KR20160043460A - The low sodium fish sauces and method for preparing the same - Google Patents

Abstract

The present invention relates to a low sodium fish soy sauce and a method for preparing the same. According to the low sodium fish soy sauce and the method for preparing the same of the present invention, after adding an enzyme complex to shellfish groats in content of 1-3 w% based on the total weight of the shellfish groats, a high-pressure enzyme process is performed at a temperature of 40-60°C under 65-80 MPa for 12-20 hours. Next, enzyme inactivation, cooling aging, and oil removing steps are performed to significantly reduce the content of salt, to reduce the content of peptide providing a bitter taste, and to increase the content of peptide in a salty taste enhancer and a seasoning ingredient.

Description

[0001] The present invention relates to a low salt fish sauce and a method for preparing the same,

More particularly, the present invention relates to a process for producing a low salt fish paste, and more particularly, to a process for producing a low salt fish sauce and a method for producing the same, And more particularly, to a method for producing a low salt concentrate.

In the process of rapid industrialization and urbanization, food consumption such as eating out, fast food, pizza, western food and instant food increased due to income level increase, nuclear family, increase of single generation. In particular, Koreans are taking foods with high sodium content, such as tang, stew, kimchi, soy sauce, and salted fish. The daily intake of sodium is 6,000 to 8,000 mg, more than twice the recommended amount of the World Health Organization (WHO) It is known to consume a high concentration of sodium. Excess intake of sodium is a major cause of chronic diseases such as hypertension, heart disease, brain cardiovascular disease, kidney disease and gastric cancer.

In recent years, attention has been paid to the reduction of sodium intake in order to prevent chronic diseases and to promote health, and as a result, research on the development of low sodium salt or saltiness enhancer is underway. As an example, natural seasoning substitutes for salt using natural extracts such as natural salt enhanced with minerals, natural extract such as kelp, kelp, gugija, onion, shiitake mushroom, radish, garlic, etc .; sodium salt, potassium chloride, calcium chloride, magnesium chloride, Salt, or chemical seasoning such as aspartate, monosodium glutamate (MSG). However, low sodium salt has less salty taste, so it uses more sodium than usual. As a result, it consumes a lot of sodium. When salt of potassium or calcium is added to kidney patients, it causes problems such as dyspnea, chest pain and heart attack . In addition, the chemical seasoning produced by an artificial synthesis is absorbed into the nerve tissue when it is ingested a lot and destroys the cell membrane to exhibit such symptoms as headache, vomiting, nausea and tongue paralysis. Recently, as a cancer inducing substance, stability against chemical seasoning And there is a problem of harmfulness.

On the other hand, soy sauce is seasoned food that can be contacted in any form in our table every day. In general, soy sauce takes meju as a source of flavorful amino acids. Due to the restriction of this source, There was a limit. Accordingly, there has been an effort to find an amino acid source other than meju as an alternative to this, and a representative example thereof is an eel.

The above-mentioned eel is one of the representative fermented foods of Korea with Kimchi, and has been used to supply high protein and salt regardless of season. Specifically, the eelgrass is a salt product in a wide sense, salted fish and shellfish, fermented by aging, and then the supernatant is separated and filtered. Salt is added to fish, shellfish, shellfish, etc. to preserve it. Various enzymes present in fish and shellfish cause self-digestion and fermentation. Various amino acids are produced to give special flavor and richness, and rich in amino acids and inorganic components. It is nutritionally superior, and it is known that digestion and absorption rate is also good because it is composed of small molecules. Despite these unique flavors and potential utility values, however, they are avoided due to high salt concentrations, long term aging and spoilage. In addition, proteolytic enzymes are used for the properties of the fish paste. In this case, the protein degradation rate of the fish is accelerated, but the quality of the product deteriorates due to the decrease in the flavor due to the bitter peptides, When enzymes for the removal of proteolytic enzymes or bitter peptides are used, it is difficult to maintain the activity due to the high salt content, and thus it is necessary to increase the amount of enzymes to be added, resulting in a lack of competitiveness due to a rise in cost, resulting in difficulties in mass production and generalization of products. In order to overcome the above problems, various attempts have been made to develop technologies, and core technologies thereof include enzyme engineering, separation and purification technology, fermentation engineering, microbiology, cultivation engineering, and advanced minimum processing technologies (ultra high pressure technology, nanotechnology, Technology, microencapsulation).

Among the above technologies, high pressure technology (HPT) applies a high pressure of 100 to 900 MPa without using heat to affect the non-covalent bonding (ionic bonding, hydrogen bonding) and hydrophobic bonding of spoilage microorganisms and pathogenic microorganisms, It is a technology that collapses to kill microorganisms or denature protein and minimize the destruction of nutrients. Such ultra high pressure treatment consumes significantly less heat energy than heat treatment and can be carried out at room temperature or low temperature and can maintain the taste, flavor, color, freshness, and nutritional content of the natural food, In addition to microbial death, it can induce protein denaturation or deformation, enzyme activation or inactivation, enzymatic substrate specificity change, change in carbohydrate and fat properties, and covalent and hydrogen bonding And can be used in a state of being packed in a pouch-shaped bag such as a plastic film, thereby facilitating the experiment.

Accordingly, the present inventors have made efforts to develop a method for producing a low salt crab starch wherein the content of the bitter peptide specific to seafood is reduced while the content of sodium is decreased by using the high pressure enzyme treatment technique, and the peptide content of the salty taste enhancing substance and the seasoning ingredient is improved , Thereby completing the present invention.

It is an object of the present invention to provide a method for producing a low salt fish paste using high pressure enzyme treatment technology.

Another object of the present invention is to provide a low salt fish paste prepared by the above method.

In one aspect, the present invention provides a method for removing foreign matter from a fishery product, 1 to 3% by weight of a complex enzyme is added to the crushed seafood product obtained in the pre-treatment step (S100), and the mixture is subjected to high pressure enzyme treatment for 12 to 20 hours at 40 to 60 DEG C and 65 to 80 MPa A processing step (S200) of hydrolyzing by treating; An enzyme inactivation step (S300) of inactivating the enzyme of the crushed seafood product hydrolyzed in the processing step (S200); A cooling aging step (S400) for cooling and aging the hydrolyzed fish and shellfish whose enzyme activity is stopped in the enzyme inactivation step (S300); (S500) of removing the oil of the hydrolyzate of the fish and shell hydrolyzate which has been cooled and aged in the cooling aging step (S400); (S600) for collecting only the supernatant of the fish and shell hydrolyzate from which the oil is removed in the maintaining and removing step (S500).

Hereinafter, the method for producing a low salt fish paste starch according to the present invention will be described in detail with reference to FIG.

(1) a pretreatment step of removing foreign substances from the fish and shellfish and crushing them (S100).

In the present invention, the fish and shellfish can be used as raw materials of fish and shellfish products produced in Korea, and can be used as fish and shellfish products. For example, fish, shellfish, anchovy, horseshoe, pollack, nogari, mackerel, reference fish, squid, oyster, And a reference group, and preferably anchovy.

In the present invention, the method of removing the foreign substances from the fish and shellfish is not limited to the above, as long as the surface of the fish or shellfish is cleaned with water to remove salt and foreign matter. For example, Lt; 0 > C for 1 hour to remove impurities and salts.

Next, the fish and shellfish from which the foreign substance is removed are crushed. The pulverization may be carried out by a conventional method, for example, pulverization by a pulverizer. In such a crushing process, as the fish crust is crushed finely, the action of the enzyme in the fish and shellfish is facilitated during the high-pressure enzyme treatment performed at a later stage to produce various amino acids or peptides, which are high-temperature components, Preferably has a particle size of 150 to 200 mu m.

In the present invention, the crushed fish and shellfish are crushed at 14,000 to 18,000 rpm for 3 to 5 minutes.

At this time, it is preferable that crushing is performed by adding 1 to 3 times, preferably 2 times, water to the total weight of the fish and shellfish. If the amount of water is less than the total weight of fish and shellfish, it takes a long time to crush fish and shellfish. When the amount of water is more than 3 times the total weight of fish and shellfish, the amount of water is added and pulverized The time for pulverizing fish and shellfish or the decrease in the size of the fish and shellfish particles is insufficient, which is uneconomical.

In one specific embodiment, the anchovy which was caught in the sea area of Jeju Island in early March and stored at -20 캜 was thawed at room temperature, and then immersed in water at 10 to 15 캜 for one hour and washed with water to remove salts and impurities. Then, water was added twice as much as the weight of the anchovy, and the mixture was pulverized twice for 2 minutes using a household blender and then pulverized at 16,000 rpm for 5 minutes using a homogenizer to prepare anchovy pulverized product. As a result, the particle size of the anchovy pulverized by the primary pulverization using a household blender was less than about 1,000 占 퐉, the size of the anchovy pulverized by the homogenizer was about 180 占 퐉, more than 16,000rpm and more than 5 minutes It was confirmed that the size of particles was not significantly different when crushed under the conditions.

(2) In step S200, the complex enzyme is added to the ground fish meal crushed in the pre-treatment step (S100), followed by high pressure enzyme treatment for a certain temperature, pressure and time to hydrolyze the crushed fish meal.

Protein hydrolases are enzymes that make a variety of amino acids or peptide mixtures and are used to hydrolyze proteins in plants and animals. In particular, when a protein hydrolyzate of fish and shellfish is to be produced, it is possible to produce a protein hydrolyzate of vegetable or microbial origin (pancreatin, trypsin, pepsin, papain, bromelain, Fungus-derived protein hydrolytic enzymes, etc.) are used. The kinds of enzymes are selected and carried out by a person skilled in the art in consideration of efficiency and economy.

For example, enzymes (alcalase, flavorzyme, corolase, etc.) have been used to produce protein hydrolysates of fish and shellfish. To solve this problem, the enzyme decomposes the proteins of fish and shellfish in a short period of time. To solve this problem, selective extraction using activated charcoal or alcohol, chromatography using various other matrices And graphical removal methods have been used. However, the methods for removing the bitter taste include various problems such as removal of useful amino acids from the hydrolyzate, low recovery, necessity of specific equipment, and high cost.

Therefore, in the present invention, a complex enzyme having an endopeptidase or a proteinase and an exopeptidase is used to produce a low salt fish paste having a high nitrogen recovery while reducing the bitter taste.

Specifically, the complex enzyme used in the present invention is a complex enzyme in which an endo-type protease and an endo-type and an exo-aminopeptidase are mixed. Preferably, the complex enzyme is a protease and an aminopeptidase ) Is mixed at a weight ratio of 0.8 to 1.2: 4.8 to 5.2, preferably 1: 5.

The protease may be any one or more selected from the group consisting of alcalase (TM), neutrase (TM), protamex (TM), and bromelin (TM), preferably alcalase ). The aminopeptidase may be any one or more selected from the group consisting of flavorzyme (TM), peptidase (R), N-glycosidase (TM), and flavorzyme )to be.

In one specific embodiment, anchovy is washed and pulverized to give powdered anchovy, which contains 5: 1, 4: 2, 3: 3, 2: 4 or 1: 5, respectively, and reacted at 50 ° C for 3 hours to obtain a Peak II fraction, which is expected to have a protein hydrolysis activity, a tryptophan content and a salty taste enhancer And the content thereof was measured. As a result, the protein hydrolysis activity was the highest in the alkalase, and the complex enzyme in which the alkalase and the flavor were mixed at the weight ratio of 1: 5 showed a hydrolytic activity similar to that of the alkalase. In addition, the complex enzyme in which the alkalase and the flavor were mixed at a weight ratio of 1: 5 decreased the content of tryptophan, a bitter peptide, and the peak content of Peak II fraction was higher than that of the anchovy- (See Examples 1-2 and 1-3).

Accordingly, it can be seen that the complex enzyme of the present invention is very effective in easily forming a low salt fish paste containing the content of the salty taste enhancing substance while reducing the production of the bitter peptide by easily hydrolyzing the protein of the fish and shellfish.

In the present invention, the complex enzyme is added to crushed fish and shellfish in an amount of 1 to 3% by weight, preferably 1.5 to 2.5% by weight, more preferably 2% by weight, based on the total weight of crushed fish and shellfish products . When the content of the complex enzyme is less than 1% by weight, hydrolysis of the fish protein is not easily performed, or the efficiency of liberating amino acids, especially glutamine, asparagine, etc., , There is a problem in that the content of the amino acid in the hydrolyzed fish is insufficient or the content of the bitter peptide is increased and the flavor of the low salt fish paste is lowered.

In the present invention, the high-pressure enzyme treatment refers to hydrolysis of a substrate by an enzyme at a high pressure and at a specific temperature for a certain period of time.

Specifically, the specific temperature for the high pressure enzyme treatment is 40 to 60 DEG C, preferably 45 to 55 DEG C, more preferably 50 DEG C, and the high pressure is 65 to 80 MPa, preferably 70 to 80 MPa, The following is 75 MPa, and the predetermined time is 10 to 20 hours, preferably 12 to 18 hours, more preferably 16 hours. In addition, the enzyme is the complex enzyme described above, and the substrate is the fish meal crushed product described above. There is a problem that it takes a long time to hydrolyze the protein of the fish or shellfish or the content of the protein hydrolysis degree, the nitrogen recovery rate, the yield and the fraction that is expected to contain the salty taste enhancing substance is lowered under conditions other than the above conditions.

In one specific embodiment, the anchovy crushed material is treated with 1 or 2% by weight of the complex enzyme (alkalase: flavozyme = 1: 5) relative to the total weight of the anchovy crushed product, and then subjected to a pressure of 50, 75 or 100 MPa , 40, 50 or 60 < 0 > C for 12, 24 or 48 hours. (Degree of hydrolysis, DH,%), nitrogen recovery (NR,%), yield, molecular weight (%) obtained by removing enzyme inactivation, cooling aging and maintenance of the high pressure enzyme treated anchovy crushed product And S / N ratio (signal - to - noise ratio) for each high - pressure enzyme condition were measured. As a result, the degree of hydrolysis (DH,%) was increased with the addition of complex enzyme or reaction time, and the recovery of nitrogen (NR,%) was increased with the addition of complex enzyme. While the amount of complex enzyme was increased. In addition, it was confirmed that the content of Peak II fraction, which is expected to have a salty taste enhancing substance, increases as the amount of enzyme added increases (see Example 2).

Therefore, the optimal condition for the high-pressure enzyme treatment of the present invention is that the crushed product of fish and shellfishes is treated with 2% by weight of the complex enzyme based on the total weight of the crushed fish and shellfish and then treated at 75 MPa and 50 ° C for 12 to 16 hours, (50 MPa and 48 hours treatment at 50 < 0 > C, Okazaki meat al . , 2003), it is possible to produce a free amino acid of about 2.5 times the shortened time and four times shorter time than the hydrolyzed fish meal crushed product.

(3) an enzyme inactivation step (S300) for inactivating the enzyme of the hydrolysates of fish and shellfish produced in the processing step (S200).

In the present invention, the enzyme inactivation is not limited to the method of stopping the activity of the enzymes present in the hydrolyzed fish meal crushing product, but may include heat treatment, high pressure treatment under enzyme inactivation conditions, and the like. As a specific example, the enzyme activity can be stopped by heat treatment at a temperature of 70 to 90 DEG C, preferably at a temperature of 80 DEG C for 10 to 30 minutes, preferably 20 minutes.

(4) a cooling and aging step (S400) of cooling and hydrolyzing the hydrolyzed fish and shellfish whose enzyme activity is stopped in the inactivation step (S300) of the enzyme at a low temperature.

In the present invention, the cooling and aging is preferably carried out at a low temperature of 0 to 5 DEG C for 6 to 12 hours, preferably 6 to 10 hours. If the cooling aging is carried out at a temperature exceeding 5 캜 or less than 6 hours, suspensions (such as fat) are generated in the low-salt seasoning gauges to be finally prepared and already dried or odors are generated. It is uneconomical that the decrease in the offensive odor is less than that in the case of the low salt crab prepared by aging at a higher temperature or lower.

(5) a maintenance removing step (S500) for removing fats and fatty oils of the fish and shell hydrolyzate aged in the cooling aging step (S400).

When hydrolyzed fish, such as acid, alkali, enzyme or high pressure, are used as raw materials for soy sauce, soybean paste, and aged cheese, the protein hydrolyzate is generally sterilized, centrifuged, evaporated and dried Filtration using a centrifugal separator, a plate and frame filter press, or a microfiltration system. However, the final product produced through the filtration process is already contaminated with oil and fat components inherent in fish and shellfish, and it is also possible to remove the useful substances contained in fish and shellfish through repetitive filtration processes such as a salty taste enhancing substance, There is a problem that the quality of a final product is deteriorated.

Accordingly, in the present invention, it is aimed to minimize the loss of useful components contained in fish and shellfish by omitting the filtration process while eliminating the oil of the hydrolyzed fish meal crumbs and reducing the amount of imaged fish in the final fish cakes.

In the present invention, the fish and shellfish oil-retaining is carried out by cooling and aging the hydrolyzed fish and shellfish crushing product, cooling and agitating the floating fish oil by using a wool fabric, a synthetic sponge, a degreasing pad, an oil paper, a cloth or a sieve, Can be removed.

The maintenance removal of the present invention shows the effect of improving the quality and the odor of the low salt fish paste finally produced.

In one specific implementation, the high pressure enzyme treated anchovy lysate was inactivated at 90-100 ° C for 10 min and quenched in a 0 ° C chiller to stop the reaction of additional enzymes. The solid content of fat was removed by centrifugation, and the fat content of frozen and frozen dices were measured. The fat content of the frozen dices was measured at 4 ℃ for 12 hours. As a result, it was confirmed that the oil in the eel was not floated during the storage period, and the fat content of the oil eel removed from the oil was found to be about 0.1% or less. (See Examples 1-5).

(6) Separation step (S600) of separating the fish and shell hydrolyzate from which the fat is removed in the above-described maintenance and removal step (S500) to obtain a clear supernatant.

In the present invention, the separation is not particularly limited as long as it is a method for separating solids and a supernatant from seaweed hydrolyzate commonly used in the art to obtain only a supernatant. For example, a solid or a solid is removed using a cloth or sieve Centrifugal separation or the like. In one specific example, the oil-and-fat hydrolyzate from which fat has been removed can be centrifuged at a speed of 15,000 to 20,000 rpm, preferably 16,000 to 18,000 rpm using a tubular centrifuge to obtain only the supernatant.

In the present invention, the low salt seasoning gland may further include any one of (i) a refining step or (ii) a decoloring and deodorizing step, if necessary, after the step (S500) And (ii) a decoloring and deodorizing process. Whether or not these processes are added can be selected depending on the intended use of the final eulogang, and the purification process can remove impurities, salts, and the like contained in the eulogy or remove substances including salty taste, seasoning and / And the like. The decolorizing and deodorizing process may be used to provide a deep-sea bowl to a person sensitive to the flavor and color of fish or shellfish such as patients and children, or to improve the sensory properties of food, food, etc. when used as a seasoning or substitute salt.

The purification process may be performed through a purification process commonly known in the art. There are, for example, ultrafiltration, nanofiltration, freezing filtration, various chromatography (size, charge, hydrophobicity or affinity chromatographic separation), electrodialysis systems, etc., having a constant molecular weight cut- May be accomplished through an ultrafiltration system, a nanofiltration system, or an electrodialysis system.

In one specific embodiment, an ultrafiltration system having a molecular weight cut-off value of 5 to 10 kDa can be used to separate and purify euglenoids that have been removed from the hydrolyzed material, euglenoids containing a large amount of salty taste enhancers , The purified eulogy field was electrodialysed in a nanofiltration system having a cutoff value of 250 Da molecular weight or a voltage of 12 V, a current of 21 A, a time of 40 minutes and a cross current of 0.22 A / hour to obtain a salt-free eelhouse (See Examples 7 and 8).

The decolorization and deodorization process may be performed by a decolorization and deodorization process commonly known in the art. For example, centrifugation, repeated filtration or activated carbon treatment may be used, and preferably 1 to 3% by weight of activated carbon may be treated.

In one specific embodiment, when 2% of activated carbon is treated in a low salt eel of a 2.5% concentration prepared by adding a complex enzyme to anchovy crumbs and subjected to a high pressure treatment, an annex in which the intensity of yellowish, (See Example 9).

The low salt fish gyoza prepared according to the present invention contains 4.3 to 4.7 parts by weight of sodium based on 100 parts by weight of the building of the eelhouse. In addition, the low salt fasting starch is characterized in that tryptophan which gives a bitter taste of amino acid is contained in an amount of 0.6 to 1% by weight based on the total weight of the amino acid, glutamic acid which gives a rich taste is contained in an amount of 8 to 15% .

In addition, the low salt seasoning gland exhibits at least 10%, preferably at least 15%, more preferably at least 20%, and even more preferably at least 30% higher salinity than a 45 mM salt solution.

In one specific embodiment, the anchovy crushed material was treated with 2% by weight of complex enzyme (alkalase: flavorzyme = 1: 5) based on the total weight of the anchovy crushed product, and then subjected to high pressure After the treatment, the enzyme was inactivated for 80 to 20 minutes. Then, the mixture was aged at 4 ° C for 12 hours, centrifuged using a continuous centrifuge, and solidified to remove floating fat. The resulting solution was filtered through an ultrafiltration membrane having a molecular weight cutoff of 10 kDa to obtain a molecular weight of 10 kDa or more (MW < 10 kDa). Subsequently, the sodium content of unpeeled fish paste (APH; MV ≥ 10 kDa), the content of L-arginine and arginyldipeptide, which are salty taste enhancing substances, and the sensory evaluation of salty taste enhancement were applied to the ultrasound membrane and ultrafiltration membrane Respectively. As a result, it was confirmed that 4.5 g of sodium silicate was contained per 100 g of the building, in which the unfiltered ultrafiltration membrane (APH; MW ≥ 10 kDa) and the material having a molecular weight of 10 kDa or more were removed. 1 (RY, Arg-Tyr) and STD-6 (RA / AR-Tyr) among the nine standard products were excluded from the eugenol (MW <10 kDa) from which substances having a molecular weight of 10 kDa or more were removed , And Arg-Ala). Epitope (APH; MW ≥ 10 kDa), unfiltered in the ultrafiltration membrane, was detected in 8 diepeptides except STD-1 (RY, Arg-Tyr) And STD-4 (EQ, Glu-Gln) was the most detected in both of the two fisheries areas. (MW < 10 kDa) in which a substance having a molecular weight of 10 kDa or more was removed was dissolved in a salt solution and a model solution (monosodium L-glutamate monohydrate 1.9 g / L, maltodextrin 6.375 g / L and yeast extract 2.1 g / L) (See Example 10). &Lt; tb &gt; &lt; TABLE &gt;

The production method of the low salt edible island can shorten the production time by 4 to 8 hours by omitting the pH control and the filtration process of the enzyme compared to the conventional method of producing the eel seasoning and increase the content of the peptides of the salty taste enhancer and the seasoning ingredient And at the same time, the content of the bitter peptide is reduced to enhance the salty taste and at the same time, the salt interacts with the tasty ingredient to exhibit a synergistic effect of taste, so that it can be used as a mass production system capable of mass production of a low salt fish paste.

In another embodiment, the present invention provides a low salt fish paste prepared by adding a complex enzyme to fish meal crumbs and subjecting the mixture to high pressure enzyme treatment at 40 to 60 DEG C and 65 to 80 MPa for 12 to 20 hours.

In the present invention, the low salt fish gyoza contains sodium in an amount of 4.3 to 4.7 parts by weight based on 100 parts by weight of the building of the eel storehouse. In addition, the low salt fasting starch is characterized in that tryptophan which gives a bitter taste of amino acid is contained in an amount of 0.6 to 1% by weight based on the total weight of the amino acid, glutamic acid which gives a rich taste is contained in an amount of 8 to 15% .

In addition, the low salt seasoning gland exhibits at least 10%, preferably at least 15%, more preferably at least 20%, and even more preferably at least 30% higher salinity than a 45 mM salt solution.

In the present invention, the low salt fish paste can be used as a substitute salt for processed food and food or as a natural spice.

The production method of the present invention efficiently hydrolyzes fish and shellfish in a short time to increase the content of the peptides of the salty taste enhancing substance and the seasoning ingredient and reduces the content of the bitter taste peptide, thereby enhancing the salty taste and simultaneously interacting with the tasting ingredients, It is possible to provide a low salt fish starch showing a synergistic effect. The low salt fish paste prepared according to the above method can be used as a substitute salt for processed food and food cooking or as a natural spice. In addition, the above production method can be utilized as a mass production system capable of mass-producing a low salt fish paste which has improved salt content of salty taste and seasoning ingredients by hydrolyzing fish and shellfish.

FIG. 1 shows the result of measuring the content of tryptophan in the hydrolyzed anchovy pulp according to the type of enzyme and the reaction time.
Fig. 2 shows the result of measurement of the change in the molecular weight of the hydrolyzed anchovy pulp according to the kind of the enzyme.
FIG. 3 shows the results of measurement of free amino acid content of the hydrolyzed anchovy crushed product by high pressure enzyme treatment according to parameters of pressure, temperature, time and enzyme concentration set using MINITAB, which is an orthogonal array table of L ₉ (3 ⁴ ) to be.
4 is L ₉ (3 ⁴⁾ orthogonal array of the set pressure by using a MINITAB, temperature, time and hydrolysis of the anchovy pulverized pressure treatment High pressure enzyme according to the parameters of the enzyme concentration, temperature, and specific time and the enzyme of And the signal-to-noise ratio (S / N ratio) for the degree of hydrolysis.
Figure 5 is L ₉ (3 ⁴⁾ orthogonal array of the set pressure by using a MINITAB, temperature, time and hydrolysis of the anchovy pulverized pressure treatment High pressure enzyme according to the parameters of the enzyme concentration, temperature, and specific time and the enzyme of And the signal-to-noise ratio (S / N ratio) to the nitrogen recovery rate.
6 is L ₉ (3 ⁴⁾ orthogonal array is set by using the MINITAB pressure, temperature, time, and the high-pressure enzyme treatment according to the parameters of the enzyme concentration of the hydrolyzed anchovy grinding water pressure, temperature and specific time and the enzyme of This is the result of measuring the signal-to-noise ratio (S / N ratio) for the yield.
FIG. 7 shows the result of measuring the change in the molecular weight of the hydrolyzed anchovy crushed product by high pressure enzyme treatment according to the parameters of pressure, temperature, time and enzyme concentration set using MINITAB, which is an orthogonal array table of L ₉ (3 ⁴ ) .
8 is L ₉ (3 ⁴⁾ orthogonal array is set by using the MINITAB pressure, temperature, time, and the high-pressure enzyme treatment according to the parameters of the enzyme concentration of the hydrolyzed anchovy grinding water pressure, temperature and specific time and the enzyme of This is the result of measuring the signal to noise ratio (S / N ratio) for the salty taste enhancing substance.
FIG. 9 shows the results of comparative measurement of proteolytic activity of anchovy according to enzyme types at normal pressure and high pressure.
FIG. 10 shows the result of comparative measurement of the degree of hydrolysis of anchovy pulp hydrolyzed according to enzyme types at normal pressure and high pressure.
Fig. 11 shows the results of measuring the degree of hydrolysis of hydrolyzed anchovy pulp according to yield, nitrogen recovery, hydrolysis, and optimal high-pressure enzyme treatment conditions suitable for Peak II fraction.
FIG. 12 shows the results of measurement of the nitrogen recovery rate of hydrolyzed anchovy pulp according to yield, nitrogen recovery rate, hydrolysis degree and optimal high-pressure enzyme treatment conditions suitable for Peak II fraction.
13 is a schematic diagram of a process for manufacturing a low salt eutectic field on a pilot plant scale.
14 shows the result of the addition of 2% of the weight of the anchovy crushed product to the anchovy crushed product and the complex enzyme (alkalase: flavozyme = 1: 5) at a pressure of 75 MPa and high pressure treatment at a temperature of 50 캜 for 16 hours And the composition / free amino acid content of the low salt fish paste obtained by hydrolysis and centrifugation.
Fig. 15 shows the result of the addition of 2% of the weight of the anchovy crushed product to the anchovy crushed product, and the complex enzyme (alkalase: flavozyme = 1: 5) was treated at a pressure of 75 MPa and a high pressure for 16 hours at a temperature of 50 캜 The results are obtained by measuring the molecular weight of the purified low molecular weight umbilical artery obtained by hydrolysis and centrifuging, ultrafiltration with 5 to 10 kDa and purification using a 250 Da nanofiltration system.
FIG. 16 shows the results of the addition of 2% of the weight of the anchovy crushed product to the anchovy crushed product and the complex enzyme (alkalase: flavozyme = 1: 5) was treated at a pressure of 75 MPa and a high pressure for 16 hours The results are obtained by visually observing the color of tablets purified by ultrafiltration of 5 to 10 kDa and nanodispersion system of 250 Da after the hydrolysis and centrifugation.
Fig. 17 shows the results obtained by adding the complex enzyme (alkalase: flavozyme = 1: 5) to the anchovy crushed product in an amount of 2% by weight of the anchovy crushed product, treating the mixture at a pressure of 75 MPa, The effect of decolorization on the concentration of activated carbon treated by hydrolysis and centrifugal separation was observed with naked eyes.
FIG. 18 shows the results obtained by adding the complex enzyme (alcalase: flavozyme = 1: 5) to the anchovy crushed product in an amount of 2% by weight of anchovy crushed product, treating the mixture at a pressure of 75 MPa, The results of visual observation of decolorizing effect of 2% of activated carbon treatment on 2.5%, 5%, and 10.0% low salt water content by centrifugation after hydrolysis.
Fig. 19 shows the results of the addition of 2% of the weight of the anchovy crushed product to the anchovy crushed product, and the complex enzyme (alcalase: flavozyme = 1: 5) was treated at a pressure of 75 MPa and a high pressure for 16 hours After hydrolyzing and then centrifuging, 2% of activated carbon was treated with 2.5% low salt water content, and the discoloration effect was observed with the naked eye.
20 shows the result of adding 2% of the weight of the anchovy crushed product to the anchovy crushed product and adding the complex enzyme (alkalase: flavozyme = 1: 5) to the anchovy crushed product at a pressure of 75 MPa and a high pressure for 16 hours at a temperature of 50 캜 (MW < 5 kDa) purified by ultrafiltration membranes of 10 kDa and 5 kDa and the ultrafiltration membranes obtained by centrifuging and then hydrolyzed, The purified product (APHN-1) obtained by refining the refined product with a 250 Da nanofiltration membrane was visually observed for discoloration effect due to 2% activated carbon treatment.
21 shows the result of the addition of 2% of the weight of the anchovy crushed product to the anchovy crushed product and the complex enzyme (alcalase: flavozyme = 1: 5) was treated at a pressure of 75 MPa and a high pressure for 16 hours at a temperature of 50 캜 (MW < 5 kDa) purified by ultrafiltration membranes of 10 kDa and 5 kDa and the ultrafiltration membranes obtained by centrifuging and then hydrolyzed, This is the result of measuring the content of volatile components by 2% activated carbon treatment on the purified product (APHN-1) obtained by purifying the purified product with a nanofiltration membrane of 250 Da.
FIG. 22 shows that an anchovy crushed product was added with a complex enzyme (alcalase: flavozyme = 1: 5) in an amount of 2% by weight of the anchovy crushed product and treated at a pressure of 75 MPa and a high pressure for 16 hours at a temperature of 50 DEG C (MW < 5 kDa) purified by ultrafiltration membranes of 10 kDa and 5 kDa and the ultrafiltration membranes obtained by centrifuging and then hydrolyzed, The formula of the volatile substance removed by the 2% activated carbon treatment on the purified product (APHN-1) obtained by purifying the purified product with a nanofiltration membrane of 250 Da is shown.
23 shows the result of the addition of 2% of the weight of the anchovy crushed product to the anchovy crushed product and the complex enzyme (alcalase: flavozyme = 1: 5) at a pressure of 75 MPa and high pressure treatment at a temperature of 50 캜 for 16 hours (APH) obtained by hydrolysis and centrifugation using an ultrafiltration system of 5-10 kDa to evaluate flavor sensory evaluation of tablets having a molecular weight of 5 kDa or less.
24 shows the results of the addition of 2% of the weight of the anchovy crushed product to the anchovy crushed product based on the salt solution of 25 to 45 mM and the standard solution having the concentration of 45 mM (alkalase: flavorzyme = 1: 5) , And the salty taste of 0.1%, 0.3% 0.5% and 1% low salt confectionery obtained by hydrolysis and hydrolysis at a pressure of 75 MPa and a temperature of 50 캜 for 16 hours under high pressure was measured.

Hereinafter, the present invention will be described in more detail with reference to Production Examples and Examples. It is to be understood that both the foregoing preparations and examples are for the purpose of illustrating the present invention more specifically and that the scope of the present invention is not limited by these preparations and examples according to the gist of the present invention, It will be obvious to those who have knowledge of.

Preparation Example 1 Preparation of Enzyme and Anchovy Embedding Enzyme Solution

1-1. Preparation of enzymes and complex enzymes

Protease and amino-peptidase were selected among the enzymes that could be easily obtained in Korea, and these were purchased and prepared. Specifically, the protease can be obtained by purchasing alcalase and protamex in novozymes (Denmark) and flavorzyme in daemyungcorp (Korea) in aminopeptidase Prepared. In addition, complex enzymes were prepared by mixing alcalase and flavor in the enzymes at a weight ratio of 5: 1, 4: 2, 3: 3, 2: 4 or 1: 5. Information on enzymes which can be easily obtained in Korea is shown in Table 1 below, and the characteristics of the three enzymes selected first among them are shown in Table 2 below.

Enzyme name product name origin type Mechanism of action Protease
(Subtilisin) Alcalase 2.4L Bacillus licheniformis endo
(serine endoprotease) Serine action. Gelatin Extraction Protease
(neutral) Neutrase 0.8L Bacillus amyloliquefaciens endo
(metalloendoprotease) gluten weakening. Excellent properties of dough Trypsin PTN 6.OS porcine pancreas It breaks the peptide bond on the carboxyl side of arginine and lysine residues. Amino-
peptidase Flavourzyme 500MG Aspergillus oryzae exo (aminopeptidase), endo Amino-
peptidase Flavourzyme 1000L Aspergillus oryzae exo, endo mix It is easy to manufacture peptides such as cheese, yeast, fish and meat. Flavor enhancement Protease
(Subtilisin) Protamex Bacillus endo
(serine endoprotease) Unlike other endoproteases, they do not produce bitter taste protein degradation even at low hydrolysis rates. protease Bromeline 1200 GDU pineapple protease Collupullin MG carica papaya Multi-enzyme complex Viscozyme L Aspergillus aculeatus cell wall destruction

product name Molecular Weight
(kDa) Optimum pH Temperature (℃) Metal ion requirement Active amino acid A major active inhibitor Alcalsae 2.4L 18 ~ 35 6.5 to 8.5 55 ~ 70 Ca ^{2 +} cool phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate Flavourzyme 500 mg 30 ~ 45 5.5 to 7.5 45 ~ 65 - N-terminal N-bromophenacyl bromide, Iodine, potassium permanganate Protamex 1.5 MG 18 ~ 35 5.5 to 7.5 35 to 60 Ca ^{2 +} cool phenylmethylsulfonyl fluoride (PMSF), EDTA

1-2. Preparation of anchovyte internal enzyme solution

Anchovies kept at -20 ℃ and caught in the sea area of Cheju Island in early March were purchased from East Sea Fisheries. The anchovy was divided into a whole anchovy (WA), a viscera anchow (VA), and a muscle anchow portion by separating into an intestine and a flesh after thawing. Then, the whole anchovy (WA), visceral (VA) and muscle (MA) were mixed with 20 mM phosphate buffer (pH 7.0) at a weight ratio of 1: 2 and homogenizer (IKA, Germany ) At 16,000 rpm for 5 minutes. Then, the supernatant was separated by centrifugation at 17,500 x g for 20 minutes in a centrifuge maintained at 4 ° C, and the supernatant was filtered as filter paper (Whatman paper No. 4) to be used as coenzyme.

Example  One : Eel  Preliminary experiments for manufacturing process

1-1. Pretreatment process: Setting condition of raw material crushing

Anchovies kept at -20 ℃ and caught in the sea area of Cheju Island in early March were purchased from East Sea Fisheries. The purchased anchovy was thawed at room temperature and then immersed in water at 10 to 15 ° C for 1 hour and washed with water to remove salts and impurities. Then, the anchovy was added twice as much water as the anchovy weight, pulverized twice for 2 minutes using a household blender, and then pulverized at 16,000 rpm for 5 minutes using a homogenizer to prepare anchovy crush Respectively.

As a result, although not shown in the drawing, the particle size of anchovy pulverized by primary pulverization using a household blender was about 1,000 μm or less, and the size of anchovy pulverized by homogenizer was about 180 μm. In the preliminary experiment, the pulverization rate affected the decrease of particle size, and it was confirmed that particle size was not significantly different when pulverized at more than 16,000 rpm and at least 5 minutes.

1-2. Processing: Optimal enzyme selection (1)

In order to select the enzyme having the best hydrolytic activity, the protein hydrolysis activity of the complex enzyme prepared according to the single enzyme and mixing ratio prepared in Preparation Example 1-1, and the anchovy extract coenzyme prepared in Preparation Example 1-2 was measured Respectively. First, casein (Sigma C-7078, USA) powder was dissolved in 0.03M phosphate buffer (pH 7.5) to prepare a concentration of 20 mg / ml. Then, 1 ml of the complex enzyme prepared according to the single enzyme and mixing ratio prepared in Preparation Example 1-1 and the anchovy stock enzyme prepared in Preparation Example 1-2 were added to 5 ml of the casein, and the mixture was incubated at 50 ° C for 30 minutes Lt; / RTI &gt; Then, 5 ml of 0.44 M TCA solution was added, mixed, and then allowed to stand at room temperature for 30 minutes to stop the enzyme reaction. After the enzyme reaction was stopped, the sample was centrifuged at 12,000 × g for 10 minutes, and the supernatant was filtered with a 0.2 μm syringe filter. Next, 2.5 ml of 0.55 M sodium carbonate (Na ₂ CO ₃ ) solution and 0.5 ml of 1N polynuclear reagent (Sigma, USA) were added to 1 ml of the filtrate, followed by color development at 37 ° C for 30 minutes . Then, the color-developed sample was allowed to stand at room temperature for 10 minutes and absorbance was measured at 660 nm. The hydrolytic activity was determined from the measured values as 1 unit in which 1 ml of the enzyme solution produced tyrosine corresponding to 1 간 per minute. L-Tyrosine (Sigma, USA) was used as a standard. The results are shown in Table 3.

enzyme Protein hydrolysis activity (U / g) Alcalase 15,100.00 ProTamex 13,402.63 Flavor 8,400.00 Complex enzyme
(Alcalase: flavor) 5: 1 17,800.00 4: 2 19,050.00 3: 3 17,375.00 2: 4 16,100.00 1: 5 15,425.00 Anchovy-derived coenzyme WA (all) 44.74 MA (muscle) 34.21 VA (built-in) 163.16

As a result of the experiment, the hydrolysis activity of alcalase was the highest, and the complex enzyme in which 1: 5 weight ratio of alkalase and flavor was mixed showed hydrolytic activity similar to alkalase.

1-3. Processing: Optimal enzyme selection (2)

To 100 g of anchovy powder was added 2 ml of the complex enzyme prepared according to the single enzyme and mixing ratio prepared in Preparation Example 1-1 and the anchovy inner coenzyme prepared in Preparation Example 1-2, Lt; / RTI &gt; At this time, the anchovy powder was collected every 30 minutes, and the collected anchovy powder was heated at 100 ° C for 10 minutes to inactivate the enzyme, cooled in a chiller kept at 0 ° C, And the supernatant was separated by centrifugation for 20 minutes at a speed of x g. The separated supernatant was filtered with a filter paper (Whatman paper No. 4) and bitterness degree of the anchovy powder hydrolyzed was measured by HPLC and GPC chromatogram.

The degree of bitter taste was determined by measuring the content of tryptophan, known as the bitter taste amino acid. Samples were further processed for the above HPLC (Hewlett-Agilent, 1100-series, USA) analysis. Specifically, each of the filtered samples was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was separated. Then, 0.5 ml of 12% perchloric acid (HClO ₄ ) was added to 1 ml of the supernatant, and the mixture was centrifuged at 10,000 rpm for 30 minutes. The supernatant was then separated and passed through a 0.22 μm syringe filter and injected into the HPLC. The column used for the sample analysis was C18 Hypersil ODS (4.0 × 250 mm), and the mobile phase was flowed at a flow rate of 1 mL / min with methanol and sodium acetate (8.5 mM) Respectively. The sample was analyzed by measuring the absorbance at 280 nm and DL-tryptophan (Sigma, USA) was used as a standard. The results are shown in Figs. 1 and 2. Fig.

The results showed that alcalase and flavozyme reduced the tryptophan production and the percentage of Peak Ⅱ fraction, which is expected to contain salty taste enhancer, As a raw material for the mixed enzyme.

Thus, the results of Examples 1-2 and the results of the present experiment are combined, and it is confirmed that Alkalase and Plavarase, which can produce a large amount of Peak Ⅱ fraction having a good hydrolysis activity but minimizing a bitter taste and containing a salty taste enhancing substance Were used in a weight ratio of 1: 5.

In order to maximize the action of the enzyme, it is required to adjust the pH, but in the present experiment, the pH control is omitted for the purpose of simplifying the process in consideration of the industrial production of the liquefied product.

1-4. Machining process: High pressure processing condition setting

The complex enzyme prepared according to the single enzyme and mixing ratio prepared in Preparation Example 1-1 was treated with 100 ml of the crushed anchovy crusher under the conditions of Example 1-1, Pressure treatment for 50 hours at a pressure of 50 to 200 MPa.

As a result, the enzyme activity was shown to proceed up to 48 hours. In the case of commercial enzymes, it was reported that the optimum activity was observed at an elevated temperature of about 5 ° C under the pressure, It was decided to proceed liquefaction at a temperature of 60 占 폚.

Further, when the pressure is set to 100 MPa or more, a problem arises in the temperature control, so it was decided to set the range of the optimum pressure to 50 to 100 MPa.

1-5. Post-treatment process: Enzyme inactivation and post-treatment condition setting

The high pressure / enzyme treated hydrolyzed anchovy crushed product was inactivated at 90-100 ° C for 10 minutes and then cooled in a 0 ° C chiller to stop further enzyme reaction. After 12 hours of aging at 4 ° C, solidification of the fat in the sample was induced, and solidified fat was removed through centrifugation and filtration and then stored frozen.

As a result, the fat content of the hydrolyzed anchovy crumbs having undergone cooling aging was found to be about 0.1% or less, and this proved to be an important factor for the prevention of the already produced liquefaction and the generation of offensive odor. That is, it has been confirmed that if sufficient cooling is not performed, suspended substances (fat, etc.) are generated in the stored sample, sufficient aging is necessarily required.

Therefore, the temperature (40-60 ° C), pressure (50-100 MPa), time (12-48 hours) and enzyme use concentration (0-2%) were used as variables for the optimization of high- Respectively.

Example  2 : Low salt Eccentrics  For manufacturing High-pressure enzyme hydrolysis  Establish processing conditions

2-1. Material preparation

Anchovies kept at -20 ℃ and caught in the sea area of Cheju Island in early March were purchased from East Sea Fisheries. The purchased anchovy was thawed at room temperature and then immersed in water at 10 to 15 ° C for 1 hour and washed with water to remove salts and impurities. Then, the anchovy was added twice as much water as the anchovy weight, followed by pulverization twice for 2 minutes using a household blender, followed by secondary pulverization at 16,000 rpm for 5 minutes using a homogenizer to prepare anchovy crush Respectively.

The enzymes used for liquefaction of the anchovy crush were prepared by mixing alcalase (novozymes, Demark) and flavorzyme (Daemyung Co., Korea) at a weight ratio of 1: 5.

2-2. Establish high-pressure treatment conditions

First, that the number of experiments to be reduced as much as possible by using the orthogonal array of MINITAB of L ₉ (3 ⁴⁾ was finished, as shown in Table 4, all the experiments were conducted in accordance with procedures established in preliminary tests in Example 1. At this time, the experiment of repeating the experiment of 9 conditions 3 times and the verification experiment 3 times was performed 30 times in total.

Parameters Pressure (MPa) Temperature (℃) Time (hrs) Enzyme concentration (%) One 50 40 12 0 2 50 50 24 One 3 50 60 48 2 4 75 40 24 2 5 75 50 48 0 6 75 60 12 One 7 100 40 48 One 8 100 50 12 2 9 100 60 24 0

2-3. Analysis of general components and physicochemical properties of hydrolyzed anchovy pulp according to high pressure / enzyme treatment conditions

The moisture, crude fat, crude protein and ash content of the anchovy pulp hydrolyzed by high pressure / enzyme treatment under the conditions of Example 2-2 were analyzed by AOAC general component analysis method. The pH, salinity and water solubles content of the anchovy crushed product were measured with a pH meter (Orion 4-star Plus, Thermo Sci., USA), a salinity meter (Master meter, S28M, Atago, Japan) and a master refractometer Master-M, 2M and 3M, Atago, Japan). The results are shown in Table 5 and Table 6.

1) Moisture

3 to 5 g of the hydrolyzed anchovy crushed material was subjected to high pressure / enzyme treatment under the conditions of Example 2-2, and then dried in a vacuum oven at 98 to 100 ° C. for 3 to 5 hours, and then dried in a desiccator It was cooled for 30 minutes and weighed. Then, the weighing dish was again dried for 1 to 2 hours and repeatedly dried until the weight became constant, and moisture content was measured using the following Equation 1 (AOAC method 926.08 and 925.09, 1990).

[Experimental Equation 1]

Water (%) = [(W ₁ -W ₂ ) / (W ₁ -W ₀ )] × 100

W ₁ : Weighing plate and anchovy crushing weight

W ₂ : Weight after weighing plate and anchovy crush

W ₀ : constant weight of weighing dish

2) crude fat

The crude fat content was measured by soxhlet (Raypa, SX-6MP) method. Specifically, 10 to 20 g of the hydrolyzed anchovy pulverized product subjected to high pressure / enzyme treatment under the condition of Example 2-2 was put into a blender, and 100 ml of methanol was divided into two portions and mixed. Then, the mixture was put in a funnel, . Then chloroform was also added to funnel in the same amount and same way, shaken vigorously, then 90 ml of distilled water was added and shaken vigorously. Then, when the funnel was left to separate layers, a funnel was added to a constant weight (weighing bottle), and the chloroform layer was concentrated using anhydrous sodium sulfate. Thereafter, 90 ml of chloroform was added to the funnel, and the mixture was shaken. Then, the layer was separated, and the chloroform layer was obtained by the same method as described above and concentrated. After that, the concentrated layer was dried, and the water content was heated to 105 ° C or higher, and the crude fat content was determined using the following Equation 2.

[Experimental Equation 2]

Crude fat content (%) = [(W ₁ -W ₀ ) / S] × 100

W ₁ : constant weight after fat extraction (g)

W ₀ : constant weight of hand (g)

S: Weight of sample (g)

3) Crude protein

The crude protein content was analyzed using the micro-Kjeldahl method. About 0.5 g of the hydrolyzed anchovy crushed product was subjected to high pressure / enzyme treatment under the conditions of the above Example 2-2, placed in a proteolytic tube (250 ml digestion tube, Buchi), dissolved with Kjeltabs tablets containing potassium sulfate and a catalyst, Foss. Denmark) was added thereto, and 20 to 25 mL of sulfuric acid (95%, Kanto, 0172-00438, Japan) was added thereto, and the flask was shaken while mixing. A proteolytic tube containing a sample and reagent was placed in a proteolytic unit (Raypa-R. Espinar, S.L., Spain), and the lysate of the digestion apparatus was covered and then decomposed at 410 ° C. for about 12 hours until the final sample was cleaved transparently. After completion of the decomposition, the flask was taken out to room temperature, allowed to cool for 15 minutes, and then distilled in 60 mL of 3% boric acid in a distillation apparatus (Raypa-R. Espinar, Then, titration was carried out with a 0.1N hydrochloric acid solution (Samchun, H0241, Korea) by an automatic periodical (Crison Instruments, S.A., Titromatic, Spain) and calculated using Equation 3 shown below.

[Experimental Equation 3]

Crude protein (% N) = {[(V ₁ - V ₂ ) x f x 0.0014] / S} x 100

V ₁ : consumption of 0.1N hydrochloric acid solution (ml)

V ₂ : consumption of 0.1 N hydrochloric acid solution in blank test (ml)

f = 0.1 N hydrochloric acid solution Titer

S = weight of anchovy crushed

4) minutes

The hydrolyzed anchovy crushed material was subjected to high pressure / enzyme treatment under the condition of Example 2-2 and ignited at 550 ± 10 ° C to remove volatile organic substances. After determining the weight of the residue, the ash content was calculated (AOAC method 923.03, 1990).

5) Experimental results

moisture(%) Crude fat (%) Crude protein (%) Views min (%) One 95.78 ± 0.06 0.10 ± 0.01 3.98 ± 0.01 0.14 0.20 2 94.46 ± 0.10 0.07 ± 0.00 4.36 + 0.02 0.28 ± 0.39 3 93.42 ± 0.20 0.09 ± 0.00 5.18 ± 0.00 0.43 + - 0.60 4 93.48 + 0.13 0.03 ± 0.00 4.90 ± 0.00 0.41 + - 0.57 5 94.96 ± 0.01 0.04 ± 0.00 4.21 + - 0.01 0.16 + 0.22 6 94.62 ± 0.06 0.01 ± 0.00 4.41 ± 0.01 0.27 ± 0.38 7 94.48 ± 0.07 0.01 ± 0.00 4.45 ± 0.01 0.28 ± 0.38 8 93.45 + 0.14 0.00 ± 0.00 5.08 ± 0.02 0.40 + - 0.56 9 95.21 + 0.02 0.01 ± 0.00 4.18 ± 0.00 0.14 0.20

pH Salinity (%) Soluble solids (Brix,%) One 6.17 ± 0.02 0.08 ± 0.00 5.80 ± 0.00 2 6.51 ± 0.01 0.37 ± 0.01 6.40 ± 0.00 3 6.35 ± 0.00 0.74 ± 0.01 8.00 ± 0.00 4 6.45 ± 0.01 0.73 ± 0.01 7.60 ± 0.00 5 6.55 ± 0.01 0.08 ± 0.01 6.00 ± 0.00 6 6.40 ± 0.00 0.33 ± 0.00 5.33 ± 0.12 7 6.44 ± 0.00 0.45 ± 0.00 6.80 ± 0.00 8 6.36 ± 0.00 0.66 ± 0.00 6.40 ± 0.00 9 6.50 ± 0.00 0.06 ± 0.00 4.47 0.12

As a result, crude fat content was less than 0.1% and salinity was less than 0.1%. The pH was constant in the range of 6.1 ~ 6.6. However, only crude protein content and water soluble solid content showed significant changes according to experimental conditions.

2-4. Determination of Free Amino Acid Content of Hydrolyzed Anchovy Grind with High Pressure / Enzyme Treatment Conditions

Analysis of the free amino acids was performed using an HPCL system (Agilent Technologies Inc., Santa Clara, CA). Samples were separated on a C18 column (4.6 × 150 mm, 5; Youngjinbiochrom, Korea), and the separated samples were analyzed with two detectors (fluorescence detector, UV detector). The hydrolyzed anchovy crushed by the high pressure / enzyme treatment under the condition of Example 2-2 was diluted to a proper concentration with distilled water and then diluted with a sealing paper filter (DISMIC-13CP 0.45 micron syringe filter, Advantec Co., Saijo, Japan) Filtered. The mobile phase was a mixture of 20 mM sodium phosphate buffer (pH 7.8), 45% (v / v) acetonitrile and 45% (v / v) methanol solution. The sample was reacted with 9-fluorenylmethyl chloroformate (FMOC) in a fluorescence detector (450 nm emission, 305 nm excitation) when reacted with o -phthalaldehyde (OPA) And analyzed with a UV detector (338 nm). The results are shown in FIG. 3 and Table 7.

(mg / L) Classification One 2 3 4 5 6 7 8 9 Glutamate 1,853.94 2,336.31 3,177.16 3,926.92 24,16.45 2,661.21 3,247.31 3,158.00 1,770.11 Asparagine 2,007.80 2,397.26 2,880.68 3,329.25 2,640.57 2,575.29 3,183.72 2,904.26 2,056.83 Serine 0.00 205.90 0.00 0.00 159.01 297.71 80.10 69.12 190.78 Glutamine 1,291.27 1,442.93 1,958.10 2,136.44 1,515.97 1,730.82 1,849.28 1,937.89 1,252.86 Histidine 3,345.68 2,576.31 648.34 4,509.64 2,110.83 2,779.53 3,485.74 3,853.03 1,426.46 Glycin 943.75 1,038.05 1,689.60 1,845.14 724.80 1,249.80 1,195.03 1,633.53 593.66 Threonine 703.89 1,071.28 1,167.74 1,408.02 1,158.61 1,011.74 1,382.54 1,209.29 804.72 Arginine 1,222.47 1,455.38 1,981.70 2,167.88 1,512.86 1,767.33 1,911.45 1,960.42 1,277.10 Alanine 2,088.37 2,124.21 2,775.38 2,903.18 2,270.69 2,641.43 2,569.81 2,720.68 2,200.49 GABA 2,163.44 2,179.34 2,874.90 3,147.71 2,446.71 2,534.83 2,914.38 2,838.40 1,999.13 Tyrosine 608.49 517.29 744.70 708.85 597.91 668.89 600.25 665.12 562.55 Valine 432.63 456.93 541.54 516.04 545.67 494.51 535.39 505.85 470.08 Methionine 1,541.99 1,829.54 2,495.44 2,637.13 1,836.54 2,183.58 2,222.65 2,442.98 1,586.83 Tryptophan 1,112.12 1,220.92 1,505.25 1,515.77 1,138.04 1,263.97 1,297.92 1,463.45 1,086.36 Phenyl-
field 4,222.73 4,758.35 6,510.15 6,574.62 4,692.94 5,271.15 5,328.27 6046.98 4193.87 Isoleucine 1,350.66 1,534.65 1,996.89 2,021.14 1,487.70 1,740.71 1,697.32 1,936.70 1,370.07 Leucine 1,511.85 2,012.07 2,426.38 2,505.83 1,865.24 2,068.11 2,164.58 2,412.00 1,588.37 Lysine 2,818.21 3,315.97 3,907.03 3,966.16 3,150.27 3,401.41 3,412.07 3,825.85 2,828.99 Proline 2,244.41 2,431.90 3,664.01 3,849.42 2,597.35 3,035.24 3,180.91 3,417.81 2,495.62 Total 32,174.40 35,308.14 43,432.76 50,561.26 35,324.25 39,794.81 43,254.86 45,605.36 30,033.37

As a result of the experiment, it was confirmed that the amino acid content of hydrolyzed anchovy crushed product was the highest by treatment of 2% of complex enzyme in anchovy crushed product and high pressure treatment at a pressure of 75 MPa and 40 ° C. for 24 hours.

2-5. Measurement of degree of hydrolysis (DH) of hydrolyzed anchovy according to high pressure / enzyme treatment conditions

The hydrolysis degree of hydrolyzed anchovy crushed by the high pressure / enzyme treatment under the conditions of Example 2-2 was measured by using the content of free amino acids contained in the supernatant after high pressure / enzyme treatment based on the content (51.16 mg / mL) of anchovy amino acid Total contents were analyzed. Amino acid content in anchovy was analyzed by hydrolysis of crushed anchovy diluted with water to acid, and free amino acid content was analyzed by filtration (2 ㎛) with a sealing paper filter without pretreatment. The degree of hydrolysis was calculated using Equation 5 below. Amino nitrogen was analyzed by formalin titration and 2,4,6-trinitrobenzenesulfonic acid (Sigma, USA) method for analysis of hydrolysis. 2 mL of 0.2 M phosphate buffer (pH 8.2) and 2 mL of 0.05% TNBS were added to 0.25 mL of the sample, and the mixture was incubated at 50 ° C for 60 minutes Lt; / RTI &gt; To stop the reaction, 4 mL of 0.1 N HCl was added and reacted for 30 min. The absorbance was measured at 340 nm with a UV / Vis spectrophotometer (Beckman, DU530, USA). In the Formol titration method, the sample was diluted 40 times with distilled water and the pH was adjusted to 7.0. Then, 10 mL of formalin was added to the sample, and the pH was adjusted to 8.5 with NaOH. From the amount of NaOH added, the content of formol nitrogen was calculated using the following equation (4). In addition, pressure, temperature, time, and S / N ratio (signal to noise ratio) for each enzyme were also determined. The results are shown in FIG. 4 and Table 8.

[Experimental Equation 4]

Amino nitrogen (mg) = NaOH (ml) x Normal concentration of NaOH x 14

[Experimental Equation 5]

Hydrolysis (DH,%) = (Amino nitrogen / Total nitrogen) × 100

2-6. Determination of nitrogen recovery (NR) of hydrolyzed anchovy crushed according to high pressure / enzyme treatment conditions

The nitrogen recovery of the hydrolyzed anchovy crushed by the high pressure / enzyme treatment under the conditions of Example 2-2 was calculated as the total nitrogen content, the nitrogen content in the water-soluble protein, and the nitrogen content in the free amino acid form Respectively. The contents of water soluble protein and formol nitrogen were analyzed to calculate the total nitrogen content as a percentage and the unknown nitrogen content as unknown. The water soluble protein was quantified by the lowry method. BSA (Bovine Serum Albumin, Sigma, USA) was used as a standard sample. 1 mL of the sample was dissolved in 1 mL of Lori reagent (Sigma, TP0300-1KT Kit, USA) And react for 20 minutes. The reaction mixture was reacted for 30 minutes with 0.5 ml of a Folin-ciocalteu's phenol reagent (Sigma, TP0300-1KT Kit, USA), and the mixture was reacted at 570 nm with a UV / Vis spectrophotometer (Beckman, Was measured. The unknown includes volatile non-volatile nitrogen and precipitated nitrogen. In addition, pressure, temperature, time, and S / N ratio (signal to noise ratio) for each enzyme were also determined. The results are shown in FIG. 5 and Table 8.

2-7. Determination of the yield of hydrolyzed anchovy according to high pressure / enzyme treatment conditions

The hydrolyzed anchovy crushed product was subjected to high pressure / enzyme treatment under the conditions of Example 2-2 to centrifuge at 17,000 rpm to obtain a supernatant, and the anchovy crush product yield was expressed as a percentage by the following Equation (6). The results are shown in FIG. 6 and Table 8.

[Experimental Equation 6]

Yield (%) = {Weight after centrifugation of the hydrolyzed anchovy crushed product (g) / Weight (g) before hydrolyzed anchovy crushed product} × 100

Total nitrogen
(mg / mL) Formalin nitrogen
(mg / mL) Water soluble protein nitrogen (mg / mL) DH (%) S / N1 NR (%) S / N2 yield(%) S / N3 One 30.88 4.32 ± 0.04 5.12 ± 0.03 13.99 22.91 62.86 35.97 86.30 38.72 2 34.24 6.25 + 0.14 2.61 0.36 18.25 25.23 68.98 36.77 74.15 37.40 3 35.52 6.51 ± 0.03 3.27 ± 0.12 18.33 25.26 84.85 38.57 67.76 36.62 4 28.16 7.15 ± 0.28 3.44 ± 0.09 25.4 28.10 98.78 39.89 70.91 37.01 5 20.48 5.69 + 0.14 3.89 ± 0.34 27.8 28.88 69.01 36.78 50.34 34.03 6 31.52 5.77 ± 0.15 3.64 0.14 18.29 25.24 77.74 37.81 71.94 37.14 7 24.64 6.16 ± 0.06 2.77 ± 0.01 24.99 27.96 84.50 38.54 47.99 33.62 8 25.44 7.07 + - 0.45 2.66 ± 0.01 27.81 28.88 89.09 39.00 98.62 39.88 9 29.92 5.17 ± 0.06 3.69 ± 0.07 17.3 24.76 58.67 35.37 75.58 37.57

As shown in FIG. 4, the hydrolysis degree was affected by all factors such as pressure, temperature, time and amount of enzyme. Pressure and temperature were found to be optimal and enzyme addition and reaction times were more or less good. Based on the S / N ratio, the best conditions for hydrolysis were the pressure 75 MPa, the temperature 50 ℃, the reaction time 48 hours and the addition amount of complex enzyme 2%.

As shown in FIG. 5, the nitrogen recovery rate was not influenced by time, temperature, and pressure, but it was found that the optimum condition was obtained in the case of pressure, and the more the amount of enzyme added, the better. Based on the S / N ratio, the optimal conditions for nitrogen distribution were 75 MPa at the temperature, 40 ℃ for the reaction time, 48 hours for the reaction time and 2% for the complex enzyme.

As shown in FIG. 6, the effect of pressure and temperature was small, and the longer the time, the less effective the yield. The more the added amount of enzyme, the better the yield. Based on the S / N ratio, the optimal conditions for the best yield were 50 MPa pressure, 50 ℃ temperature, 12 h reaction time and 2% addition of complex enzyme.

2-8. Determination of molecular weight distribution of hydrolyzed anchovy crushed according to high pressure / enzyme treatment conditions

Gel permeation chromatography (GPC, GE healthcare, AKTAP prime plus) was used to examine the possibility of optimizing the production of the salty taste enhancing substance in the hydrolyzed anchovy pulp by high pressure / enzyme treatment under the conditions of Example 2-2 And analyzed by GPC chromatogram. Sephadex G-10 (Sigma, G10120) was packed in a glass column (GE healthcare, XK16 / 70) as a stationary phase and separated by molecular weight. The mobile phase used was 1% formic acid, and the sample was injected at a flow rate of 0.5 mL / min and detected at 214 nm. The percentage of Peak II fraction and the S / N ratio (signal-to-noise ratio) of salted tastants expected to be present in the detected fractions were also determined. The results are shown in FIG. 7, FIG. 8, and Table 9.

Peak II
(%) S / N ratio One 43.40 32.75 2 49.18 33.84 3 53.70 34.60 4 90.17 39.10 5 42.66 32.60 6 51.89 34.30 7 54.72 34.76 8 97.77 39.80 9 39.61 31.966

As a result of the experiment, it was found that the possibility of producing the salty taste enhancing substance has the greatest effect on the enzyme. Concretely, the content of Peak Ⅱ fraction, which is expected to have a salty taste enhancing substance, increased with increasing pressure, but remained constant at above 75 MPa, and remained constant up to 50 캜 and decreased at a higher temperature. In addition, the content of Peak II decreased when the reaction was performed for 12 hours or more.

Example  3: Eel  Pilot plant for mass production ( pilot plant ) Enzyme stability verification at the level

3-1. Anchovy and enzyme preparation

Anchovies were prepared in the early march of Jeju Island by purchasing the anchovies frozen at -20 ℃ in East Sea of Korea. Then, the anchovy was thawed at room temperature and then immersed in water at 10 to 15 ° C for 1 hour to remove salts and impurities. Then, water of twice the weight of anchovy was added, and the mixture was pulverized twice using a household blender for 2 minutes and then pulverized at 16,000 rpm for 5 minutes using a homogenizer to prepare anchovy pulverized product.

The enzyme was prepared in the same manner as in Production Example 1-1 except that commercially available commercial enzymes selected for hydrolysis of anchovy were alcalase, flavoruzyme, neutrase, and protamex. (Novonodisk Bioindustrials, Inc., Denmark). In addition, a complex enzyme (A: F = 1: 5) was prepared by mixing alcalase and flavor in the enzyme at a weight ratio of 1: 5. The optimum conditions and origins of each enzyme are shown in Table 10 below.

Enzyme name Optimum condition Material origin Temperature (℃) pH Alcalase 2.4 L 55-70 6.5-8.5 Bacillus licheniformis Flavourzyme 500 mg 50 7.0 Aspergillus oryzae Neutrase 0.8 L 45-55 6.0 Bacillus amyloliiensquefaciens Protamex 1.5 MG 40 6.0-7.0 Bacillus sp .

3-2. Enzyme activity measurement at normal or high pressure

The anchovy crush prepared in Example 3-1 was supplemented with alcalase, flavorzyme, neutrase, protamex, and alcalase and flavorzyme 1, 2, 6, 12 and 24 hours at ambient pressure (0.1 MPa, 50 ℃) or high pressure (100 MPa, 50 ℃) after adding 2% of complex enzyme mixed with flavorzyme at a weight ratio of 1: And the protein hydrolyzing activity of the enzyme was measured in the same manner as in Example 1-2. As a control group, anchovy pulverized material which had been treated with high pressure without enzyme was used. The results are shown in Fig.

As a result, it was confirmed that the stability of five enzymes was maintained at both high pressure and atmospheric pressure.

3-3. Measurement of hydrolysis rate by enzyme at normal or high pressure

The anchovy crush prepared in Example 3-1 was supplemented with alcalase, flavorzyme, neutrase, protamex, and alcalase and flavorzyme (0.1 MPa, 50 ° C) or high pressure (100 MPa, 50 ° C) in the presence of 2% of a complex enzyme (A: F = 1: 5) mixed with a flavorzyme at a weight ratio of 1: , 2, 6, 12 and 24 hours, and the degree of hydrolysis of the hydrolyzed anchovy crushed product was analyzed in the same manner as in Example 2-5. As a control group, anchovy pulverized material which had been treated with high pressure without enzyme was used. The results are shown in Fig.

As a result of the experiment, the five hydrolyzed enzymes showed high hydrolysis rate at high pressure compared with the normal pressure, and the hydrolysis degree of the hydrolyzate containing the enzyme was higher than that of the control group when the hydrolysis degree of the enzyme containing the enzyme was compared there was. These results indicate that hydrolysis is promoted at high pressure (100 MPa, 50 ℃) rather than atmospheric pressure (0.1 MPa, 50 ℃).

3-4. Determination of the bitter taste of hydrolyzed anchovy ground by enzymatic treatment at high pressure

The anchovy crush prepared in Example 3-1 was supplemented with alcalase, flavorzyme, neutrase, protamex, and alcalase and flavorzyme 1, 2, 6, 12, and 24 at a high pressure (100 MPa, 50 ° C) condition after addition of 2% each of a complex enzyme (A: F = 1: 5) mixed with a flavorzyme at a weight ratio of 1: And the tryptophan content of the hydrolyzed anchovy crushed product was measured in the same manner as in Example 1-3. As a control group, anchovy pulverized material which had been treated with high pressure without enzyme was used. The results are shown in Table 11.

Tryptophan (mM) Time Control group Alcalase Flavor Ntrease ProTamex A: F = 1: 5 0 1.11 1.15 1.62 1.15 1.52 1.61 One 6.12 4.59 7.60 7.27 7.56 4.61 2 8.96 8.41 8.61 9.83 13.10 8.73 6 15.10 14.92 12.71 16.71 18.45 12.81 12 17.12 16.06 13.69 19.66 19.60 15.19 24 16.98 17.03 12.61 20.59 21.92 16.90

Experimental results showed that tryptophan was less abundant in the hydrolyzate added with flavorzyme than in the control, and then complex enzyme (A: F = 1: 5), alkalase alcalase, neutrase, and protamax, respectively. The complex enzyme (A: F = 1: 5) was determined to be most suitable considering these results and the price of the enzyme.

Example  4 : Eel  Optimal for mass production High-pressure enzyme hydrolysis  Process condition verification experiment

Based on the S / N ratio of Example 2, hydrolyzed anchovy crushed material was prepared by high pressure / enzyme treatment under the conditions of optimal high pressure enzyme hydrolysis determined according to pressure, temperature, time and enzyme, Recovery rate and molecular weight were measured in the same manner as in Examples 2-5, 2-6 and 2-8, respectively. The optimum conditions for each measurement are shown in Table 12 below. The results are shown in Figs. 11 and 12 and Table 13.

Measure Pressure (MPa) Temperature (℃) Time (hrs) enzyme(%) yield(%) 50 50 12 2 Nitrogen recovery (NR,%) 75 40 48 2 Hydrolysis (DH,%) 75 50 48 2 Peak (%) 75 50 12 2

GPC fraction (%) Peak I Peak II Peak III 1 (Yield,%) 7.65 50.17 42.18 2 (NR,%) 4.79 48.92 46.29 3 (DH,%) 5.42 48.99 45.59 4 (Peak,%) 5.67 47.57 46.76 Peak I: 1,300 Da or more, Peak II: 250-1,300 Da, Peak III: 250 Da or less

The hydrolysis rate of hydrolyzed anchovy was highest at the optimum conditions of nitrogen recovery (75 MPa, 40 ℃, 48 hours, enzyme concentration 2%) according to the optimum conditions of high pressure enzyme hydrolysis Nitrogen recovery by high pressure enzyme hydrolysis optimization condition was the highest in hydrolysis degree (75 MPa, 50 ℃, 48 hours, enzyme concentration 2%).

The molecular weight of the hydrolyzed anchovy crushed by the high pressure enzyme hydrolysis optimization conditions was not significantly different according to the hydrolysis conditions.

It is important to reduce the amount of enzyme in order to produce a low-salt fish paste at the pilot plant scale and to match the reaction time of the high-pressure treatment time with the working time.

Therefore, it was decided to apply to the pilot plant scale production by adjusting the time to 16 hours under the treatment conditions of 75 MPa, 50 ℃, 2% complex enzyme and 12 hours, which are optimum conditions for producing the salty taste enhancing substance.

Example  5: Hydrolysis of high-pressure enzymes  According to the optimum condition Low salt Eccentrics ( APH ) Produce

(APH) was prepared according to the optimum conditions of high-pressure enzyme hydrolysis and the process of FIG. Specifically, we purchased anchovies from the East Sea Fisheries (Captain) kept in-sea at -20 ℃ and caught in Cheju Island in early March. The purchased anchovy was thawed at room temperature and then immersed in water at 10 to 15 ° C for 1 hour and washed with water to remove salts and impurities. Then, the anchovy was added twice as much water as the anchovy, and the resulting mixture was pulverized twice for 2 minutes using a household blender, followed by secondary pulverization at 16,000 rpm for 5 minutes using a homogenizer to obtain anchovy crush . Then, 2% by weight of the weight of the anchovy crushed material was added to the pulverized anchovy powder and the complex enzyme (alkalase: flavorzyme = 1: 5) was added to the pouch in 2 kg units and sealed. The enzyme was then inactivated by high pressure system (INOSHELLE KOREA, Korea) at a pressure of 75 MPa and a high pressure for 16 hours at a temperature of 50 DEG C, followed by heat treatment for 80 to 20 minutes. Then, the mixture was aged at 4 ° C for 12 hours, centrifuged at 17,000 rpm using a tubular centrifuge (A-V10675G, TO-MOE ENGINEERING, Japan) to remove floating oil, Respectively.

Example  6: Low salt Eel  Composition / free amino acid content and protein extraction efficiency measurement

6-1. Constitution of low salt fish paste / free amino acid content measurement

The composition / free amino acid content of the low salt crab paste (APH) prepared in Example 5 was measured in the same manner as in Example 2-4. The mobile phase used was 10 mM Na ₂ HPO ₄ and 10 mM Na ₂ B ₄ O ₇ 10 H ₂ O (pH 8.2) for the constituent amino acids and 20 mM sodium phosphate solution phosphate buffer, pH 7.8), 5% (v / v) acetonitrile and 45% (v / v) methanol solution were used. The sample was reacted with 9-fluorenylmethyl chloroformate (FMOC) in a fluorescence detector (450 nm emission, 305 nm excitation) when reacted with o -phthalaldehyde (OPA) And analyzed with a UV detector (338 nm). The results are shown in Fig. 14 and Table 15.

Condition Constituent amino acid Free amino acid HPLC equipment Ultimate 3000 (Thermo Dionex, USA) FL Detector Emission 450 nm, Excitation 340 nm (OPA) Emission 305 nm, Excitation 266 nm (FMOC) UV Detector 338 nm Standard product 1 nmol / μL Amino acids 17 std (0.1 N-HCL dissolved) and 6 additional species 1 nmol / μL Amino acids 17 std (0.1 N-HCL dissolution) Column Inno C18 column (4.6 mm.times.150 mm, 5 .mu.m / Youngjinbiochrom, Korea) Mobile Phase A Na ₂ HPO ₄ 10 mM, Na ₂ B ₄ O ₇ 10H ₂ O 10 mM, pH 8.2 20 mM sodium phosphate monobasic, pH 7.8 Mobile Phase B 3D.W. / Acetonitrile / Methanol (10: 45: 45 v / v%) Injection Volume 1 μL 0.5 μL Column Temperature 40 ℃ Sample Temperature 20 ℃

No. Ret.Time
min Peak Name
Height
LU Area
LU * min Rel.Area
% Content (mg / kg) One 2.79 Aspartic acid 0.059 0.014 0.23 860.897 2 4.33 Glutamic acid 0.068 0.007 0.12 542.059 3 7.65 Serine 0.104 0.012 0.21 525.731 4 8.89 Histidine 0.417 0.045 0.77 5,166,471 5 9.50 Glycine 5.514 0.621 10.71 18,294,064 6 9.68 Threonine 0.760 0.082 1.41 4,598,326 7 10.84 Arginine 2.795 0.312 5.38 20,143,196 8 11.57 Alanine 10.958 1.247 21.51 44,303.244 9 11.89 Taurine 1.522 0.186 3.21 7,551,130 10 13.38 Tyrosine 0.434 0.048 0.82 3,912,426 11 16.13 Novaline 5.698 0.687 11.85 22,076,667 12 16.49 Methionine 1.466 0.178 3.07 9,231,835 13 18.31 Phenylalanine 1.944 0.245 4.23 14,594,773 14 18.55 Isoleucine 4.286 0.554 9.57 24,941,859 15 19.54 Leucine 5.860 0.726 12.52 32,930,612 16 20.06 Lysine 1.076 0.138 2.37 36,236,801 17 21.40 Hydroproline 0.198 0.050 0.86 1,462,020 18 26.21 proline 3.449 0.646 11.15 20,034,288 Total: 46.609 5.796 100.00 267,406.400

Asparagine and glutamine were found to be converted into aspartic acid and glutamine acid, which are free amino acids, and tryptophane, which is a bitter taste amino acid, was detected. I did.

6-2. Protein Extraction Efficiency of Low Salt Urethane (APH)

The low-salt crabs and the low-salt crabs prepared in Example 5 were centrifuged and the nitrogen content was measured, and the protein extraction efficiency was determined using Equation (7). Freshwater anchovy was used as a control. The results are shown in Table 16.

[Experimental Equation 7]

Protein extraction efficiency (CIP,%) = (Nitrogen content after centrifugation (per 1 ml of supernatant) / Nitrogen content before centrifugation (per 1 ml of APH)) × 100

Anchovy: Water (1: 2) The protein content (g / kg) 51.90 Protein extraction efficiency (%) 63.50

As a result of the experiment, the protein content of the live fish was 51.90 g / kg and the total protein content of the fish paste was 1,307.68 g. The protein extraction efficiency was 63.50% because 830.43 g of the protein was present as a water soluble protein. From this value, it can be confirmed that the process developed through the present invention is very efficient.

Example  7: Eel  Separation and purification process for mass production

7-1. Production of purified water from low-salt fish paste using ultrafiltration system and nanofiltration system

(5 kDa, proflux M60 tangential fow filtration system, Millipore, USA and 10 kDa, Lab U / F system) and nanofiltration system (250 Da, Prolab system , USA).

Specifically, the low salt confectionery prepared in Example 5 was passed through an ultrafiltration membrane (MWCO 10 kDa) to obtain a purified product (hereinafter referred to as APHU-1) having a size of 10 kDa or more. Then, the permeate (MW < 10 kDa) permeated to the ultrafiltration membrane was dissolved in water at a ratio of 1:10 and then passed through an ultrafiltration membrane (MWCO 5 kDa) to separate the purified product of 5 kDa or more and less than 10 kDa Hereinafter, referred to as APHU-2). Then, a permeate (MW <5 kDa) permeated to the ultrafiltration membrane was passed through a nanofiltration membrane (MWCO 250 Da) to obtain a purified product of 250 Da or more and less than 5 kDa (hereinafter referred to as APHN- (MW < 250 Da, hereinafter referred to as APHN-2) was fractionated. Each of the tablets was frozen at -70 ° C for 24 hours, then dried and stored at -20 ° C.

7-2. Determination of the yield and protein content of the purified product obtained through ultrafiltration and nanofiltration

The weight of the low salt crab paste (APH) prepared in Example 5, the APHU-1 tablets prepared in Example 7-1, the APHU-2 tablets, the APHN-1 tablets and the APHN-2 tablets were weighed, The extraction yield was measured on the basis of unhydrolyzed live anchovy. The protein content of the purified product was measured using a micro-Kjeldahl method. Specifically, crude protein content analysis method of Example 2-3 was used. The results are shown in Table 17.

Control group Purified water Fresh Anchovy Low salt eel (APH) Ultrafiltration Nanofiltration APHU-1 APHU-2 APHN-1 APHN-2 weight 8.39
Wet wt (kg)
2.10
Dry wt (kg) 1,110.20
Dry wt (g) 130.20
Dry wt (g) 94.40
Dry wt (g) 309.99
Dry wt (g) 59.51
Dry wt (g) yield(%) 100 52.86 6.20 4.64 14.76 2.83 Protein content (g) 1,493.42 830.43 95.57 71.37 244.27 ND Protein yield (%) 100 55.61 6.40 4.78 16.36 ND

As a result of the experiment, 8.39kg (dry weight 2.1kg) of fresh fish was hydrolyzed by high pressure / enzyme treatment and then centrifuged to obtain 1.1kg of low salinity arsenic (APH). Using ultrafiltration system and nano filtration system, 594.1 (APHU-1, APHU-2, APHN-1 and APHN-2). On the other hand, the yields of the hydrolyzed proteins of the above low salt confectioneries and tablets were also similar to the yields of low salt esters and tablets.

7-3. Molecular weight measurement of purified water obtained through ultrafiltration and nanofiltration

The salty taste enhancing substance in the low salt crab paste (APH) prepared in Example 5, the APHU-1 purified product, the APHU-2 purified product, the APHN-1 purified product and the APHN-2 purified product prepared in Example 7-1 Gel permeation chromatography (GPC) was performed to investigate the possibility of the presence. First, a GPC column (YMC pack diol-60, 300 mm x 20 mm, ID S-5 m, 6 nm) was equilibrated with 1% acetic acid. Thereafter, the purified APHU-1, APHU-2, APHN-1 and APHN-2 purified were diluted to a concentration of 18 mg / ml. Then, 1 ml of the diluted purified water was filled in the column, Min, and the molecular weight distribution and area of each low salt starch residue and each purified water were measured by measuring at 214 nm. Vitamin B ₁₂ (MW 1,355.37 Da), vitamin B ₁ (MW 337.27 Da) and L-glutamic acid (MW 147.13 Da) were used as reference materials. The area of the molecular weight of each purified product was calculated by multiplying the ordinate axis and the ordinate axis (min x mAu) of the molecular weight distribution diagram. The results are shown in FIG. 15, Table 18, and Table 19.

GPC Peak distribution for each tablet GPC peak APH (%) APHU-1 (%) APHU-2 (%) APHN-1 (%) APHN-2 (%) Ⅰ 5.8 24.6 5.7 6.0 0.0 Ⅱ 47.8 38.0 51.3 48.4 35.3 Ⅲ 46.4 37.4 43.0 45.4 64.7 Distribution 100 100 100 100 100 peak I: 1,300 Da or more, peak II: 250 Da to 1,300 Da, peak III: 250 Da or less

GPC peak area per tablet GPC peak APH (%) APHU-1 (%) APHU-2 (%) APHN-1 (%) APHN-2 (%) Ⅰ 1,766.8 9,683.0 1,686.3 2,535.7 4.0 Ⅱ 14,516.2 14,961.2 15,144.0 20,306.1 4,370.2 Ⅲ 14,092.7 14,735.7 12,692.3 19,093.2 8,020.9 Total area 30,375.6 39,379.9 29,522.6 41,935.0 12,395.05 peak I: 1,300 Da or more, peak II: 250 Da to 1,300 Da, peak III: 250 Da or less

As a result, three major peaks were identified as shown in FIG. 15, and purified tablets based on the molecular weight of the low salt epidermis (APH) had molecular weights of less than 1,500 Da and similar molecular weight distributions and patterns.

Peak Ⅰ fraction was significantly higher in APHU-1 tablets compared to other tablets. Peak Ⅰ fraction decreased gradually as the limiting molecular weight of the membrane decreased, and it was found that APHN-2 was almost completely disappeared. On the other hand, it was confirmed that the ratio of Peak Ⅱ and Ⅲ fraction was high. Peak I, II and III fractions of APHN-2 purified water were sharply reduced and the APHN-1 purified water content was only 30% of APHN-1 purified water.

7-4. Analysis of Physicochemical Properties of Purified Water from Ultrafiltration and Nanofiltration

The APHU-1 purified product, the APHU-2 purified product, the APHN-1 purified product and the APHN-2 purified product prepared in Example 7 were dissolved in 2.5% (Master meter, Master-M, 2M and 3M), and a pH meter (Orion 4-star Plus, Thermo Sci., USA) , Atago, Japan) was used to measure solubility, pH, water solids content and salinity. In addition, the low salt crab paste (APH) prepared in Example 5, the APHU-1 purified product, the APHU-2 purified product, the APHN-1 purified product and the APHN-2 purified product prepared in Example 7-1 were dissolved in distilled water , And 1 ml of the solution filtered through a 0.2 μm sealing paper filter (DISMIC-25JP, Advantec, Tokyo, Japan) was used as the sample solution. The test solution was analyzed using an ion chromatography (IC) (Dionex ICS-900, Thermo Scientific, Japan) at a flow rate of 1 ml / min, a pressure of 1,200-1,400 psi and a current of 60 mA. The results are shown in Table 20.

Solubility (%) pH Soluble solids (Brix) Salinity (%) g / 100 g of powder salt Nitrogen, NK Protein, NK * 6.25 APH 100 7.1 2.9 0.32 4.5 11.97 74.80 APHU-1 100 6.4 2.8 0.07 4.4 11.74 73.40 APHU-2 100 6.6 2.4 0.18 4.7 12.10 75.60 APHN-1 100 6.2 2.5 0.00 1.1 12.61 78.80 APHN-2 100 5.9 2.2 1.13 15.6 ND ND

As a result, the pH of APHN-2 purified product was the lowest at 5.9 and the other samples were in the range of 6.2 to 7.1, similar to commercial soy sauce and fish paste. The solubility was according to the solution dissolved at 2.5% and all the samples were found to be very good in solubility. The salinity of APHN-2 purified water was much higher than that of the other samples. APHN-1 purified water permeated through 5 kDa ultrafiltration membranes was completely desalted. Therefore, the nanofiltration system has a great effect on the desalting as well as on the fraction of the peptide.

On the other hand, the protein content of 100 g of APHU-1 purified product, APHU-2 purified product and APHN-1 purified product was very high, ie, 73 to 79%.

7-5. Measurement of color of purified water obtained through ultrafiltration and nanofiltration

A sample of the low salt crab paste (APH) prepared in Example 5, the APHU-1 purified product prepared in Example 7-1, the APHU-2 purified product, the APHN-1 purified product and the APHN-2 purified product prepared in Example 7-1 was dissolved in water To give a concentration of 2.5%. The turbidity and browning of the low-salt eelgrass and each tablet were measured at 660 nm and 430 nm using a spectrophotometer (UV-140-02, Shimadzu Co., Japan). The L, lightness, redness, and yellowness of the low-salt crab shell and each of the tablets were measured using a colorimeter (CM-3500d, Minolta, Japan) . The standard white plate was based on L = 89.74, a = 1.24, b = -7.26. The results are shown in FIG. 16 and Table 21.

Turbidity (OD ₆₆₀ nm) Browning degree (OD ₄₃₀ nm) Chromaticity Brightness (L) Redness (a) Yellow color (b) APH 0.025 0.537 54.77 -0.52 32.72 APHU-1 0.087 1.162 41.39 7.75 43.27 APHU-2 0.018 0.396 56.33 -1.83 25.71 APHN-1 0.029 0.665 56.17 0.34 37.88 APHN-2 0.001 0.017 67.12 -0.08 -3.83

As a result, the turbidity of APHN-2 was the lowest, and the other tablets were not significantly different from each other. In case of browning, the value of APHU-1 tablets was 68 times higher than that of APHN-2 tablets, which was darker than the other tablets. The color intensity of APHN-2 was slightly higher than that of other tablets, and redness and yellowness were highest in APHU-1 tablets.

7-6. Analysis of glass / constituent amino acids of purified water obtained through ultrafiltration and nanofiltration

The free / constituent amino acid content (%) of the low salt crab paste (APH) prepared in Example 5, the APHU-1 purified product, the APHU-2 purified product, the APHN-1 purified product and the APHN-2 purified product prepared in Example 7-1 Was measured in the same manner as in Example 6-1. The results are shown in Table 22.

(Unit: mg / kg) APH APHU-1 APHU-2 APHN-1 APHN-2 Asp 2,910 29,874 22,802 39,890 3,921 Glu 54,765 39,170 39,505 83,311 3,406 Asn 806 626 538.7 205 495 Ser 1,007 16,675 10,604 311 29,002 Gln 1,437 14,973 14,777 7,412 9,868 His 20,003 17,403 18,179 20,973 7,837 Gly 17,640 11,751 11,847 709 34,819 Thr 28,727 22,460 23,407 10,055 21,729 Arg 1,432 33,963 17,701 36,624 11,489 Ala 44,043 33,353 35,620 6,495 64,358 Tarurine 8,024 6,642 7,809 1,010 15,317 Gaba 4,497 - - - - Tyr 7,392 17,193 51,743 5,771 8,208 Val 53,794 28,536 29,778 26,188 18,870 Met 22,311 19,384 19,503 8,625 24,513 Trp 3,659 3,246 3,582 2,015 3,454 Phe 25,187 25,183 28,782 12,141 28,747 Ile 32,177 28,703 27,883 35,103 14,280 Leu 51,929 50,500 49,886 54,282 32,402 Lys 16,246 48,333 50,688 100,376 12,209 Hydroproline 697 493 410 680 156 Pro 7,503 7,326 8,094 2,598 10,248 Total 388,187 455,785 473,136 454,772 355,329

As a result, the content of tryptophan (Trp) in bitter tastes was lowest in APHN-1 tablets, while the content of glutamic acid (Glu) was highest in APHN-1 tablets. On the other hand, the sweetness of glycine (Gly), proline (Pro), alanine (Alanine, Ala) and serine (Ser) was the most abundant in the APHN-2 tablets.

Example  8 : Eel  For mass production Desalination  Fairness

8-1. Desalination using nanofiltration membrane

(5 kDa) was passed through the ultrafiltration system (MWCO 10 kDa and 5 kDa), and the permeate (5 kDa permeate) was subjected to a desalting process using a nanofiltration system (MWCO 250 Da) Respectively. APHN-1 and APHN-2, which have been subjected to the desalting process by the nano filtration system, APHN-2, and APHU-1, 2) were lyophilized and then sodium content was measured by ion chromatography (IC). The lyophilized sample was dissolved in water at a concentration of 2.5%, and the salt content was measured using a salt measuring instrument (YK-31SA, Lutron, Taiwan), and the salinity was calculated in terms of a percentage. The results are shown in Table 23.

APH APHU-1 APHU-2 APHN-1 APHN-2 Sodium (g / 100 g) 4.5 4.4 4.7 1.1 15.6 Salt content (g / 100 g) 11.4 11.2 11.9 2.8 39.6 Salinity (%) 12.8 2.8 7.2 0.0 45.2

As a result, the salt content of APH, APHU-1 and APHU-2 before passing through the nanofiltration system was 11.2 ~ 11.9 g per 100g. However, the salt content of APHN-1 separated by nanofiltration system was 2.8 g per 100 g and the salt content was reduced to 1/5. The APHN-2 sample was 39.6 g per 100 g, which was lower than the sample before passing through the nanofiltration system And APHN-1, respectively. It can be seen that desalting was performed in the APHN-1 sample in which salts having a small molecular weight did not pass through the filtration membrane while passing through the nanofiltration membrane (250 Da).

8-2. Desalination using electrodialysis

The APHN-1 liquid sample passed through the ultrafiltration system (MWCO 5 kDa) and the nano filtration system (MWCO 250 Da) prepared in Example 5 was transferred to an electrodialyzer S3 (AC-220, Astom, Japan) was installed in a desalting machine under conditions of a voltage of 12 V, a current of 21 A, a manipulation time of 40 minutes and a mutual current of 0.22 A / h, Respectively. Next, sodium and total nitrogen contents of the sample before and after the electrodialysis treatment were measured using ion chromatography and micro-Kjeldahl method. The results are shown in Table 24.

Before Electrodialysis After electrodialysis Electrical Conductivity (mS / cm) 11.2 5.9 Sodium (g / 100 g) 1.10 0.85 Total nitrogen (g / 100 g) 13.0 6.70

As a result, the electrical conductivity of the APHN-1 sample before electrodialysis was 11.2 mS / cm, but the electrical conductivity of the sample dropped to 5.9 mS / cm after electrodialysis. On the other hand, the electrical conductivity of 0.5% NaCl was about 2 times increased from 4.8 mS / cm to 11.9 mS / cm. The sodium content decreased to 1.1 g / 100 g before electrodialysis and to 0.85 g / 100 g after electrodialysis. Nitrogen content was also detected in the 0.5% NaCl sample after electrodialysis. As a result, the desalting process showed that the materials other than the salt (including nitrogen) were removed and moved together.

In conclusion, the desalting process using the nano filtration system and the electrodialysis equipment removes 76.67% and 22.73% salt, respectively, compared to the untreated desalting process. However, if a pilot plant scale system produces large quantities of products, electrodialysis is not cost effective and can lead to nitrogen loss, so if the desalination process is absolutely necessary, the nanofiltration system will be utilized.

Example  9: Eel  Decolorization and deodorization process for mass production

9-1. Determination of decolorization rate of low salinity arsenic (APH) according to activated carbon concentration

L-glutamate monohydrate (1.9 g / L), maltodextrin (6.375 g / L) and yeast extract (2.1 g / L) were dissolved in 2.5% , Purified active carbon (No. 2524, SHIN KI CHEMICAL, Korea) of 1.0, 2.0 and 3.0% was added and reacted for 5 minutes at room temperature. OD value was measured at 420 nm absorbance to calculate discoloration rate. The decoloring rate is expressed as a percentage using the following Equation (8). As a control group, a low salinity irrigation well without activated carbon was used. The results are shown in FIG. 17 and Table 25.

[Experimental Equation 8]

Discoloration rate (%) = (absorbance of untreated sample - absorbance of treated sample) / (absorbance of untreated sample)} x 100

Activated carbon concentration (%) Control group 0.5 1.0 2.0 3.0 OD 0.570 0.084 0.038 0.005 0.001 Decolorization rate (%) - 85.3 93.3 99.1 99.8

As a result, the decolorization rate was more than 99% when the concentration of activated carbon was 2% or more. Based on the results, the reaction conditions were determined with the concentration of activated carbon of 2% and the reaction time of 5 minutes.

9-2. Determination of decolorization rate according to the concentration of low-salt umbilical artery (APH)

5.0% and 10.0% of the low salt crab paste (APH) prepared in Example 5 was added to the model solution (monosodium L-glutamate monohydrate 1.9 g / L, maltodextrin 6.375 g / L and yeast extract 2.1 g / (No. 2524, SHIN KI CHEMICAL, Korea) was added, and the reaction was allowed to proceed at room temperature for 5 minutes. Then, the OD value was measured at 420 nm absorbance and the discoloration rate was calculated. The decoloring rate is expressed as a percentage using Equation (7). As a control group, 2.5%, 5.0% and 10.0% low salinity irrigation not treated with activated carbon was used. The results are shown in FIG. 18 and Table 26.

Concentration of low salt eel (%) 2.5 5.0 10.0 Un-treatment Treatment Un-treatment Treatment Un-treatment Treatment OD 0.570 0.005 1.124 0.057 2.312 0.393 Decolorization rate (%) 99.1 94.9 83.0

As a result, the decolorization rate was the highest at 99.1% when the concentration of APH was 2.5%, and 94.9% when the concentration was 5.0%, but still showed a decolorization rate of 90% or more. However, when the concentration of APH was lowered to 10%, the decolorization rate was lowered to 83.0%, and in FIG. 18, it was found to be yellowish when compared with 2.5% and 5.0%.

9-3. Determination of decolorization rate according to reaction temperature of activated carbon

The low salt crab paste (APH) prepared in Example 5 was added to a model solution (monosodium L-glutamate monohydrate 1.9 g / L, maltodextrin 6.375 g / L and yeast extract 2.1 g / L) 2% of purified activated carbon (No. 2524, SHIN KI CHEMICAL, Korea) was added and reacted at room temperature, 40 ° C, or 60 ° C for 5 minutes. OD value was measured at 420 nm absorbance to calculate discoloration rate. The decoloring rate is expressed as a percentage using Equation (7). As a control group, a low salinity irrigation well without activated carbon was used. The results are shown in Fig. 19 and Table 27.

Room temperature 40 ℃ 60 ° C Un-treatment Treatment Un-treatment Treatment Un-treatment Treatment OD 0.570 0.005 0.570 0.005 0.570 0.005 Decolorization rate (%) 99.1 99.1 99.1

As a result of the experiment, when the activated carbon was reacted at room temperature, 40 ° C and 60 ° C, the decolorization rate was 99.1%, indicating that the decolorization effect was high even when the activated carbon was not heat treated.

9-4. Measurement of decolorization rate of purified water obtained by ultrafiltration and nanofiltration by low salt salt water storage by activated carbon treatment, low salt water storage by concentrated decompression

The anhydrous hydrolyzate of Example 5 (APH), the low concentration of concentrated low concentration salt (Evaporation APH), the purified product obtained by ultrafiltration in Example 7-1 (MW < 5 kD) The purified APHN-1 purified product was added to a model solution (monosodium L-glutamate monohydrate 1.9 g / L, maltodextrin 6.375 g / L and yeast extract 2.1 g / L) and dissolved at 2.5% Activated carbon (No. 2524, SHIN KI CHEMICAL, Korea) was added and reacted at room temperature for 5 minutes. Then OD value was measured at 420 nm absorbance and discoloration rate was calculated. The decoloring rate is expressed as a percentage using Equation (7). The control group consisted of low salt salting (APH) without treatment of activated carbon, Evaporation APH concentrated under reduced pressure, purified water (MW <5 kD) obtained by ultrafiltration and APHN-1 purified by nanofiltration (250 Da ≤ MW < 5 kDa) was used. The results are shown in FIG. 20 and Table 28.

APH Evaporated APH MW <5 kDa APHN-1 Un-
treatment Treatment Un-
treatment Treatment Un-
treatment Treatment Un-
treatment Treatment OD 0.562 0.025 0.589 0.007 0.411 0.013 0.715 0.016 Decolorization rate (%) 95.6 98.8 96.8 97.8

As a result of the experiment, the discoloration rate of all the samples was more than 95%, showing a high discoloration rate. Specifically, the decolorization rate of the low-concentration concentrated evaporated APH (98.8%) was the highest, followed by the APHN-1 purified product (97.8%) obtained by the nanofiltration, the purified product having a molecular weight of less than 5 kDa (96.8%) and low-salt fish paste (95.6%).

9-5. Determination of Volatile Flavor Components of Purified Water by Low Carbonation Treatment with Activated Carbon, Low-Concentration Low Concentration by Concentration and Concentration, and Ultrafiltration and Nanofiltration

The purified water (MW < 5 kD) obtained by ultrafiltration and the ultrafilter obtained from the ultrafiltration were subjected to nano-filtration to obtain APHN- 1 ethyl acetate (EA) and 1 g of NaCl were added to 5 ml of purified water (250 Da ≤ MW <5 kDa) and heated for 5 minutes. Next, 50 μl of pyridine (Junsei) and 50 μl of trimethyl-silane (Sigma-aldrich, USA) were added to each of the above extracts, and the mixture was reacted at 75 ° C. for 75 minutes. After that, the gas chromatograph-mass spectrometer (GC-MS, Shimadzu GC-2010 plus with GCCMS-2010 SE) was used to confirm the deodorization of each sample. The column was maintained at 50 ° C. for 15 minutes and then heated to 280 ° C. at a rate of 9 ° C./min using a Rtx-5MS column (30 m, 0.25 μm film thickness, 0.25 mm id, Restek, USA) And then kept for 10 minutes. The injector temperature was 275 ° C, the carrier gas was helium, and the flow rate was 1 ml / min. Mass spectrometry of each component was confirmed by volatile component content of each sample in TIC mode with molecular weight ranging from 50 to 500. The list of volatile components detected according to the retention time (Rt) is shown in Table 29 below. The control group consisted of low salt salting (APH) without treatment of activated carbon, Evaporation APH concentrated under reduced pressure, purified water (MW <5 kD) obtained by ultrafiltration and APHN-1 purified by nanofiltration (250 Da ≤ MW < 5 kDa) was used. The results are shown in FIG. 21, FIG. 22, Table 30 and Table 31.

Rt
(min) Compound name Rt
(min) Compound name 12.810 N, N-Diethylformamide 27.250 Mandelic acid 14.320 Dihydropyrazole 27.380 Phenylacetamide 14.650 Alkylthiols (unknown 1) 28.040 Acetophenone says. 16.520 Alkanone oxime (unknown 2) 29.005 Unknown 4 17.750 Furan-2-yl-methanol 30.250 Unknown 5 17.931 N, N-Diethylacetamide 33.145 Cyclic dipeptide 1 18.840 N-2-Ethyl-butylamine 34.255 3-Isobutyl hexa hydropyrrolo
[1,2-a] pyrazine-1,4-dione (Isomer 1) 20.350 Alkanediol (unknown 3) 34.450 3-Isobutylhexa hydropyrrolo
[1,2-a] pyrazine-1,4-dione (Isomer 2) 20.900 Hexanoic acid 34.655 Proline cyclic anhydride 22.095 Alkylamine 1 (Pentyl,?) 34.715 Hexadecanoic acid 22.295 Alkylamine 2 (Hexyl,?) 36.385 Unknown 7 23.250 Sarcosine 37.950 Cyclic dipeptide 2 24.820 Phenylacetic acid 38.865 Cyclic dipeptide 3 25.251 Octanoic acid 39.110 Cyclic dipeptide 4 26.100 Indanedione

division List of volatile components for each sample component-1 N-2-Ethyl-butylamine component-2 Alkylamine 1 (Pentyl,?) component-3 Alkylamine 2 (Hexyl,?) component-4 Phenylacetic acid component-5 Phenylacetamide component-6 3-Isobutylhexahydropyrrolo [1,2-a] pyrazine-1,4-dione (Isomer 1) component-7 3-Isobutylhexahydropyrrolo [1,2-a] pyrazine-1,4-dione (Isomer 2) component-8 Proline cyclic anhydride component-9 Cyclic dipeptide 2 component-10 Cyclic dipeptide 3 component-11 Cyclic dipeptide 4

TR: Trace level division APH
Before activated carbon treatment APH
After activated carbon treatment Evaporated APH
Before activated carbon treatment Evaporated APH
After activated carbon treatment R.T Area R.T Area R.T Area R.T Area Component-1 18.815 185679 18.827 189027 18.832 183236 18.820 229186 Component-2 22.095 TR 22.091 TR 22.081 151840 22.109 TR Component-3 22.295 TR 22.342 TR 22.332 489434 22.341 312120 Component-4 24.841 TR 24.820 TR 24.817 128696 24.817 TR Component-5 27.380 88934 27.380 TR 27.382 182737 27.382 TR Component-6 34.267 153530 34.267 TR 34.272 191029 34.272 TR Component-7 34.459 304588 34.459 TR 34.461 495932 34.461 TR Component-8 34.638 125034 34.638 TR 34.640 71671 34.640 TR Component-9 37.949 45215 37.949 TR 37.950 74656 37.950 TR Component-10 38.546 232975 38.546 TR 38.860 92981 38.860 TR Component-11 39.111 141877 39.111 TR 39.108 188337 39.108 TR division MW <5 kD
Before activated carbon treatment MW <5 kD
After activated carbon treatment APHN-1
Before activated carbon treatment APHN-1
After activated carbon treatment R.T Area R.T Area R.T Area R.T Area Component-1 18.840 194309 18.832 193190 18.826 199260 18.767 176422 Component-2 22.051 250122 22.051 TR 22.143 456162 22.165 282625 Component-3 22.300 171609 22.300 TR 22.285 176540 22.285 TR Component-4 24.817 69261 24.814 TR 24.820 TR 24.820 TR Component-5 27.383 86305 27.383 TR 27.380 TR 27.380 TR Component-6 34.269 137250 34.452 23166 34.264 47911 34.264 TR Component-7 34.458 412044 34.641 26529 34.450 TR 34.450 TR Component-8 34.636 96044 34.636 TR 34.638 33948 34.638 TR Component-9 37.951 85574 37.951 TR 37.951 44122 37.951 TR Component-10 38.856 114935 38.856 TR 38.865 TR 38.865 TR Component-11 39.110 108039 39.110 TR 39.105 46561 39.105 TR

As a result of the experiment, it was confirmed that some of the volatile components (amide, carboxylic acid, maltol, furan-2-yl-methanol) were effectively removed by the activated carbon treatment and the cyclic dipeptide dipeptide) were mostly removed by activated carbon treatment. Specifically, 30 or more kinds of substances were identified from each sample, and as a component expected to be volatile when activated carbon was treated, phenylacetic acid and phenylacetamide were removed, and other small amounts Of alkylamines were also observed. On the other hand, those substances with unknown retention time (Rt) of 30 to 40 minutes were mostly present in samples not treated with activated carbon, but they were mostly removed from samples treated with activated carbon.

9-6. Measurement of total protein content of anchovy hydrolyzate (APH), hydrolyzed anchovy by concentrated filtration, and purified water obtained by ultrafiltration and nanofiltration according to activated carbon treatment

(EF-APH), which is obtained by concentrating the low-salt spatula (APH) after filtration, and the low-salt spatula (APH) The total protein content of the sample (EFA-APH) treated with 2% of activated carbon after concentration and filtration under reduced pressure was measured by the micro-Kjeldahl method. As a control group, low salinity arteriovenous (APH) was used. The results are shown in Table 32.

Control (APH) E-APH E. F-APH E.F.A-APH Protein (g / 100 g) 74.80 72.80 74.80 63.20

As a result of the experiment, it was found that protein loss was less in EF-APH and EF-APH filtered and concentrated than in control (APH) (EFA-APH) showed a small amount of protein loss.

9-7. Sensory Evaluation of Flavor Sensory Properties of Low Salinity Crabs with Activated Carbon

The permeate purified product (MW &lt; = 5 kDa) was filtered through the ultrafiltration membrane in Example 7-1 and the model solution (monosodium L-glutamate monohydrate 1.9 g / L, maltodextrin 6.375 g / L and yeast extract 2.1 g / ) To make 1% and 2%, respectively. Then, the 1% and 2% purified water was treated with 2% activated carbon, respectively, and reacted for 5 minutes. After the reaction, the supernatant was recovered by centrifugal separation and then filtered under reduced pressure using a 0.45 μm membrane filter. A 0.1% NaCl solution was used as a reference sample, and the model solution was used as a standard sample. The tablets used in the flavor sensory evaluation were given random numbers (three digits) and then presented to the panel (Table 33).

Evaluation items were evaluated in terms of color, smell, taste, and taste. Specifically, the color shows the intensity of yellow light and the transparency of the sample. In the smell, the overall intensity and the smell of seawater, the smell of dried anchovy and algae, the overall intensity and taste of fish, the bitter taste, the salty taste and the dried anchovy taste, And odor and taste. The evaluation was conducted at 5 points. For each color, smell and taste, 5 points were very strong, 1 point was very weak, 5 points were very good, and 1 point was very disliked. The results are shown in Fig.

One% 2% Before activated carbon treatment After activated carbon treatment Before activated carbon treatment After activated carbon treatment A random number # 186 # 216 # 543 # 416

(# 216)> 2% Purified water Activated carbon treated sample (# 416)> 1% Purified water Active carbon treated sample (# 216) (# 543) before 2% purified water activated carbon treatment was significantly higher than those of other samples, followed by 1 (# 186), and the samples after the treatment with the remaining activated carbon (# 216 and # 416) showed similar strength. The bitter taste was the highest in the samples before treatment with activated charcoal (# 543) and the remaining three samples (# 186, # 216 and # 416) And the strength was remarkably decreased. In the case of salty taste, the sample (# 416) after 2% purified water activated charcoal felt stronger than the sample before the 2% purified activated carbon treatment (# 543) (# 216) after the purified water and activated carbon.

Overall acceptance was highest in the sample after treatment with 2% purified water activated carbon (# 416), and the other three samples showed similar values. (# 416) after treatment with 2% purified water activated charcoal> 1% purified sample (# 216) after the activated carbon treatment, > 2% purified water and activated carbon sample (# 543). The color of the sample was the highest before 1% purified activated carbon treatment (# 186) and the same value after 1% purified activated carbon treatment (# 216) and 2% purified activated carbon treated sample (# 416) (# 543) before 2% purified water activated carbon treatment showed the lowest value. (# 216)> 2% Purified water Activated carbon treated sample (# 416)> 1% Purified water Active carbon treated sample (# 186)> 2% Purified water Active carbon treated sample # 543), and the taste was similar for all four samples.

As a result, it was confirmed that the intensity of yellowish color, aroma, and taste decreased with the increase of the activated carbon concentration in the purified water treated before the activated carbon treatment, and that the decolorization and deodorization using the activated carbon was very effective And it was found. However, overall appearance and color were highest in 1% purified (# 186) without activated carbon treatment.

Therefore, it can be concluded that, when the natural seasoning is developed, only the concentration of the material developed in the present invention can be controlled without producing decolorization and deodorization processes.

Example  10: manufactured using an optimized process Low salt Eel  Sensory evaluation

10-1. Manufacture of low-salt fish paste (APH)

In early March, they purchased anchovies from East Sea Fisheries (Captain), which were caught in Cheju Island and stored at -20 ℃. The purchased anchovy was thawed at room temperature and then immersed in water at 10 to 15 ° C for 1 hour and washed with water to remove salts and impurities. Then, the anchovy was added twice as much water as the anchovy weight, pulverized twice for 2 minutes using a household blender, and then pulverized at 16,000 rpm for 5 minutes using a homogenizer to prepare anchovy crush Respectively. Next, 2% of the weight of the anchovy crushed material was added to the crushed anchovy powder, and the complex enzyme (alkalase: flavozyme = 1: 5) was added to the crushed anchovy powder and sealed in a pouch in 2 kg increments. The enzyme was then inactivated using a high pressure system (INOSCHEL KOREA, korea) at a pressure of 75 MPa and a high pressure for 16 hours at a temperature of 50 DEG C and then heat treatment at 80 DEG C for 20 minutes. After cooling and aging at 4 ° C for 24 hours, centrifugation was carried out at 17,000 rpm using a continuous centrifuge (Tubular centrifuge, A-V10675G, TOmoe engineering, Japan) to remove floating fat. Then, it was filtered through an ultrafiltration membrane (MWCO 10 kDa) to prepare a low salt well (MW <10 kDa) in which a substance having a molecular weight of 10 kDa or more was removed.

10-2. Determination of the content of L-arginine and arginyl dipeptide in low-salt fish liver

(MW < 10 kDa) prepared in Example 10-1 and the low salt eulogangery (APH; MW 10 kDa) prepared in Example 5 were dissolved in distilled water (SIMENS LAbostar 7 TWF-UV) at a concentration of 500 ppm After dissolution, 5 占 퐇 was applied to a liquid chromatograph-mass spectrometer and the contents of L-arginine and arginyl dipeptides were measured under the conditions shown in Table 34 below . The contents of the arginine (L-arginine) and arginyl dipeptides were determined by diluting the diluted standard peptide of the following Table 35 with 1, 2, 4, 5, 6, 8 and 10 ppm And qualitative analysis were performed. The retention time (Rt) and area of each standard product are shown in Table 36 below, and the standard calibration curve (R ² ) is shown in Table 37 below. The results are shown in Table 38.

Analysis condition Column Agilent Eclipse XDB-C8 (5 [mu] m 4.6 x 150 mm) 열 온도 25 ℃ Mobile phase Distilled water (0.1% FA): ACN (0.1% FA) (95: 5)
15 min Isocratic Detector UV / Visible (210 nm) Flow rate 0.2 mL / min Injection volume 5 μL Needle voltage 3500 volts Capillary voltage 80 volts Drying gas temp 350 ℃ Drying gas pressure (nitrogen) 30 psi Nebulizer gas pressure (nitrogen) 40 psi Scan range 90 to 600

division Dipeptide Molecular Weight manufacturer STD-1 H-Arg-Tyr-OH acetate salt 337.38 BACHEM STD-2 H-Glu (His-OH) -OH 284.27 STD-3 H-Arg-Glu-OH 303.32 STD-4 H-Glu (Gln-OH) -OH 275.26 STD-5 H-Glu (Gly-OH) -OH 204.18 STD-6 H-Arg-Ala-OH acetate 245.28 STD-7 H-Arg-Asp-OH 289.29 STD-8 H-Arg-Gly-NH2 Sulfate 328.35 STD-9 H-Glu (Glu-OH) -OH 276.25

Dipeptide Retention time (Rt) area H-Arg-Tyr-OH acetate salt 8.631 7,024,000 H-Glu (His-OH) -OH 6.605 185,824 H-Arg-Glu-OH 6.441 2,987,000 H-Glu (Gln-OH) -OH 7.092 1,257,000 H-Glu (Gly-OH) -OH 7.127 254,204 H-Arg-Ala-OH acetate 6.413 2,645,000 H-Arg-Asp-OH 6.372 2,483,000 H-Arg-Gly-NH2 Sulfate 6.140 1,496,000 H-Glu (Glu-OH) -OH 7.817 1,406,000

Dipeptide linear regression Standard calibration curve (R ² ) H-Arg-Try-OH acetate salt (RY) y = 14755x 0.9864 H-Glu (His-OH) -OH (EH) y = 3467.2x 0.9689 H-Arg-Glu-OH (RE) y = 60022x 0.9795 H-Glu (Gln-OH) -OH (EQ) y = 26721x 0.9781 H-Glu (Gly-OH) -OH (EG) y = 5609.8x 0.933 H-Arg-Ala-OH acetate (RA) y = 53458x 0.9784 H-Arg-Asp-OH (RD) y = 50766x 0.9837 H-Arg-Gly-NH2Sulfate (RG) y = 24432x 0.8255 H-Glu (Glu-OH) -OH (EE) y = 29484x 0.9635

Compound Concentration (mol / L) Low salt eel (APH)
(MW &gt; 10 kDa) Low salt eel (APH)
(MW < 10 kDa) STD-1 (RY) 0 0 STD-2 (EH) 0.80 1.10 STD-3 (RE / ER) 0.75 0.69 STD-4 (EQ) 4.64 4.59 STD-5 (EG) 0.26 3.24 STD-6 (RA / AR) 0.06 0 STD-7 (RD) 0.43 0.23 STD-8 (RG / GR) 2.98 1.62 STD-9 (EE) 1.70 2.46

As a result, eight kinds of dipeptides except for STD-1 (RY, Arg-Tyr) among 9 kinds of the standard products were detected in the low-salt ellageway (MW ≥ 10 kDa) which was not filtered with the ultrafiltration membrane. Seven dipeptides except STD-1 (RY, Arg-Tyr) and STD-6 (RA / AR, Arg-Ala) were detected in the low salt medium (MW <10 kDa). On the other hand, STD-4 (EQ, Glu-Gln) was the most detected among the dipeptides to be analyzed in both of the above low-salt fish tanks.

10-3. Sensory evaluation of low salt fish paste

(NaCl) was dissolved in the mineral water so as to compensate for the effect of imidazole and odor, and a salt solution and a salt prepared at concentrations of 25, 35 and 45 mM were added thereto to prepare a model solution (monosodium L- 0.3, 0.5 and 1.0% by adding the low salt eulogyangel prepared in Example 10-1 to glutamate monohydrate 1.9 g / L, maltodextrin 6.375 g / L and yeast extract 2.1 g / L) , And the rate of increase in salinity was calculated as a percentage using the following Equation (9). The results are shown in Fig.

[Experimental Equation 9]

(%) = {(Concentration of purified water with salty taste - concentration of actual purified water) / (concentration of actual purified water)} × 100

As a result, the salt solution (25, 35, and 45 mM) prepared by adding NaCl to the fresh water was used as a standard sample. In the salt solution 25 mM, the salt concentration of 0.1%, 29.40 mM, 0.3% The concentration of salt was 35.5 mM and the concentration of sodium salt of 0.5% was 39.75 mM. The salinity of 35 mM of salt solution was 40.05 mM at 0.1%, 44.87 mM at 0.3%, and 47.74 mM at 0.5% respectively. Finally, in the salt solution 45 mM, the concentration of low salt starch was 0.1%, 50.4 mM, 0.3% of low salt starch was 56.58 mM, and 0.5% of low salt starch was 59.22 mM.

The salting enhancement rate of low salt saline was 15.6% at 25 mM, 14.42% at 35 mM, and 12.00% at 45 mM at each salt solution concentration of 0.1% At the NaCl solution concentration of 0.3%, NaCl solution concentration of low salt was 50.24% at 25 mM, 28.20% at 35 mM and 25.73% at 45 mM. At the concentration of 0.5% NaCl solution in low NaCl solution, the salinity improvement rate of low salt fish paste was 58.88% at 25 mM, 36.40% at 35 mM and 31.60% at 45 mM. Therefore, the salty taste enhancing effect was increased as the concentration of low salt fish paste was increased, but the intensity of the salty taste felt by the panels did not increase in proportion to the amount added.

On the other hand, as a result of evaluating the strength of each low concentration salt solution with a salt concentration of 45 mM as a standard sample, the concentration of low salt salt in 1%, 59.83 mM in low salt salt, 51.23 mM in low salt salt in 0.5%, 55.21 in low salt salt in 0.3% mM, and 0.1% of the low salt concentration was 53.13 mM. In terms of the improvement rate of saltiness, it was 32.96% for 1% low salted crab, 13.84% for low salt salted crab 0.5%, 22.69% for 0.3% low salted crab, and 18.07% for low salinity crab 0.1%. The above results show that the salt increase rate was about 18% lower than that of the salt solution, but the salty taste was still improved.

Example  11: Economical analysis of optimized process

The production efficiency and the average cost of production of the low salt fish paste prepared in Example 10-1 were calculated and the economic efficiency was analyzed. Freshwater anchovy was used as a control. The results are shown in Table 39 and Table 40.

Materials Fresh Anchovy Low salt eel (APH) weight 8.39 wet weight (kg) 1.11 FD weight (kg) Total nitrogen content (g) 239.6 132.87 Yield (%) (wet basis) 100 13.23 Nitrogen recovery (%) (dry basis) 100 55.61

Category Cost per unit Cost (won) Enzyme 66,333 / kg 13,267 Anchovy (10 kg)
Transportation
Washing & Soaking
Electricity
Water
Labor
Mincing & Packaging
Electricity
Pre-packaging
Water
Labor 1500 / kg
20 / kg

185 / kWh
125 / L
1300 / h

185 / kWh
35 / kg
125 / L
1300 / h 15,000
200

481
2,500
2,600

204
1,050
3,750
7,800 Hydrolysis at 50 under high pressure
Electricity
Water
185 / kWh
125 / L
66,600
1,875 Termination of hydrolysis
Electricity
Water
Labor
185 / kWh
125 / L
1300 / h
7,400
3,750
1,300 Cooling & Aging
Electricity
185 / kWh
111 Centrifugation
Electricity
Labor
185 / kWh
1300 / h
342
1,300 Membrane separation (UF)
Water
Electricity
125 / L
185 / kWh
1,850
139 Packaging 70 / kg 63 Waste-treatment credit 41 / kg 328 Total 131,910

As a result of the experiment, the unit price per kilogram of low salt index was estimated to be about 131,910 won. (100kg / day anchovy 300kg - Consider high pressure system capacity), Continuous production (Unit price adjustment according to monthly production), Various salty taste improvement by using excipient in powdering process, Considering the inhibition of the use of enzymes, the suppression of water use to increase the yield, and the improvement of the energy management efficiency, which are greatly influencing the price, it is possible to produce products with 1,500 ~ 5,000 KRW per kilogram of live koji, .

Claims (11)

A pretreatment step (S100) for removing foreign substances from the fishery products and pulverizing the fishery products; 1 to 3% by weight of a complex enzyme is added to the crushed seafood product obtained in the pre-treatment step (S100), and the mixture is subjected to high pressure enzyme treatment for 12 to 20 hours at 40 to 60 DEG C and 65 to 80 MPa A processing step (S200) of hydrolyzing by treating; An enzyme inactivation step (S300) of inactivating the enzyme of the crushed seafood product hydrolyzed in the processing step (S200); A cooling aging step (S400) for cooling and aging the hydrolyzed fish and shellfish whose enzyme activity is stopped in the enzyme inactivation step (S300); (S500) of removing the oil of the hydrolyzate of the fish and shell hydrolyzate which has been cooled and aged in the cooling aging step (S400); And a separating step (S600) of collecting only the supernatant of the fish and shell hydrolyzate from which the oil is removed in the maintaining and removing step (S500).

[4] The method of claim 1, wherein the low salt fish gyoza contains 4.3 to 4.7 parts by weight of sodium per 100 parts by weight of the building.

[Claim 3] The method according to claim 1, wherein the low salt crab dish contains 0.6 to 1% by weight of tryptophan which gives a bitter taste to amino acids, and glutamic acid which gives a rich taste is added to the total weight of amino acids By weight to 15% by weight.

The method according to claim 1, wherein the low saline irrigation field is improved in salinity by 10 to 30% as compared with a 45 mM salt solution.

The method according to claim 1, wherein the complex enzyme in the processing step (S200) is a mixture of protease and aminopeptidase at a weight ratio of 0.8 to 1.2: 4.8 to 5.2.

The method according to claim 5, wherein the protease is an endo type, and the anminopeptidase is an endo type or an exo type.

The method according to claim 1, wherein the step (S500) further comprises: (i) a purification step, or (ii) a decoloring and deodorization step, Deodorization process, and the deodorization process.

A complex enzyme is added to crushed fish and shellfish and subjected to high-pressure enzyme treatment at 40 to 60 占 폚 and 65 to 80 MPa for 12 to 20 hours to obtain low-salt crab dipping sauces containing 4.3 to 4.7 parts by weight of sodium, .

[Claim 9] The method according to claim 8, wherein the low salt crab jar contains 0.6 to 1% by weight of a tryptophan which produces a bitter taste of amino acids, and glutamic acid which gives a rich taste is added to the total weight of the amino acids By weight to 15% by weight.

[9] The low salt crab hanging dish of claim 8, wherein the low salt crab dish is improved in salinity by 10 to 30% as compared with a salt solution of 45 mM.

[9] The low salt water content as set forth in claim 8, wherein the complex enzyme is a mixture of protease and aminopeptidase at a weight ratio of 0.8 to 1.2: 4.8 to 5.2.

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