KR101918267B1 - Method for production of fermented fishery product with reduced histamine content - Google Patents

Abstract

The present invention relates to a method for producing a fermented aquatic product in which histamine is reduced while ensuring high antioxidant activities, by using Bacillus sp. YMF-001 (KFCC11768P), and a use thereof. According to the present invention, the fermented aquatic product ensures reduced histamine in a microbial fermentation process compared to previously published techniques, antioxidant activities are increased, and fermentation time was shortened while having functionality similar to traditional fish sauces. The fermented aquatic product, according to the present invention, can be made into finished products such as fermented anchovy sauce, a fermented sand eel sauce, a fermented shrimp sauce, and fish sauce based on raw materials, and can also be applied as a food seasoning through additional concentration, blending, and pulverization.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing fermented aquatic products having reduced histamine,

The present invention is Bacillus (Bacillus) sp. YMF-001 (KFCC11768P), and has high antioxidative activity, and its use.

It is produced by fermentation of anchovy, canary, etc. for 6 ~ 12 months. It contains a lot of tastes such as various amino acids and nucleic acid such as glutamic acid, glycine, alanine and lysine. . Peptides contained in fish sauces have not only physiological activities such as antioxidative effect and antimicrobial effect but also immunoregulatory function, hormone and neurotransmitter, and also biological control function.

However, fish sauces prepared by conventional methods cause discomfort due to fish smell and deterioration of external sensuality such as formation of sediments. As a result, the number of young housewives who are avoiding the use of salted fish is increasing. In addition, histamine, a kind of biogenic amine, is produced in a large amount through unsanitary aging process, threatening food safety of consumers.

Biogenic amines are amine-based substances that are produced by the chemical action of free amino acids during fermentation and fermentation, such as decarboxylase enzymes and amino-transferases. They are used in fermentation products such as meat, fish, cheese, and wine Found. One of the best known biogenic amines is histamine. Histamine in the human body is involved in allergic reactions or inflammation. Histamine released by antigen-antibody reaction in immune cells causes bronchoconstriction (allergic asthma), capillary vasodilation, and increased permeability (nasal discharge or edema). Fish, especially sardines such as sardines, mackerel, and saury, contain a large amount of amino acids called histidine, which are degraded by bacteria during storage, fermentation and maturation, transforming it into an allergen called histamine.

Korean Patent Laid-Open Publication No. 10-2012-0097650 discloses a method of reducing the histamine content by mixing Aspergillus oryzae asparagus, which is used for soy sauce production, in anchovy and aging for 12 to 24 months, thereby producing histamine at a level of 1,100 to 1,600 ppm In the range of 5 ~ 400ppm. However, as described in this patent, the presence or inoculation of Aspergillus strains did not affect the reduction of histamine at all, and only the effect of increasing the total nitrogen release of fish was described. Korean Patent Registration No. 10-1382230 discloses a technique for reducing histamine levels using a high-temperature fermentation technology. In this technique, considering that histamine-producing bacteria are mostly mesophilic bacteria, they are fermented at high temperature by using Bacillus strain which is active at high temperature. However, since most of the anchovy, canary, and other aquaculture tanks are 20 to 50 tons in size, low-temperature aging and temperature aging of 40 degrees or more, avoiding mid-temperature, which is the production temperature of histamine, are industrially impossible. Especially, It is almost impossible to do.

As pointed out in Korean Patent No. 10-1382230, in order to prevent the contamination of the nascent bacteria and inhibit histamine production, a high-temperature fermentation process at 50 ° C or higher, in which proliferation of histamine-degrading bacteria can be active, should be applied. However, the fermentation level should be at the level of commercially available fermentation tank of about 5 tons, so that the production capacity can not be less than the existing production amount. In addition, fermentation can not be carried out for 6 to 12 months at 50 ° C. Thus, the fermentation time should be shortened in a short period of time. In spite of the shortened period of time, Remains.

To solve this problem, in the present invention, fresh anchovy and salt were mixed and immediately crushed to a size of about 10 μm to prevent contamination of germs caused by salt, and inoculated with a salt-tolerant culture of Bacillus sp. In addition, after the fermentation, proteolytic enzymes were added to effectively shorten the fermentation time, while inoculation of the fermented soy sauce and aging of the fermented soy sauce reduced the histamine and produced similar products in the conventional traditional fermented soy sauce. In addition, products with an increased antioxidative activity by the Bacillus strain could be produced.

(A) mixing salt with aquatic products to prepare a mixture; (B) after said step (a), breaking the mixture; After step (b), the shredded aquatic products are treated with Bacillus , sp. And (c) mixing and fermenting a culture solution of YMF-001 (KFCC11768P) in a fermentation broth.

In the method for producing a fermented aquatic product according to the present invention, the method for producing the fermented aquatic product preferably further comprises the step (d) of hydrolyzing the protease after the step (c). In this case, the fermented aquatic product may further comprise (e) a step of adding the fish sauce to the fermented aquatic product after the step (d).

In the method for producing a fermented aquatic product according to the present invention, the aquatic product may be one selected from among a green anchovy, a canary and a shrimp.

In the method for producing a fermented aquatic product according to the present invention, the fermentation is preferably fermented at 28 to 32 ° C, pH 5.0 to 7.0 for 12 to 36 hours.

In the method for producing a fermented aquatic product according to the present invention, the hydrolysis is preferably carried out at 45 to 55 ° C for 48 to 96 hours.

In the method for producing a fermented aquatic product according to the present invention, the fish sauce may be, for example, anchovy sauce.

In the method for producing a fermented aquatic product according to the present invention, the aging is preferably performed for 10 days to 12 months.

The fermented fish product according to the present invention showed reduced histamine and antioxidative activity in the microbial fermentation process compared to the known technology, and the sensory properties of the conventional fish sauces were similar to those of the conventional fish sauces. The fermented fish product according to the present invention can be made into finished products such as anchovy sauce, canned fish sauce, shrimp sauce, and fish sauce according to the raw materials, and can be fully utilized as food seasoning through additional concentration, blending, and pulverization. Based on this, the fermented fish products produced according to the present invention can contribute to the improvement of the productivity of the fish sauce, the securing of the food safety, and the consumption increase.

Bacillus sp. The histamine content (Example 1) according to fermentation time in the production of anchovy sauce using YMF-001 is shown.
2 shows the histamine content (Comparative Example 1) according to the fermentation time in the preparation of the traditional anchovy sauce.
FIG. 3 is a graph showing the results of five-point scale sensory evaluation of Example 3 and Comparative Example 1 (all samples were diluted with a 3% solution and subjected to sensory evaluation, and the salt contents of the two solutions were adjusted to 2% Respectively.
4 is a comparison result of DPPH scavenging effect analysis of Example 3 and Comparative Example 1

The present invention is to produce a fermented fish product similar to the known technology, in which the histamine is reduced and the antioxidative activity is increased in the microbial fermentation process and the fermentation time is reduced as well as the sensory properties of the conventional fish sauce. A mixture of and more particularly, anchovy fish sauce, sand eel fish sauce, fish sauce shrimp, seafood that is used as the material of the eoganjang with salt and then a constant rate, then using a chopper broken so 10㎛, Bacillus sp. YMF-001 (KFCC11768P) is added to the fermentation, and the proteolytic enzyme and the fermented fish sauce are further added to the fermentation process. Thus, histamine is reduced, antioxidative activity is increased through the production of a specific coloring matter of bacteria, and flavor and aroma are enhanced through fermentation during the fermentation process of additional proteolytic enzyme and fermented fish sauce. Thus, a commercially available fermented product can do. The fermented fish product according to the present invention can be made into finished products such as anchovy sauce, canned fish sauce, shrimp sauce, fish sauce, and the like, and can be used as a food seasoning through additional concentration, blending, and pulverization.

The present invention,

(1) a step of mixing salt at a ratio of 80:20 to aquatic products such as live anchovy, canary, shrimp, etc. (a)

(2) a step (b) of crushing to a size of about 10 mu m using a chopper machine,

(3) Bacillus sp. (C) mixing the culture medium of YMF-001 and fermenting at 30 DEG C and pH 6.0 for 24 hours,

(4) a step of adding proteolytic enzyme and decomposing at 50 DEG C for 72 hours (d)

(5) adding a fermented fish sauce and aging the fermented aquatic product for 10 to 20 days or more.

In addition, the present invention provides fish sauce, salted fish, eel, and seasoning prepared using the fermented aquatic products.

In step (a) of the present invention, it is preferable that seafood such as live anchovy, canary, shrimp and the like be mixed with salt as soon as possible. Although it is not possible to specify a specific time in the process of obtaining and distributing aquatic products, it is not preferable that aquatic products are present at room temperature, and it is preferable to mix with salt and store at room temperature. In addition, it is preferable that at least the salt is mixed with 20% or more of the weight of the marine product to establish the preservability thereof. More preferably, the aquatic products and the salt are mixed at a ratio of 80:20.

In the present invention, in step (b), the chopper may use a chopper with a double-edged blade, wherein the first-stage blender can be cut to a size of 1 cm or less, and the second- . More preferably, it is preferable to cut all the marine products to 10 탆 or less since the production of histamine can be initially suppressed therefrom. If the size of the aquatic product to be cut is larger than 10 탆, the mixing with salt may be uneven and the growth of the spoilage bacteria may become large, and the histamine level may increase therefrom. Therefore, it is necessary to control the chopper's cutting performance to the greatest extent and to densely cut it, thereby suppressing the growth of the rot fungus which can produce histamine through uniform mixing with salt.

In step (c), the present invention relates to a method for producing Bacillus sp. The culture medium of YMF-001 (KFCC11768P) was Bacillus sp. It is preferable to use YMF-001 (KFCC11768P) strain which is cultured at a temperature of 28 to 32 ° C and a pH of 5 to 7.

Bacillus sp. YMF-001 is a strain deposited by the present inventors on March 15, 2018, separated from anchovy sauce, and has a deposit number of KFCC11768P. The carbon source, the nitrogen source, and the inorganic source used in the culture of the strain may be usually those suitable for the raw materials of the food. More preferably, molasses, which is most commonly used as a carbon source, is suitable. The nitrogen source and the inorganic source, It is preferred to use a protein concentrate. If the bacteria are cultured for 24 hours or more under the above conditions and the bacteria are sufficiently activated, they are applied to the present fermentation. At this time, the Bacillus sp. It is preferable that the YMF-001 culture medium is mixed with salt and mixed with crushed aquatic products in an amount of 1 to 50% based on the total weight. The fermentation of step (c) is preferably carried out at a temperature of 28 to 32 ° C and a pH of 5 to 7. More preferably, fermentation is carried out at 30 DEG C and pH 6.0 for 24 hours. Bacillus sp. YMF-001 is a pigment-forming microorganism. As the incubation time progresses, the culture medium becomes dark brown, and protease activity starts to decompose the aquatic protein.

In the step (d) of the present invention, it is preferable that the fermentation broth is fermented by mixing an endo, exo type protease in the fermentation broth to accelerate the enzyme reaction in earnest. The amount of the enzyme may be 0.01 ~ 1% (relative to the weight of anchovy), more preferably 0.05 ~ 0.5% (relative to anchovy weight). The addition of less than 0.05% of the enzyme can cause precipitation by the undegraded protein in the final product, and the addition of more than 0.5% of the enzyme has a cost that is not commercially feasible due to the commercial price of the enzyme. In addition, Bacillus sp. In order to activate the protease eluted from YMF-001 and additionally added proteolytic enzymes, the reaction temperature during the hydrolysis is preferably 45 to 55 ° C, and the reaction is preferably performed for 48 to 96 hours. More preferably, the reaction is carried out at 50 DEG C for 3 days. The protease has a characteristic that the survival rate is drastically lowered at a high temperature, and it is not preferable to react at a temperature exceeding 55 캜, and after 96 hours at a temperature of 45 캜 or higher, activity of less than 10% is recorded.

In the present invention, in step (e), it is preferable to add a fish sauce and mature it. In this case, the liquid used is a kind of a fermented sauce. The fish sauce (fish sauce) used in the present invention can be anchovy sauce, canned fish sauce, and shrimp sauce prepared by aging for more than one year by a conventional method. In order to produce a traditional taste and flavor, various microbial actions are required. The fermentation process by a single microorganism as in the present invention is limited in the number of microorganisms to obtain the flavor of a conventional fish sauce. Therefore, it is necessary to carry out the process of obtaining the taste and flavor of the traditional fish sauce by mixing and fermenting the fermented fish sauce. The fermented sea urchin (fermented sea urchin) is preferably mixed with 1 to 50% and can be aged for 10 days to 1 year. However, it is more preferable to mix 1 to 10% of fermented soy sauce for 10 to 20 days or more preferably commercially. At this time, it is recommended to have sufficient ventilation conditions and to introduce clean air once a day so that the microbial aging can be smoothly performed. When the mixing ratio of the fermented fish sauce exceeds 10%, the histamine concentration of the fish product of the present invention may be too high due to the histamine contained in the seaweed sauce itself. Taking the fermentation period for more than 20 days, Is not large and thus is not preferable due to the characteristics of the low productivity process.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following embodiments and experimental examples, and includes modifications of equivalent technical ideas.

[Example 1: Bacillus sp. Preparation of anchovy sauce using YMF-001 (KFCC11768P)

50 kg of molasses and 50 kg of anchovy fish sauce concentrate were mixed with 900 kg of purified water and sterilized at 95 ° C for 30 minutes. After cooling to 30 ° C, the cells were incubated with Bacillus sp. 1 kg of YMF-001 (KFCC11768P) culture was added and cultured at 30 占 폚 for 24 hours.

On the other hand, 1,000 kg of anchovy mixture was prepared by mixing 800 kg of salted anchovy and 200 kg of salted salmon, and then anchovy was crushed to 10 탆 using a chopper having a two-stage blender and transferred to a 5-ton reactor. Thereafter, the above-prepared 24-hour cultured Bacillus sp. YMF-001 medium, and fermented at 30 DEG C and pH 6.0 for 24 hours.

[Comparative Example 1: Preparation of anchovy sauce by a conventional method]

A mixture of 800 kg of fresh anchovy and 200 kg of sun salmon was mixed and 1,000 kg of anchovy mixture was prepared and transferred to a storage tank. Then, they were naturally matured for 6 to 12 months in a natural state.

[Experimental Example 1: Bacillus sp. Comparison of histamine in anchovy sauce and traditional anchovy sauce using YMF-001]

100 ml of each of Example 1 and Comparative Example 1 was sampled, sterilized at 85 ° C for 30 minutes, filtered using No. 4 filter paper, and centrifuged at 5,000 rpm to prepare a sample. Then, the histamine contents in the samples were compared according to the following procedure.

(1) 150 μl of a buffer (buffer) and sample / std (0,1,5,10,15,20 ppm) were added to 96 wells of a histamine kit (Ridascreen histamine No. R1605), followed by shaking.

(2) The OD value was measured at 450 nm after 3 minutes. (OD value: A1)

(3) Add 10 μl of the enzyme solution of the kit and shake it.

(4) The OD value was measured at 450 nm after 10 minutes. (OD value: A2)

(5) A2-A1: Final OD value.

(6) The histamine value in the sample was quantified using the OD value of std.

Experimental results are shown in Figs. 1 and 2. Bacillus sp. (Example 1) according to fermentation time in the production of anchovy sauce using YMF-001. Fig. 2 shows the result of measurement of the histamine content (Comparative Example 1) according to the fermentation time in the production of the conventional anchovy sauce.

As shown in Fig. 1, in accordance with Example 1, Bacillus sp. In the preparation of anchovy sauce using YMF-001, the histamine content was measured to be 10 ppm to a maximum of 65 ppm (Fig. 1). However, the histamine content of Comparative Example 1 prepared by the conventional method was increased from 524 ppm at the first month of fermentation to 978 ppm at the 12-month fermentation as shown in FIG. 2 (FIG. 2).

From the above results, it was confirmed that histamine was lowered at least 9 times and at most 15 times in Example 1 as compared with Comparative Example 1.

[Example 2: Enzyme reaction step by adding commercial enzyme to Example 1]

Bacillussp. One endo-type protease and one exo-type protease were mixed in a commercially available protease in the fermented product of Example 1, which had been fermented using YMF-001. The amount of enzyme added was 4kg of enzyme, which is 0.5% of anchovy weight. The enzyme reaction temperature was 50 ° C, and the reaction was continued for 3 days at pH 6.0.

[Experimental Example 2: Measurement of hydrolysis degree according to fermentation time of Examples 1 and 2 and Comparative Example 1]

100 ml of the solution prepared in each of Examples 1 and 2 and Comparative Example 1 was taken and sterilized at 85 캜 for 30 minutes to prepare a sample. The degree of hydrolysis was determined by adding the same amount of trichloroacetic acid to each sample, centrifuging at 5,000 rpm, measuring the amount of nitrogen in the supernatant (semi-micro Kjeldahl method), dividing the total amount of nitrogen before hydrolysis Respectively. Table 1 shows the results of hydrolysis measurement of Examples 1 and 2, and Table 2 shows the results of hydrolysis measurement of Comparative Example 1.

Time (hr) Hydrolysis (%) Bacillus sp. YMF-001 Fermentation Stage
(Example 1) One 5.4 5 6.4 9 6.8 13 7.1 17 14.5 20 16.8 24 20.4 Enzymatic decomposition step
(Example 2) One 25.7 10 37.5 24 49.7 34 54.6 48 59.8 58 65.4 72 68.7

The degree of hydrolysis of Comparative Example 1 Time (month) Hydrolysis (%) Traditional fermentation
(Comparative Example 1) One 25.1 2 32.3 3 48.7 4 50.2 5 53.4 6 58.6 7 60.5 8 62.4 9 63.4 10 65.4 11 65.7 12 66.4

The total nitrogen content of the supernatant after removal of the undegraded protein precipitated in TCA solution in total nitrogen was calculated and the degree of hydrolysis was calculated. As a result, in Example 2, 24 hours of fermentation and 72 hours of enzymatic decomposition were performed, About 68.7% was decomposed to be released as a flavor component.

However, in the case of anchovy sauce prepared by the conventional method as in Comparative Example 1, 66.4% of the protein was released as a flavor component during the fermentation period of 12 months.

From the above results, it was confirmed that the hydrolysis degree of the fermented product of Example 2, which had undergone the fermentation for 1 day and the enzymatic decomposition for 3 days, was superior to the fermentation for 12 months. These results indicate that hydrolyzate of about the same degree as that of 12-month aging can be obtained by the additional enzymatic treatment, even though the aging time is shortened.

[Example 3: Preparation of aged product by adding seafood sauce to Example 2]

The fermented sauce used in this example is anchovy sauce prepared by aging for more than one year by the conventional method. The fermentation broth obtained in Example 2 was mixed with 10% by weight of fermented fish sauce and aged for 20 days. A sufficient aging tank was provided with ventilation conditions, so that clean air was introduced once a day, so that the microbial aging was smoothly performed. Table 3 below shows the histamine content, the degree of hydrolysis and the free amino acid content of Example 3 and Comparative Example 1 in comparison.

designation Example 3 Comparative Example 1 Histamine content (ppm) 152.40 978.50 Hydrolysis (%) 70.50 66.40 Free amino acid
(mg / 100g) Aspartic acid 45.31 41.69 Threonine 10.34 9.51 Serine 11.14 10.25 Glutamic acid 488.99 449.87 Glycine 27.17 25.00 Alanine 64.58 59.41 Valine 37.37 34.38 Cystine 21.08 19.39 Methionine 21.93 20.18 Isoleucine 32.25 29.67 Leucine 48.47 44.59 Phenylalanine 18.89 17.38 Lysine 62.44 57.44 Histidine 10.94 10.06 Arginine 0.00 0.00 Total free amino acid content
(mg / 100g) 900.90 828.83

As shown in Table 3, the histamine content of Example 3 was 6.4 times lower than that of Example 3 and Comparative Example 1, which was a conventional fermented fish sauce. Hydrolysis was also performed by Bacillus sp. YMF-001 fermentation, enzyme decomposition and addition of fermented seaweed showed a degree of hydrolysis of about 10% higher than that of Comparative Example 1. Also, the content of free amino acid according to the high hydrolysis rate was also high in Example 3 as a whole.

On the other hand, based on the results of the above-described physicochemical analysis, the sensory evaluation was performed on a 5-point scale as shown in Fig. 3. The sensory evaluations of Example 3 were carried out in the same manner as in Comparative Example 1 except that sweetness, meat taste, Respectively. FIG. 3 is a graph showing the results of five-point scale sensory evaluation of Example 3 and Comparative Example 1 (all samples were diluted with a 3% solution and subjected to sensory evaluation, and the salt contents of the two solutions were adjusted to 2% Respectively.

[Experimental Example 3: Comparative Experiment of Antioxidative Activity - Analysis of DPPH scavenging effect of Example 3 and Comparative Example 1]

Example 3 and Comparative Example 1 After extracting the sample with methanol, extracts of 32 mg and 27 mg, respectively, were obtained through extraction of ethyl acetate from the total extracts obtained. Each extract was redissolved in 10 ml DMSO and used for antioxidant experiments. 16 mg of DPPH reagent was dissolved in 100 ml of ethanol, and 100 ml of distilled water was mixed and sonication was performed to prepare a reagent. After mixing 0.1 ml of the sample (1% of each vinegar ethyl acetate extract powder dissolved in DMSO reagent) in 0.9 ml of reagent and reacting in the dark room for 30 minutes, the absorbance was measured using a spectrophotometer at 528 nm. The DPPH radical scavenging effect (Control absorbance - sample absorbance) / control absorbance × 100.

FIG. 4 shows the results of DPPH scavenging analysis of Example 3 and Comparative Example 1. FIG. DPPH scavenging effect The tocopherol used as a positive control showed a DPPH scavenging ability of 100% over the whole range. Bacillus sp. The DPPH scavenging antioxidative effect of YMF-001 fermentation, enzyme digestion, and fermentation of the fermented soy sauce was significantly different from that of the conventional fermented soy sauce. Respectively.

This is because Bacillus sp. YMF-001 was associated with the production of antioxidant-related pigment substances, and Bacillus sp. YMF-001 was found to have beneficial effect on the coloring of fish sauce, which is scientifically helpful and also related to the health promotion of consumers.

Name of depository: Korea Microorganism Conservation Center (Domestic)

Accession number: KFCC11768P

Checked on: 2018315

Claims (8)

(A) mixing salt with aquatic products to prepare a mixture;
(B) after said step (a), crushing the mixture to 10 mu m;
After the step (b), 1 to 50% (w / w) of the culture broth of Bacillus sp. YMF-001 (KFCC11768P) is added to the crushed aquatic products at 28 to 32 ° C, (C) fermenting for 12 to 36 hours;
(D) adding the proteolytic enzyme after the step (c) and hydrolyzing the proteolytic enzyme at 45 to 55 DEG C for 48 to 96 hours; And
And (e) adding the fish sauce after the step (d) and aging the fermented aquatic product for 10 to 20 days.
delete delete The method according to claim 1,
Preferably,
Wherein the fermented aquatic product is at least one selected from the group consisting of green tea, green tea, green tea, green tea, green tea, green tea, green tea, green tea, green tea,
delete delete The method according to claim 1,
In the sauce,
Anchovy sauce, and fermented seafood.
delete

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