Agents and methods for inhibiting the hardening of fish eggs, agents and methods for softening fish eggs, and methods for producing processed fish egg products.

Abstract

The objective is to provide a technology that suppresses the hardening of fish roe during storage and maintains an appropriate texture. Furthermore, it aims to provide a technology that can effectively soften hard fish roe. [Solution] A hardening inhibitor and softening agent for fish roe, comprising chymotrypsin as the active ingredient. According to the present invention, the hardening of fish roe during storage can be suppressed, maintaining an appropriate texture. This can prevent a decline in the commercial value of processed fish roe products. Furthermore, according to the present invention, hard fish roe can be softened to a texture suitable for consumption. This makes it possible to utilize raw eggs that were previously unsuitable for consumption or had low commercial value, contributing to the reduction of food waste and the improvement of the value of processed fish roe products.

Description

Technical Field

[0001] The present invention relates to a novel use of chymotrypsin, and more particularly, to an agent and method for suppressing the hardening of fish eggs, an agent and method for softening fish eggs, and a method for producing a processed food of fish eggs, characterized by using chymotrypsin as an active ingredient.

Background Art

[0002] Various fish eggs such as salmon roe, cod roe, flying fish roe, and fish fry are favorably consumed as food. On the other hand, as is particularly notable in the case of salmon roe with high demand, as the maturity of the raw eggs progresses, the egg membrane hardens, and the egg grains become hard and rubbery, resulting in a significant decline in texture and taste, which is a major problem. Therefore, technologies for softening the egg membrane have been researched and developed. For example, Patent Document 1 discloses a method for softening the egg membrane of overripe salmon roe, characterized by treating overripe salmon roe with a protease.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, the protease described in Patent Document 1 is to be allowed to act at 50°C for 10 to 20 minutes and at 10°C for 10 to 20 hours (Examples). Such relatively high treatment temperatures and long treatment times have problems such as being unfavorable from the viewpoint of food hygiene, increasing the energy cost for heat retention, or lengthening the product manufacturing cycle.

[0005] Furthermore, the present inventors have now discovered a novel problem: even processed salmon roe products (salted or soy sauce-marinated salmon roe) that have an appropriate texture immediately after manufacturing become hardened during refrigerated storage after thawing, resulting in a hard texture when consumed. No technology capable of suppressing this hardening of fish roe during storage is disclosed in Patent Document 1.

[0006] This invention was made to solve these problems and aims to provide a technology that can suppress the hardening of fish roe during storage and maintain an appropriate texture. It also aims to provide a technology that can effectively soften hard fish roe even when applied for a short time or at low temperatures. [Means for solving the problem]

[0007] As a result of diligent research, the inventors have discovered that chymotrypsin significantly suppresses the hardening of fish roe during storage and maintains an appropriate texture. They also found that chymotrypsin can significantly soften hard fish roe even when applied for a short time or at low temperatures. Based on these findings, the inventors have completed the following inventions.

[0008] (1) The fish roe hardening inhibitor according to the present invention is an agent for inhibiting the hardening of fish roe, and comprises chymotrypsin as an active ingredient.

[0009] (2) The fish roe softening agent according to the present invention is an agent for softening fish roe, and comprises chymotrypsin as an active ingredient.

[0010] (3) The method for suppressing the hardening of fish eggs according to the present invention comprises the step of contacting the fish eggs with chymotrypsin.

[0011] (4) The method for softening fish eggs according to the present invention comprises the step of contacting the fish eggs with chymotrypsin.

[0012] (5) A method for producing processed fish roe food according to the present invention comprises the step of contacting the fish roe with the fish roe hardening inhibitor of (1) or the fish roe softener of (2). That is, the method comprises the step of contacting the fish roe with chymotrypsin in order to suppress the hardening of the egg grains, or the step of contacting the fish roe with chymotrypsin in order to soften the egg grains. [Effects of the Invention]

[0013] According to the present invention, the hardening of fish roe during storage can be suppressed, maintaining an appropriate texture. This helps to prevent a decline in the commercial value of processed fish roe products. Furthermore, according to the present invention, hard fish roe can be softened to a texture suitable for consumption. This makes it possible to utilize raw eggs that were previously unsuitable for consumption or had low commercial value, contributing to the reduction of food waste and the improvement of the value of processed fish roe products. [Brief explanation of the drawing]

[0014] [Figure 1] This bar graph shows the percentage change in hardness (%) between the hardness on day 1 and day 3 when salted salmon roe treated with enzymes A-D during the loosening process is refrigerated for 3 days after thawing. [Figure 2] This bar graph shows the percentage change in hardness (%) between the hardness on day 1 and day 2 when salted salmon roe, treated with enzymes B to D during the pickling process, is refrigerated for two days after thawing. [Figure 3] This bar graph shows the breaking load of salted salmon roe made from pinko fish, after being treated with enzymes B to D as a softening process. [Modes for carrying out the invention]

[0015] The present invention will be described in detail below.

[0016] This invention is characterized by inhibiting the hardening of fish eggs or softening fish eggs using chymotrypsin, and in this invention, chymotrypsin may be referred to as the active ingredient.

[0017] In the present invention, fish eggs refer to eggs of fishery products (including egg cells). Here, the eggs may be those before spawning or after spawning. Also, the eggs may be unfertilized eggs or fertilized eggs. Further, they may be in the state of ovaries (in the state where multiple eggs are wrapped in a membrane), or in the state where individual eggs (egg grains) are scattered.

[0018] The species of fishery products from which the fish eggs are derived may be any as long as the fish eggs are used for food. Specifically, for example, salmonidae fish (masago and ikura), herring (shumagoko), cod (tarako, karashi mentaiko), flying fish (tobikko, tobiko), cuttlefish (takomanma), sturgeon (caviar), crab, sea bass, carp, shrimp, flatfish, sea bream, etc. can be exemplified.

[0019] "Processed fish egg food" refers to food processed using fish eggs as raw materials. Representative processed fish egg foods can be exemplified by pickling fish eggs in salt, soy sauce, or miso. More specifically, for example, salted or soy-sauce pickled masago, salted or soy-sauce pickled ikura, salted shumagoko, tarako, karashi mentaiko, tobiko, salted takomanma, caviar, etc. can be exemplified.

[0020] In the present invention, "suppressing the hardening of fish eggs" or "suppressing the hardening of egg grains" means preventing the egg grains (egg membranes) of fish eggs from becoming hard, or even if they become hard, reducing the degree of hardness. Note that the hardening of fish eggs targeted by the present invention is not due to high-temperature heating (for example, 60°C or higher) that causes heat denaturation of proteins, but rather hardening that progresses at a lower temperature (around 50°C or lower), for example, at room temperature (1 - 35°C), normal temperature (15 - 25°C), or low temperature (0 - 10°C) environments.

[0021] In the present invention, "softening (soften) fish eggs" or "softening (soften) egg grains" means making the egg grains (egg membranes) of fish eggs soft.

[0022] Chymotrypsin (EC.3.4.21.1, EC.3.4.21.2) is an endopeptidase and a type of serine protease. In the human body, it is known as a digestive enzyme contained in pancreatic juice, and it hydrolyzes peptide bonds having aromatic side chains (such as tryptophan, tyrosine, phenylalanine, histidine, etc.) or large hydrophobic side chains (such as leucine, methionine, etc.) at the carboxyl terminus of the bond. In the present invention, chymotrypsin refers to an enzyme having the above reaction characteristics or substrate specificity, and its origin is not particularly limited. For example, chymotrypsin may be derived from mammals, or may be derived from microorganisms or produced by microorganisms.

[0023] In the present invention, commercially available food enzymes can be used as chymotrypsin. Examples of commercially available products of chymotrypsin include, for example, "Formea Sol" (Novozymes).

[0024] In the present invention, the enzyme activity can be confirmed according to a conventional method, for example, it can be confirmed by the following method. 《Serine endopeptidase activity (PROT)》 Definition: Relative value with respect to the enzyme activity of the standard substance "standard enzyme" (Novozymes). Measurement principle: The synthetic substrate Suc-Ala-Ala-Pro-Phe-pNA is hydrolyzed by the enzyme, and the absorbance of the generated pNA (yellow chromogenic substance) is measured. Since the magnitude of the absorbance correlates with the magnitude of the enzyme activity, the activity is determined thereby. Measurement method: The standard enzyme is diluted in 7 steps (0.060 - 0.300 mKMCU / mL, 10 mM citrate buffer) to obtain an enzyme solution. 160 μL of 0.1 M Tris buffer (pH 9.0) containing 0.56 mg / mL of Suc-Ala-Ala-Pro-Phe-pNA is incubated at 37°C for 480 seconds, then 40 μL of the enzyme solution is added and incubated for 60 seconds. Subsequently, the absorbance is measured at 405 nm for 190 seconds. A standard curve of the activity concentration is created based on the measurement results of the standard enzyme. The results of measuring the enzyme of the sample in the same manner are applied to the standard curve to determine the enzyme activity.

[0025] Protease activity (AU(A)) Definition: Relative value to the enzyme activity of the standard substance "Protease A standard" (Novozymes). Measurement principle: Dimethyl casein is hydrolyzed using an enzyme as a substrate, and the resulting primary amino group is reacted with trinitrobenzenesulfonic acid (TNBS) to form a chromogenic substance. The absorbance of this chromogenic substance is measured. Since the magnitude of the absorbance correlates with the magnitude of the enzyme activity, the activity is determined accordingly. Measurement method: Standard protease A is diluted in seven stages (0.072 to 0.216 mAU-A / mL, sodium sulfite solution) to prepare the enzyme solution. 180 μL of sodium tetraborate / sodium dihydrogen phosphate / Brij35 solution (pH 8.3) containing 0.25% dimethylcasein is incubated at 50°C for 480 seconds, then 36 μL of 0.1% TNBS aqueous solution is added and incubated for 60 seconds. Subsequently, 18 μL of the enzyme solution is added and incubated for 60 seconds, and the absorbance is measured at 405 nm for 120 seconds. A standard curve of activity concentration is created based on the measurement results of standard protease A. The results of similar measurements of the sample enzyme are applied to the standard curve to determine the enzyme activity.

[0026] Leucine aminopeptidase activity (LAPU) Measurement principle: The synthetic substrate L-leucine-p-nitroanilide is hydrolyzed enzymatically, and the absorbance of the resulting p-nitroaniline (a yellow coloring substance) is measured. Since the magnitude of the absorbance correlates with the magnitude of the enzyme activity, the activity is determined accordingly.

[0027] (I) Definition: 1 LAPU is defined as the amount of enzyme that hydrolyzes 1 μmol of L-leucine-p-nitroanilide per minute.

[0028] (II) Definition: Relative value to the enzyme activity of the standard substance "standard enzyme" (novozymes). Measurement method: Dilute the standard enzyme in seven stages (0.021~0.083 LAPU / mL, zinc chloride / Brij L23 solution) to prepare the enzyme solution. Prepare an ethanol / Tris buffer containing 6.532 g / L of L-leucine-p-nitroanilide to prepare the substrate solution. Add 40 μL of the enzyme solution to 150 μL of 0.1 M Tris buffer (pH 8.0) and incubate at 37°C for 594 seconds. Then, add the substrate solution to a total concentration of 1.136 g / L and incubate for 144 seconds. Subsequently, measure the absorbance at 405 nm for 297 seconds. Create a standard curve of activity concentration based on the measurement results of the standard enzyme. Apply the results of similar measurements of the sample enzyme to the standard curve to determine the enzyme activity.

[0029] Chymotrypsin is used by bringing it into contact with fish eggs. The method and timing of contact can be any means usable in food processing, and can be appropriately set according to the type and maturity of the raw fish eggs, the time and temperature required for various operations, the type of processed fish egg food, and the desired taste and texture. Examples of contact methods include immersing the fish eggs (ovaries or egg grains, etc.) in a solution of the active ingredient, adding and mixing the active ingredient in liquid or powder form to the fish eggs, and coating the fish eggs with the active ingredient by application or spraying.

[0030] For example, salted or soy sauce-pickled salmon roe and flying fish roe are typically produced through a process of immersing them in warm water to loosen the ovarian membrane-bound eggs (loosening process) and then immersing them in a seasoning solution containing salt and soy sauce (soaking process). Chymotrypsin may be added to the warm water used in the loosening process, or to the seasoning solution used in the soaking process, or used in both processes. Alternatively, chymotrypsin may be brought into contact with fish roe products that have been completed through the usual manufacturing process.

[0031] As will be shown in the examples described later, chymotrypsin exhibits a high hardening inhibitory effect and softening effect in a short time, about 1 minute at relatively high temperatures (45°C) and about 1 hour even at low temperatures (4°C). Therefore, the temperature and time for which chymotrypsin is applied to fish eggs are not particularly limited and can be set appropriately according to the type and maturity of the raw fish eggs, the time and temperature required for various operations, the type of processed fish egg food, and the desired taste and texture. In other words, processed fish egg food can be manufactured by known conventional methods, other than bringing the active ingredient into contact with the fish eggs.

[0032] For example, possible contact temperatures for chymotrypsin with fish eggs (action temperature) include 0 to less than 80°C, 0 to less than 70°C, 0 to less than 60°C, and 0 to less than 50°C. Furthermore, possible contact times for chymotrypsin with fish eggs (action time) include 1 second to 48 hours or less, 1 second to 36 hours or less, 1 second to 24 hours or less, 1 second to less than 20 hours, 1 second to 12 hours or less, 1 second to less than 10 hours, 1 second to 60 minutes or less, 1 second to less than 20 minutes, and 1 second to less than 10 minutes (excluding frozen storage time). The closer the temperature is to the optimal temperature, or the higher the enzyme concentration, the shorter the contact time required.

[0033] The amount of chymotrypsin used can also be appropriately set according to the type and maturity of the raw fish roe, the time and temperature required for various operations, the type of processed fish roe product, and the desired taste and texture. For example, the amount used could be 0.01 to 50 PROT / g in the liquid in which the fish roe is soaked (such as warm water or seasoning liquid used for loosening) and 0.001 to 50 PROT / g in the processed fish roe product.

[0034] Fish roe processed foods may contain, in addition to fish roe and active ingredients, any ingredients used in food manufacturing. Examples of such ingredients include seasonings such as salt, sugar, mirin, and sake; spices such as pepper, mustard, and chili peppers; meats, seafood, legumes such as soybeans; eggs such as chicken eggs; vegetables, fruits, seaweed, and grains; and, if necessary, thickening polysaccharides, starch, flours such as wheat flour, food additives such as sucrose fatty acid esters, and edible oils.

[0035] In the present invention, the method for suppressing the hardening of fish eggs, the method for softening fish eggs, and the method for producing processed fish eggs may include other steps as long as they do not impair the features of the present invention. Examples of such steps include cutting or crushing ingredients, washing, ingredient preparation, seasoning, maturation, molding, heating, sterilization, cooling, container filling, packaging, freezing, and thawing.

[0036] The present invention will be described below based on various examples. The technical scope of the present invention is not limited to the features shown in these examples. [Examples]

[0037] <Testing Method> Unless otherwise specified, this embodiment was carried out by the following methods (1) to (4). (1) Enzymes Four commercially available enzyme products (abbreviated as Enzymes A to D) were used. The specifications of these enzymes are shown in Table 1. Enzyme A is subtilisin, Enzyme B is chymotrypsin, Enzyme C is trypsin, and Enzyme D is a mixture of aminopeptidase and carboxypeptidase. The amount of enzyme used in each example is the weight of the product. [Table 1]

[0038] (2)Fish eggs The fish eggs used were raw salmon roe from trout. In this example, eggs that are enclosed in the ovarian membrane and connected together are called "sujiko," raw salmon roe that has been removed from the fish's abdominal cavity and is unprocessed is called "nama-sujiko," and eggs that have been removed from the ovarian membrane and the individual eggs are separated are called "ikura."

[0039] (3) Production of salted salmon roe Salted salmon roe was produced by the following steps [1] to [6]. [1] Thawing: The frozen salmon roe was thawed by transferring it to the refrigerator (approximately 4°C) and leaving it overnight. [2] Washing: The salmon roe was washed twice using 150g of 5% by mass salt water (10°C) per 100g. [3] Separation: Enzymes were added to 3% by mass salt water (45°C) to make separation water. 150g of the salmon roe from [2] was immersed in the separation water and stirred for 1 minute in a constant temperature bath while maintaining a temperature of 45°C. This operation separated the egg grains from the ovarian membrane to make ikura. [4] Washing: After draining the liquid from the salmon roe from [3], the roe was washed twice using 150g of 2% by mass salt water (4°C). Then, it was passed through a sieve to remove any remaining fibers (residue of the ovarian membrane, etc.). [5] Pickling: Prepare a 3% by mass salt solution and use it as the pickling water. Immerse the salmon roe from [4] in 80% by mass of the pickling water and pickle it in the refrigerator (4°C) for 1 hour. [6] Frozen storage: After draining the liquid from the salmon roe [5], it was rapidly frozen at -40°C for 1 hour and then stored in a freezer at -20°C.

[0040] (4) Evaluation of the firmness of salted salmon roe Salt-cured salmon roe that had been frozen was thawed and stored in a refrigerator (approximately 4°C). The day after being moved to the refrigerator was counted as day 1. For salt-cured salmon roe on days 1 to 3, a creep meter RE2-33005C (Sanyo Electric) with a 0.5 cm diameter cylindrical plunger was used to compress the roe at a compression speed of 1.0 cm / second until the roe grains burst, and the maximum load (N) was measured. For each sample, the average value of the maximum load for 10 to 15 roe grains was calculated.

[0041] Next, the percentage change in hardness of salted salmon roe stored in the refrigerator for two or three days was calculated using Equation 1 below. In other words, a larger percentage change in hardness indicates that the salted salmon roe has become harder. Equation 1: Hardness increase / decrease rate (%) = {Average maximum load on day 3 or day 2 (N) / Average maximum load on day 1 (N) × 100} - 100

[0042] <Example 1> Hardening inhibition effect: Investigation using loosening water Salted salmon roe was prepared as described in Test Method (3). However, as the loosening water in step [3], a 3% by mass brine containing enzymes A to D in external amounts of 0.01%, 0.02%, or 0.04% by mass was used (water: 145.5g, salt: 4.5g, enzymes A to D: 0.015g each (0.01% by mass), 0.030g each (0.02% by mass), or 0.060g each (0.04% by mass)). Subsequently, the maximum load was measured on the salted salmon roe on day 1 and day 3 after thawing, as described in Test Method (4), to determine the hardness increase / decrease rate. The hardness increase / decrease rate is shown in Figure 1.

[0043] As shown in Figure 1, in the case of loosening water containing enzyme A, enzyme C, or enzyme D, the hardness increase / decrease rate exceeded 20% at enzyme concentrations of 0.01% by mass, 0.02% by mass, and 0.04% by mass. In contrast, in the case of loosening water containing enzyme B, the hardness increase / decrease rate was significantly smaller, less than 1%, at enzyme concentrations of 0.01% by mass, 0.02% by mass, and 0.04% by mass.

[0044] Specifically, salted salmon roe treated with subtilisin, trypsin, aminopeptidase, or carboxypeptidase during the loosening process became noticeably hard during refrigerated storage after thawing. In contrast, salted salmon roe treated with chymotrypsin remained almost completely soft even after 3 days of refrigerated storage after thawing. These results clearly demonstrate that chymotrypsin can suppress the hardening of salmon roe during storage.

[0045] <Example 2> Hardening Inhibition Effect: Investigation using immersion water Salted salmon roe was prepared as described in Test Method (3). However, as the pickling water in step [5], either 3% by mass brine without enzymes, or 3% by mass brine containing 0.1% by mass of enzymes B, C, or D (water: 145.5g, salt: 4.5g, enzymes B-D: 0.15g each) was used. Furthermore, for all samples, the loosening water in step [3] was prepared by adding enzyme D at a ratio of 0.015g (0.01% by mass) to 150g of 3% by mass brine. Subsequently, the maximum load was measured on the salted salmon roe on day 1 and day 2 after thawing, as described in Test Method (4), to determine the hardness increase / decrease rate. The hardness increase / decrease rates are shown in Figure 2.

[0046] As shown in Figure 2, the hardness increase / decrease rate was approximately 50% in both cases: when no enzyme was added to the pickling water, and when enzyme C or enzyme D was added. In contrast, when enzyme B was added to the pickling water, the hardness increase / decrease rate was significantly smaller at 6.18%.

[0047] Specifically, salted salmon roe treated with trypsin, aminopeptidase, or carboxypeptidase during the pickling process became noticeably harder during refrigerated storage after thawing. In contrast, salted salmon roe treated with chymotrypsin remained almost completely soft even after two days of refrigerated storage after thawing. These results clearly demonstrate that chymotrypsin can suppress the hardening of salmon roe during storage.

[0048] <Example 3> Softening effect (1) Production of salted salmon roe Salted salmon roe was produced using mature, hardened egg membranes (so-called "pinko") as the raw material, according to test method (3). However, process [3] was as follows. [3] Separation: 3% by mass salt water was used as the separation water. 150g of the salmon roe from [2] was immersed in the separation water and stirred for 1 minute. This procedure separated the egg grains from the ovarian membrane and separated them to form ikura.

[0049] (2) Measurement of softening treatment and breaking load The salted salmon roe of Example 3(1), which had been frozen and stored, was thawed by transferring it to a refrigerator (approximately 4°C) and leaving it overnight. The maximum load (N) of the thawed salted salmon roe was measured using the method described in Test Method (4), and the average value of 10 to 15 salmon roe was calculated and defined as the "breaking load before softening treatment."

[0050] Next, the thawed salted salmon roe was divided into four groups. Each group was then immersed in either a 3% by mass brine solution without enzymes, or a 3% by mass brine solution containing 0.1% by mass of enzymes B, C, or D (water: 145.5g, salt: 4.5g, enzymes B-D: 0.15g each), and softened by refrigeration (4°C) for 1 hour. Subsequently, the maximum load (N) was measured for each group of salted salmon roe using the method described in test method (4), and the average value of 10-15 salmon roe was calculated and defined as the "breaking load after softening treatment." The results are shown in Figure 3.

[0051] Figure 3 also shows, for reference, the breaking load of salted salmon roe produced using the "3% by mass brine without enzymes" from Example 2 as the pickling water, on the day after being transferred to the refrigerator (Day 1 of thawing). Since the raw material for Example 2 is raw salmon roe that is not overripe and has a relatively soft egg membrane, the breaking load of this sample can be considered a guideline value for the breaking load of ordinary salted salmon roe suitable for consumption. Therefore, in this example, this sample will be abbreviated as "ordinary salmon roe".

[0052] As shown in Figure 3, the breaking load of salted salmon roe before softening treatment was 1.317 N. In contrast, the breaking loads of salted salmon roe softened with brine without enzymes, brine containing enzyme C, and brine containing enzyme D were 1.426 N, 1.337 N, and 1.343 N, respectively, showing a slight increase compared to before softening treatment. On the other hand, the breaking load of salted salmon roe softened with brine containing enzyme B was 0.691 N, which was significantly lower than before softening treatment and close to the value of normal salmon roe (0.520 N).

[0053] Specifically, when salted salmon roe made from pinko was immersed in brine containing trypsin, aminopeptidase, or carboxypeptidase at 4°C for 1 hour, the egg membrane did not soften. However, when immersed in brine containing chymotrypsin at 4°C for 1 hour, the egg membrane softened significantly, and the egg grains became as soft as those of regular edible salmon roe. From these results, it became clear that chymotrypsin can effectively soften hard salmon roe grains to a degree suitable for consumption, even when applied for a short time or at low temperatures.

Claims

1. A fish roe hardening inhibitor containing chymotrypsin as its active ingredient.

2. A fish roe softener containing chymotrypsin as its active ingredient.

3. A method for suppressing the hardening of fish eggs, comprising the step of contacting the fish eggs with chymotrypsin.

4. A method for softening fish eggs, comprising the step of contacting the fish eggs with chymotrypsin.

5. A method for producing processed fish roe food, comprising the step of bringing the agent described in claim 1 or 2 into contact with fish roe.

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