGACAGCGTCGGCCACGGTACCCATGTTGCCGGCACT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> NAAGGQHVDSVGHGTHVAGT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 610 620 630 640 650 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ATCGGTGGCAAAACCTACGGTGTTTCCAAGAAGGCCAATCTTCTCTCTGTCAAGGTCTTC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> IGGKTYGVSKKANLLSVKVF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 670 680 690 700 710 720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> CAGGGCGAATCTAGCAGCACCCTCCATTATCCCTCGATGGCTACAACTGGGCTGCTAACGAC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> QGESSSTSIILDGYNWAAND<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 730 740 750 760 770 780<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ATTGTAAGCAAGGGCCCGCACCGGCAAGTCGGCAATCAATCTGAGTTTGGTGTGGTGGATAC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> IVSKGRTGKSAINLSLGGGY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 790 800 810 820 830 840<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TCTTTATGCTTTCAACAATGCAGTCGAAAGCGCATTTGATGAGGGGGTTCTCTCTGTTGTA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SYAFNNAVESAFDEGVLSVV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 850 860 870 880 890 900<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GCAGCCGGCAACGAAAACGTGGATGCATCGAACACCAGTCCCGCTTCTGCACCTAACGCT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> AAGNENVDASNTSPASAPNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 910 920 930 940 950 960<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> CTTACTGTTGCGGCTAGCACAGAAAGAAATGCTCGCGCCTCGTTCTCCAACTATGGTAGT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LTVAASTERNARASFSNYGS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 970 980 990 1000 1010 1020<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GTGGTCGACATCTTTGCCCCCGGTGAAGACATCCTCTCGGCCTGGATTGGCGGAACCACT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VVDIFAPGEDILSAWIGGTT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1030 1040 1050 1060 1070 1080 GCCACTAACACCATTTCTGGCACATCTATGGCCACTCCCCATATTGTTGGTTTGTCCCTC ATNTISGTSMATPHIVGLSL 1090 1100 1110 1120 1130 1140 TACTTGATCGCTCTTGAGGGGCTCTCCACTCCCGCCGATGTGACTTCCCGAATCAAGGAG YLIALEGLSTPADVTSRIKE 1150 1160 1170 1180 1190 1200 CTGGCTACCCAGGACGTTCTCTCAAACGTGTCTGGCAGCCCAACGCGCTTGCCTACAAC LATQDVLSNVSGSPNALAYN 1210 GGCGCTGAATAA GAE*

[0098] A homology search by Protein BLAST for the amino acid sequence deduced from the 7Pro2 nucleotide sequence revealed that it shared 84% identity with keratinolytic serine protease from Aspergillus nidulans FGSC A4 (accession number, Q00208). Therefore, like 5Pro2(7Pro1), 7Pro2 is also classified as an endo-type peptidase and is predicted to hydrolyze the interior of the sequence of bonito muscle-derived proteins.

[0099] The culture obtained by inoculating and culturing Aspergillus sydowii MA0196 revealed that the significant increase in the amounts of free amino acids and soluble polypeptides was due to the following: first, soluble (poly)peptides were produced from bonito meat-derived proteins by the action of endo-type peptidases such as 5Pro2 (7Pro1) and 7Pro2, and then the amount of free amino acids was increased by the further action of exo-type peptidases such as 7Pro1. From the above, the use of the MA0196 strain demonstrated the superiority of the microbial fermentation production method of dried bonito extract residue, in which multiple proteolytic enzymes derived from this bacterium act in concert.

[0100] [Table 6] Table 6 shows the relationship between sequence ID numbers and sequence names in the sequence listing attached to this application. [Industrial Applicability]

[0101] According to the production method of the present invention, by fermenting the residue of dried fish extract used to make soup stock, which was previously discarded as industrial waste, it is possible to provide foods containing a variety of active ingredients, which can be widely applied to issues such as food crisis. [Explanation of symbols]

[0102] 1. Fermented food composition 2. Control for fermented food composition without filamentous fungi 3 Lactobacillus acidophilus NBRC13951 lactic acid bacteria 4,6,8,10,12,14,16 Control for fermented food composition without lactic acid bacteria 5 Lactobacillus brevis NBRC107147 7 Lactobacillus casei NBRC15883 9 Lactobacillus delbrueckii NBRC3202 11 Lactobacillus fermentum NBRC15885 13 Lactobacillus lactis subsp. lactis NBRC 100933 15 Lactobacillus plantarum NBRC 15891 17 Solubility points 18. The soluble bacteria are classified using the コントロール for comparison. M Myoglobin (molecular mass (Da), 17,000; dissolution time, 8.9 min) C cytochrome c (molecular mass (Da), 12,400; dissolution time, 9.6 min) B bacitracin (molecular mass (Da), 1,422; dissolution time, 11.8 min) H N-hippuryl His Leu hydrate (molecular mass (Da), 429; dissolution time, 12.8 min) G glycylglycylglycine (molecular mass (Da), 189; dissolution time, 13.55 min) BM broad range protein marker LM low range prptein marker

Claims

1. A step of extracting stock from the fish flakes; Separating a fish flakes extraction residue from the mixture of fish flakes and stock; A method for producing a fermented food composition, comprising a step of fermenting dried fish extract residue using a xerophilic filamentous fungus belonging to the genus Aspergillus.

2. The method includes a step of subjecting a fish flakes extraction residue to a proteolytic enzyme, The method for producing a fermented food composition according to claim 1 , wherein the protease is at least one of an endo-type peptidase and an exo-type peptidase derived from a xerophilic filamentous fungus belonging to the genus Aspergillus.

3. The method includes a step of subjecting a fish flakes extraction residue to a proteolytic enzyme, The protease contains an endopeptidase derived from a xerophilic filamentous fungus belonging to the genus Aspergillus, The method for producing a fermented food composition according to claim 2 , wherein the endopeptidase is two types of serine protease.

4. The method includes a step of subjecting a fish flakes extraction residue to a proteolytic enzyme, The protease contains an exopeptidase derived from a xerophilic filamentous fungus belonging to the genus Aspergillus, The method for producing a fermented food composition according to claim 2 , wherein the exopeptidase is an aminopeptidase.

5. 2. The method for producing a fermented food composition according to claim 1, wherein the xerophilic filamentous fungus belonging to the genus Aspergillus has a proteolytic enzyme activity of at least one of serine protease and aminopeptidase of 0.3 units / g of culture or more.

6. 2. The method for producing a fermented food composition according to claim 1, wherein the fermentation period using the xerophilic filamentous fungus belonging to the genus Aspergillus is in the range of 1 to 3 weeks.

7. 2. The method for producing a fermented food composition according to claim 1, wherein the xerophilic filamentous fungus belonging to the genus Aspergillus is Aspergillus glaucus MA0196, which is deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center under the accession number NITE AP-02122.

8. The method for producing a fermented food composition according to claim 1, comprising a production method for a fermented food composition for at least one use selected from the group consisting of fermented food compositions for radical scavenging activity, fermented food compositions for antioxidant activity, fermented food compositions for angiotensin-converting enzyme inhibitory activity, and fermented food compositions for promoting the growth of lactic acid bacteria.

9. A step of extracting stock from the fish flakes; Separating a fish flakes extraction residue from the mixture of fish flakes and stock; The method comprises the step of fermenting the extract residue of dried fish using a xerophilic filamentous fungus belonging to the genus Aspergillus. A method for improving the quality of a fermented food composition, comprising at least one method selected from the group consisting of a method for improving the taste of a fermented food composition, a method for improving the radical scavenging activity of a fermented food composition, a method for improving the antioxidant activity of a fermented food composition, a method for improving the angiotensin-converting enzyme inhibitory activity, and a method for improving the growth-promoting activity of lactic acid bacteria.

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