Method for preparing liquid koji with high protease production

Abstract

To provide a method for producing liquid koji containing protease sufficient for the decomposition of raw material, and a liquid medium for use in the method.SOLUTION: A liquid medium for koji mold protease production contains at least defatted soybeans and livestock by-products or processed products thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the production of liquid koji. [Background technology]

[0002] To date, "liquid koji," made by cultivating koji in liquid, has attracted attention and numerous studies have been conducted on it due to its ease of controlling quality and microbial contamination.

[0003] In order to culture koji, which is necessary for brewing miso and soy sauce, in liquid, a culture method with suitable pH and medium composition has been developed, particularly as a technique for producing proteases (see Patent Document 1). Furthermore, a method for continuously culturing koji mold in liquid and producing enzymes has been developed based on the efficiency of enzyme production (see Patent Document 2).

[0004] Protease production is limited to the stationary phase of the growth curve, during which it is difficult to control the carbon and nitrogen sources and the enzyme is susceptible to catabolite repression.

[0005] By contrast, if liquid koji was cultivated continuously, the above-mentioned problems could be overcome, but the shape of the koji mold hyphae meant that it was prone to clogging, which gave rise to a new problem: it was unsuitable for continuous production. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 61-007317 [Patent Document 2] Special Publication No. 7-121218 Summary of the Invention [Problem to be solved by the invention]

[0007] Liquid koji, which is made by adding meat extract and milk casein, which are common culture medium components, to defatted soybeans, does not produce a sufficient amount of enzymes to break down the ingredients, making it impossible to brew miso or soy sauce. An object of the present invention is to provide a method for producing liquid koji that stably produces protease with a high titer sufficient to decompose fermentation raw materials, and a liquid medium for use in said method. [Means for solving the problem]

[0008] In the present invention, when culturing koji mold with high protease production ability in liquid, in addition to using defatted soybeans in the medium as in the conventional method, gelatin was added as a substrate for inducing protease production. As a result, it was found that protease production was more induced than with conventional medium components. It was also found that liquid koji with higher protease titer could be prepared more stably by using meat extract and gelatin in combination in a certain ratio in addition to defatted soybeans.

[0009] That is, the present invention is as follows. [1] A liquid medium for producing koji mold protease, comprising at least defatted soybeans and livestock by-products or processed products thereof. [2] A liquid medium for producing Aspergillus oryzae protease according to [1], wherein the livestock by-product or its processed product is dried and ground organ or gelatin. [3] A liquid medium for producing Aspergillus oryzae protease according to [1] or [2], which contains a livestock by-product or a processed product thereof as well as a meat extract, and the mass ratio of the livestock by-product or a processed product thereof to the meat extract (converted into a dry matter content) is 1:19 to 19:1. [4] A liquid medium for producing a koji mold protease according to [3], wherein the total content of livestock by-products or processed products thereof and livestock meat extract (concentration on a dry basis) is 1.6 to 2.5% by weight based on the total weight of the liquid medium. [5] A liquid medium for producing a koji mold protease according to any one of [1] to [4], wherein the content of livestock by-products or processed products thereof is 0.1 to 2.0% by weight based on the total weight of the medium. [6] A liquid medium for producing a koji mold protease according to any one of [1] to [5], wherein the amount of fat or oil relative to the liquid medium is 0.1 mass% or less. [7] Liquid koji cultivated using any of the liquid media for producing koji mold protease [1] to [6]. [8] A fermented food or fermented seasoning brewed using the liquid koji described in [7]. [9] A fermented food or fermented seasoning according to [8], in which the content of grains derived from koji contained in the entire fermented food is 0.75% by weight or less.

[10] Foods containing fermented foods and / or fermented seasonings as described in [8] or [9].

[11] A method for producing liquid koji with high protease production, comprising culturing koji fungus in any one of the liquid media for producing koji fungus protease described in [1] to [6].

[12] The method for producing liquid koji with high protease production according to

[11] , wherein the titer of the produced protease in the medium is 500 U / mL or more.

[13] A method for producing a liquid koji with high protease production according to

[11] or

[12] , in which the shape of the mycelium is less likely to become pellet-like.

[14] A method for producing liquid koji with high protease production according to any of

[11] to

[13] , wherein the koji fungus is Aspergillus oryzae. Effect of the Invention

[0010] By producing liquid koji using the liquid medium of the present invention, the pH 7 protease titer of the liquid koji becomes higher and more stable than when produced by a conventional culture method using defatted soybeans. This makes it possible to produce liquid koji with enzyme activity that is comparable to that of solid koji, greatly increasing the possibility of industrial use. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows pH 7-protease titers in liquid media containing each main component. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in detail below. The present invention relates to a method for preparing liquid koji with high protease production. The term "liquid koji with high protease production" refers to liquid koji with high protease production ability. The present invention also relates to a method for culturing a koji mold with high protease production ability in a liquid. Liquid koji is used in various fermented foods and the like, and is obtained by adding raw materials that serve as nutrient sources to water to prepare a liquid medium, and then inoculating and culturing koji mold in the prepared liquid.

[0013] 1. About Koji mold The koji mold used for culturing in the present invention includes koji molds belonging to the genus Aspergillus. The koji molds belonging to the genus Aspergillus are strains belonging to the genus Aspergillus used in producing fermented foods and fermented seasonings such as sake, miso, vinegar, pickles, soy sauce, shochu, awamori, and dried bonito, and include yellow koji mold, white koji mold, black koji mold, and katsuobushi mold. Examples of yellow koji mold include Aspergillus oryzae and Aspergillus sojae, white koji mold includes Aspergillus kawachii, black koji mold includes Aspergillus luchuensis (formerly Aspergillus awamori) and Aspergillus saitoi, and katsuobushi mold includes Aspergillus glaucus. Either wild-type or mutant strains of these may be used.

[0014] One type of strain of these koji molds may be inoculated, or two, three, four, five or more types of strains may be inoculated and mixed and cultured.

[0015] The koji mold to be inoculated may be spores or pre-cultured hyphae, but it is preferable to inoculate hyphae since this shortens the time required for the logarithmic growth phase.

[0016] For example, wheat bran (the husk of wheat removed during the milling process) is added with water, and the koji mold is inoculated and cultivated to obtain a solid seed, which can then be diluted and inoculated as a starter. A solid seed is a koji made from wheat bran or grains such as rice. The solid seed is made by following the procedure below.

[0017] When koji is spread on grains and left to stand at room temperature and high humidity, the white hyphae grow and penetrate the grains, starting to produce enzymes and generate heat. If the mixture is stirred to release heat and ventilate, spores will form, and if cultured further, it will become colored and become a good solid koji. Koji mold can be cultured for 1 to 7 days, preferably 3 to 5 days. Because solid koji has formed spores, it will last for 2 to 3 months if refrigerated, but it is best to use it within 1 month.

[0018] The amount of koji mold to be inoculated into the liquid medium is not limited, but in the case of spores, 1 x 10 2 ~10 6 / mL, and more preferably 1×10 4 ~10 6 The liquid medium can be inoculated to a density of 100 / mL. In the case of mycelium obtained by preculture, the preculture liquid can be added to the liquid medium at 0.1 to 10% by weight (w / v%). Here, preculture refers to culture in a small amount of medium carried out as a preliminary step to main culture.

[0019] 2. Liquid medium The liquid medium used for preparing liquid koji is a liquid medium based on a pH 5.0 to 7.0 buffer solution and containing various nutrient sources. The buffer solution is not limited, but examples of the buffer solution that can be used include phosphate buffer, citrate buffer, HEPES (N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid) buffer, Good buffer, etc. The liquid medium used to prepare the liquid koji of the present invention is called a liquid medium for producing a koji mold protease.

[0020] The liquid medium of the present invention contains at least defatted soybeans and livestock by-products or processed products thereof as nutrient sources. The liquid medium of the present invention may further contain a meat extract. Livestock by-products or processed products thereof and livestock meat extracts are sometimes called N sources because they contain a lot of nitrogen.

[0021] Defatted soybeans are the residue left over after soybean oil has been extracted from soybeans, and are also called soybean meal. For example, "ZFS Soya" by Nissin Shokai Co., Ltd. can be used as a commercially available product.

[0022] Livestock by-products refer to the remains of carcasses harvested from livestock and bones removed from the carcasses, including internal organs, skin, and raw hide. Livestock here refers to cattle, pigs, horses, sheep, goats, deer, wild boars, rabbits, donkeys, mules, buffaloes, yaks, llamas, alpacas, reindeers, dogs, etc. Livestock generally refers to animals bred and raised for human use, but in the present invention also includes wild animals (game) obtained by hunting. Processed livestock by-products refer to those livestock by-products further processed by extraction, heating, enzyme treatment, crushing, etc. Processed livestock by-products may be in the form of powder or liquid.

[0023] Livestock by-products and their processed products include gelatin and livestock by-products containing gelatin. Gelatin is extracted by applying heat to collagen contained in the skin, bones, tendons, internal organs, etc. of animals, and has a linear polymer structure of amino acids.

[0024] Gelatin may be derived from cow or pig skin or bone. Examples of such gelatin include gelatin that has been acid-treated with an isoelectric point of 6 to 9, and gelatin that has been alkali-treated with an isoelectric point of about 5. Powdered gelatin and granular gelatin may be used. Commercially available gelatin products have different jelly strengths, and any jelly strength may be used. For example, "Gelatin R", "APH-250", "APH-200", "APH-150", "APH-100", "PSV (pig skin treated with acid)", "GBL-250", "GBL-200", "GBL-150", "GBL-100", "PGD (pig bone treated with acid)", "#300", "#250", "#200", "#150", "#100", "Gelatin 21", "RR", "New Silver (cow bone treated with alkali)" and the like from Nitta Gelatin Co., Ltd. may be used.

[0025] Livestock by-products that contain gelatin include organ meats such as tomozune, achilles, scarlet oyster, daicho, chokucho, tail, knuckle, monk, liver, heart, beef tripe (first stomach), honeycomb tripe (second stomach), omasum (third stomach), giara (fourth stomach), pig guts, and harabura. When using these organ meats, it is desirable to defat and dry them before use. Dried powder products of these organ meats are called dried organ pulverized products.

[0026] Gelatin derived from fish may be used, in which case fish-derived gelatin may be used instead of livestock by-products or processed products thereof. Examples of fish-derived gelatin that can be used include "Fish Collagen" from Nichie Co., Ltd., "Marine Gelatin" from Entech Co., Ltd., and "FGL-250TS" and "FGL-200SP" from Nitta Gelatin Co., Ltd.

[0027] From the viewpoint of improving and stabilizing the amount of protease produced, it is preferable to use gelatin derived from animals, more preferably from cows or pigs, and particularly preferably from cows.

[0028] Meat extract is also called meat extract. Meat extract is a water-soluble component extracted from meat or seafood, and is a concentrated extract obtained by extracting meat with hot water, or a concentrated liquid obtained by decomposing meat with enzymes and / or acids. Meat extract contains a large amount of peptided medium to low molecular weight proteins.

[0029] Any meat extract can be used as long as it contains peptides derived from animal or seafood meat. For example, meat extracts with a weight average molecular weight of 500 to 5000 (Mw), preferably 600 to 4000 (Mw), and more preferably 840 to 3600 (Mw) can be used. Commercially available products include "LAB LEMCO POWDER" from Kanto Chemical Co., Ltd., "Beef Extract 4543M" from DSP Gokyo Food & Chemical Co., Ltd., "Beef Extract SE" from Nikken Food Co., Ltd., "Chicken Extract BS" from Tsukasa Food Industry Co., Ltd., and "Pork Extract TH-K" from Nippon Pure Food Co., Ltd.

[0030] Preferably, the food product contains a mixture of a livestock by-product or a processed product thereof containing gelatin, which is a high molecular weight protein, and a meat extract. The livestock by-product or the processed product thereof may contain two, three, four, five, or more kinds of livestock by-products or processed products thereof.

[0031] Preferably, a liquid medium containing the following nutrient sources in combination is used: (1) Defatted soybeans and gelatin (2) Defatted soybeans, gelatin and meat extract (3) Defatted soybeans, livestock by-products or processed products thereof, including gelatin, and meat extract The content of defatted soybeans in the liquid medium is 1 to 5% by weight, preferably 1 to 3% by weight, and more preferably 1.2 to 3% by weight.

[0032] The total content of livestock by-products or processed products thereof and meat extract in the liquid medium is 1.0 to 2.3% by weight, preferably 1.1 to 2.3% by weight, more preferably 1.2 to 2.3% by weight, even more preferably 1.3 to 2.3% by weight, even more preferably 1.4 to 2.3% by weight, even more preferably 1.5 to 2.3% by weight, and particularly preferably 1.6 to 2.2% by weight. The total content of livestock by-products or processed products thereof and meat extract in the liquid medium is also referred to as the total content of livestock by-products or processed products thereof and meat extract in the entire liquid medium.

[0033] The content of livestock by-products or processed products thereof in the liquid medium is 0.1 to 2.0% by weight, preferably 0.5 to 2.0% by weight, and more preferably 1.0 to 2.0% by weight.

[0034] The meat extract (meat extract) content in the above content is calculated on a dry basis. For example, when 2 g of meat extract with a water content of 50% is added, it is calculated as 1 g of meat extract being added.

[0035] When the liquid medium contains livestock by-products or processed products thereof and meat extract, the mass ratio of the livestock by-products or processed products thereof to the meat extract is 1:19 to 19:1, preferably 1:3 to 3:1, and more preferably 1: 1. When the mass ratio of the livestock by-products or processed products thereof to the meat extract is within the above-mentioned range, protease is produced with a high titer and in a more stable manner.

[0036] When the Aspergillus oryzae is cultured in the above liquid medium, catabolite repression can also be avoided.

[0037] If fats and oils such as beef tallow are contained in the medium, they affect the protease production in liquid koji. In other words, the higher the fat and oil content, the more it inhibits protease production. Therefore, it is better to add less fat and oil to the medium, preferably 0.1% by weight or less, more preferably 0.05% by weight or less, and even more preferably 0.01% by weight or less.

[0038] 3.Culture method (conditions) After sterilizing the liquid medium of 2, the koji mold is inoculated as a starter and cultured. The sterilization is not particularly limited as long as it is performed by autoclave sterilization (121°C, 15 minutes or more) or the treatment conditions have an equivalent sterilization effect.

[0039] The culture temperature is 25 to 45° C., preferably 25 to 40° C., and more preferably 25 to 37° C. The culture time is 24 to 72 hours, preferably 36 to 60 hours, and more preferably 42 to 54 hours. The pH of the liquid medium is 4.0 to 7.0, and preferably 5.0 to 6.0.

[0040] Since koji mold is an aerobic fungus, the culture is carried out under conditions in which oxygen or air can be supplied to the liquid medium. For example, shaking culture may be performed using a thermostatic shaker. The shaking conditions are not limited as long as oxygen can be supplied, but may be, for example, 100 to 1000 rpm, preferably 100 to 500 rpm. When culturing using a jar fermenter, the shaking conditions are not limited as long as oxygen can be supplied, but for example, the stirring speed is 0 to 1000 rpm, preferably 100 to 500 rpm, and oxygen is preferably supplied aseptically using a compressor or the like. The oxygen aeration rate should be increased in proportion to the volume. In addition, when the volume of the jar fermenter is several hundred liters or more, the amount of dissolved oxygen is unlikely to be affected even if the stirring speed is increased, so the amount of dissolved oxygen may be controlled only by the aeration rate.

[0041] The hyphae of koji mold cultivated under the above conditions are not pellet-like (spherical hyphae), but pulp-like (small irregular hyphae are formed, with uniformly dispersed fibrous hyphae).

[0042] 4.Effects By culturing under the above conditions, a liquid koji with high protease production, high protease content, and improved protease activity can be obtained.

[0043] The protease includes a plurality of proteases, including those having an optimum pH in the neutral pH range, those having an optimum pH in the weakly alkaline pH range, and those having an optimum pH in the weakly acidic pH range.

[0044] In the culture method using the liquid medium of the present invention, protease production can be evaluated by titration at pH 7. That is, titration at pH 7 reflects the production amounts of multiple types of proteases.

[0045] The titer of the protease can be measured, for example, by the following method. Add 10 ml of 66.7 mM phosphate buffer to 0.5 ml of the cultured solution and stir for 10 minutes with a stirrer. Filter with filter paper (Advantec No. 131 filter paper) to obtain the filtrate. Place 5 ml of substrate solution in a test tube and pre-incubate for 5 minutes at 37°C in a thermostatic water bath (Yamato Scientific Co., Ltd.). Then add 1 ml of filtrate (if there are multiple samples, add them at equal intervals to make the incubation time for each sample constant) and incubate at 37°C for 10 minutes. After 10 minutes, add 5 ml of precipitating reagent (similarly at equal intervals for multiple samples) to stop the enzyme reaction. After further incubation at 37°C for 30 minutes, filter with filter paper to obtain the reaction solution. On the other hand, since background is required for each sample, add precipitating reagent to 1 ml of filtrate first and stir, then add substrate solution and incubate at 37°C for 30 minutes to coagulate, and filter in the same way to obtain the background reaction solution for each sample.

[0046] After the reaction, 2 ml of the filtrate is accurately dispensed into a new test tube, 5 ml of sodium carbonate test solution is added and mixed. 1 ml of 3-fold diluted Folin's test solution is added and mixed, and the mixture is incubated in a thermostatic water bath at 37°C for 30 minutes. The colored solution is measured at an absorbance of 660 nm using a spectrophotometer (Shimadzu Corporation).

[0047] The pH7-protease titer is calculated as follows: (Abs·S-Abs·BG)×Σ×N / n×M / m (U / ml). (Abs·S: absorbance of sample, Abs·BG: background absorbance, N: total volume of reaction mixture 11, n: amount dispensed from reaction mixture 2, M: amount of phosphate buffer used for dilution 10, m: amount of solution after incubation used for dilution 0.5)

[0048] The coefficient Σ was calculated as follows using a tyrosine standard solution. That is, 5 ml of sodium carbonate solution and 1 ml of phenol test solution were added to 1 ml of each tyrosine standard solution, and color development was carried out for 30 minutes at 37°C. A calibration curve was then created by measuring the absorbance at 660 nm using an absorbance cell with a 10 mm path length for a solution that did not contain tyrosine. One unit of pH7-protease activity is the activity that produces color equivalent to 1 μg of tyrosine in 1 minute at 37°C.

[0049] (Regarding preparation of each reagent) 66.7mM phosphate buffer is 0.2M Na 2 HPO 4 The solution contains 0.2M KH 2 PO 4 After adding the solution to adjust the pH to 7.0, dilute the adjusted 0.2 M phosphate buffer (pH 7.0) three times with distilled water (60 ml to 180 ml). The substrate solution is made by adding 200 ml of distilled water containing Na 2 HPO 4 Dissolve 1.42 g of casein and dispense 160 ml of it. Weigh out 1.26 g of casein into a 300 ml Erlenmeyer flask and add the dispensed 160 ml of 50 mM sodium monohydrogen phosphate solution. Dissolve by boiling in a water bath for 4 minutes, cool, and then add KH 2 PO 4 (The remainder of the 66.7 mM phosphate buffer was used.) Finally, the volume was filled up to 200 ml with distilled water.

[0050] The precipitation reagent is prepared by adding 1.8 g of trichloroacetic acid (TCA), 1.8 g of sodium acetate anhydride, and 5.5 mL of 6M acetic acid TS and filling up to 100 mL with distilled water. The sodium carbonate solution is prepared and used as 0.55 mol / L sodium carbonate test solution.

[0051] The titer measured by this method is called the pH7-protease titer. When cultured using the liquid medium of the present invention, the pH7-protease titer in the liquid medium is 500 U / mL or more, preferably 600 U / mL or more, and more preferably 700 U / mL or more.

[0052] 5.About Liquid Koji The liquid koji obtained by culturing koji mold in the above liquid medium contains koji mold and a protease with a pH 7-protease titer of 500 U / mL or more.

[0053] The obtained liquid koji can be used for the brewing production of fermented seasonings such as miso, soy sauce, sake, etc. Because the protease activity in the obtained liquid koji is high, it can efficiently decompose the raw materials of the fermented seasonings.

[0054] It can also be used to produce fermented foods made by breaking down meat. For example, beef can be minced using a food processor or the like, and the liquid koji of the present invention can be added to the minced meat and fermented. The liquid koji of the present invention contains almost no grain ingredients other than defatted soybeans, so fermented foods can be produced that make the most of the characteristics of the ingredients themselves.

[0055] The grain content derived from the koji of the present invention is 0.75% by weight or less, preferably 0.45% by weight or less, and more preferably 0.25% by weight or less.

[0056] The present invention also includes fermented foods and fermented seasonings brewed using the liquid koji of the present invention. Furthermore, the present invention also includes foods that include the fermented foods and / or fermented seasonings. Examples of foods include, but are not limited to, meat seasonings (e.g., yakiniku sauce, shogayaki sauce, demi-glace sauce, yakitori sauce, steak sauce), hot pot seasonings (e.g., kimchi hot pot, tonkotsu hot pot, tantan sesame hot pot), vegetable seasonings, noodle seasonings (e.g., ramen soup, udon soup, pasta sauce), rice seasonings (e.g., fried rice, pilaf, porridge), and processed foods (e.g., gyoza, shumai, hamburger steak, fried chicken, gratin, croquette). EXAMPLES

[0057] The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.

[0058] [Comparative Example 1] Confirmation of reproducibility of prior art 1. Purpose The effect of adding peptone and meat extract was also confirmed, while also confirming the protease titer of the prior art.

[0059] 2. Experimental equipment and operation (1) Strain: Aspergillus oryzae (2) species As a seed, solid bran koji was prepared by adding 1 g of wheat bran to 1 ml of deionized water, autoclaving, and culturing for 3 days. (3) Preparation of starter A starter was prepared by diluting 1 g of solid bran koji in 100 ml of buffer solution. (a) Buffer: 370mM KH 2 PO 4 , 30 mM K 2 HPO 4 , 0.5% NaCl, 0.012% MgSO 4 7H 2 Buffer solution O was prepared and dispensed in 15 ml portions into 100 ml baffled Erlenmeyer flasks. (b) The main ingredients shown in Table 1 (defatted soybeans ("ZFS Soya", manufactured by Nisshin Shoji Co., Ltd.), peptone ("Hipolypeptone", manufactured by Nippon Seiyaku Co., Ltd.), and meat extract ("LAB LEMCO POWDER", manufactured by Kanto Chemical Co., Ltd.)) were added to (a) and sterilized in an autoclave. (c) After cooling, 0.9 ml of starter was added. (d) The cells were cultured in a thermostatic shaker at 33°C and 200 rpm for 48 hours. (e) After the culture was completed, the cells were harvested and the pH7-protease titer was measured.

[0060] The titer of pH7-protease was measured by the following method. 10 ml of 66.7 mM phosphate buffer was added to 0.5 ml of the cultured solution, and the mixture was stirred with a stirrer for 10 minutes. The mixture was filtered with filter paper (Advantec No. 131 filter paper) to obtain the filtrate. 5 ml of the substrate solution was placed in a test tube and pre-incubated at 37 ° C for 5 minutes in a thermostatic water bath (manufactured by Yamato Scientific Co., Ltd.). Then, 1 ml of the filtrate was added (if there are multiple specimens, add them at equal intervals to make the incubation time for each specimen constant), and incubated at 37 ° C for 10 minutes. After 10 minutes, 5 ml of the precipitation reagent was added (similarly at equal intervals for multiple specimens) to stop the enzyme reaction. After further incubation at 37 ° C for 30 minutes, the reaction solution was obtained by filtering with filter paper. On the other hand, since background is required for each specimen, the precipitation reagent was added to 1 ml of the filtrate first and stirred, and then the substrate solution was added and incubated at 37 ° C for 30 minutes to coagulate, and the same filtration was used as the background reaction solution for each specimen.

[0061] After the reaction, 2 ml of the filtrate was accurately dispensed into a new test tube, and 5 ml of sodium carbonate test solution was added and stirred. 1 ml of Folin's test solution diluted 3 times was added and stirred, and the mixture was incubated in a thermostatic water bath at 37°C for 30 minutes. The colored solution was measured at an absorbance of 660 nm using a spectrophotometer (Shimadzu Corporation).

[0062] The pH7-protease titer is calculated as follows: (Abs·S-Abs·BG)×Σ×N / n×M / m (U / ml). (Abs·S: absorbance of sample, Abs·BG: background absorbance, N: total volume of reaction mixture 11, n: amount dispensed from reaction mixture 2, M: amount of phosphate buffer used for dilution 10, m: amount of solution after incubation used for dilution 0.5)

[0063] The coefficient Σ was calculated using a tyrosine standard solution as follows: 5 ml of sodium carbonate solution and 1 ml of phenol test solution were added to 1 ml of each tyrosine standard solution, and color development was carried out for 30 minutes at 37°C. A calibration curve was then drawn by measuring the absorbance at 660 nm using a 10 mm light path length absorbance cell for a solution that did not contain tyrosine. One unit of pH7-protease activity was defined as the activity that showed color development equivalent to 1 μg of tyrosine in 1 minute at 37°C.

[0064] (Regarding preparation of each reagent) 66.7mM phosphate buffer is 0.2M Na 2 HPO 4 The solution contains 0.2M KH 2 PO 4 After adding a solution to adjust the pH to 7.0, the adjusted 0.2 M phosphate buffer (pH 7.0) was diluted 3-fold with distilled water (60 ml to 180 ml).

[0065] The substrate solution was prepared by adding 200 ml of distilled water to Na 2 HPO 4 1.42 g of casein was dissolved and 160 ml was dispensed from it. 1.26 g of casein was weighed into a 300 ml Erlenmeyer flask and 160 ml of 50 mM sodium monohydrogen phosphate solution was added. The solution was dissolved by boiling in a water bath for 4 minutes, cooled, and then added to KH 2 PO 4 (The remainder of the 66.7 mM phosphate buffer was used.) Finally, the volume was filled up to 200 ml with distilled water.

[0066] The precipitation reagent was prepared by filling up 1.8 g of trichloroacetic acid (TCA), 1.8 g of sodium acetate anhydride, and 5.5 mL of 6M acetic acid solution with distilled water to 100 mL. The sodium carbonate solution used was 0.55 mol / L sodium carbonate test solution.

[0067] 3.Results The pH7-protease titers in liquid media containing each main component are shown in Table 1.

[0068] [Table 1]

[0069] In the culture using the liquid medium of this comparative example, the mycelium pelleted once in several times, and the enzyme production was significantly reduced due to the formation of clumps of mycelium. It is believed that the reason why the enzyme titer did not improve significantly even when a N source was added, as in the conventional technology, was due to the large effect of the pelleting.

[0070] [Example 1] Search for substrates that produce high levels of proteases 1. Purpose Until now, protease production using liquid koji has been mainly achieved using defatted soybean powder. In addition, it is known from prior art that protease activity can be increased by adding meat extract or peptone in addition to defatted soybean powder. Therefore, we investigated whether a medium to high molecular weight N source such as meat extract or peptone would contribute more to protease production.

[0071] 2. Experimental equipment and operation (1) Strain: Aspergillus oryzae (2) Main components of medium: defatted soybeans ("ZFS Soya", manufactured by Nisshin Shoji Co., Ltd.), gelatin ("Gelatin R", manufactured by Nitta Gelatin Co., Ltd.), milk casein (manufactured by Kanto Chemical Co., Ltd.), peptone ("Proteose Peptone", manufactured by Difco Laboratories), peptone ("Tryptose", manufactured by Difco Laboratories), peptone ("Select Soytone", manufactured by Difco Laboratories), yeast powder extract (manufactured by BD) (3) species As a seed, solid bran koji was prepared by adding 1 g of wheat bran to 1 ml of deionized water, autoclaving, and culturing for 3 days. (4) Preparation of starter A starter was prepared by diluting 1 g of solid bran koji in 100 ml of buffer solution. (a) Buffer: 370mM KH 2 PO 4 , 30 mM K 2 HPO 4 , 0.5% NaCl, 0.012% MgSO 4 7H 2 Buffer solution O was prepared and dispensed in 15 ml portions into 100 ml baffled Erlenmeyer flasks. (b) The main components of the medium shown in Table 2 were added to (a) and sterilized in an autoclave. (c) After cooling, 0.9 ml of starter was added. (d) The cells were cultured in a thermostatic shaker at 33°C and 200 rpm for 48 hours. (e) After the culture was completed, the cells were harvested and the pH7-protease titer was measured.

[0072] 3.Results The pH 7-protease titers in liquid media containing each main component are shown in Table 2. In Table 2, the weight average molecular weight is the value for gelatin, milk casein, proteose peptone, tryptose, select soytone and yeast powder extract.

[0073] [Table 2]

[0074] Regarding the medium components examined in this example, it was found that the use of gelatin significantly improved the amount of protease produced.

[0075] Among the N sources used in this example, the enzyme titer was low even in liquid koji using N sources with relatively large molecular weights (proteose peptone and tryptose). This is thought to be because these N sources were enzymatically treated, which left few cleavage sites for protease to recognize as substrates, and protease production was not induced. Conversely, it was found that low molecular weight N sources such as milk casein reduced activity.

[0076] [Example 2] Verification of gelatin type 1. Purpose We investigated whether the amount of protease produced was affected by the origin and manufacturing method of gelatin. 2. Experimental equipment and operation (1) Strain: Aspergillus oryzae (2) Main components of medium: defatted soybeans ("ZFS Soya", manufactured by Nisshin Shoji Co., Ltd.), beef gelatin ("Gelatin R", manufactured by Nitta Gelatin Co., Ltd.), beef gelatin ("#300", manufactured by Nitta Gelatin Co., Ltd.), pork gelatin ("APH-100", manufactured by Nitta Gelatin Co., Ltd.), pork gelatin ("APH-250", manufactured by Nitta Gelatin Co., Ltd.), fish gelatin ("Marine Gelatin", manufactured by Entec Co., Ltd.), fish gelatin ("Fish Collagen", manufactured by Nichiace Co., Ltd.) (3) species Seeds were prepared in the same manner as in Example 1. (4) Preparation of starter A starter was prepared in the same manner as in Example 1, and after completion of the culture, the pH7-protease titer was measured. 3.Results The pH 7-protease titers in liquid media containing each main component are shown in Table 3.

[0077] [Table 3]

[0078] It was found that gelatin from sources other than beef, such as pork gelatin and fish gelatin, had the same effect. It was found that the effect of gelatin on improving protease titer was highest for gelatin derived from cow, followed by gelatin derived from pig and then from fish.

[0079] [Example 3] Verification of stabilization of protease production 1. Purpose Since the amount of protease produced can be unstable when using only a high molecular weight N source such as gelatin, we investigated whether protease production could be stabilized by combining it with a medium to low molecular weight N source. 2. Experimental equipment and operation (1) Strain: Aspergillus oryzae (2) Main components of medium: defatted soybeans ("ZFS Soya", manufactured by Nisshin Shoji Co., Ltd.), beef gelatin ("Gelatin R", manufactured by Nitta Gelatin Co., Ltd.), fish gelatin ("Marine Gelatin", manufactured by Entec Co., Ltd.), fish gelatin ("Fish Collagen", manufactured by Nichiace Co., Ltd.), meat extract (LAB LEMCO POWDER [weight average molecular weight: 840 Mw], manufactured by Kanto Chemical Co., Ltd.), meat extract (pork extract TH-K, manufactured by Nippon Pure Food Co., Ltd.) (3) species Seeds were prepared in the same manner as in Example 1. (4) Preparation of starter A starter was prepared in the same manner as in Example 1, and after completion of the culture, the pH7-protease titer was measured. 3.Results The pH7-protease titers in liquid media containing each main component are shown in Table 4 and FIG.

[0080] [Table 4]

[0081] It was found that the pH 7-protease titer increased by combining gelatin and meat extract. When using only a single N source, it was easy to pellet (pelletize once every few times), but when N sources were combined, such as gelatin and meat extract, it formed a stable pulp and the amount of protease produced was also stable. In that respect, there is also an advantage to using a combination of N sources.

[0082] The pH 7-protease activity also increased when fish gelatin was used in combination with meat extract or when beef gelatin was used in combination with other meat extracts.

[0083] [Example 4] Verification of stabilization of protease production 1. Purpose It was verified whether other medium to low molecular weight N sources different from Example 3 could stabilize the amount of protease produced by gelatin. 2. Experimental equipment and operation (1) Strain: Aspergillus oryzae (2) Main components of medium: Defatted soybeans (manufactured by Nisshin Shoji Co., Ltd.), beef gelatin ("Gelatin R", manufactured by Nitta Gelatin Co., Ltd.), meat extract (beef extract 4543M [weight average molecular weight: 3600Mw]), DSP manufactured by Gokyo Food & Chemical Co., Ltd.) (3) species Seeds were prepared in the same manner as in Example 1. (4) Preparation of starter A starter was prepared in the same manner as in Example 1, and after completion of the culture, the pH7-protease titer was measured. 3.Results The pH7-protease titers in liquid medium are shown in Table 5.

[0084] [Table 5]

[0085] For other meat extracts, the protease activity was stable at a high titer by adjusting the gelatin:meat extract (dry content) ratio to 1:19 to 19:1.

[0086] [Example 5] Examination of the amount of main components added to the medium for high protease production 1. Purpose The total amount of main components in the medium was examined when gelatin and meat extract were used in combination. 2. Experimental equipment and operation (1) Strain: Aspergillus oryzae (2) Main components of medium: defatted soybeans ("ZFS Soya", manufactured by Nisshin Shoji Co., Ltd.), beef gelatin ("Gelatin R", manufactured by Nitta Gelatin Co., Ltd.), meat extract (LAB LEMCO POWDER [weight average molecular weight: 840 Mw], manufactured by Kanto Chemical Co., Ltd.) (3) species Seeds were prepared in the same manner as in Example 1. (4) Preparation of starter A starter was prepared in the same manner as in Example 1, and after completion of the culture, the pH7-protease titer was measured. 3.Results The pH7-protease titers in liquid media containing each main component are shown in Tables 6 and 7.

[0087] [Table 6]

[0088] [Table 7]

[0089] If the total amount of gelatin and meat extract is 1.0% or more, protease is relatively produced, but if it is 2.4% or more, protease production drops sharply. This peak indicates that the addition of gelatin and meat extract induces protease production, and if it exceeds the allowable range, protease production is suppressed due to catabolite inhibition. It was found that in order to produce more protease, it is desirable for the total amount of gelatin and meat extract to be 1.6 to 2.2%.

[0090] [Example 6] Verification of other media 1. Purpose They also tested whether meat by-products such as gelatin could have a similar effect. 2. Experimental equipment and operation (1) Strain: Aspergillus oryzae (2) Main medium components: In addition to the main medium components of Example 5, dried powder of Tomozune ※ , dried mino powder ※ , dried omasum powder ※ , phenylalanine, and tryptophan were used. *Each raw meat (from domestic beef) was boiled, and the fat and water fractions were removed. The residue was then dried in a blower dryer and powdered in a grinder to create the dried powder product. (3) species Seeds were prepared in the same manner as in Example 1. (4) Preparation of starter A starter was prepared in the same manner as in Example 1, and after completion of the culture, the pH7-protease titer was measured. 3.Results The pH7-protease titers in liquid media containing each main component are shown in Table 8.

[0091] [Table 8]

[0092] In this example, it was found that the test group in which the dried meat powder was used had an effect equal to or greater than that in which gelatin was added, demonstrating that the dried meat powder has the effect of inducing pH 7-protease production in liquid koji, similar to gelatin.

[0093] In addition, it was found that by combining dried meat powder with a medium to low molecular weight N source, the protease activity of liquid koji was stabilized at a high titer, similar to that of gelatin.

[0094] It was also found that the addition of amino acids such as phenylalanine and tryptophan reduced the protease titer.

[0095] It was also confirmed that when raw meat was used instead of each dry powder product, the protease titer decreased.

[0096] [Example 7] Examination of the amount of fats and oils in the medium 1. Purpose We investigated whether the amount of fat or oil contained in the medium affects the protease production in liquid koji. 2. Experimental equipment and operation (1) Strain: Aspergillus oryzae (2) Main components of the medium: In addition to the same medium composition as in Example 5, beef fat was used. The beef fat used was made by boiling minced beef shank in a water bath for 30 minutes and then collecting the fat that floated to the top of the pot. (3) species Seeds were prepared in the same manner as in Example 1. (4) Preparation of starter A starter was prepared in the same manner as in Example 1, and after completion of the culture, the pH7-protease titer was measured. 3.Results The pH7-protease titers in liquid medium are shown in Table 9.

[0097] [Table 9]

[0098] Beef tallow tended to inhibit protease production in an amount-dependent manner. When fats and oils were added at a concentration of 0.16%, a significant decrease in protease activity was confirmed.

[0099] [Example 8] Production of fermented foods using this liquid koji 1. Purpose We investigated whether liquid koji with high protease activity could be used to break down meat and produce a fermented food with excellent flavor. 2. Experimental equipment and operation As koji raw materials, liquid koji (mainly composed of 1.5% defatted soybeans, 1.0% beef gelatin, and 1.0% meat extract as in Example 3), dried rice koji (manufactured by Bioc) and dried barley koji (manufactured by Bioc) were used, and beef, mozune and salt were added to these koji raw materials to produce fermented foods. (1) Mince the beef using a food processor. (2) 240g of minced meat was placed in a jar that had been sterilized with alcohol. (3) The bottle was sterilized in an autoclave at 121°C for 20 minutes. (4) After cooling, 30 g of each koji ingredient and 30 g of salt were added and mixed well. (5) Place in a 42°C incubator and leave for approximately three weeks. (6) They were then taken out and subjected to sensory evaluation.

[0100] 3. Results and Discussion The fermented food made with liquid koji had a sweet aroma and umami flavor reminiscent of Wagyu beef, and had a beefy flavor. The minced meat turned liquid in 2-3 days, after which a savory aroma was gradually released from low molecular weight amino acids and Maillard. In contrast, the fermented foods made with rice koji and barley koji used as controls had a cereal flavor similar to miso or soy sauce, and the beef aroma was almost completely absent. This is thought to be because solid koji such as rice koji and barley koji contain a variety of organic acids and aroma components produced by the metabolism of koji mold during koji production.

[0101] In addition, these solid koji produced in an open system contain yeast-like bacteria that produce aromatic compounds, which is thought to be one of the reasons for the increase in aroma components specific to miso and soy sauce, including 4-hydroxy-2(or5)-ethyl-5(or2)-methyl-3(2H)-furanone (HEMF), during brewing and pasteurization.

[0102] Because liquid koji does not contain grain ingredients and can be produced in a sterile environment, it has been found that it is possible to produce fermented foods that make better use of the characteristics of the ingredients themselves than conventional fermented foods. In addition, when the histamine in fermented foods using liquid koji was measured, it was below the detection limit (lower limit of quantification: 0.5 mg / 100 g). [Industrial Applicability]

[0103] High-quality fermented foods and fermented seasonings can be produced using the liquid koji prepared by the method of the present invention. In addition, since the liquid koji contains almost no grains as raw materials, fermented foods and fermented seasonings that make the most of the flavor of the ingredients to be fermented can be produced more effectively than with general rice koji, etc. In addition, by using this brewing method, it is possible to produce fermented foods and fermented seasonings that do not contain allergens such as histamine.

Claims

1. A liquid medium for producing a protease from Aspergillus oryzae, comprising at least defatted soybeans, dried and ground organs or gelatin, and a meat extract, the content of defatted soybeans being 1-5% by weight relative to the total medium, the content of dried and ground organs or gelatin and the meat extract being 1.0-2.3% by weight (calculated as dry matter), and the mass ratio of the dried and ground organs or gelatin and the meat extract being 1:19-19:

1.

2. 2. The liquid medium for producing a protease according to claim 1, wherein the content of the dried pulverized organ or gelatin is 0.1 to 2.0% by weight based on the total weight of the medium.

3. 3. The liquid medium for producing a koji mold protease according to claim 1, wherein the amount of fat or oil relative to the liquid medium is 0.1% by mass or less.

4. A method for producing liquid koji with high protease production, comprising culturing koji fungus in the liquid medium for producing the koji fungus protease according to any one of claims 1 to 3.

5. The method for producing a liquid koji with high protease production according to claim 4, wherein the titer of the produced protease in the medium is 500 U / mL or more.

6. The method for producing a liquid koji that highly produces proteases according to claim 4 or 5, wherein the shape of the mycelium is unlikely to become pellet-like.

7. The method for producing a liquid koji that highly produces protease according to any one of claims 4 to 6, wherein the koji fungus is Aspergillus oryzae.

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