Fish sauce manufacturing method and fish sauce

Abstract

To provide a manufacturing method capable of manufacturing a fish sauce at a low cost and in a short time while combining salt reduction and decay suppression, and a fish sauce having excellent flavor.SOLUTION: A method for manufacturing a fish sauce comprising an enzyme reaction step using protease, and a fermentation step using a salt-resistant yeast, in which a malt is not used, in the fermentation step, a sugar is added two or more times. The fish sauce is characterized by containing 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone in the range of 0.5 to 100 ppm, and 2-phenylethanol in the range of 0.5 to 50 ppm, and lactic acid ethyl in the range of 0.5 to 50 ppm.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing fish sauce and fish sauce. [Background technology]

[0002] In recent years, there has been a steady demand for fish sauce in Japan. The main uses of fish sauce are for commercial use, such as as a seasoning in processed foods and in the food service industry.

[0003] In the traditional method of producing fish sauce, a large amount of salt is added to seafood to prevent spoilage, and the ingredients are decomposed by the action of autolytic enzymes and microorganisms derived from the environment and the ingredients, over a long period of time, usually more than one year. This traditional method has many problems, such as the generation of oxidized odors derived from lipids contained in seafood, unpleasant odors due to short-chain fatty acids and trimethylamine, and a lack of umami flavor due to insufficient maturation (Non-Patent Document 1).

[0004] One method for solving these problems is a manufacturing method using koji. There have been several reports on flavor improvements using koji, such as promoting the decomposition of proteins in the raw materials, increasing the free amino acid content and enhancing the umami taste (Non-Patent Document 2), reducing the fishy odor by suppressing the production of short-chain fatty acids (Non-Patent Document 3), and imparting kokumi (a persistent, rich, strong taste and flavor) by Maillard peptides (Non-Patent Document 4).

[0005] On the other hand, it was known that the use of koji provides sugars that are not originally contained in the raw materials of fish sauce, which accelerates browning due to the Maillard reaction (Non-Patent Document 2). Furthermore, koji is expensive and is used in a large proportion of the raw materials used, which is one of the reasons for the high production costs. In addition, the production period for fish sauce using koji is at least two months, which is also a problem.

[0006] Therefore, a method has been proposed to accelerate the decomposition of moromi by using protein-degrading enzymes (proteases).

[0007] Patent Document 1 discloses a method for producing fish sauce by enzymatically hydrolyzing raw seafood using a protease derived from Aspergillus melleus in the presence of an inorganic salt concentration of 3 to 25% (w / w) at a temperature of 25 to 45°C.

[0008] Patent Document 2 discloses a method for producing a seasoning composition, which includes the steps of allowing raw seafood to act on the raw material with decomposing enzymes, protease, and an effective amount of yeast for maturation, at a salt concentration that is effective in preventing spoilage and does not inhibit yeast growth, for a period of time effective for decomposing the raw material, to obtain a decomposition product, and then obtaining a liquid from the decomposition product.

[0009] Patent Document 3 discloses a method for producing a seasoning, which includes a first-stage fermentation in which grain koji, an enzyme agent (protease), and water are added to a minced main ingredient and then the mixture is retained, and then salt is added and retained; and a second-stage fermentation in which glucose is added to the decomposition product obtained in the first-stage fermentation and thoroughly dissolved, and then a culture solution of a salt-tolerant yeast is added and the mixture is fermented. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Japanese Patent Application Publication No. 10-42828 [Patent Document 2] JP 2011-120484 A [Patent Document 3] Japanese Patent Application Publication No. 11-178540 [Non-patent literature]

[0011] [Non-Patent Document 1] Noda, F. (1993). Fish sauces in Southeast Asia. Journal of the Brewing Society of Japan, 88, 531-536. [Non-Patent Document 2] Takeshima, F. et al. (2001). Development of fish sauce made from Bunazake salmon. Toyama Prefectural Food Research Institute Research Report, 4, 1-8. [Non-Patent Document 3] Tokiko Kojima et al. (2000). Construction of a new fish sauce manufacturing system and practical application test. Japan Soy Sauce Research Institute Journal, 26, 11-16 [Non-Patent Document 4] Saito, T. (2004). The richness and fullness of food. Journal of the Japanese Society for Taste and Smell Studies, 11, 165-174. Summary of the Invention [Problem to be solved by the invention]

[0012] However, although the method of Patent Document 1 reduces raw material costs by not using koji and reduces the fishy odor, it has the problem that it cannot impart a pleasant soy sauce-like flavor while maintaining the original seafood flavor.

[0013] Furthermore, the method of Patent Document 2 requires unheated raw materials because it uses enzymes derived from the raw materials in addition to the protease, which makes the process complicated and leaves the problem of the susceptibility to spoilage due to the use of unheated raw materials.

[0014] Furthermore, the method of Patent Document 3 uses koji and multiple microorganisms, and thus similarly has been viewed as problematic in that it increases the cost of raw materials.

[0015] The present invention has been made in consideration of the above circumstances, and aims to provide a method for producing fish sauce at low cost and in a short period of time while simultaneously reducing salt content and inhibiting spoilage, and to provide a fish sauce having a good flavor. [Means for solving the problem]

[0016] In order to achieve the above object, a first aspect of the present invention is a method for producing fish sauce, comprising the steps of: an enzymatic reaction step using a protease; a fermentation step using a salt-tolerant yeast; Including, No koji is used, In the fermentation step, sugar is added in two or more portions. It is characterized by:

[0017] For example, in the enzyme reaction step, the protease is added in two or more portions.

[0018] For example, in the enzyme reaction step, salt is added in an amount of 5 to 12% (w / w) relative to the hydrated raw material.

[0019] The fish sauce according to the second aspect of the present invention comprises: 0.5 to 100 ppm of 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone, 2-phenylethanol at 0.5 to 50 ppm, and Ethyl lactate 0.5 to 50 ppm It is characterized in that it contains

[0020] For example, the salt content is 5-12% (w / w).

[0021] For example, the free amino acid content is greater than or equal to 6% (w / w) and the organic acid content is greater than or equal to 0.7% (w / w).

[0022] For example, the chromaticity is between 23 and 52 inclusive of the soy sauce standard color scale. Effect of the Invention

[0023] According to the present invention, it is possible to provide a production method for producing fish sauce at low cost and in a short period of time while simultaneously reducing salt content and inhibiting spoilage, and to provide fish sauce having a good flavor. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a graph showing the effect of the amount of added water on the amount of free amino acids. [Diagram 2] FIG. 1 is a graph showing the effect of reaction time on the extract recovery amount. [Diagram 3]FIG. 1 is a graph showing the effect of fermentation temperature, where (a) is the number of salt-tolerant yeasts, (b) is the ethanol concentration, and (c) is the glucose concentration. [Figure 4] FIG. 1 is a graph showing the effect of culture temperature, where (a) is the number of salt-tolerant yeasts, (b) is the ethanol concentration, and (c) is the glucose concentration. [Diagram 5] FIG. 1 is a diagram showing schemes of the method of the present embodiment and a conventional method (comparative example). [Figure 6] FIG. 1 is a graph showing the results of an investigation into the moromi of fish sauce fermented by the method of this embodiment and a conventional method (comparison example), where (a) is the number of salt-tolerant yeasts, (b) is the ethanol concentration, and (c) is the glucose concentration. [Figure 7] FIG. 1 is a graph verifying the extract recovery rate of the fish sauce moromi fermented by the method of this embodiment and the conventional method (Comparative Example), where (a) is the extract recovery rate and (b) is the net extract recovery rate. [Figure 8] FIG. 1 is a photograph of fish sauce produced by the present method (Example) (right side) and fish sauce produced by a conventional method (Comparative Example) (left side). [Figure 9] FIG. 1 is a diagram showing a production scheme of fish sauce according to the present embodiment. [Figure 10] FIG. 1 shows the test methods for the group in which protease was added in portions and the group in which protease was added all at once. [Figure 11] FIG. 1 is a graph showing the extract recovery rate in the case of the protease added in separate portions or the case of the protease added all at once. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] First, the method for producing fish sauce according to the present invention will be described in detail.

[0026] In the method for producing fish soy sauce according to the present invention, by promoting the decomposition of moromi using protease without using koji, it is possible to reduce the cost of raw materials and significantly shorten the production period. In addition, by utilizing the ethanol produced by fermentation with yeast, it is possible to reduce salt while suppressing spoilage (maintaining shelf life) through the synergistic effect of salt and ethanol, and to produce aroma components to improve the aroma.

[0027] The method for producing fish sauce according to the present invention includes: (a) an enzyme reaction step using a protease; (b) a fermentation step using a salt-tolerant yeast, wherein no koji is used, and (b) in the fermentation step, sugar is added in two or more portions.

[0028] The raw material used in the method for producing fish soy sauce according to the present invention can be any fish or shellfish that can be used as a raw material for fish soy sauce, without any particular limitations, and examples of such fish or shellfish include salmon, mackerel, sea urchin, sardine, shrimp, Alaska pollock, surf clams, octopus, squid, etc. These fish or shellfish can be used after being cut into thin strips.

[0029] In the method for producing fish sauce according to the present invention, koji is not used. In this specification, koji refers to koji generally used in food products, for example, koji obtained by propagating microorganisms, mainly molds effective for food fermentation, such as Aspergillus oryzae, on grains such as rice, wheat, and soybeans. In the production method of the present invention, koji is not used, but this also includes "substantially no koji is used," and "substantially no koji is used" refers to no koji being used for the purpose of promoting protein decomposition in raw materials. Therefore, the production method of the present invention allows (i) the use of koji for purposes other than promoting protein decomposition (for example, promoting fermentation, improving flavor, etc.), and (ii) the use of koji in an amount that does not promote protein decomposition, etc.

[0030] (a) Explain the enzymatic reaction process.

[0031] As the protease, any protease capable of promoting the decomposition of moromi may be used without any particular limitation, and examples thereof include Sumiteam LP-50 (Shin-Nihon Kagaku), Protease M (Amano Enzyme), Protease P (Amano Enzyme), FS-1 (glutaminase-containing protease, Kyowa Kasei), and FS-2 (glutaminase-containing protease, Kyowa Kasei). In the method of the present invention, by using protease without using koji, moromi can be efficiently decomposed and umami can be increased. In addition, since koji is not used, raw material costs can be reduced and the production period can be significantly shortened. In addition, liquefaction of moromi can be promoted, and the generation of bad odors caused by spoilage microorganisms can be prevented.

[0032] From the viewpoint of efficiency in decomposing the moromi, the protease may be added in two or more portions. For example, by adding the protease in two portions, the decomposition of the moromi can be efficiently promoted.

[0033] The amount of protease added can be, for example, 0.1% (w / w) or more per use relative to the raw material when the aforementioned Sumizyme LP-50, Protease M or Protease P is used, and 0.05% (w / w) or more per use relative to the raw material when FS-1 or FS-2 is used.

[0034] The enzyme reaction temperature can be set to, for example, 50 to 60° C. from the viewpoint of the enzyme activity of the protease.

[0035] The enzyme reaction time can be set to, for example, 1 to 5 hours from the viewpoint of efficiency in decomposing the moromi.

[0036] The amount of water added can be, for example, 10 to 40% (w / w) relative to the raw materials. The addition of water reduces the viscosity of the moromi in the early stage of fermentation, and the enzymes are uniformly dispersed, allowing the moromi to be efficiently decomposed. At the same time, the salt is uniformly dispersed in the moromi, suppressing the proliferation of putrefactive microorganisms and preventing spoilage.

[0037] Salt can be added in an amount of, for example, 5 to 12% (w / w), preferably 7 to 11% (w / w), based on the ratio of hydrated raw material. In the present invention, by utilizing the bacteriostatic effect of ethanol produced by salt-tolerant yeast in the fermentation step (b) described below, it is possible to reduce salt while suppressing spoilage due to the synergistic effect of salt and ethanol.

[0038] (b) Explain the fermentation process.

[0039] The sugar to be used is not particularly limited as long as it is decomposed by fermentation to produce ethanol and carbon dioxide, and examples of the sugar include glucose, fructose, and sucrose.

[0040] Sugar is added in two or more portions. Sugar is essential for the growth of salt-tolerant yeast and the production of ethanol, but seafood contains very little sugar, so sugar chaptalization is necessary. On the other hand, sugar is a substrate for the Maillard reaction, so adding a large amount of sugar at once may cause browning of fish soy sauce. Therefore, in the present invention, sugar is added in two or more portions to suppress browning of fish soy sauce caused by the Maillard reaction. It is also known that ethanol produced during fermentation is used to synthesize ethyl esters such as ethyl lactate, which is an aroma component (Watanabe Jun (2018). Research on the mechanism of aroma component production in soy sauce yeast, Journal of Bioengineering, 96, (3), 106-112.). In order to reduce salt while suppressing spoilage (maintaining preservation) through the synergistic effect of salt and ethanol by using the ethanol produced by fermentation with salt-tolerant yeast, and to produce aroma components to improve the aroma, it is important to continue producing ethanol (maintaining the ethanol concentration). Adding sugar once may cause ethanol production by the salt-tolerant yeast to cease, but in the present invention, adding sugar multiple times makes it possible to continue producing ethanol (maintain the ethanol concentration).

[0041] Regarding the timing of sugar addition, in the case of adding sugar twice, for example, (i) adding it before adding the salt-tolerant yeast and (ii) adding it several days after fermentation can be mentioned. In the case of adding sugar three times, for example, (i) adding it before adding the salt-tolerant yeast, (ii) adding it several days after fermentation and (iii) adding it several days or 2 to 3 weeks after (ii).

[0042] The amount of sugar added at one time can be, for example, 3 to 7% (w / w) relative to the hydrated raw material.

[0043] As the salt-tolerant yeast, any yeast that can exhibit the effects of the present invention can be used without particular limitation. For example, the following commercially available products can be used. Zygosaccharomyces rouxii: Bioc (main fermentation yeast for soy sauce) (concentrated culture liquid, slant (agar medium solidified in a test tube in a slant shape, on which the bacteria is grown)) Zygosaccharomyces rouxii: Akita Konno Shoten (soy sauce yeast) (concentrated culture, slant) Zygosaccharomyces rouxii: Higuchi Matsunosuke Shoten (Diamonden) (Slant) Zygosaccharomyces rouxii: Hishiroku (M6, etc.) (slant) Candida versatilis: Bioc (post-ripening yeast) (concentrated yeast liquid, slant) · Candida versatilis: Higuchi Matsunosuke Shoten (Versatilis) (Slant) ·Candida etschelsii (Candida etschelsii): Higuchi Matsunosuke Shoten (Eschelsii) (Slant)

[0044] The amount of salt-tolerant yeast added is, for example, 1 × 10 4 cfu / g moromi ~1×10 8 The amount of moromi can be expressed as cfu / g.

[0045] It is preferable that the ethanol concentration is maintained at 2.0% (w / w) or more for 50% or more of the entire fermentation process, and is 2.0% (w / w) or more at the end of fermentation. With an ethanol concentration in this range, the salt content of the fish sauce can be reduced while preventing spoilage.

[0046] In the method for producing fish soy sauce according to the present invention, after (a) the enzyme reaction step and (b) the fermentation step, fish soy sauce is obtained through the steps of squeezing moromi, pasteurization, removing sediment, filtration, etc., which are generally performed in the production process of fish soy sauce. Note that, before the addition of the salt-tolerant yeast, the pH may be adjusted to a level suitable for the growth of the salt-tolerant yeast using a pH adjuster, etc.

[0047] An example of a method for producing fish sauce according to the present invention is shown below. Fish and shellfish raw materials are cut into strips, water is added at a ratio of 30% (w / w) to the raw materials, and the mixture is heated to 70°C or higher for sterilization. The evaporated water is added, and 10% (w / w) salt is added relative to the water-added raw materials, and the mixture is cooled to 55-60°C. 0.1% (w / w) protease is added, the mixture is kept warm for 4 hours, and then cooled to 35°C. 0.1% (w / w) protease is added again, 5% (w / w) glucose, and salt-tolerant yeast are added at a final concentration of 1×106 cfu / g moromi, and the mixture is kept warm for 28 days. During this time, 5% (w / w) glucose is added on the third day of fermentation, and 5% (w / w) glucose is added on the tenth day of fermentation. The moromi is then pressed, pasteurized (85°C, 30 minutes), and the fish sauce is obtained after removal of sediment and filtration.

[0048] Next, the fish sauce according to the present invention will be described in detail.

[0049] The fish sauce according to the present invention contains 0.5 to 100 ppm of 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone, 0.5 to 50 ppm of 2-phenylethanol, and 0.5 to 50 ppm of ethyl lactate. These components are aroma components, and the fish sauce according to the present invention has a preferable and good aroma (pleasant smell) produced by fermentation, mainly consisting of the soy sauce aroma familiar to Japanese people. The fish sauce according to the present invention preferably contains 10 to 100 ppm of 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone, 5 to 50 ppm of 2-phenylethanol, and 1 to 50 ppm of ethyl lactate. In addition, since 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone and its isomer 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone are interconverted, in this specification, "HEMF" may refer to "4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone".

[0050] The above components can be measured, for example, by GC-MS using a sample extracted from fish sauce by solid phase extraction.

[0051] The salt content of the fish sauce according to the present invention is, for example, 5 to 12% (w / w), preferably 7 to 11% (w / w). Since the salt content is about the same as that of reduced-salt soy sauce, the fish sauce according to the present invention meets the needs of health-conscious people.

[0052] The salt content can be measured by a known method, for example, using a salinity meter.

[0053] The free amino acid content is, for example, 6% (w / w) or more, and the organic acid content is, for example, 0.7% (w / w) or more. The richness of free amino acids enhances the umami taste and has the effect of reducing the saltiness. The richness of organic acids also has the effect of reducing the saltiness.

[0054] The free amino acid content can be measured by a known method using, for example, an amino acid analyzer, and the organic acid content can be quantified by, for example, HPLC using a post-column pH buffered electrical conductivity detection method.

[0055] The color tone is, for example, within the range of standard soy sauce colors 23 to 52 in the "Japanese Agricultural Standards for Soy Sauce." Because it is a light color, it can be used for a wide variety of food applications.

[0056] The fish sauce according to the present invention can be produced by the above-mentioned production method of the present invention, although it is not particularly limited thereto.

[0057] As described above, the method for producing fish soy sauce according to the present invention does not use koji and efficiently decomposes moromi using protease, so that it is possible to reduce the cost of raw materials and to significantly shorten the production period. In addition, it is possible to promote the liquefaction of moromi, prevent the generation of bad odors caused by spoilage microorganisms, and accelerate the generation of umami. Furthermore, by adding sugar multiple times in the fermentation process, it is possible to make the salt-tolerant yeast continue to produce ethanol (maintain the ethanol concentration), so that it is possible to achieve both salt reduction and spoilage prevention (maintaining shelf life) due to the synergistic effect of salt and ethanol. In addition, it is possible to suppress browning due to the Maillard reaction, obtain a light-colored fish soy sauce, and impart a good flavor through continuous ethanol production. According to the present invention, it is possible to produce fish soy sauce having a preferable and good flavor (pleasant aroma) generated by fermentation, mainly consisting of the soy sauce flavor familiar to Japanese people. EXAMPLES

[0058] The present invention will be specifically described below with reference to examples, although the present invention is not limited to these examples.

[0059] In the following examples, percentages (%) are by mass unless otherwise specified.

[0060] Example 1 A method for producing fish sauce using protease was investigated as follows.

[0061] (1) Raw materials The raw material used was a round frozen product of Chum Salmon (Oncorhynchus keta) caught in Erimo Town, Hidaka District in 2016. The grade was C crucian carp, and the sex was male. The frozen raw material was thawed and separated into head, innards, and flesh (fish skin and muscle), which were then minced using a meat grinder (C4, Higashimoto Kikai) through a 5mm screen and frozen at -20°C. For the test, the required amount of each part was left to thaw overnight at 5°C, and then remixed according to the composition ratio of the raw material.

[0062] (2) Consideration of the amount of water to be added In investigating the use of proteases to increase the umami components of fish sauce, we expected that the reaction would differ depending on the amount of water added to the fish meat during the enzyme reaction, so we investigated the effect of the amount of water added. Distilled water equivalent to 0-50% (w / w) of the raw material ratio and protease (Sumiteam LP-50 (Shin Nippon Chemical Industry Co., Ltd.), hereinafter referred to as LP) equivalent to 0.2% (w / w) were added to the raw material prepared in (1) above, and the enzyme reaction was carried out in a water bath at 55°C. After the reaction, the amount of extract recovered, the amount of solubilization, and the amount of free amino acids (Figure 1) were measured using the methods described below.

[0063] (3) Examination of enzyme reaction conditions In order to enhance the umami components by using proteases, the type, concentration and reaction time of the enzyme were investigated. Protease M (Amano Enzyme, hereafter referred to as AM), Protease P (Amano Enzyme, hereafter referred to as AP) and LP were used. The amount of protease added was 0.1, 0.2 and 0.3% (w / w) relative to the raw material. The reaction temperatures were 50, 55 and 60°C, and the reaction times were 1, 2, 3 and 4 hours. In addition, since it was expected that the amount of salt added would affect the enzyme reaction, the effect of the amount of salt added was confirmed. After adding 30% (w / w) of water to the raw material, salt was added at 0, 4, 8, 12 and 16% (w / w) relative to the hydrated raw material, and 0.2% (relative to the raw material) of enzyme LP was added to each group and reacted at 55°C for 4 hours. In examining the enzyme reaction conditions described above, the extract recovery amount (Figure 2), Brix, and free amino acid amount (Tables 1 and 2) were measured after the reaction using the methods described below.

[0064] (4) Fermentation conditions using salt-tolerant yeast In order to reduce the salt content of fish sauce by utilizing the bacteriostatic effect of ethanol produced by yeast, the effects of starter type and fermentation temperature on ethanol production were investigated. A method for increasing the ethanol concentration by sugar addition was also investigated. Two strains of concentrated liquid starter of commercially available salt-tolerant yeast (Zygosaccharomyces rouxii) used in soy sauce production were used as starters: soy sauce yeast (Akita Konno Shoten, hereafter referred to as strain A) and soy sauce main fermentation yeast (Bioc, hereafter referred to as strain B). Instead of fish sauce moromi, 0.5% (w / v) Nutrient Broth (Kyokuto Pharmaceuticals), 0.5% (w / v) Bacto Peptone (BD), 5% (w / v) glucose, 10% (w / v) salt, and 0.1% (w / v) LP were used as the medium, and 300 mL was prepared per test. Cultivation was performed three times by static cultivation at 30 to 40°C. The culture liquid was thoroughly mixed and samples were collected to measure the number of salt-tolerant yeast cells (Figure 3(a)), ethanol concentration (Figure 3(b)), and glucose concentration (Figure 3(c)). In addition, when the glucose concentration in the moromi mash reached the 1% (w / v) range, 5% (w / v) sugar was added, and samples were collected during the fermentation period to measure the number of salt-tolerant yeast cells (Figure 4(a)), ethanol concentration (Figure 4(b)), and glucose concentration (Figure 4(c)).

[0065] (5) Fish sauce fermentation test using enzyme reaction and yeast fermentation in combination It was confirmed whether the desired salt reduction, lightening of color, and suppression of fishy odor could be achieved by combining the enzymatic hydrolysis and fermentation conditions examined in (2) to (4) above. Water (raw material ratio 30% (w / w)), salt (same 10% (w / w)), and LP (same 0.1% (w / w)) were added to the raw material and subjected to an enzymatic reaction at 55°C for 4 hours. After cooling to 35°C, LP (same 0.1% (w / w)) and salt-tolerant yeast (A strain, final cell count 1 x 10 6 cfu / g) and glucose (5% (w / w)) were added and fermented at 35°C for 4 weeks. The same amount of glucose was added on the 7th and 14th days after fermentation. The fish sauce was prepared by heating the juice obtained by squeezing the moromi after the fermentation was completed at 85°C for 30 minutes, adding a 10-fold diluted solution of a lees reducer (Coporoc SA, Otsuka Foods) in an amount of 1 / 20 of the weight of the supernatant and diatomaceous earth (Celpure S300, Advanced Minerals Corporation) in an amount of 0.1% of the weight of the supernatant, leaving it to stand for 3 days, and filtering under reduced pressure (test group "LP" in Figure 5). Part of the culture solution was sampled during the fermentation period, and the number of salt-tolerant yeasts (Figure 6(a)), ethanol concentration (Figure 6(b)), and glucose concentration (Figure 6(c)) were measured. The free amino acids and organic acids (Table 3) of the fish sauce samples were also measured, as well as the aroma components (Table 4).

[0066] (6) Component analysis and measurement of salt-tolerant yeast count The component analysis and measurement of the number of salt-tolerant yeasts were carried out by the following methods. (6-1) Extract recovery volume The moromi was centrifuged at 3,500 rpm for 20 minutes in a centrifuge (CR-4, Hitachi Koki) and the weight of the supernatant was recorded. The weight obtained by subtracting the amount of water added from the amount of extract recovered was recorded as the solubilized amount. (6-2)BRIX The supernatant prepared in (6-1) above was measured five times using a BRIX densitometer (PAL-1, Atago) and the average value was calculated. (6-3) Free Amino Acids The supernatant prepared in (6-1) above was diluted 10-fold with distilled water, and an equal volume of 2% (w / v) aqueous sulfosalicylic acid solution was added to denature the proteins. The solution was then filtered through a cellulose acetate filter with a pore size of 0.45 μm and analyzed with an amino acid analyzer (L-8900, Hitachi High-Technologies). (6-4) Color of fish sauce This was done by comparing the color of the prototype with a color sample of soy sauce color numbers (the standard soy sauce colors in the "Japanese Agricultural Standards for Soy Sauce"), and the number closest to the color was selected. (6-5) Number of salt-tolerant yeasts According to a previously reported detection method (Yoshikawa S., Kurihara H., Kawai Y., Yamazaki K., Tanaka A., Nishikiori T., and Ohta T. (2010). Effect of halotolerant starter microorganisms on chemical characteristics of fermented chum salmon (Oncorhynchus keta) sauce. J. Agric. Food Chem., 58, 6410-6417.), the detection medium was prepared by adding chloramphenicol to a final concentration of 100 mg / L to potato dextrose agar medium (Nissui Pharmaceutical Co., Ltd.) containing 0.5% (w / w) yeast extract and 10% (w / w) salt. The moromi was diluted 10-fold in steps with sterilized 0.85% saline, and the number of bacteria was measured by the plate spreading method (incubated at 30°C for 3 days). (6-6)Glucose The moromi was appropriately diluted with distilled water, and the glucose content was measured using the glucose CII Test Wako (Wako Pure Chemical Industries) by the glucose oxidase method. (6-7) Organic acid amount Quantification was performed using HPLC (Prominence, Shimadzu Corporation) with post-column pH buffered electrical conductivity detection. 1 mL of sample was centrifuged at 10,000 rpm, 4°C, 5 min, and 50 μL of the supernatant was diluted 50-fold with water and passed through a 0.45 μm cellulose acetate filter before being used in the test. Two Shim-pack SCR-102H (300 mm x 8.0 mm id, Shimadzu Corporation) columns were connected in series, and a Shim-pack SCR 102 HG (50 mm x 6.0 mm i.d., Shimadzu Corporation) guard column was used. The mobile phase was a 20 mM aqueous bis(2-hydroxyethyl)-iminotris(hydroxymethyl)methane solution (containing 5 mM p-toluenesulfonic acid and 100 μM EDTA), the flow rate was 0.8 mL / min, the column temperature was 40°C, the sample injection volume was 10 μL, and the detector was an electrical conductivity detector (CDD-10A VP, Shimadzu Corporation). (6-8) Aroma components The method of Suezawa (Suezawa Yasuhiko (1998). Rapid analysis of 2-phenylethanol, 4-ethylguaiacol and 4-ethylphenol in soy sauce using solid-phase extraction. Journal of the Japan Soy Sauce Research Institute, 24, pp. 21-25) was modified and samples extracted from fish sauce by solid-phase extraction were measured by GC-MS (GCMS-QP2010 Plus, Shimadzu Corporation). 3 g of salt was added to 7 mL of fish sauce and the mixture was shaken at 300 rpm for 10 minutes to prepare a salt-saturated sample. 5 mL of this was taken, mixed with 50 μL of 1 mg / mL 1-decanol as an internal standard, and adsorbed onto a SepPak C18 Plus cartridge (Waters) conditioned with 20 mL each of methanol and distilled water. The elution was performed with 0.8 mL of methyl acetate and subjected to GC-MS. The measurement conditions for GC-MS were as follows: That is, the capillary column used was DB-Wax (30 m × 0.32 mm id, film thickness 0.25 μm, J&W Scientific), and the heating conditions were 40 °C hold for 2 min, 40 °C to 160 °C rise at 6 °C per minute, 160 °C to 240 °C rise at 10 °C per minute, and 240 °C hold for 6 min. The carrier gas was helium, the flow rate was 3 mL / min, the injection port temperature was 250 °C, and the injection volume was 1 μL, and the sample was introduced in splitless mode. (6-9) Ethanol Measurements were performed using a gas chromatograph (Model 263-70, Hitachi, Ltd.). 0.25 volumes of acetone was added to the fish sauce as an internal standard substance to prepare a measurement sample. Measurements were performed using an FID detector under the following conditions: glass column φ3 mm × 2.1 m, packing material 5% polyethylene glycol 1000 (GL Science), column temperature 75°C, injection port and detector temperatures 230°C, carrier gas nitrogen, and flow rate 40 mL / min. (6-10) Salinity 9 g of distilled water was added to 1 g of fish sauce and mixed thoroughly, and the average value was calculated after measuring three times using a salinity meter (PAL-SALT, Atago).

[0067] (7) Technology demonstration tests by companies In order to confirm the effectiveness of the manufacturing method (hereinafter referred to as this method) (Figure 9) developed in this study, which combines enzymatic decomposition and fermentation processes, on a practical production scale, a technical demonstration test was conducted at two fish sauce manufacturing companies in Hokkaido, using salmon fillets as the raw material and repeating two replicates of approximately 50 kg each (one of the companies added a test area using mackerel as the raw material).

[0068] (8) Experimental planning and statistical processing Using the statistical processing software JMP (SAS-Japan Ver.13.1), the test areas were set based on the experimental design. Statistical processing (significance test) was performed using the Tukey-Krammer HSD test at a significance level of 5%.

[0069] (result) (1. Examination of the moromi enzyme decomposition process) The amount of water added to the raw material was examined. As the amount of water added increased, the net solubilized amount of moromi, calculated by subtracting the amount of water added from the liquid volume obtained by centrifugation, decreased (data not shown). In addition, compared to no water addition (comparison example), the amount of free amino acids in the extract increased when the amount of water added was 10% and 30% of the raw material, and reached a maximum at 30% water addition (Figure 1) (in Figure 1, error bars indicate standard deviation (n = 3)). This is thought to be because the viscosity of the moromi at the beginning of fermentation was reduced by the addition of water, allowing the enzymes to disperse evenly and enzymatic decomposition to proceed efficiently. On the other hand, with 50% water addition, the amount of free amino acids in the extract decreased compared to no water addition. In addition, the amount of extract recovered with 30% water addition increased by about two times after 1 hour of reaction and remained flat at 4 hours of reaction time (Figure 2).

[0070] Factors affecting the free amino acid concentration of the extract were examined, including the type of enzyme used to decompose the moromi, reaction temperature, reaction time, amount of enzyme added, and amount of salt added. The results are shown in Table 1.

[0071] [Table 1]

[0072] Regarding the type of enzyme, the free amino acid concentration in the extract was high in the AP and LP application groups. However, there was no significant difference between the AP and LP application groups.

[0073] Regarding reaction temperature, the free amino acid concentration in the extract was highest at 55°C.

[0074] Regarding the enzyme reaction time, the free amino acid concentration of the extract increased significantly as the enzyme reaction time increased, reaching the highest value after 4 hours of reaction.

[0075] The free amino acid concentration of the extract increased as the amount of enzyme added increased. There was no significant difference between 0.2% and 0.3% in terms of the ratio to the raw material.

[0076] From the above results, it was considered that the enzymatic hydrolysis would proceed more smoothly by adding 30% water, 0.2% AP or LP enzyme, setting the enzyme reaction time to 4 hours, and setting the reaction temperature to 55°C.

[0077] Under the above enzyme reaction conditions, the relationship between the amount of salt added and the amount of free amino acids in the extract was examined. The results are shown in Table 2.

[0078] [Table 2]

[0079] When salt was added at 8% or more, the amount of free amino acids was significantly lower than in the control without salt (comparative example). No significant difference in the amount of free amino acids was observed between concentrations of 8% or more.

[0080] (2. Fermentation process considerations) In order to popularize this method and to make it possible for companies that do not have facilities for culturing microorganisms to manufacture the product, we investigated the suitability and fermentation temperature using two concentrated strains of commercially available salt-tolerant yeast (Z. rouxii) so that the work of culturing the bacteria would be unnecessary. The results are shown in Figure 3.

[0081] The number of salt-tolerant yeast strains (Figure 3(a)) showed good proliferation in the early stage of fermentation at 30°C and 35°C for both strains. Furthermore, at 40°C, the proliferation rate of strain B was slower than that of strain A at the early stage of fermentation, but ultimately reached the same level of proliferation.

[0082] The maximum ethanol concentration (Figure 3(b)) was approximately 2% regardless of the fermentation temperature. In addition, the time it took for the ethanol to reach its maximum concentration tended to be shorter at fermentation temperatures of 30°C and 35°C.

[0083] The glucose concentration (Figure 3(c)) tended to decrease in association with an increase in the number of salt-tolerant yeast strains, and at 35°C, it tended to decrease more rapidly in the early stages of fermentation.

[0084] In this test, it was confirmed that when strain A was used for fermentation, a maximum of 2% ethanol was produced. It is known that ethanol has an osmotic pressure similar to that of salt, and that the combined use of ethanol and salt produces a higher osmotic pressure (Ogawa Toshio (1989). Salt and Pickles, "Pickle Manufacturing Studies", Korin, Tokyo, pp. 25-56.). It has also been reported that in order to stably produce fish sauce using yamame salmon as the raw material, it is necessary to add 10-12% salt (Kojima Tokiko et al. (2000). Construction of a new fish sauce manufacturing system and practical application tests. Japan Soy Sauce Research Institute Journal, 26, appended 11-16).

[0085] Therefore, because it is expected that the ethanol produced by the salt-tolerant yeast will reduce the salt content of fish sauce, a trial was carried out with the salt concentration reduced to 10%. However, as mentioned above, the ethanol produced gradually decreases after reaching 2%, so an attempt was made to maintain the ethanol concentration by adding glucose during the fermentation period.

[0086] The results of glucose chaptalization are shown in Figure 4. The number of salt-tolerant yeasts (Figure 4(a)) did not differ significantly depending on the fermentation temperature, and the number of salt-tolerant yeasts at the end of fermentation was 1.1 × 10 at 30°C. 8 CFU / mL, 3.9 × 10 at 35°C 7 CFU / mL.

[0087] The ethanol concentration (Figure 4(b)) reached a maximum of 3.6% by performing chaptalization twice, and was maintained at 2% or higher until the end of fermentation.

[0088] The glucose concentration (Figure 4(c)) remained at around 1% even when sugar was added, suggesting that browning due to the Maillard reaction could be suppressed.

[0089] From the above results, it was found that by using strain A in the fermentation process, setting the fermentation temperature at 35°C, and adding glucose twice, it was possible to maintain the ethanol concentration at the end of fermentation at 2% or higher, which was more effective in reducing salt and preventing browning.

[0090] (3. Small-scale fish sauce fermentation test using a combination of enzyme reaction and yeast fermentation) Based on the enzyme reaction conditions in 1. above and the fermentation conditions in 2. above, the present method (Example) combining the enzyme decomposition step and the fermentation step was compared with a conventional method using koji (hereinafter, the conventional method) (Comparative Example). The scheme of each test section is shown in FIG. 5 (in FIG. 5, the present method (Example) is the test section of "LP"). Regarding the addition of protease, taking into consideration that the enzyme decomposition is almost flat depending on the reaction time as shown in FIG. 2, it was decided to add it in two separate steps, that is, 0.1% (w / w) of LP was added when the mash was prepared, and the mash was cooled after the enzyme decomposition step was completed, and the same amount was added again at the time of adding yeast. In addition, during the period of "heating at 35°C for 4 weeks" in FIG. 5, the first chaptalization was performed at the start of yeast fermentation, the second chaptalization was performed 3 to 7 days after the yeast was added, and the third chaptalization was performed 19 to 26 days after the yeast was added (all 5% (w / w) glucose of the mash weight). The verification of the divided addition of enzymes will be described later in Example 2.

[0091] The results are shown in Figure 6. The number of salt-tolerant yeasts in the moromi mash by this method (Example) peaked on the third day of fermentation, then dropped rapidly, but was detected again after the 24th day of fermentation (Figure 6(a)). The ethanol concentration reached the target concentration of 2% on the seventh day after fermentation by this method, and reached about 3% at the end of fermentation (Figure 6(b)). On the other hand, in the conventional method (Comparative Example), the ethanol concentration never exceeded 0.4% throughout the fermentation period (Figure 6(b)). In the present method (Example), the glucose concentration in the moromi mash was maintained at 0.5% or less from the 10th day, and the added glucose was quickly consumed (Figure 6(c)). On the other hand, in the conventional method (Comparative Example), the concentration was 10% or more from the 3rd day (Figure 6(c)), suggesting that the darkening due to the Maillard reaction progresses.

[0092] Regarding the extract recovery rate (Figure 7), the moromi produced by this method (Example) was higher than that of the conventional method (Comparative Example) throughout the test period. In this method (Example), water was added at 30% of the raw material ratio when preparing the moromi, so even in the net extract recovery rate after subtracting this amount, the method had a recovery rate that was about 9% higher.

[0093] The organic acid and free amino acid compositions of the fish sauces produced by this method (Example) and the conventional method (Comparative Example) are shown in Table 3. With regard to free amino acids, the fish sauce produced by this method had a higher total amount of free amino acids than that produced by the conventional method, and by type, lysine, glutamic acid, asparagine, etc. were abundant, with the asparagine content being particularly high, but leucine being low. With regard to organic acids, the fish sauce produced by this method had a slightly 20% increase in organic acids compared to that produced by the conventional method, with lactic acid being particularly abundant.

[0094] [Table 3]

[0095] The salt content of the fish sauce produced by this method (Example) was 9.2%, whereas that of the fish sauce produced by the conventional method (Comparative Example) was 14.7%. As for color, the fish sauce produced by this method had a soy sauce color number of 35, which was equivalent to white soy sauce, whereas the fish sauce produced by the conventional method had a soy sauce color number of 13, which was equivalent to dark soy sauce. Photographs of the fish sauce produced by this method (Example) and the fish sauce produced by the conventional method (Comparative Example) are shown in FIG. 8. In FIG. 8, the left side is the fish sauce produced by the conventional method (Comparative Example), and the right side is the fish sauce produced by this method (Example). The fish sauce produced by this method (Example) was obviously lighter in color than that of the conventional method (Comparative Example).

[0096] Table 4 shows the aroma components of fish sauce produced by this method (Example) and the conventional method (Comparative Example). In the fish sauce produced by this method, due to the addition of salt-tolerant yeast, aroma components such as furanones such as HEMF (4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone), 2-phenylethanol, and ethyl lactate, which are hardly contained in the fish sauce produced by the conventional method, were detected. In addition, components derived from lipid oxidation of seafood, such as 1-penten-3-ol, which cause unpleasant odors, were not detected. In Table 4, "HEMF" refers to 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone.

[0097] [Table 4]

[0098] (4. Technology demonstration tests by companies) To confirm the effectiveness of this method on an actual production scale, a technical demonstration test was conducted at two fish sauce manufacturing companies in Hokkaido, based on the process shown in Figure 9. As a result, compared to the conventional method, the fermentation period was shortened from two months to one month, and it was confirmed that the color was lighter, the salt content was reduced (10%), and aromatic components were added; similar effects were confirmed when mackerel was used as the raw fish (data not shown). The cooperating companies estimated that this method can reduce costs by about 20% compared to the conventional method. From the above, the effectiveness of this method was confirmed even on an actual production scale.

[0099] Example 2 The effect of adding protease in portions during the enzyme reaction process was examined.

[0100] The schemes for the test section where protease was added in two separate portions and the test section where protease was added all at once are shown in Figure 10. In Figure 10, ethanol was added in order to verify the effect of adding protease in separate portions under similar conditions (in the presence of ethanol), in consideration of the fact that yeast (originally used in the production of fish sauce) produces ethanol.

[0101] 13 g of the moromi was taken and centrifuged at 3200 rpm for 20 minutes. The weight of the supernatant obtained was taken as the amount of recovered extract, and the ratio of the amount of recovered extract to the weight of the moromi before centrifugation was taken as the extract recovery rate.

[0102] The results are shown in Figure 11. When adding protease all at once (0.2%) in the enzyme reaction process, compared to adding it in separate portions at 0.1% each at the start of the enzymatic hydrolysis process and the fermentation process, the extract recovery rate was shown to be approximately 8% higher when added in separate portions.

[0103] Example 3 The amount of aroma components in the fish sauce produced by the method of Example 1 was quantified, and a test for determining the optimum concentration of the aroma components was carried out.

[0104] (Test Method) The test was carried out using fish sauce produced by the scheme in FIG. 5 and “4. Technical demonstration experiment by a company” shown in Example 1. In FIG. 5, “LP” is Sumiteam LP-50 (Shin-Nihon Kagaku), and “FS-1” and “FS-2” are glutaminase-containing proteases (Kyowa Kasei). Regarding the addition of proteases, LP was added at 0.1% (w / w) and FS-1 or FS-2 at 0.05% (w / w) when the moromi was prepared, and the moromi was cooled after the enzymatic decomposition process was completed, and the same amount was added again when the yeast was added. In addition, during the period of “heating at 35° C. for 4 weeks” in FIG. 5, the first chaptalization was carried out at the start of yeast fermentation, the second chaptalization was carried out 3 to 7 days after the yeast was added, and the third chaptalization was carried out 19 to 26 days after the yeast was added (all 5% (w / w) glucose of the moromi weight).

[0105] Quantitative analysis of aroma components in fish sauce produced by the method of Example 1 0.1% 1-decanol was added to the fish sauce as an internal standard substance, and quantitative analysis was performed by gas chromatography in the same manner as in Example 1.

[0106] Testing to determine the appropriate concentration of aroma components The aroma components to be detected were HEMF (4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone), 2-phenylethanol, and ethyl lactate, the presence of which had been confirmed in advance by GC-MS analysis. Samples were prepared by adding the respective standards (HEMF: SIGMA, 2-phenylethanol: SIGMA, ethyl lactate: Kanto Chemical) to final concentrations of 0.1, 0.5, 1, 5, 10, 50, 100, and 500 ppm to Atka mackerel fish sauce (Katakura Foods) that did not contain these components. Four panelists (one male, three females; two in their 30s, one in their 40s, and one in their 50s) whose ability to distinguish odors had been confirmed in advance evaluated the aroma components using a five-point scale of no difference, slight difference, some difference, difference, and very difference, and also answered whether or not they felt any discomfort with the aroma. The appropriate concentration range was determined as the concentration range in which more than half of the panelists could detect a difference in odor from the control and did not feel any discomfort.

[0107] The ability to distinguish odors was confirmed by a preliminary test based on the 5-2 method, an olfactory test method used in the National Examination for Odor Evaluators, using the standard odor for panel selection (Daiichi Pharmaceutical Industry) used in the same examination. The 5-2 method involves soaking two of five pieces of perfumed filter paper with the odor component to be distinguished, providing them to the subject at random and asking them to answer whether the odor was present or not. The subject was deemed to have the ability to distinguish odors if they could distinguish two of the five scented filter papers. The kit was composed of five odor components with different scent characteristics: 2-phenylethanol, methylcyclopentenolone, isovaleric acid, γ-undecalactone, and skatole. Subjects who could distinguish all of these odors were deemed to have the ability to distinguish odors.

[0108] (Test Results) Quantitative analysis of aroma components in fish sauce produced by the method of Example 1 The aroma components were ethyl lactate at 2.6 to 7.5 ppm, 2-phenylethanol at 9.7 to 31.7 ppm, and HEMF at 19.8 to 95.0 ppm (Table 5). In Table 5, "HEMF" refers to 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone.

[0109] [Table 5]

[0110] Testing to determine the appropriate concentration of aroma components The addition of ethyl lactate, 2-phenylethanol, and HEMF (4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone), which are not present in the conventional method, was confirmed by this manufacturing method. Therefore, the effect of the added aroma components on the sensory evaluation was examined by adding them at various concentrations to fish sauce that does not contain these three components. As a result, the aroma of HEMF was not felt at 0.1 ppm, the difference from the control was recognized at 0.5 ppm or more, and an unpleasant smell like a chemical odor occurred at 500 ppm. The aroma of 2-phenylethanol was not felt at 0.1 ppm, the difference from the control was recognized at 0.5 ppm or more, and an unpleasant smell occurred at 100 ppm or more. The difference from the control was not felt at less than 0.1 ppm, the difference from the control was recognized at 0.5 ppm or more, and an unpleasant smell occurred at 100 ppm or more (Table 6). In Table 6, the area surrounded by a thick line indicates the appropriate concentration range that does not cause an unpleasant smell from a sensory perspective. Also, in Table 6, "HEMF" refers to 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone.

[0111] [Table 6]

[0112] From the above, it was revealed that the desirable aroma component concentrations for HEMF (4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone) are 0.5 ppm or more and 100 ppm or less, and for 2-phenylethanol and ethyl lactate, 0.5 ppm or more and 50 ppm or less. The aroma components generated by the production method of this example were 2.6 to 7.5 ppm for ethyl lactate, 9.7 to 31.7 ppm for 2-phenylethanol, and 19.8 to 95.0 ppm for HEMF, all of which were within the appropriate aroma component concentration range. From this, it was revealed that the production method of this example is an excellent production method that imparts a good aroma to fish sauce.

[0113] It was shown that the method for producing fish sauce in this example can produce a light-colored fish sauce that is rich in umami, has low salt content (less than 10%), and contains three types of aromatic components, in a short period of one month. It is expected that this method will reduce production costs by cutting raw material costs and shortening the production period.

Claims

1. an enzymatic reaction step using a protease; a fermentation step using a salt-tolerant yeast; Including, No koji is used, In the fermentation step, sugar is added in two or more portions to suppress browning of the fish sauce due to the Maillard reaction, and the ethanol concentration is maintained at 2.0% (w / w) or more for 50% or more of the entire period of the fermentation step, and the ethanol concentration is set to 2.0% (w / w) or more at the end of the fermentation step. The method for producing fish sauce is characterized by the above.

2. In the enzyme reaction step, the protease is added in two or more portions. The method for producing fish sauce according to claim 1 .

3. In the enzyme reaction step, salt is added in an amount of 5 to 12% (w / w) based on the hydrated raw material. The method for producing fish sauce according to claim 1 or 2.

4. 0.5 to 100 ppm of 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone or 4-hydroxy-5-ethyl-2-methyl-3(2H)-furanone, 2-phenylethanol at 0.5 to 50 ppm, and The method further comprises obtaining a fish sauce containing 0.5 to 50 ppm of ethyl lactate. The method for producing fish sauce according to claim 1 .

5. The salt content of the fish sauce is 5 to 12% (w / w). The method for producing fish sauce according to claim 4 .

6. The free amino acid content of the fish sauce is 6% (w / w) or more, and the organic acid content is 0.7% (w / w) or more. The method for producing fish sauce according to claim 4 or 5.

7. The color of the fish soy sauce is a soy sauce standard color of 23 or more and 52 or less. The method for producing fish sauce according to claim 4 .

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