KR101712492B1 - Natural salty taste enhancer and method for preparing the same - Google Patents

Abstract

The present invention relates to a method for producing a fish meal, which comprises adding 1 to 3% by weight of a complex enzyme to the crushed fish and shellfish product based on the total weight of the crushed fish and shellfish, treating the fish with the enzyme under high pressure for 12 to 20 hours at 40 to 60 ° C and 65 to 80 MPa, A natural salty enhancer having a taste similar to or higher than that in the case of adding bitter taste and adding salt at the time of cooking after extracting the salty taste enhancer from the fish and shellfish through the activation, cooling, aging, purification, ≪ / RTI >

Description

Natural salty taste enhancer and method for preparing same

The present invention relates to a natural salinity enhancer and a method for preparing the same, and more particularly, to a method for producing a salmonid enhancer and a method for preparing the same, which comprises extracting a salty enhancer from fish and shellfish using a high- To a natural salinity enhancer having a higher degree of preference and a method for producing the same.

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.

High pressure technology (HPT), on the other hand, exerts high pressure of 100 to 900 MPa without using heat to affect noncovalent bonding (ionic bonding, hydrogen bonding) and hydrophobic bonding of 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 salty taste enhancing agent having increased salty taste as compared with salt containing ordinary salt or salty taste enhancing agent, without adverse effect even if it is taken from a natural product for a long period of time. As a result, The saltiness enhancing substance was extracted from fish and shellfish, and the saltiness enhancing substance showed bitterness reduced and similar to or higher than that in the case of adding salt at the time of cooking, and the present invention was completed.

It is an object of the present invention to provide a method for producing a natural salinity enhancer using a high pressure enzyme treatment technique.

Another object of the present invention is to provide a natural salinity enhancer produced 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); A purifying step (S600) of separating and purifying the salt-enhancing substance from the fish or shell hydrolyzate from which the oil is removed in the maintaining and removing step (S500); And a formulation step (S700) of drying and pulverizing the salty enhancer obtained in the purifying step (S600).

Hereinafter, the method for producing the natural salinity enhancer of 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 crumbs are crushed finely, the action of the enzymes in the fish and shellfishes is facilitated during the high-pressure enzyme treatment at the subsequent stage to produce various amino acids or peptides as the high-boiling components. 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, Fungal free protein hydrolytic enzymes, etc.) have been used, and the types 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 in which endopeptidase or proteinase and exopeptidase are mixed is used to produce a salty enhancer 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).

Therefore, it can be seen that the complex enzyme of the present invention is very effective in easily forming a salty taste enhancer containing salty taste enhancing substance while reducing the production of bitter taste peptide by easily hydrolyzing proteins of 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 salty enhancer 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. The degree of hydrolysis (DH,%), nitrogen recovery (NR,%), yield and molecular weight of anchovy hydrolyzate obtained by enzymatic inactivation, cooling aging and filtration of the high pressure enzyme- And S / N ratio (signal - to - noise ratio) for each high - pressure enzyme condition were measured. As a result, the hydrolysis degree (DH,%) of the anchovy hydrolyzate increased with the addition amount of the complex enzyme or the reaction time, and the nitrogen recovery (NR,%) increased with the addition amount of the complex enzyme. It was confirmed that the longer the reaction time, the more 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. When the cooling aging is carried out at a temperature exceeding 5 ° C or less than 6 hours, floating fat is generated in the salty enhancer to be finally produced, and if it occurs at a temperature lower than 0 ° C or longer than 12 hours Is less economical than a salty enhancer prepared by aging at a temperature of or less than the above-mentioned time or a decrease in the offensive odor.

(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.

Therefore, in the present invention, it is aimed to minimize the loss of useful components contained in fish and shellfishes by omitting the filtration process while removing the sebaceous crumbs of the hydrolyzed crumbs, thereby reducing the amount of already existing salty enhancers and odors.

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. As a specific example, the hydrolyzate of the cooled and aged fish and shellfish can be centrifuged at a speed of 15,000 to 20,000 rpm using a tubular centrifuge to remove the fish and shellfish.

The maintenance removal of the present invention has an effect of improving the already prepared salty enhancer and the odor.

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. And then aged at 4 캜 for 12 hours to induce solidification of the oil in the sample. The solidified oil was removed through centrifugation, and the fat content of the anchovy hydrolyzate which was stored frozen and the anhydrous hydrolyzate without oil were measured . As a result, it was confirmed that the anchovy hydrolyzate which did not remove the fat was suspended during the storage period, and the fat content of the anchovy hydrolyzate without the fat was found to be about 0.1% or less. It was confirmed that the generation of odor was suppressed (see Example 1-5).

(6) a purifying step (S600) for separating and purifying the semen enhancement material from the fish and shell hydrolyzate in which the oil is removed in the maintaining and removing step (S500).

In the present invention, the separation and purification of the salt-enhancing substance can be carried out through a purification method 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- Can be separated and purified by using an ultrafiltration system and a nanofiltration system.

In one specific implementation, a salt enhancement material can be obtained by stepwise separation and purification using an ultrafiltration system having a molecular cut-off value of 5 to 10 kDa and a nanofiltration system having a 250 Da molecular weight cut-off value (See Examples 7 and 8).

In the purifying step (S600) of the present invention, the salty enhancing substance separated and purified contains 0.8 to 1.3 parts by weight of sodium based on 100 parts by weight of the building. In addition, the salty enhancer may contain 0.2 to 0.6% by weight of tryptophan which gives a bitter taste to the amino acid, and the glutamic acid which gives a rich taste is contained in an amount of 15 to 20 wt% %.

In one specific embodiment, 2% by weight of a complex enzyme (alcalase: flavozyme = 1: 5) was added to the anchovy crushed product with respect to the weight of the anchovy crushed product, placed in a pouch, sealed, After 16 hours of high pressure treatment, the enzyme was inactivated at 80 < 0 &gt; C for 20 minutes. Thereafter, the mixture was cooled and aged at 4 ° C for 12 hours and then centrifuged using a continuous centrifuge to solidify and remove the floating oil to prepare anchovy hydrolyzate (APH). The anchovy hydrolyzate was added to 10 kDa and 5 kDa (APHU-1, APHU-2, APHN-1 and APHN-2) were prepared by filtration stepwise into an ultrafiltration membrane having a molecular weight cut-off value and a nanofiltration membrane having a cutoff value of 250 Da. Respectively. (APHU-1: MW <10 kDa; APHU-2: 5 kDa? MW <10 kDa; APHN-1: 250 Da? MW <5 kDa and APHU-2: anchovy hydrolyzate fraction PH, soluble solids content, sodium content, nitrogen content and free / constituent amino acid content of Peak II, which are expected to have a salty taste enhancing substance, were measured . As a result, anchovy hydrolyzate (APH) obtained by centrifugation was the highest in protein yield of the anchovy, and the protein yield of APHN-1 was high among the fractions purified through ultrafiltration and / or nanofiltration, Peak II molecular weight expected to be present also was highest in APHN-1 among the fractions purified through ultrafiltration and / or nanofiltration, while sodium content was the lowest at 1.1 g per 100 g of building. The pH of the fractions purified through anchovy hydrolyzate (APH) and ultrafiltration and / or nanofiltration were found to be in the range of 6.2 to 7.1, with the exception of the APHN-2 fraction in the fractions, and the bitter taste tryptophan content was found to be APHN-1 While the content of glutamic acid showing the highest tastiness was the highest (see Example 7).

In the present invention, the seaweed enhancing substance separated and purified from the seaweed hydrolyzate (APH) is obtained by filtering the seaweed hydrolyzate into the ultrafiltration membrane and then filtering it into the nanofiltration membrane to refine the material deposited on the nanofiltration material. Therefore, the APHN- .

In the present invention, the salty-growth enhancing substance may be obtained by adding a decoloring and deodorizing process as needed after the purifying step (S600). The addition of the decolorizing and deodorizing process may be used to provide a salt-enhancing substance to a person sensitive to the flavor and color of fish or shellfish such as a patient or a child, or to improve the sensuality of food, food, Can be selected and used.

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 can be used, and preferably 1 to 3% by weight of activated carbon can be treated.

In one specific embodiment, when 2% of activated carbon is treated on the fraction obtained by treating the anchovy crush with high pressure enzyme treatment and then fractionated using the ultrafiltration membrane, it is possible to obtain a salty enhancing substance in which the intensity of yellowish, (See Example 9).

(7) The step (S700) is a formulating step (S700) of drying and pulverizing the salty enhancing substance obtained in the purification step (S600).

In the present invention, the drying is not limited as long as it is a method of removing moisture of the salt enhancement substance. For example, the drying can be performed by hot air drying, freeze drying, spray drying, fluidized bed drying, fluidized bed spray drying, Preferably by fluid bed spray drying.

In one specific example, the salt enhancement material of the present invention has an inlet temperature of 150 캜 to 200 캜, preferably 160 캜 to 190 캜, more preferably 180 캜, and an inlet temperature of 15 x 10 kPa to 25 x 10 kPa, It may be formulated by fluidized bed drying at an injection pressure of 16 x 10 kPa to 20 x 10 kPa, more preferably 18 x 10 kPa. There is a problem that the recovery rate of the salting-out enhancer is lowered when drying is carried out under the conditions of the inlet temperature and the injection pressure.

In one specific embodiment, the anchovy hydrolyzate prepared by treating the anchovy crush with high pressure enzyme was filtered through an ultrafiltration membrane (MWCO 10 kDa), and the permeate was passed through a fluidized bed spray drier at an ejection rate of 0.5 m ³ / The recovery rate, moisture content and particle size of the dried enhancement material at 500 mL / hour, inlet temperature of 180 and 200 ° C and injection pressure of 180 and 240 kPa were determined. As a result, the salt enhancement material dried at an inlet temperature of 180 ° C. and an injection pressure of 180 kPa had the highest recovery rate compared to the salt enhancement material spray dried at an inlet temperature of 200 ° C. or an injection pressure of 240 kPa. In addition, the salty enhancing substance showed a lighter hue than the lyophilized salty enhancing substance (control group), and the water content was increased about twice (see Example 10).

In the present invention, the dried salting-enhancing material exhibits an average particle size of 20 to 30 μm ² . When the particle size is less than 20 탆 ² , the strength of salty taste and salty taste are similar to those of a salty taste enhancing substance having a larger grain size. When the particle size is more than 30 탆 ² , The strength of the seasoning can be reduced.

The salty taste enhancer produced according to the present invention is at least two times, preferably at least five times, more preferably at least two times higher than the peeled seaweed hydrolyzate (APH) in which the content of dipeptide, which is a salty taste enhancing substance, More preferably at least 10-fold, even more preferably at least 20-fold, and the salt content is at least 0.1%, preferably at least 1%, more preferably at least 5%, even more preferably at least 0.1% Shows an improvement of 10% or more, and more preferably 13%.

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 at 80 DEG C for 20 minutes. Then, it was cooled and aged at 4 ° C for 12 hours, and centrifuged using a continuous centrifuge to solidify and remove floating fat to prepare anchovy hydrolyzate (APH). Then, the fraction (10 kDa permeate) permeating the anchovy hydrolyzate (APH) through an ultrafiltration membrane having a molecular cut-off value of 10 kDa and the fraction permeated through the ultrafiltration membrane were treated with ultrafiltration membrane having a cut-off value of 5 kDa (APHN-1) which had not been permeated by filtration through a nanofiltration membrane having a cut-off value of 250 Da was obtained, and a fraction (10 kDa permeate) permeated through the ultrafiltration membrane was obtained through a nanofiltration membrane Sensory evaluation of salty taste enhancement of one APHN-1 fraction was carried out. As a result, the fractions (10 kDa permeate) permeated to the ultrafiltration membrane increased 10 to 35% in salinity compared to the standard sample having a salt concentration of 45 mM and the APHN-1 fraction had a salt concentration of 0.4 to 13 % Salinity, and the intensity of the salty taste was increased as the amount of 10 kDa permate and APHN-1 fractions was increased (see Example 11-3).

APHU-2: 5 kDa? MW <10 kDa; APHN-1: 250 Da? MW <5 kDa and APHU-2 (APHU-1: MW <10 kDa; fractions of anchovy hydrolyzate Arginine and arginine dipeptides contained in the APHU-1 fraction, APHU-2 fraction and APHN-1 fraction of the anchovy hydrolyzate (APH), APHU-1 fraction and APHN- Eight kinds of dipeptides were detected except for STD-1 (RY, Arg-Tyr) in the branch, and the largest number of dipeptides were detected in the APHN-1 fraction obtained by nanofiltration (see Example 11-2).

In another aspect, the present invention provides a natural salinity enhancer prepared by subjecting crushed seafood to high-pressure enzyme treatment, followed by purification and drying.

In the present invention, the natural salinity enhancer may be prepared according to the above-described method.

In the present invention, the natural salinity enhancer contains 0.8 to 1.3 parts by weight of sodium per 100 parts by weight of the building. In addition, the natural salinity enhancer may include a tryptophan which produces a bitter taste of amino acids in an amount of 0.2 to 0.6% by weight based on the total weight of amino acids, and the glutamic acid which gives a rich taste is contained in an amount of 15 to 20 wt% %.

In the present invention, the natural salinity enhancer is at least two times or more, preferably five times or more, more preferably at least two times, more preferably at least two times, more preferably at least two times, more preferably at least two times greater than the peel- , More preferably at least 10-fold, even more preferably at least 20-fold, and the salt content is at least 0.1%, preferably at least 1%, more preferably at least 5%, even more preferably at least 0.1% , And more preferably 10% or more, and more preferably 13%.

In the present invention, the natural salinity enhancer can be used as a substitute salt for processed food and food or as a natural spice because it is separated from fish and shellfish and has no side effects even if taken for a long period of time.

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, A natural salinity enhancer exhibiting a synergistic effect can be provided. The natural salty enhancer prepared according to the above method can be used as a substitute salt for processed foods and food cooking or as a natural spice. In addition, the above-described production method can be utilized as a mass production system capable of mass-producing saltiness enhancing substances from fish and shellfish.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a process for producing the natural salinity enhancer of the present invention. FIG.
FIG. 2 shows the result of measurement of the tryptophan content of hydrolyzed anchovy pulp according to the type of enzyme and the reaction time.
Fig. 3 shows the result of measurement of the change in the molecular weight of the hydrolyzed anchovy crushed product according to the kind of the enzyme.
FIG. 4 is a graph showing the content of free amino acids in hydrolyzed anchovy crumbs treated with high pressure enzymes according to parameters of pressure, temperature, time, and enzyme concentration set using MINITAB, an orthogonal array table of L ₉ (3 ⁴ ) to be.
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) for the degree of hydrolysis.
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 And the signal-to-noise ratio (S / N ratio) to the nitrogen recovery rate.
7 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.
8 is a result of measuring the L ₉ (3 ⁴⁾ orthogonal array of the established using the MINITAB pressure, temperature, time and hydrolysis of the anchovy pulverized molecular weight change of the treated high-pressure enzyme according to the parameters of the enzyme concentration in the .
9 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 This is the result of measuring the signal to noise ratio (S / N ratio) for the salty taste enhancing substance.
FIG. 10 shows the results of comparative measurement of proteolytic activity of anchovy according to enzyme types at normal pressure and high pressure.
Fig. 11 shows the result of comparative measurement of the hydrolysis degree of hydrolyzed anchovy pulp according to the kind of enzyme at normal pressure and high pressure.
Fig. 12 shows the hydrolysis degree of the hydrolyzed anchovy crushed product according to yield, nitrogen recovery rate, hydrolysis degree and optimum high-pressure enzyme treatment conditions suitable for Peak II fraction.
FIG. 13 shows the results of measurement of the nitrogen recovery rate of hydrolyzed anchovy pulp according to yield, nitrogen recovery, hydrolysis, and optimum high-pressure enzyme treatment conditions suitable for Peak II fraction.
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 The results of hydrolysis and centrifugation of the anchovy hydrolyzate obtained from the composition / free amino acid content of the anchovy hydrolyzate.
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 hydrolyzate obtained by hydrolysis and centrifugation was subjected to ultrafiltration with 5-10 kDa and fractionation 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 anhydrous hydrolyzate obtained by hydrolysis and centrifugation was subjected to ultrafiltration at 5 to 10 kDa and to a fraction of the fraction obtained using a 250 Da nanofiltration system.
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 anchovy hydrolyzate obtained by hydrolysis and centrifugation according to the concentration of activated carbon treated was visually observed.
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 the decolorizing effect of 2% activated carbon treatment on the anchovy hydrolysates of 2.5%, 5% and 10.0% obtained by hydrolysis and centrifugation.
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 centrifuging, 2.5% of anchovy hydrolyzate was treated with 2% of activated carbon, and the discoloration effect was observed visually according to the reaction temperature.
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 (APH) obtained by hydrolysis and then centrifuged, anhydrous anhydrous hydrolyzate (Evaporation APH), fraction (MW < 5 kDa) obtained by filtering the anchovy hydrolyzate with an ultrafiltration membrane of 10 kDa and 5 kDa, (APHN-1) filtered 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 (APH) obtained by hydrolysis and then centrifuged, anhydrous anhydrous hydrolyzate (Evaporation APH), fraction (MW < 5 kDa) obtained by filtering the anchovy hydrolyzate with an ultrafiltration membrane of 10 kDa and 5 kDa, (APHN-1), which was filtered through a 250 Da nanofiltration membrane, was measured for the content of volatile components by 2% activated carbon treatment.
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 (APH) obtained by hydrolysis and then centrifuged, anhydrous anhydrous hydrolyzate (Evaporation APH), fraction (MW < 5 kDa) obtained by filtering the anchovy hydrolyzate with an ultrafiltration membrane of 10 kDa and 5 kDa, The fraction filtrated with a filtration membrane was subjected to 2% activated carbon treatment on a fraction (APHN-1) filtered through a 250 Da nanofiltration membrane to show the chemical formula of the volatile substance.
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 The APHN-1 fraction obtained by hydrolyzing and then centrifuging the anchovy hydrolyzate (APH) fractionated by filtration through an ultrafiltration membrane and a nanofiltration membrane was the result of measuring the rate of increase in salinity according to 2% activated carbon treatment.
24 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 at a temperature of 50 캜 (APH) obtained by hydrolysis and centrifugation using an ultrafiltration system of 5 to 10 kDa to evaluate the flavor sensory evaluation of a fraction having a molecular weight of 5 kDa or less.
Fig. 25 shows the result of the addition of 2% of the weight of the anchovy crushed product to the anchovy crushed product with the complex enzyme (alkalase: flavozyme = 1: 5) and the mixture was subjected to high pressure treatment at a pressure of 75 MPa and a temperature of 50 캜 for 16 hours The fraction (10 kDa permate) obtained by drying fractionated (10 kDa permate) by using an ultrafiltration system of 10 kDa obtained by hydrolysis and centrifugation of the anchovy hydrolyzate (APH) (10 kDa permate) were observed under a microscope.
26 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 fraction (10 kDa permate) obtained by drying fractionated (10 kDa permate) by using an ultrafiltration system of 10 kDa obtained by hydrolysis and centrifugation of the anchovy hydrolyzate (APH) (10 kDa permate) obtained by the method of the present invention.
27 is a graph showing the effect of adding a complex enzyme (alkalase: flavozine = 1: 5) to anchovy crumbs in an amount of 2% by weight based on the standard solution having a concentration of 45 mM, (10 kDa permate) and a 250 Da nanofiltration system using an ultrafiltration system (10 kDa) were prepared by hydrolysis and hydrolysis at high temperature for 16 hours. And the salty taste of the filtered APHN-1 fraction 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) by 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 anchovy-derived coenzyme

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  1: From anchovies Salty-boosting  Preliminary experiments for extraction

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 grinding speed affects the decrease of the particle size, and it was confirmed that the particle size was not significantly different when pulverized at 16,000 rpm for more than 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. Subsequently, 1 ml of the complex enzyme prepared according to the single enzyme, mixing ratio prepared in Preparation Example 1-1 and the anchovy stock enzyme prepared in Preparation Example 1-3 were added to 5 ml of 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. 2 and 3.

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

After the high pressure / enzyme treatment, the anchovy crushed material was inactivated at 90 to 100 ° C for 10 minutes and then cooled in a 0 ° C chiller to stop the reaction of the additional enzyme. 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, if sufficient cooling is not carried out, it is confirmed that floating fat is generated in the storage sample, and sufficient aging is necessarily required.

Therefore, the temperature (40 to 60 ° C), the pressure (50 to 100 MPa), the time (12 to 48 hours) and the enzyme use concentration (0 to 2 %).

Example  2 : Salty-boosting  For extraction 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 treated with high pressure / enzyme 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]

Fat (%) = [(W ₁ -W ₀ ) / S] × 100

W ₁ : the weight of fat in hand (g),

W ₀ : the constant amount of fat water after the fat extraction (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, followed by addition of 20 to 25 mL of sulfuric acid (95%, Kanto, 0172-00438, Japan), and the flask was shaken while mixing. The proteolytic tube containing the sample and the reagent was placed in a proteolysis apparatus (Raypa-R. Espinar, SL, Spain), and the lid was covered with the lysis apparatus. The lysate was decomposed at 410 ° C. for about 1 to 2 hours Respectively. 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, SL, Spain) for 5 minutes. After that, titration with a 0.1 N hydrochloric acid solution (Samchun, H0241, Korea) was carried out by an automatic periodic system (Crison Instruments, SA, 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 4.2 ± 10.01 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 diluted with a sealing paper filter (DISMIC-13CP 0.45 micron syringe filter, Advantec Co., Saijo, Japan) And filtered before analysis. 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. 4 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. 5 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. 6 and Table 8.

2-7. Determination of the yield of hydrolyzed anchovy crushed by 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. 7 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. 5, 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. 6, 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. 7, the effect of pressure and temperature was small and the effect was decreased with time, and the yield of enzyme was increased as the amount of enzyme was increased. 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. 8, FIG. 9, 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: Pilot plant ( 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 obtained 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 room temperature (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 treated with high pressure without using 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 was analyzed in the same manner as in Example 2-5. As a control group, anchovy treated with high pressure without using 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 crumbs 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 treated with high pressure without using 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: Pilot plant scale Sulfate-promoting substances  Optimal for extraction 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. 12 and 13 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 the hydrolyzed anchovy crushed by the optimum conditions of high pressure enzyme hydrolysis according to the measured values was the highest at the optimum condition of nitrogen recovery (75 MPa, 40 ℃, 48 hours, enzyme concentration 2%), 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 product was not significantly different depending on the conditions of high - pressure enzyme hydrolysis according to the measured values.

It has been found that it is very important to reduce the amount of enzyme in order to extract the salty substances from anchovy on 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  Anchovy according to optimum conditions Hydrolyzate ( APH ) Produce

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, 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, . Then, the complex enzyme (alcalase: flavozyme = 1: 5) was added to the crushed anchovy powder in an amount of 2% of the weight of the anchovy crush, and the mixture was put in a pouch in 2 kg units and sealed. 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. Then, the mixture was aged at 4 ° C for 12 hours and then centrifuged at 17,000 rpm using a tubular centrifuge (A-V10675G, TOmoe engineering, Japan) to remove the floating oil and remove the anchovy hydrolyzate .

Example  6: Anchovies Hydrolyzate  Composition / free amino acid content and protein extraction efficiency measurement

6-1. Composition of anchovy hydrolysates / Determination of free amino acid content

The composition / free amino acid content of the anchovy hydrolyzate (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 anchovy hydrolyzate (APH)

The anchovy hydrolyzate prepared in Example 5 and the sample obtained by centrifuging the anchovy hydrolyzate were respectively measured for nitrogen content and the protein extraction efficiency was calculated 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

The protein content of the anchovy hydrolyzate was 1,307.68 g, and the protein extraction efficiency was 63.50% since 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: Salty-boosting  Establish separation and purification conditions for mass production

7-1. Preparation of fraction of anchovy hydrolyzate using ultrafiltration system and nano filtration system

(5 kDa, proflux M60 tangential fow filtration system, Millipore, USA and 10 kDa, Lab U / F system) and a nanofiltration system (250 Da, Prolab) using the ultrafiltration system system, USA) was used to prepare anchovy hydrolyzate fractions.

Specifically, the anchovy hydrolyzate prepared in Example 5 was passed through an ultrafiltration membrane (MWCO 10 kDa) to obtain a separated fraction (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 obtain a fraction of 5 kDa or more and less than 10 kDa , APHU-2). Then, the filtrate (MW < 5 kDa) permeated to the ultrafiltration membrane was passed through a nanofiltration membrane (MWCO 250 Da) to separate fractions of 250 Da and less than 5 kDa (hereinafter referred to as APHN-1) and a nanofiltration membrane The permeated permeate (MW < 250 Da, hereinafter referred to as APHN-2) was fractionated. Each of the above fractions was frozen at -70 ° C for 24 hours, then dried and stored at -20 ° C.

7-2. Measurement of yield and protein content of fractions obtained by ultrafiltration and nanofiltration

The weights of the anchovy hydrolyzate (APH) prepared in Example 5, the APHU-1 fraction, the APHU-2 fraction, the APHN-1 fraction and the APHN-2 fraction prepared in Example 7-1 were measured, The extraction yield was measured on the basis of the unopened anchovy. Protein content of the fractions 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 Fraction Fresh Anchovy Anchovy hydrolyzate (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, the hydrolyzate of 8.39kg (dried weight 2.1kg) of fresh live anchovy was hydrolyzed by high pressure / enzyme treatment and centrifuged to obtain 1.1kg of anchovy hydrolyzate (APH). Using an ultrafiltration system and a nano filtration system 594.1 g fractions (APHU-1, APHU-2, APHN-1 and APHN-2) were obtained. On the other hand, the yields of hydrolyzed proteins of the anchovy hydrolyzate and fractions were also similar to those of anchovy hydrolyzate and fractions.

7-3. Molecular weight measurement of fraction obtained from ultrafiltration and nanofiltration

The possibility of the presence of a salty taste enhancer in the anchovy hydrolyzate (APH) prepared in Example 5, the APHU-1 fraction, the APHU-2 fraction, the APHN-1 fraction and the APHN-2 fraction prepared in Example 7-1 Gel permeation chromatography (GPC) was performed to find out. First, a GPC column (YMC pack diol-60, 300 mm 20 mm, ID S-5 탆, 6 nm) was equilibrated with 1% acetic acid. Thereafter, the APHU-1 fraction, the APHU-2 fraction, the APHN-1 fraction and the APHN-2 fraction were diluted to a concentration of 18 mg / ml. Then, 1 ml of the diluted fraction was loaded into each column, and eluted at a flow rate of 5 ml / And analyzed the molecular weight distribution and area of the anchovy hydrolyzate and each fraction. 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 molecular weight area of each fraction was calculated by multiplying the horizontal axis and the vertical axis (minmau) of the molecular weight distribution. The results are shown in FIG. 15, Table 18, and Table 19.

Distribution of GPC Peak for each fraction 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: more than 1,300 Da, peak Ⅱ: 250 Da to 1,300 Da, peak Ⅲ: less than 250 Da

GPC peak area for each fraction 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: more than 1,300 Da, peak Ⅱ: 250 Da to 1,300 Da, peak Ⅲ: less than 250 Da

As shown in FIG. 15, three major peaks were identified, and hydrolyzate fractions fractionated on the basis of the molecular weight of anchovy hydrolyzate (APH) had molecular weights of less than 1,500 Da and similar molecular weight distributions and patterns .

Peak Ⅰ fraction of APHU-1 fraction was significantly higher than that of other fractions. The peak Ⅰ fraction decreased gradually as the membrane molecular weight decreased, indicating that APHN-2 fraction disappeared, whereas Peak Ⅱ and Ⅲ showed a higher ratio. The peak area of APHN-1 fraction was the largest in APHN-2 fraction and the peak of APHN-1 fraction was only 30% in Peak I, II and III fractions.

7-4. Analysis of Physicochemical Properties of Fractions Obtained by Ultrafiltration and Nanofiltration

The anchovy hydrolyzate (APH) prepared in Example 5 and the APHU-1 fraction, APHU-2 fraction, APHN-1 fraction and APHN-2 fraction prepared in Example 7-1 were dissolved in water at a concentration of 2.5% After dissolution, a pH meter (Orion 4-star Plus, Thermo Sci., USA), a salinity meter (Master meter, S28M, Atago, Japan) and a master meter (Master-M, 2M and 3M, Japan) was used to measure solubility, pH, soluble solids content and salinity. The anchovy hydrolyzate (APH) of Example 5 and the APHU-1 fraction, APHU-2 fraction, APHN-1 fraction and APHN-2 fraction prepared in Example 7-1 were diluted 1000 times with distilled water Then, 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 fraction was the lowest at 5.9 and the other samples ranged from 6.2 to 7.1, similar to commercial soy sauce and anchovy hydrolyzate. 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 fraction was significantly higher than that of other samples. APHN-1 fraction, which was permeated through 5 kDa ultrafiltration membrane, showed complete desalination. 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 the anchovy hydrolyzate (APH), APHU-1 fraction, APHU-2 fraction and APHN-1 fraction was very high, ie, 73 to 79%.

7-5. Color measurement of fractions obtained through ultrafiltration and nanofiltration

The anchovy hydrolyzate (APH) prepared in Example 5 and the APHU-1 fraction, APHU-2 fraction, APHN-1 fraction and APHN-2 fraction prepared in Example 7-1 were dissolved in water to give a concentration of 2.5% . The turbidity and browning of the hydrolyzate and each fraction were measured at 660 nm and 430 nm using a spectrophotometer (UV-140-02, Shimadzu Co., Japan). The lightness, lightness, redness, and yellowness of the hydrolyzate and fractions 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 fraction was the lowest and the other fractions were not significantly different from each other. In the case of browning, the APHU-1 fraction had a value 68 times higher than that of the lowest APHN-2 fraction, and was darker than the other fractions. The fluorescence intensity of APHN-2 fraction was higher than that of other fractions. The redness and yellowness values of APHU-1 fraction were the highest.

7-6. Analysis of free / constituent amino acids of fractions obtained by ultrafiltration and nanofiltration

The free / constituent amino acid contents of the anchovy hydrolyzate (APH) prepared in Example 5 and the APHU-1 fraction, APHU-2 fraction, APHN-1 fraction and APHN-2 fraction prepared in Example 7-1 were measured 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 388,187 455,785 473,136 454,772 335,329

As a result, the bitter taste tryptophane content was lowest in the APHN-1 fraction, while the glutamic acid content in the APHN-1 fraction was the highest. On the other hand, sweetness glycine, proline, alanine and serine were the most abundant in the APHN-2 fraction.

Example  8 : Salty-boosting  For mass production Desalination  Fairness

8-1. Desalination using nanofiltration membrane

The anchovy hydrolyzate (APH) prepared in Example 5 was sequentially passed through an 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 the desalting was performed in the APHN-1 fraction, which is a fraction of salts with low molecular weight that 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, which passed through an ultrafiltration system (MWCO 5 kDa) and passed through a nanofiltration system (MWCO 250 Da) prepared in Example 5, was electrodialysed A cartridge (AC-220, Astom, Japan) was attached to a desalting apparatus at a voltage of 12 V, a current of 21 A, a manipulation time of 40 minutes and a cross 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 fraction 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, anchovy hydrolyzate treated with desalting process using nano filtration system and electrodialysis equipment removed 76.67% and 22.73% salt, respectively, compared to anchovy hydrolyzate without 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: Salty-boosting  Decolorization and deodorization process for mass production

9-1. Determination of decolorization rate of anchovy hydrolyzate (APH) according to activated carbon concentration

The anchovy hydrolyzate (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) After the addition of 0.5, 1.0, 2.0 and 3.0% of purified activated carbon (No. 2524, SHIN KI CHEMICAL, Korea), the reaction was allowed to proceed at room temperature for 5 minutes, and 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, anchovy hydrolyzate 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 discoloration rate according to concentration of anchovy hydrolyzate (APH)

The anchovy hydrolyzate (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) %), And 2% purified activated carbon (No. 2524, SHIN KI CHEMICAL, Korea) was added. After reacting at room temperature for 5 minutes, 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). 2.5%, 5.0% and 10.0% of anchovy hydrolysates not treated with activated carbon were used as controls. The results are shown in FIG. 18 and Table 26.

Concentration of anchovy hydrolyzate (%) 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 of the anchovy hydrolyzate (APH) at the concentration of 2.5% was the highest at 99.1%, and at the concentration of 5.0%, the decolorization rate of the anchovy hydrolyzate (APH) was lowered to 94.9%. However, when the concentration of anhydrous hydrolysates of anchovy (APH) was 10%, the decolorization rate was lowered to 83.0%, and in FIG. 18, it was found to be yellowish visually compared with 2.5% and 5.0%.

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

The anchovy hydrolyzate (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) After adding 2% purified activated carbon (No. 2524, SHIN KI CHEMICAL, Korea) and reacting at room temperature, 40 ° C or 60 ° C for 5 minutes, 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, anchovy hydrolyzate 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. Determination of decolorization rate of anchovy hydrolyzate (APH), hydrolyzed anchovy by concentrated filtration, and fractions obtained by ultrafiltration and nanofiltration according to activated carbon treatment

The anhydrous hydrolyzate (APH), the concentrated anhydrous hydrolyzate (Evaporation APH) prepared in Example 5, the fraction obtained by ultrafiltration in Example 7-1 (MW < 5 kD) and the APHN-1 Were 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) CHEMICAL, Korea) was added and reacted at room temperature 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). The control group was anhydrous anhydrous hydrolyzate (APH), anhydrous anhydrous anhydrous hydrolyzate (EVOH APH), ultrafiltrate fraction (MW <5 kD) and APHN-1 (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 concentrated anhydrous hydrolyzate (Evaporated APH) was the highest at 98.8%, followed by the APHN-1 fraction (97.8%) obtained by nanofiltration, the fraction obtained by ultrafiltration with a molecular weight of less than 5 kDa 96.8%) and anchovy hydrolyzate (95.6%).

9-5. Measurement of volatile flavor components of anchovy hydrolyzate (APH), hydrolyzed anchovy by concentrated filtration, and fractions obtained by ultrafiltration and nanofiltration according to activated carbon treatment

The anhydrous hydrolyzate (APH), the concentrated anhydrous hydrolyzate (Evaporation APH) prepared in Example 5, the fraction obtained by ultrafiltration in Example 7-1 (MW < 5 kD) and the APHN-1 5 ml of ethyl acetate (EA) and 1 g of NaCl were added to 5 ml of fraction (250 Da ≤ MW <5 kDa) and the mixture was extracted by heating 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 Rtx-5MS (30m, 0.25 film thickness, 0.25mm id, Restek, USA) under the GC-MS analysis conditions and maintained at 50 ° C for 15 minutes and then heated to 280 ° C at a rate of 9 ° C / It was maintained for the next 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 was anhydrous anchovy hydrolyzate (APH), anhydrous concentrated anchovy hydrolyzate (Evaporation APH), ultrafiltrate fraction (MW <5 kD) and APHN-1 fraction 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 fractions obtained by ultrafiltration and nanofiltration according to activated carbon treatment

(EF-APH) and anchovy hydrolyzate (APH) obtained by concentrating the anchovy hydrolyzate (APH) of Example 5 under reduced pressure and concentrated (Evaporation APH, E-APH), anchovy hydrolyzate ) Was concentrated under reduced pressure, and the total protein content of the sample (EFA-APH) treated with 2% of activated carbon was measured by the micro-Kjeldahl method. A low salt anchovy hydrolyzate (APH) was used as a control. 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 the protein loss was less in the case of the control (APH) than in the control (APH) and the activated hydrolyzate of the anchovy (EF-APH) after the filtration of the concentrated anhydrous hydrolyzate The hydrolyzate (EFA-APH) showed a small amount of protein loss.

9-7. Measurement of the rate of increase of salinity of APHN-1 fraction by activated carbon treatment

The APHN-1 fraction obtained in Example 7-1 was added to a model solution having a salt concentration of 45 mM (monosodium L-glutamate monohydrate 1.9 g / L, maltodextrin 6.375 g / L and yeast extract 2.1 g / L) , 0.3, 0.5 and 1%, and then the intensity of the salty taste was measured by the activated carbon treatment. The salinity increase rate was calculated as a percentage using the following Equation (9). The standard sample was the model solution. The results are shown in Fig.

[Experimental Equation 9]

(%) = {(Concentration of salty flavor fraction - concentration of actual fraction) / (concentration of actual fraction)} × 100

As a result, the APHN-1 fraction after the activated carbon treatment showed a decrease in the intensity of the salty taste compared to the APHN-1 fraction before the activated carbon treatment, but the intensity of the salty taste was found to be proportional to the concentration. Specifically, the addition of 1% APHN-1 fraction before the treatment with activated carbon resulted in 49.63 mM, 48.40 mM for 0.5%, 48.81 mM for 0.3%, and 45.19 mM for 0.1%. When this value is converted into the rate of increase of saltiness, 1% is 10.29%, 0.5% is 7.56%, 0.3% is 8.47%, and 0.1% is 0.42%. When 1% APHN-2 fraction was added after the treatment with activated carbon, 48.60 mM, 46.79 mM at 0.5%, 45.48 mM at 0.3% and 44.94 mM at 0.1%, respectively. When this value is converted into the rate of increase of the salinity, it is 8.00% for 1%, 3.98% for 0.5%, 1.07% for 0.3% and 0.13% for 0.1%.

9-7. Sensory Evaluation of Flavor Sensory Properties of Activated Carbon Treated by Ultrafiltration of Anchovy Hydrolyzate

The fraction (MW5 kDa) obtained by ultrafiltration in Example 7-1 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) 1% and 2%, respectively. The 1% and 2% fractions were then treated with 2% activated carbon and reacted for 5 minutes. After the reaction, the supernatant was recovered using a centrifugal separator, and the filtrate was filtered through 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 fractions used in the flavor sensory evaluation were given random numbers (three digits) to the panels (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

First, the transparency of color, odor and taste was evaluated as follows: 1% fraction activated carbon treated sample (# 216)> 2% fraction activated carbon treated sample (# 416)> 1% fraction activated carbon treated sample (# 186) (# 543), and the intensity of yellow light was significantly higher than that of the other samples before the 2% fraction activated carbon treatment, followed by 1% fraction treated with activated carbon The sample (# 186) and the samples after the remaining activated carbon treatment (# 216 and # 416) showed similar strength. The bitter taste was highest in the samples before treatment with activated charcoal (# 543) and the remaining three samples (# 186, # 216 and # 416) Was significantly decreased. In the case of salty taste, the sample (# 416) after 2% fraction activated charcoal felt stronger than the sample (# 543) before 2% fraction activated carbon treatment. Next, 1% fraction activated carbon fraction (# 186) And the sample (# 216).

Overall acceptance was highest in the sample after treatment with 2% fraction activated carbon (# 416), and the other three samples showed similar values. (# 416)> 1% fraction after treatment with activated charcoal (# 216)> 2% after 2% fraction activated carbon treatment, (# 543) before treatment with activated carbon fraction. The color (1%) showed the highest value before the 1% fraction activated carbon treatment (# 186) and the fraction after 1% fraction activated carbon treatment was similar to the fraction after the 2% fraction activated carbon treatment (# 543) before the activated carbon treatment showed the lowest value. Fractions of 1% fractions after fractionation of activated carbon (# 216)> 2% fractions fraction of activated carbon sample (# 416)> 1% fractions fraction of activated carbon sample (# 186)> fraction of 2% And the taste was similar for all four samples.

In conclusion, when the activated carbon is treated with the fractions, the intensity of yellowishness, aroma, and taste are decreased as the concentration of activated carbon is decreased, and the decolorization and deodorization using activated carbon is very effective Could know. However, overall appearance and color were highest in the 1% fraction (# 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: Salty-boosting Formulation  Establishing drying conditions for

The anchovy hydrolyzate (APH) prepared in Example 5 was filtered through an ultrafiltration membrane (MWCO 10 kDa), and 375 ml of the permeated permeate (10 kDa permeate) was filtered through a fluidized bed spray drier (SD-1000, EYELA, Japan) And spray-dried under the conditions shown in Table 34. The moisture content and particle size of the spray dried sample were then checked. In addition, the weight of the dried fraction compared to the solid (18.38 g / 375 ml) present in the sample before spray drying was calculated as a percentage, and the color of the sample dried by naked eyes was confirmed. As a control, the permeate (10 kDa permeate) was poured into a pouch and frozen at 70 ° C for 24 hours. Then, the lyophilized permeate (10 kDa permeate) was permeabilized using a freeze dryer (PYTF20R, ) Were used. The results are shown in Fig. 25, Fig. 26 and Table 35.

Sample 1 Sample 2 Sample 3 Sample 4 Inflow temperature (℃) 180 180 200 200 Injection pressure (kPa) 180 240 240 180 Discharge speed (m ³ / min) 0.50 0.50 0.50 0.50 Infusion rate (mL / h) 500 500 500 500

Spray drying Freeze-dried Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
(Control group) Recovery (g / 375 mL) 6.15 4.10 4.72 5.09 16.96 Recovery rate
(%) 34.01 22.31 25.61 27.70 92.30 Moisture content
(%) 9.11 ± 1.05 9.33 ± 1.22 9.66 + - 2.52 9.44 ± 1.30 4.86 ± 0.23 Particle size (탆 ² ) 28.06 + 14.39 26.39 ± 12.58 28.35 + 17.61 23.52 ± 13.72 27.90 +/- 14.30

As a result, the yields of the permeates (samples 1 to 4) dried according to the spray conditions were 34.01%, 22.31%, 25.61%, and 27.70%, respectively. The yield of freeze-dried permeate was 92.30%, which was much higher than that of spray-dried permeate. On the other hand, the water content was about 2 times higher than that of the lyophilized permeate.

On the other hand, the particle size of spray - dried and freeze - dried permeate showed no significant difference, and the color of spray - dried permeate was found to be higher than that of freeze - dried permeate.

Example  11: Anchovy prepared using optimized process Hydrolyzate ( APH ) And Fraction As a salty enhancer  Explore and identify possibilities

11-1. Preparation of anchovy hydrolyzate and its fractions using optimized process

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, 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, . 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 at 4 ° C for 24 hours, the suspension was centrifuged at 17,000 rpm using a continuous centrifuge (Tubular centrifuge, A-V10675G, TOmoe engineering, Japan) to remove the floating fat and remove the anchovy hydrolyzate APH). The APHU-1 fraction, APHU-2 fraction, APHN-1 fraction and APHN-1 fraction were successively filtered through an ultrafiltration membrane (MWCO 10 kDa and 5 kDa) and a nanofiltration membrane (MWCO 250 Da) Two fractions were obtained and spray dried at an inlet temperature of 180, an injection pressure of 180 kPa, an ejection rate of 0.5 m &lt; ³ &gt; / min and an injection rate of 500 ml / h.

11-2. Determination of the content of L-arginine and arginyl dipeptides in anchovy hydrolysates and fractions thereof

The anchovy hydrolyzate (APH), APHU-1 fraction, APHU-2 fraction, APHN-1 fraction and APHN-2 fraction prepared in Example 11-1 were dissolved in distilled water (SIMENS LAbostar 7 TWF-UV) at a concentration of 500 ppm , 5 μl was applied to a liquid chromatograph-mass spectrometer and the content of L-arginine and arginyl dipeptides was measured under the conditions shown in the following Table 36 Respectively. The contents of the arginine (L-arginine) and arginyl dipeptides were determined by diluting the diluted standard peptide of Table 37 at 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 38 below, and the standard calibration curve (R ² ) is shown in Table 39 below. The results are shown in Table 40.

Analysis condition Column Agilent Eclipse XDB-C8 (5 [mu] m 4.6150 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) APH APHU-1 APHU-2 APHN-1 APHN-2 STD-1 (RY) 0 0 0 0 0 STD-2 (EH) 0.80 1.10 0.79 9.90 0 STD-3 (RE / ER) 0.75 0.69 1.40 4.01 0 STD-4 (EQ) 4.64 4.59 6.14 12.42 0 STD-5 (EG) 0.26 3.24 2.30 6.54 0 STD-6 (RA / AR) 0.06 0 0.61 1.28 0 STD-7 (RD) 0.43 0.23 0.35 1.51 0 STD-8 (RG / GR) 2.98 1.62 3.54 6.41 0 STD-9 (EE) 1.70 2.46 2.08 5.42 0

As a result, 8 kinds of dipeptides were detected except for STD-1 (RY, Arg-Tyr) among 9 kinds of standard products in anchovy hydrolyzate (APH), APHU-1 fraction, APHU-2 fraction and APHN- The greatest amount of dipeptide was detected in the APHN-1 fraction obtained by nanofiltration.

11-3. Sensory Evaluation of Fractions Obtained by Ultrafiltration and Nanofiltration

The fraction (10 kDa permeate) permeating the anchovy hydrolyzate (APH) prepared in Example 11-1 through an ultrafiltration membrane having a cut-off value of 10 kDa and the fraction permeated through the ultrafiltration membrane were cut into a 5 kDa molecular weight cut -Off value and then filtered through a nanofiltration membrane having a cut-off value of 250 Da to obtain a fraction (APHN-1) which was not permeated.

Then, a model solution (monosodium L-glutamate monohydrate, 1.9 g / L, maltodextrin (1 g / L), and the like) was added so that the fraction (10 kDa permeate) permeated through the ultrafiltration membrane and the APHN- (10 kDa permeate) permeated to the ultrafiltration membrane and a APHN (10 kDa permeate) solution obtained through a nanofiltration membrane were added to a standard sample prepared at a concentration of 45 mM by adding salt (NaCl) -1 fractions were added thereto, and the salty taste was measured at 0.1, 0.3, 0.5 and 1.0%, and then the salty taste was calculated as a percentage by using the above formula (9). Standard specimens for strength evaluation and each sample were prepared according to the contents of Tables 41 and 42 below. The results are shown in Fig.

(g / L) Ingredients Standard sample SE solution 10 kDa permate APHN-1 monosodium L-glutamate monohydrate 1.900 1.900 1.900 Maltodextrin 6.375 6.375 6.375 Yeast extract 2.100 2.100 2.100 NaCl 2.630 - - Salt Enhancer (SE, hydrolyzate) - 18.300 91.600

(ml) Solution for each fraction 0.1% 0.3% 0.5% 1.0% Standard sample SE Soln. Standard sample SE Soln. Standard sample SE Soln. Standard sample SE Soln. 10 kDa permate 935.76 64.24 87.29 192.71 678.82 321.18 357.64 642.36 APHN-1 988.76 11.24 966.29 33.71 943.82 56.18 887.64 112.36

As a result, the 10 kDa permate fraction showed a higher salting effect than the APHN-1 fraction, which was found to have the highest amount of dipeptide known to exhibit the salty taste enhancing effect.

Specifically, the standard samples with a salt concentration of 45 mM were 53.13 mM for the 0.1% 10 kDa permate fraction, 55.21 mM for the 0.3% 10 kDa permate fraction, 51.23 mM for the 0.5% 10 kDa permate fraction and 59.83 mM for the 1% 10 kDa permate fraction And 0.1% was 18.07%, 0.3% was 22.69%, 0.5% was 13.84%, and 1% was 32.96% in terms of the rate of increase of the saltiness.

In addition, 45.19 mM of 0.1% APHN-1 fraction, 48.81 mM of 0.3% APHN-1 fraction, 48.40 mM of 0.5% APHN-1 fraction and 49.63 mM of 1% APHN-1 fraction were obtained, Conversely, 0.1% was 0.42%, 0.3% was 8.47%, 0.5% was 7.56%, and 1% was 10.29%.

From the above results, the 10 kDa permate fraction and the APHN-1 fraction were judged to have an effect of improving the salty taste, and it seems to be possible as a salt enhancement substance in the future. In particular, when 1% APHN-1 fraction fractioned by the nanofiltration system is added, it is estimated that the use of 2% APHN-1 fraction can reduce sodium by about 20% considering that the increase rate of saltiness is 10.29% do.

Example  12: Economical analysis of optimized process

The economical efficiency of APHN-1 fraction obtained by filtration with anchovy hydrolyzate (APH) prepared in Example 11-1 and ultrafiltration and nanofiltration system was calculated by calculating the production efficiency and the average cost for production. The results are shown in Tables 43 and 44.

Materials Fresh Anchovy APH APHN-1 weight 8.39 wet weight (kg) 1.11 FD weight (kg) 0.91 FD weight (kg) Total nitrogen content (g) 239.6 132.87 104.83 Yield (%) (wet basis) 100 13.23 10.85 Nitrogen recovery (%) (dry basis) 100 55.61 43.75

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 Spray Drying
Electricity
185 / kWh
34,188 Packaging 70 / kg 63 Waste-treatment credit 41 / kg 328 Total 166,098

As a result, the unit price per kg of natural salinity enhancer was estimated to be about 166,098 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 have a great effect on the price, it is possible to produce products of about 2,000 ~ 5,000 KRW per kg of live koji, .

Claims (13)

A pretreatment step (S100) for removing foreign substances from the fishery products and pulverizing the fishery products; The complex enzyme in which protease and aminopeptidase are mixed at a weight ratio of 0.8 to 1.2: 4.8 to 5.2 to the pulverized fish products obtained in the pretreatment step (S100) is added in an amount of 1 to 3 wt% (S200) for performing hydrolysis by high-pressure enzyme treatment at 40 to 60 DEG C and 65 to 80 MPa for 12 to 20 hours; 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); A purifying step (S600) of separating and purifying the salt-enhancing substance from the fish or shell hydrolyzate from which the oil is removed in the maintaining and removing step (S500); And a formulation step (S700) of drying and pulverizing the saltiness enhancing substance obtained in the purification step (S600), wherein the natural saltiness enhancing agent has an average particle size of 20 mu m to ² mu m to 30 mu m ² Of the total weight of the amino acid, 0.2 to 0.6% by weight of tryptophan which gives a bitter taste among the amino acids and 0.2 to 0.6% by weight of the total weight of the amino acids, glutamic acid wherein the glutamic acid is contained in an amount of 15 to 20 wt% based on the total weight of the amino acid.
delete delete The method according to claim 1, wherein the natural salinity enhancer has an increase in the content of dipeptide by 2 to 20 times as compared to that of the fish and shell hydrolyzate in which the depigment is removed in the maintenance and removal step (S500) 13%. &Lt; / RTI &gt;
delete The method according to claim 1, wherein the protease is an endo type, and the aminopeptidase is an endo type or an exo type. .
2. The method of claim 1, wherein the purification and purification of the salty-growth material in the purification step (S600) comprises using an ultrafiltration system having a cut-off value of 5 to 10 kDa molecular weight and a nanofiltration system having a cutoff value of 250 Da molecular weight By weight or more by weight, based on the total weight of the composition.
The method of claim 1, further comprising a decolorizing and deodorizing step after the purifying step (S600).
A complex enzyme in which a protease and an aminopeptidase are mixed in a weight ratio of 0.8 to 1.2: 4.8 to 5.2 is added to the crushed seafood, and the mixture is subjected to high pressure treatment for 12 to 20 hours at 40 to 60 DEG C and 65 to 80 MPa Wherein the tryptophan having a bitter taste in amino acids is contained in an amount of 0.2 to 0.6 wt% based on the total weight of amino acids, Wherein the glutamic acid is contained in an amount of 15 to 20% by weight based on the total weight of the amino acid.
delete delete [Claim 11] The natural salinity enhancer according to claim 9, wherein the natural salinity enhancer is 2- to 20-fold increased in the content of dipeptide as compared to the hydrolyzate of fish and shellfishes in which fat is removed, and the salinity is improved by 0.1 to 13% Antiseptic agent.
delete

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