KR102189029B1 - Manufacturing method for fermented fish sauce using novel Bacillus velezensis L2 - Google Patents

Abstract

The present invention relates to a method for preparing fermented fish sauce and, more particularly, to a method for preparing fermented fish sauce using a novel Bacillus velezensis L2 strain, which is obtained by adding low salt to raw materials of fish and shellfish to mix the same, and then adding a novel Bacillus velezensis L2 (KCCM 12498P) as a starter strain to ferment the mixture, thereby reducing a fermentation period, reducing the content of histamine which is a component involved in an allergic reaction or inflammation and the content of volatile basic nitrogen which is a component indicating freshness, and increasing the content of total nitrogen and amino type nitrogen, thereby providing high-quality fermented fish sauce.

Description

Manufacturing method of fish sauce using novel Bacillus velezensis L2 strain {Manufacturing method for fermented fish sauce using novel Bacillus velezensis L2}

The present invention relates to a method for preparing fish sauce, and more specifically, a low salt is added to raw materials for fish and shellfish and mixed, and then a new Bacillus velezensis L2 (KCCM12498P) is added as a seed and fermented, thereby fermentation period. By reducing the content of histamine and the content of volatile basic nitrogen, and increasing the content of total nitrogen and amino nitrogen, a new Bacillus belesensis L2 strain that can provide high quality fish sauce is used. It relates to a method of manufacturing fish sauce.

Fish sauce, along with kimchi, is one of Korea's representative fermented foods and has been used since ancient times to supply high protein and salt regardless of the season.

Specifically, fish sauce is a salted product in a broad sense, obtained by separating and filtering the supernatant after fermentation by salting fish and shellfish. Salt is added to the muscles, intestines, eggs, etc. By doing so, it is known that various amino acids are produced to give a special flavor and umami, as well as nutritionally excellent because it is rich in amino acids and inorganic components, and it is composed of low molecules, so that the digestion and absorption rate is also good.

However, despite these unique flavors and potential utility values, conventional fish sauce generally takes a fermentation period of 3 months to 24 months, so production efficiency decreases. In order to increase the production efficiency of fish sauce, It is necessary to develop new strains with high proteolytic activity.

In addition, conventional fish sauce contains more than a predetermined content of histamine, one of the biogenic amine components produced during the fermentation process, and this histamine is known to be a component involved in allergic reactions and inflammation, and contains histamine produced in large amounts. When eating fish and shellfish food, it has a problem that causes acute poisoning with headache, hives, and seizures, so it is necessary to develop a new fish sauce manufacturing method that can minimize the amount of histamine produced.

The present invention was conceived by this necessity, and an object of the present invention is to develop a new strain and use it as a seed for fermentation of fish sauce, so that the fermentation period can be shortened and the content of free amino acids is increased due to excellent protein decomposition ability. It is to provide a method for preparing fish sauce that can minimize the amount of histamine produced.

Another object of the present invention is to provide a fish sauce prepared by using the method for producing the fish sauce.

Objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a raw material preparation step of preparing raw fish and shellfish raw materials; A semi-drying step of drying the prepared raw material to a moisture content of 30% to 55%; Salt mixing step of mixing by adding a salt to the semi-dried raw material; A fermentation step of fermenting the salt-mixed raw material under anaerobic conditions; And a filtrate obtaining step of filtering the fermented product generated through the fermentation step to remove the residue and obtain a filtrate; wherein the strain introduced in the fermentation step is 16S rRNA to obtain the nucleotide sequence shown in SEQ ID NO: 1 It includes, and is separated from the anchovy fermentation broth, and has a proteolytic activity Bacillus velezensis ( Bacillus velezensis ) L2 (KCCM1 2498P).

In a preferred embodiment, the strain shows a positive reaction to lactose (Lactose), amygdalin (Amygdalin), melibiose (Melibiose) and genthiobiose (gentiobiose).

In a preferred embodiment, in the fermentation step, the strain suppresses the production of volatile basic nitrogen and histamine contained in the fermentation product.

In a preferred embodiment, in the fermentation step, the strain increases the content of total nitrogen and amino nitrogen.

In a preferred embodiment, in the fermentation step, the strain increases the content of sweet-based amino acids and umami-based amino acids.

In a preferred embodiment, in the fermentation step, the strain increases the content of amino acids including Serine, Glutamic acid, Valine and Proline.

In a preferred embodiment, the fermentation step is processed at a constant temperature in a temperature range of 45°C to 55°C, and stirring is performed at 50rpm to 300rpm.

In a preferred embodiment, the salt mixing step is mixed by adding less than 10 parts by weight of salt based on 100 parts by weight of the raw material.

In a preferred embodiment, after the filtrate obtaining step, a sterilization treatment step of sterilizing the filtrate is further performed.

In a preferred embodiment, the sterilization treatment step is heat-treated for 5 minutes to 30 minutes at a constant temperature in the range of 70 ℃ to 100 ℃.

In a preferred embodiment, the raw material is any one selected from the group consisting of canary, anchovy, tuna, shrimp, herring, mackerel, and combinations thereof.

In addition, the present invention further provides a fish sauce manufactured by the method for manufacturing the fish sauce.

The present invention has the following excellent effects.

First, according to the manufacturing method of the fish sauce of the present invention, by fermentation by the Bacillus velezensis L2 (KCCM1 2498P) strain, compared to the standard strain Bacillus velezensis LMG 22478, about It has 1.5 times higher proteolytic activity, so it can shorten the fermentation period, and the content of histamine, a component involved in allergic reactions or inflammation, and the content of volatile basic nitrogen, a component that is an indicator of freshness, are drastically reduced. There is an effect that can be manufactured.

In addition, according to the method for producing a fish sauce of the present invention, it is possible to manufacture a fish sauce having an increased content of sweet-based amino acids and umami-based amino acids, thereby improving taste preference.

1 is a step diagram illustrating a method of manufacturing a fish sauce according to the present invention.
2 is a photograph showing that Bacillus velezensis L2 (KCCM1 2498P) used as a seed in the fermentation step of the present invention was cultured in a medium.
FIG. 3 is an experimental result of generating a transparent ring showing the protein resolution of Bacillus velezensis L2 (KCCM1 2498P) used as a seed in the fermentation step of the present invention.
4 is a photograph of the results of Gram staining of Bacillus velezensis L2 (KCCM1 2498P) used as a seed in the fermentation step of the present invention with an optical microscope.
Figure 5 is a diagram showing the genetic tree (Phylogenetic tree) of Bacillus velezensis L2 (KCCM1 2498P) used as a seed in the fermentation step of the present invention.
6 is a view confirming the 16S rRNA nucleotide sequence of Bacillus velezensis L2 (KCCM1 2498P) used as a seed in the fermentation step of the present invention.
Figure 7 is a graph confirming the number of viable cells by salt concentration of Bacillus velezensis L2 (KCCM1 2498P) used as a seed in the fermentation step of the present invention.
8 is a graph confirming the proteolytic activity of Bacillus velezensis L2 (KCCM1 2498P) cultured by salt concentration.

As for terms used in the present invention, general terms that are currently widely used are selected, but in certain cases, some terms are arbitrarily selected by the applicant. In this case, the meanings described or used in the detailed description of the invention are considered rather than the names of simple terms. Therefore, its meaning should be grasped.

Hereinafter, the technical configuration of the present invention will be described in detail with reference to exemplary embodiments shown in the accompanying drawings.

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. The same reference numerals denote the same elements throughout the specification.

1 is a step diagram illustrating a method for manufacturing a fish sauce according to the present invention.

Referring to FIG. 1, a method for manufacturing a fish sauce according to an embodiment of the present invention is a fish sauce for preparing a fish sauce using a new strain, Bacillus velezensis , L2 (KCCM1 2498P), as a spawn used in a fermentation process. First, a raw material preparation step (S100) of preparing a raw material for fish and shellfish is performed.

In the raw material preparation step (S100), the raw material for fish and shellfish is sufficiently washed with brine or water to be prepared.

Here, the raw materials for fish and shellfish are anchovy, octopus, squid, sardines, blackfish, herring, anchovies, mackerel, smelt, canary, yellowtail, skate, tuna, horse mackerel, saury, konja, badorachi, gulbi, pollock, yangmiri, mussels, It means any one or a mixture of two or more of the barge, shrimp and crab, and is not limited as long as it is a raw material that can be used as a raw fish sauce.

Next, a semi-drying step (S200) of drying the prepared raw material to an appropriate moisture content is performed.

The semi-drying step (S200) may be performed by natural drying, or a conventional drying means for drying the raw material may be used, and the means and methods are not limited thereto.

In addition, in the semi-drying step (S200), the raw material is preferably dried to have a moisture content of 30% to 55%.

The reason is that when the moisture content is less than 30%, the fermentation efficiency in the fermentation step to be described later decreases, and when the moisture content exceeds 55%, the amount of biogenic amines produced in the fermentation step increases, or worse, it proceeds to spoilage. Because it becomes.

Next, a salt mixing step (S300) of adding and mixing a salt to the semi-dried raw material is performed.

Here, the salt is added in less than 10 parts by weight of salt based on 100 parts by weight of the raw material for fish and shellfish.

This minimizes the amount of salt contained in the fish sauce, thereby reducing the amount of sodium intake of consumers (consumers), thereby having the advantage of increasing health.

In addition, it is preferable that the salt is a sea salt.

Next, a fermentation step (S400) of fermenting the raw material mixed with salt is performed.

In the fermentation step (S400), the raw material mixed with the salt is stirred under anaerobic conditions, and a new strain, Bacillus velezensis , L2 (KCCM1 2498P), is used as a seed.

Hereinafter, the Bacillus velezensis L2 (KCCM1 2498P) will be described in more detail.

In the present invention, excellent strains were screened from the anchovy fermentation broth prepared by the traditional method, and it was determined that there is protein decomposition ability to generate a transparent ring for some selected colonies, and the results of physiological and biochemical experiments By selecting the best strain, the Bacillus velezensis L2 (KCCM1 2498P) strain was developed.

The Bacillus velezensis L2 (KCCM1 2498P) is a 16S rRNA containing the nucleotide sequence set forth in SEQ ID NO: 1, and the standard strain B. velezensis Compared with LMG 22478 and isolates of the same genus, B. velezensis J3 and B. subtilis KCCM 11316, it has remarkably excellent proteolytic activity.

In addition, the Bacillus velezensis L2 (KCCM1 2498P) is a standard strain of B. velezensis Unlike LMG 22478, it was confirmed that it had a positive reaction to lactose, amygdalin, melibiose and genthiobiose, that is, sugar availability.

In addition, in the fermentation step (S400), the Bacillus velezensis L2 (KCCM1 2498P) suppresses the production of volatile basic nitrogen and histamine, increases the content of total nitrogen and amino nitrogen, and sweet taste series It was confirmed to increase the content of amino acids and umami amino acids. More specifically, serine and valine, which are sweet amino acids, glutamic acid, which is an amino acid that produces umami, and sweet and sour amino acids. It was confirmed to increase the content of amino acids including proline and the like.

In addition, the stirring speed in the fermentation step (S400) is preferably 50rpm to 300rpm.

The reason is to maximize the fermentation efficiency of the fermentation step, when the stirring rpm is less than 50 rpm, the effect of increasing the repellency is insignificant, and when the stirring rpm exceeds 300 rpm, the repelling efficiency is no longer increased. to be.

In the meantime, the "fermentation efficiency" here means a value calculated as "total nitrogen content of the obtained fish sauce/total nitrogen content of the injected fish and shellfish raw materials X100".

In addition, the fermentation temperature in the fermentation step (S400) is preferably processed at a constant temperature in the temperature range of 45 ℃ to 55 ℃.

The reason is that when the fermentation temperature is less than 45°C, the fermentation period is increased, and when the fermentation temperature exceeds 55°C, the fermentation period is no longer shortened and protein modification may occur during the fermentation process. to be.

Next, the fermented product generated through the fermentation step is filtered to remove the residue, and a filtrate obtaining step (S500) of obtaining a filtrate is performed.

In addition, in the fish sauce manufacturing method according to the present invention, after the filtrate obtaining step (S500), a sterilization treatment step (S600) may be further performed.

The sterilization treatment step (S600) is a process of sterilizing the aged filtrate to obtain a liquid fish sauce. The aged filtrate is heat-treated at a constant temperature in a temperature range of 70°C to 100°C for 5 to 30 minutes To obtain.

(Example 1) Bacillus velezensis L2 Selection and Isolation Identification

1-1. Selection of strains with excellent proteolytic activity

100 µl of anchovy fermentation broth produced in the Jeonnam region of Korea was plated on TSA (Trypticase Soy Agar) plate medium, and the plated plate medium was cultured at 37°C for 72 hours.

As shown in Figure 2, after culturing the medium for 72 hours, the dominant strain (W) was selected and named as L2.

In addition, the isolated L2 strain was inoculated (streaking) in a TSA (Trypticase Soy Agar) plate medium containing 1% skim milk, and then cultured for 48 hours to confirm whether or not a transparent ring was produced. .

As shown in Figure 3, the isolated L2 strain was confirmed to form a transparent ring around the colony by the protease generated in the culture process.

1-2. Identification of selected strains

For the identification of the selected isolates, morphological and biochemical investigations were primarily performed, and for final identification, 16S rRNA sequencing was performed.

The morphological investigation was performed by observing the morphology of bacteria and colonies using a microscope and whether or not Gram staining was performed. It was carried out in a way to investigate.

The results of the morphological investigation are shown in FIG. 4, and referring to FIG. 4, it was confirmed that the L2 strain was a Gram-positive bacillus.

In addition, the biochemical investigation results are shown in Table 1 below. After molecular biological identification of the L2 strain, B. velezensis , a standard strain of the same species The results for LMG 22478 were compared together.

Sugar Metabolism Sugar Metabolism Standard
Strain L2
Strain Standard
Strain L2
Strain Control - - Esculin + + Glycerol - - Salicin - - Erythritol - - Cellobiose + + D-Arabinose - - Maltose + + L-Arabinose - - Lactose - + Ribose + - Melibiose - + D-Xylose - - Sucrose + + L-Xylose - - Trehalose + + Adonitol - - Inulin - - Methyl β-D-
xylopyranoside - - Melezitose - - Galactose - - Raffinose + + Glucose + + Starch + + Fructose + + Glycogen + + Mannose + + Xylitol - - Sorbose - - Gentiobiose - + Rhamnose - - D-Turanose - - Dulcitol - - D-Lyxose - - Inositol + - D-Tagatose - - Mannitol + + D-Fucose - - Sorbitol + + L-Fucose - - Methyl α-D-mannopyranoside - - D-Arabitol - - Methyl α-D-glucoside + + L-Arabitol - - N-Acethyl-glucosamine + - Gluconate - - Amygdalin - + 2-Keto-gluconate - - Arbutin + + 5-Keto-gluconate - -

Here, "+" means positive and "-" means negative.

Referring to Table 1, the L2 strain is glucose, fructose, mannose, mannitol, sorbitol, Methyl α-D-glucoside, Amygdalin, Arbutin, Esculin, Cellobiose, Maltose, Lactose, Melibiose, Sucrose, Trehalose, Raffinose, Starch, Glycogen. In addition, it was confirmed that it has sugar utilization for Gentiobiose.

Also, the standard strain, B. velezensis Compared with LMG 22478, the L2 strain is different in that it has sugar availability for lactose, amygdalin, melibiose, and genthiobiose.

In addition, genetic identification was performed through 16S rRNA gene analysis in order to accurately identify the L2 strain.

For 16S rRNA amplification of colony samples of L2 strain, forward primer 27F (5'-AGAGTTTGATCATGGCTCAG-3') and reverse primer 1492R (5'-GGATACCTTGTTACGACTT-3') were used to amplify 16S rRNA at 95°C for 30 seconds, 52 After performing PCR (polymerase chain reaction) under the conditions of a total of 35 cycles for 30 seconds at ℃ and 90 seconds at 72 ℃, check the gene size of the PCR product amplified by electrophoresis, and then based on the sequencing analysis result through the NCBI genebank. Genetic identification was carried out.

In general taxonomically, strains showing more than 97% nucleotide sequence homology can be defined as one species. As a result of the analysis, it was identified as Bacillus velezensis with more than 99% homology, and " Bacillus velezensis L2" It was named as.

The identified Bacillus velezensis L2 was entrusted with the Korea Microbiological Conservation Center on April 18, 2019, and was given the accession number KCCM12498P.

In addition, the identified Bacillus velezensis L2 genetic phylogenetic tree (phylogenetic tree) can be represented as shown in Figure 5, the 16S rRNA base sequence is shown in SEQ ID NO: 1 and 6.

(Experimental Example 1) Confirmation of proteolytic activity of strain

The hydrolytic activity of Bacillus velezensis L2 isolated and identified in Example 1 was confirmed.

More specifically, the protein enzyme activity experiment was performed by Azocasein assay, Bacillus velezensis L2 strain, and the standard strain Bacillus velezensis LMG 22478 strain, Bacillus velezensis J3 strain and Bacillus subtilis KCCM 11316 strain, which are isolates of the same genus/species were used.

Each strain was inoculated into TSB 5ml medium, cultured at 37°C for 24 hours, and then 1ml of the culture solution was centrifuged (12,000rpm, 10 minutes, 4°C), and the supernatant was recovered, respectively.

150 µl of 0.5% azocasein dissolved in 100 mM sodium phosphate buffer (pH 7.0) was added to 50 µl of the supernatant of each strain, followed by reaction in a 30°C constant temperature water bath for 1 hour.

Thereafter, the reaction was stopped by standing for 2 minutes, followed by centrifugation (12,000 rpm, 10 minutes, 4° C.), 500 μl of the supernatant was taken, and 700 μl of 525 mM NaOH were added to mix.

Next, 1 ml of the mixed solution was put into a 48 well plate, and the absorbance was measured at 432 nm to confirm the proteolytic enzyme activity generated by each strain, and the results are shown in Table 2 below.

Bacillus velezensis LMG 22478 strain Bacillus velezensis L2 strain Bacillus velezensis J3 strain Bacillus subtilis ( Bxacillus subtilis) KCCM 11316 strain unit/ml 526 768 484 41

Here, the enzyme activity was defined as 1 unit/ml when increasing the absorbance of 0.01 at 432 nm at 30° C. for 1 hour.

As shown in Table 2, the Bacillus velezensis L2 strain of the present invention was found to have the best proteolytic activity as measured at 768 units/ml of protease activity, and the standard strain Bacillus velezensis Bacillus velezensis Compared to the LMG 22478 strain, it was confirmed to have about 1.5 times higher proteolytic activity.

(Experimental Example 2) Measurement of growth rate and protease activity by salt concentration

Using the Bacillus velezensis L2 strain isolated and identified in Example 1, the growth rate by salt concentration and proteolytic activity by salt concentration were confirmed.

In more detail, 5 ml of TSB medium to which NaCl was differently added at 0, 5, 10, 15% was prepared and prepared, and then the Bacillus velezensis L2 strain was inoculated into each medium, and 37°C, After incubation for 24 hours, the number of viable cells and protease activity were evaluated.

In the experiment to confirm the number of viable cells, the cultured culture solution was diluted 10 times, plated with 100 µl of TSA medium, and cultured at 37° C. for 24 hours, and the number of colony was measured, and the results are shown in FIG. 7.

As shown in FIG. 7, it was confirmed that the growth rate of the Bacillus velezensis L2 strain according to the salt concentration of the present invention was most suitable at a salt concentration of 5%.

In addition, the protein protein enzyme activity experiment was performed in the same manner as the Azocasein assay tested in Experimental Example 1 using the cultured culture medium, and the results are shown in FIG. 8.

As shown in FIG. 8, it was confirmed that the Bacillus velezensis L2 strain of the present invention has the most excellent proteolytic activity when cultured in 5% NaCl culture conditions.

(Example 2) Preparation of anchovy fish sauce

2-1. Preparation of test group anchovy fish sauce

Anchovy fish sauce was prepared by fermentation using the Bacillus velezensis L2 strain.

More specifically, a raw material was prepared by washing twice with brine using fresh anchovy with a length of about 17 cm caught in the southern coast as a raw material for aquatic products, and then semi-drying at a water content of 45%.

Thereafter, 5 parts by weight of salt was added to the semi-dried anchovy based on 100 parts by weight of the raw material and mixed.

Next, the Bacillus velezensis L2 strain was cultured and added so that 1 part by weight based on 100 parts by weight of the raw material was added to the raw material mixed with the salt, and fermented for 8 weeks at 50°C and anaerobic conditions. A fermented product was produced, and the fermented product was then filtered to remove the residue to prepare a fish sauce.

2-2. Preparation of control fish sauce and control fish sauce

In order to compare the quality with the anchovy fish sauce (test group) prepared using the Bacillus velezensis L2 strain, an anchovy fish sauce was prepared without using a separate strain as a control group. Cis ( Bacillus velezensis ) Anchovy fish sauce was prepared using LMG 22478 strain.

That is, as a control group, a separate strain was fermented without inoculation, and as a control group, Bacillus velezensis A fish sauce was prepared by fermentation using LMG 22478 strain, except for this, the manufacturing process of the fish sauce was carried out in the same manner as described above (Example 2-1) to prepare a control fish sauce and a control fish sauce, respectively.

(Experimental Example 3) Total nitrogen, amino nitrogen, volatile basic nitrogen, histamine content check

5 ml of fish sauce samples for the test group, control group, and control group were taken at intervals of 2 weeks for a total of 8 weeks of fermentation period. Using these fish sauce samples, total nitrogen content, amino nitrogen content, and volatile base nitrogen (VBN, Volatile Basic Nitrogen) content and histamine content were confirmed, and the results are shown in Tables 3 to 5 below.

Control (strain X) Fermentation period Week 0 2nd week Week 4 Week 6 8th week Total nitrogen (%) 0.83 1.28 1.32 1.35 1.35 Amino nitrogen (AN, mg/100g) 990 1392 1414 1436 1437 Volatile basic nitrogen (mg%) 20.8 21.6 21.4 21.2 21.2 Histamine (ppm) 123.4 128.8 136 149.1 155.5

Comparative group (using Bacillus bellegensis LMG 22478 strain) Fermentation period Week 0 2nd week Week 4 Week 6 8th week Total nitrogen (%) 0.86 1.48 1.51 1.55 1.56 Amino nitrogen (AN, mg/100g) 995 1573 1609 1620 1620 Volatile basic nitrogen (mg%) 20.7 19.4 17.6 17 16.8 Histamine (ppm) 123.7 113.3 106.4 102.4 102.1

Test group (using Bacillus belegensis L2 strain) Fermentation period Week 0 2nd week Week 4 Week 6 8th week Total nitrogen (%) 0.84 1.81 1.86 1.89 1.89 Amino nitrogen (AN, mg/100g) 989 1890 1928 1931 1932 Volatile basic nitrogen (mg%) 21 18.3 15.2 14.6 14.4 Histamine (ppm) 124.1 75 55.6 49.9 48

As shown in Tables 3 to 5, in the case of manufacturing anchovy fish sauce using Bacillus velezensis L2 strain, fermentation is completed in about 4 weeks, so the fermentation period can be shortened, and total nitrogen , Amino-nitrogen content is high, and the content of histamine, a component involved in allergic reactions or inflammation, and the content of volatile basic nitrogen, a component that serves as an indicator of freshness, are drastically reduced, which has the advantage of manufacturing high-quality fish sauce.

(Experimental Example 4) Sensory test

As an evaluation of the sensory palatability of fish sauce (control group, control group, test group) prepared by fermentation for an 8-week period, a sensory test was conducted and shown in Table 6 below.

Here, the evaluation items were overall color, scent, taste, umami, fishy taste, tail, and general preference, and a sensory test was conducted using a 9-point scale method for 30 target consumer panels who actually use the product.

Control
(Use strain X) Comparative group
(Use of Bacillus belegensys LMG 22478 strain) Test group
(Use of Bacillus belegensis L2 strain) color 6.5 7.0 8.0 incense 4.8 6.0 7.4 flavor 3.3 5.6 7.5 Umami 4.8 5.0 7.8 Fishy taste 4.8 6.2 7.5 cove 4.1 6.1 7.7 Comprehensive Symbol Chart 4.3 5.3 7.5

As a result, as shown in Table 6, the fish sauce prepared by the method for producing a fish sauce according to the present invention was compared with the fish sauce of the control group and the fish sauce of the comparative group, It was excellent in all indicators and was found to be excellent in overall acceptance.

(Experimental Example 5) Analysis of Free Amino Acid

Analysis of free amino acids was performed using fish sauce (control group, control group, test group) prepared by fermentation for 8 weeks, and the results are shown in Table 7 below.

Here, the analysis of free amino acids was confirmed using an automatic amino acid analyzer (Hitach Amino Acid Analyzer L-8900).

Amino acid (mg%) Control
(Use strain X) Comparative group
(Use of Bacillus belegensys LMG 22478 strain) Test group
(Use of Bacillus belegensis L2 strain) Remark
(Compared to the standard strain control group) Taurine 88.0 56.5 128.0 Aspartic acid 10.0 12.8 24.3 Threonine 0.0 0.0 87.9 Serine 153.8 190.0 471.9 2.5 times increase, sweet amino acid Asparagine 0.0 0.0 17.3 Glutamic acid 423.8 554.0 923.9 1.7 times increase, umami taste Glutamine 0.0 0.0 0.0 Glycine 139.6 199.1 3.8 Alanine 338.5 420.6 286.6 Valine 276.4 374.8 790.6 1.9 times increase, sweet amino acid Methionine 127.0 184.7 37.4 Isoleucine 168.2 270.9 491.1 Leucine 284.2 439.8 618.9 Tyrosine 94.9 101.7 227.4 Phenylalanine 125.3 183.8 389.2 γ-Amino-n-butyric acid 2.7 0.0 7.9 Lysine 388.2 418.4 540.0 Histidine 106.1 160.2 223.6 Arginine 5.9 124.3 478.2 Hydroxyproline 304.1 488.5 34.9 Proline 99.4 109.6 365.0 3.3 times increase,
Sweet and sour Total 3,136.1 4,289.7 6,147.9

As shown in Table 7, the fish sauce prepared by the method for producing a fish sauce according to the present invention can be prepared with an increased content of sweet-based amino acids and umami-based amino acids compared to the fish sauce of the control group and the fish sauce of the control group. It is believed that the taste preference could be improved.

More specifically, the anchovy fish sauce prepared by fermentation by the Bacillus velezensis L2 strain was compared with the fish sauce fermented by the standard strain Bacillus bellezensis LMG 22478, which is an amino acid that gives off a sweet taste. Serine is about 2.5 times higher in content, about 1.7 times higher in glutamic acid, an amino acid that gives a umami taste, and about 1.9 times content in the case of Valine, a sweet amino acid. In the case of Proline, which is a high, sweet and sour amino acid, it was confirmed that the content was about 3.3 times higher.

As described above, according to the method for producing a fish sauce according to the present invention, by using a new strain Bacillus velezensis L2 (KCCM1 2498P) as a fermentation seed, the fermentation period can be shortened, and allergic reactions In addition, the content of histamine, an ingredient involved in inflammation, and the content of volatile basic nitrogen, which is an indicator of freshness, are drastically reduced, and at the same time, it can provide a high-quality fish sauce with an increased content of sweet and umami amino acids. .

As described above, the present invention has been illustrated and described with a preferred embodiment, but is not limited to the above-described embodiment, and within the scope of the spirit of the present invention, those of ordinary skill in the art Various changes and modifications will be possible.

Korea Microorganism Conservation Center (overseas) KCCM12498P 20190418

<110> Jeonnam Bioindustry Foundation <120> Novel Bacillus velezensis L2 separated from fermented broth of anchow and Fish sauce prepared by using the same <130> DP19-0036 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1517 <212> RNA <213> Unknown <220> <223> Bacillus velezensis <400> 1 ttagagtttg attcaggctc aggacgaacg ctggcggcgt gcctaataca tgcaagtcga 60 gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta acacgtgggt 120 aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg atggttgttt 180 gaaccgcatg gttcagacat aaaaggtggc ttcggctacc acttacagat ggacccgcgg 240 cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag ccgacctgag 300 agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta 360 gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt gatgaaggtt 420 ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc cgttcaaata gggcggcacc 480 ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt 540 aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg tttcttaagt 600 ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg aacttgagtg 660 cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca 720 ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa gcgtggggag 780 cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttaggg 840 ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg gagtacggtc 900 gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt 960 aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa tcctagagat 1020 aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg 1080 tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg ccagcattca 1140 gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa 1200 tcatcatgcc ccttatgacc tgggctacac acgtgctaca atgggcagaa caaagggcag 1260 cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc gcagtctgca 1320 actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg cggtgaatac 1380 gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg 1440 gtgaggtaac cttttaggag ccagccgccg aaggtgggac agatgattgg ggtgaagtcg 1500 taacagggta aaccgta 1517

Claims (12)

A raw material preparation step of preparing raw fish and shellfish raw materials;
A semi-drying step of drying the prepared raw material to a moisture content of 30% to 55%;
Salt mixing step of mixing by adding a salt to the semi-dried raw material;
A fermentation step of fermenting the salt-mixed raw material under anaerobic conditions; And
Including; a filtrate obtaining step of filtering the fermented product generated through the fermentation step to remove the residue and obtain a filtrate; and
The strain introduced in the fermentation step is 16S rRNA, which contains the nucleotide sequence shown in SEQ ID NO: 1, is separated from the anchovy fermentation broth, has proteolytic activity, and is Bacillus velezensis L2 (KCCM 12498P). How to make a fish sauce.
The method of claim 1,
The strain is lactose (Lactose), amygdalin (Amygdalin), melibiose (Melibiose) and genthiobiose (gentiobiose), characterized in that the positive reaction to the production method of fish sauce.
The method of claim 1,
In the fermentation step, the strain is a method for producing a fish sauce, characterized in that it suppresses the production of volatile basic nitrogen and histamine contained in the fermentation product.
The method of claim 1,
In the fermentation step, the strain is a method for producing a fish sauce, characterized in that increasing the content of total nitrogen and amino nitrogen.
The method of claim 1,
In the fermentation step, the strain is a method for producing a fish sauce, characterized in that to increase the content of the sweet-based amino acids and umami-based amino acids.
The method of claim 1,
In the fermentation step, the strain is a method for producing a fish sauce, characterized in that increasing the content of amino acids including serine, glutamic acid, valine and proline.
The method of claim 1,
The fermentation step is a method for producing a fish sauce, characterized in that the fermentation step is processed at a constant temperature in the temperature range of 45 ℃ to 55 ℃, and the stirring is carried out at 50rpm to 300rpm.
The method of claim 1,
The salt mixing step is a method of producing a fish sauce, characterized in that less than 10 parts by weight of salt is added and mixed based on 100 parts by weight of the raw material.
The method of claim 1,
After the filtrate obtaining step,
A sterilization treatment step of sterilizing the filtrate; method of manufacturing a fish sauce, characterized in that further performed.
The method of claim 9,
The sterilization treatment step
A method for producing a fish sauce, characterized in that heat treatment is performed for 5 to 30 minutes at a constant temperature in a temperature range of 70 to 100°C.
The method of claim 1,
The raw material is any one selected from the group consisting of canary, anchovy, tuna, shrimp, herring, mackerel, and a combination thereof.
A fish sauce manufactured by the method for manufacturing the fish sauce according to any one of claims 1 to 11.

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