Composition for preventing discoloration of red-fleshed fish, and use thereof

Abstract

The present invention relates to a composition for preventing discoloration of red-fleshed fish, and a use thereof. According to the present invention, in order to derive optimal conditions for preventing discoloration of red-fleshed fish, optimum treatment conditions for a novel Leuconostoc citreum strain or a culture medium thereof, of the present invention, have been established using response surface methodology (RSM), and thus, productivity can be improved by enhancing palatability and a high-nutrient marine product retaining the taste, nutrition, and texture thereof can be provided.

Description

Composition for preventing discoloration of red-fleshed fish and use thereof

[0001] The present invention relates to a composition for preventing discoloration of red-fleshed fish and its use.

[0002] Red-fleshed fish undergo mutation and degeneration of chromogens, which discolors to a color tone that has a negative impact on consumers, even though it is not an indicator of freshness, and the discolored fluid leaks out, resulting in a deterioration in appearance and flavor, which significantly damages commercial value.

[0003] The color of this red fish meat is determined by myoglobin, and myoglobin is greatly affected by oxidation and reduction. When myoglobin combines with oxygen, it changes to oxymyoglobin, which makes the meat surface appear bright and red, and when oxidized, it changes to metmyoglobin, which makes the meat surface appear dark and brown. In other words, more specifically, the color of red fish meat is expressed by myoglobin. Oxymyoglobin turns red when oxygen combines with the divalent iron atom present in the molecule, and when oxidized to a trivalent iron atom, it turns into metmyoglobin, which turns brown. These changes cause problems such as lowering consumer preference, and a large amount is discarded even though there is no problem with freshness.

[0004] Currently, prior research on preventing discoloration in red fish meat to address this issue has included studies using extracts derived from fishery by-products, extending quality through irradiation, improving taste and texture through aging, utilizing packaging methods, and using synthetic antioxidants. However, technologies utilizing extracts derived from fishery by-products are currently limited in their applicability to food products in Korea, hindering their commercialization. Technologies that extend quality through irradiation may raise consumer concerns due to the negative perception of radiation. Aging technologies require a long aging process and are difficult to apply to fresh fish, limiting their applicability. Technologies utilizing packaging methods do not address the fundamental causes of discoloration. Synthetic antioxidants are limited in their applicability as food additives, and safety concerns and consumer perceptions limit their application.

[0005] Therefore, it is necessary to develop a method using effective natural materials that can overcome these problems and prevent or suppress discoloration that occurs during distribution.

[0006] Meanwhile, lactic acid bacteria, a general term for strains that produce more than 50% organic acids per glucose molecule, have been commonly utilized as probiotics, known for their beneficial effects on the intestines. Furthermore, they have been isolated and studied from various fermented foods, and their production of various organic acids has been shown to play a variety of roles, including inhibiting the growth of harmful bacteria, improving the intestinal environment, and enhancing flavor.

[0007] In addition, rosemary (Rosmarinus officinalis L.) is a herb spice widely used as an additive for food cooking and processing, and its main components such as α-pinene, apigenin, β-carotene, β-sitosterol, betulinic acid, borneol, rosmanol, rosmarinic acid, and tannin are reported to have strong antioxidant activity, fibrinolytic activity, and antibacterial activity.

[0008] Therefore, when rosemary extract is used together with lactic acid bacteria and / or their culture solution as a natural material that can prevent discoloration of red fish meat, it is expected that the quality of red fish meat during processing and distribution will be improved, the sales period will be extended, and high marketability will be achieved.

[0009] Under these circumstances, the present inventors sought to develop a method for suppressing discoloration of red-fleshed fish during distribution. As a result, a novel lactic acid bacteria strain, Leuconostoc citreum M8, with excellent antioxidant activity was isolated and identified from aquatic products. Through RSM analysis, the optimal mixing conditions for the strain culture and rosemary extract to prevent discoloration of red-fleshed fish were determined. Furthermore, statistical significance was demonstrated to demonstrate that this method not only satisfies consumer sensory and preference requirements but also maintains freshness, thereby completing the present invention.

[0010] Accordingly, one object of the present invention is to provide a composition for preventing discoloration of red-fleshed fish, comprising as active ingredients a Leuconostoc citrium strain deposited under the accession number KCTC15454BP or a culture solution thereof; and a rosemary extract.

[0011] In addition, another object of the present invention is to provide a food preservative composition comprising, as active ingredients, a Leuconostoc citrium strain deposited under the accession number KCTC15454BP or a culture solution thereof; and a rosemary extract.

[0012] In addition, another object of the present invention is to provide a method for preventing discoloration of red-fleshed fish.

[0013]

[0014] Other objects and advantages of the present invention will become more apparent from the detailed description of the invention and the claims below.

[0015] The terminology used herein is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0016] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0017]

[0018] Hereinafter, the present invention will be described in detail.

[0019]

[0020] According to one aspect of the present invention, the present invention provides a composition for preventing discoloration of red-fleshed fish, comprising as active ingredients a Leuconostoc citreum (L. citreum) strain or a culture solution thereof deposited under accession number KCTC15454BP; and a rosemary extract.

[0021] The Leuconostoc citreum (L. citreum) strain of the present invention is a novel Leuconostoc citreum strain isolated from salted hairtail squid, and was identified by the inventors as a lactic acid bacteria strain. It was named M8 and deposited with the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on May 25, 2023, and assigned the accession number KCTC15454BP.

[0022] Isolation and cultivation of the above Leuconostoc citrium strain can be performed by any method known in the art.

[0023] Additionally, the Leuconostoc citrium strain has a 16S rDNA comprising the base sequence of sequence number 1.

[0024] The Leuconostoc citreum (L. citreum) strain, a lactic acid bacteria strain used in the present invention, exhibits excellent mycological characteristics such as having the ability to produce organic acids and high antioxidant activity.

[0025] The culture solution used in the present invention may include not only the culture itself in which the strain of the present invention is cultured, but also a culture filtrate obtained by physically filtering the supernatant containing the organic acid produced by the strain of the present invention during culture, after removing a certain amount of the strain (cells) of the present invention through centrifugation or the like.

[0026] Unless otherwise specified herein, a culture means a specific microorganism cultured in a culture medium or culture solution, and the culture may include a culture containing the specific microorganism or a sterilized culture in which the microorganism has been sterilized. The formulation of the culture or the concentrate of the culture is not limited, and for example, the formulation may be a liquid or a solid.

[0027] The above medium contains nutrients required by the microorganism to be cultured, i.e., the cultured organism, for culturing specific microorganisms. It may be a mixture in which substances for special purposes are additionally added. The above medium is also called a culture medium or culture solution, and is a concept encompassing natural media, synthetic media, or selective media.

[0028] In the present invention, the effective ingredient means a mixture of the Leuconostoc citrium strain or a culture solution thereof and the rosemary extract.

[0029] The mixing ratio of the above Leuconostoc citrium strain or its culture solution (LCFS) and the above rosemary extract (RE) is 1:10 to 10:1 (v / v), preferably 1.2:1 (v / v), but is not limited thereto as long as the purpose of the present invention can be achieved.

[0030] In addition, according to a preferred embodiment of the present invention, the rosemary extract of the present invention is an extract extracted with at least one solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.

[0031] As long as the purpose of the present invention can be achieved, the type and ratio of the solvent used for extracting the effective ingredient, rosemary, are not limited.

[0032] The above solvent is at least one selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof, and is not limited thereto as long as the purpose of the present invention can be achieved.

[0033] The alcohol having 1 to 4 carbon atoms may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, and isobutanol.

[0034] The above water, lower alcohol having 1 to 4 carbon atoms, or a mixed solution thereof can be used in an amount of 1 to 10 times the weight of the extraction target.

[0035] In addition, in the present invention, the concentration of the rosemary extract is 0.01 to 10 mg / mL, preferably 0.2185 (mg / mL), but is not limited thereto as long as the purpose of the present invention can be achieved.

[0036] The culture solution of the Leuconostoc citrium M8 strain and the rosemary extract not only effectively prevent or suppress discoloration of red-fleshed fish that occurs during distribution, but also contribute to maintaining freshness, thereby having the effect of improving the nutritional value, sensory quality, consumer preference, and storability of food. In addition, the culture solution of the Leuconostoc citrium M8 strain and the rosemary extract can be applied to various purposes requiring prevention of food discoloration.

[0037] The above composition includes a culture solution of a Leuconostoc citreum (L. citreum) strain as one of the active ingredients, but, as long as a person skilled in the art can achieve the purpose of the present invention, at least one selected from the group consisting of a lysate of the strain; a fermented product of the strain; a concentrate of the strain culture solution; a dried product of the culture solution and a fraction of the culture solution may be appropriately employed and used.

[0038] The fraction of the above culture medium is fractionated using at least one solvent selected from the group consisting of hexane, ethyl acetate, butanol, and water, or fractionated using a column using an adsorbent.

[0039] Adsorbents that can be used in the above column can be selected from the group including styrene-divinylbenzene, silica gel, Sephadex, RP-18, polyamide, Toyopearl and XAD resin.

[0040] The term “comprising as an active ingredient” as used herein means that the strain of the present invention or its culture medium is included in an amount sufficient to achieve its efficacy or activity.

[0041] The effective ingredients of the composition of the present invention are a lactic acid bacteria strain isolated from fermented anchovies or a culture filtrate thereof and a rosemary extract. Since there are no side effects to the subject even when administered in excessive amounts, the quantitative upper limit included in the composition of the present invention can be selected and implemented within an appropriate range by a person skilled in the art.

[0042] In addition, as long as the purpose of the present invention can be achieved, any red-fleshed fish known in the art can be used as the red-fleshed fish.

[0043] In this specification, red meat fish is used interchangeably with red meat fish or red meat fish.

[0044] Preferably, the red meat fish is at least one selected from the group consisting of tuna, sardine, mackerel, flounder, herring, and saury, and in one embodiment of the present invention, tuna is used, but is not limited thereto.

[0045]

[0046] In addition, according to another aspect of the present invention, the present invention provides a food preservative composition comprising, as active ingredients, a Leuconostoc citreum (L. citreum) strain or a culture solution thereof deposited under accession number KCTC15454BP; and a rosemary extract.

[0047] The above preservative composition means a substance used to prevent deterioration, rancidity or decay of a preservation object that may be infected or decayed by harmful microorganisms or decaying microorganisms by inhibiting or sterilizing the growth of harmful microorganisms or decaying microorganisms such as bacteria, mold and yeast, and to maintain the state of the preservation object, and preferably means one or more selected from the group consisting of foods, feeds, cosmetics and pharmaceuticals, and more preferably means a substance that can be used in foods.

[0048] The above food includes processed food products including canned food, fish products, tofu, starch, health supplements, seasoned foods, bakery and confectionery, dairy products including dairy products, pickled foods, fermented foods or beverages including fermented milk and fermented alcoholic beverages, etc., and the above feed may be, for example, livestock feed, and its use is not limited thereto, and may be solid or liquid.

[0049] The above preservative composition may additionally contain a conventional substance included in a preservative, except for containing the above-mentioned effective ingredient. The above preservative composition may contain 0.1 to 50 parts by weight or 0.5 to 10 parts by weight of the substance based on 100 parts by weight of the total composition.

[0050] The above preservative composition can not only improve the storage properties of foods or feeds in which it is used, but also enhance their nutritional value, texture, and flavor. Furthermore, the above preservative composition can be applied to a variety of applications requiring the control of various pathogenic bacteria, molds that are the main cause of food contamination, and microorganisms that cause spoilage.

[0051] The above preservative composition may include a natural preservative, a synthetic preservative, or an organic acid mixture, as needed, within the range that maintains the effect of the present invention.

[0052]

[0053] In addition, according to another aspect of the present invention, the present invention provides a method for preventing discoloration of red meat fish, comprising the following steps:

[0054] (a) a step of washing red meat fish and removing moisture; and

[0055] (b) A step of immersing the red-fleshed fish in a composition for preventing discoloration of red-fleshed fish, comprising a Leuconostoc citreum (L. citreum) strain deposited under accession number KCTC15454BP or a culture solution thereof and a rosemary extract.

[0056] The present invention is characterized by deriving optimal conditions using response surface methodology (RSM), which is the most effective method for exploring optimal conditions of a composition for preventing discoloration. This has the advantage of saving experimental costs and being able to simultaneously predict the minimum and maximum results through the interaction of each factor.

[0057] The above red meat fish can be de-visculated prior to washing, and can be soaked and dried immediately after washing. However, in order for the discoloration prevention composition to be well absorbed into the fish, it is preferable to thoroughly remove moisture. For this purpose, a method of pre-drying the washed red meat fish for 2 to 4 hours can be used. The pre-drying step can be performed at room temperature. In this case, the room temperature can be a typical fish processing temperature of 4 to 35°C.

[0058] The above drying can be appropriately adjusted by a person skilled in the art using any drying method known in the art.

[0059] Preferably, in the step (b), the immersion time is 1 second to 100 seconds, more preferably 1 second to 60 seconds, and even more preferably 5 seconds to 30 seconds, and can be repeated 1 to 10 times at intervals of 1 to 10 hours from the time of immersing the red-fleshed fish in the composition until the end. In addition, if immersion is performed for less than 1 second or for more than 100 seconds, discoloration prevention may not be effective.

[0060] In the above step (b), the red meat fish and the discoloration prevention composition are immersed in a ratio of 1:10 to 10:1 (w / v).

[0061] The red meat fish, which is a processed material, can be immersed in the discoloration prevention composition at a ratio of 1:10 to 10:1 (w / v), but is not limited thereto.

[0062] In one embodiment of the present invention, in order to maximize the discoloration prevention effect, the mixing ratio of the red-fleshed fish and the discoloration prevention composition was explored, and it was found that it was preferable to mix the red-fleshed fish and the discoloration prevention composition at a ratio of 1:3.31 (w / v).

[0063] If the mixing ratio of red meat fish and discoloration prevention composition is less than 1:10 (w / v) or exceeds 10:1 (w / v), discoloration is not effectively prevented.

[0064] In addition, in the step (b), the Leuconostoc citrium strain or its culture solution may be included in an amount of 0.01 to 100 wt% based on the total weight of the composition. The composition may be the Leuconostoc citrium strain of the present invention or its culture solution itself, or may be prepared by mixing the strain or its culture solution with other auxiliary materials and / or additives, or may be prepared by fermenting the strain or its culture solution after mixing it with other auxiliary materials and / or additives.

[0065] In one embodiment, the culture solution of the Leuconostoc citrium strain can be prepared by inoculating the strain into a microbial culture medium and using a microbial culture method known in the art (e.g., static culture).

[0066] For the preparation of the above culture solution, the culture may be carried out under anaerobic conditions in a liquid culture at 24 to 38°C, preferably 30 to 38°C, more preferably 32 to 38°C, for 6 to 36 hours, preferably 12 to 24 hours.

[0067] In one embodiment of the present invention, the present invention has established for the first time an optimal condition for exhibiting an excellent effect of preventing discoloration and enhancing sensory and preference when treating a mixture of a culture filtrate of a Leuconostoc citreum (L. citreum) strain, which is an effective ingredient of the present invention, and a rosemary extract to a target red meat fish.

[0068] In addition, since the present invention does not go through a heat treatment step, it can process aquatic products in their original form, and has the advantage of being applicable to most aquatic products and being able to react in a liquid-liquid and liquid-solid manner.

[0069] Since the method of the present invention utilizes the composition for preventing discoloration of red-fleshed fish of the present invention described above, description of common contents between the two is omitted to avoid excessive complexity of the present specification.

[0070]

[0071] In conclusion, the present invention is expected to present a new method for preventing discoloration of various red-fleshed fish and applying lactic acid bacteria to the aquatic food industry by optimizing a bio-treatment technology that can effectively suppress discoloration of red-fleshed fish and enhance palatability.

[0072] The present invention uses response surface methodology (RSM) to establish optimal conditions for preventing discoloration of red-fleshed fish, thereby establishing optimal treatment conditions for a novel Leuconostoc citreum (L. citreum) strain or a culture solution thereof, thereby improving palatability and thereby enhancing productivity, and providing highly nutritious aquatic products with maintained taste, nutrition, and texture.

[0073] Figure 1 shows a 3D response surface model of the a* value of the rosemary extract concentration and lactic acid bacteria supernatant of the present invention according to the rosemary extract.

[0074] Figure 2 shows a 3D response surface model of the a* value of the meat surface according to the concentration of the rosemary extract of the present invention and the immersion time.

[0075] Figure 3 shows a 3D response surface model of the a* value of the meat surface according to the concentration and immersion ratio of the rosemary extract of the present invention.

[0076] Figure 4 shows a 3D response surface model of the lactic acid bacteria supernatant of the present invention: rosemary extract and the meat surface a* value according to the immersion time.

[0077] Figure 5 shows a 3D response surface model of the lactic acid bacteria supernatant of the present invention: a rosemary extract and a meat surface a* value according to the immersion ratio.

[0078] Figure 6 shows a 3D response surface model of the a* value of the meat surface according to the immersion time and immersion ratio of the present invention.

[0079] Figure 7 shows a 3D response surface model of the crude extract a* value according to the concentration of rosemary extract and lactic acid bacteria supernatant of the present invention: rosemary extract.

[0080] Figure 8 shows a 3D response surface model of the a* value of the crude extract according to the concentration and immersion time of the rosemary extract of the present invention.

[0081] Figure 9 shows a 3D response surface model of the a* value of the crude extract according to the concentration and immersion ratio of the rosemary extract of the present invention.

[0082] Figure 10 shows a 3D response surface model of the crude extract a* value according to the lactic acid bacteria supernatant of the present invention: rosemary extract and immersion time.

[0083] Figure 11 shows a 3D response surface model of the crude extract a* value according to the lactic acid bacteria supernatant of the present invention: rosemary extract and immersion ratio.

[0084] Figure 12 shows a 3D response surface model of crude extract a* value according to the immersion time and immersion ratio of the present invention.

[0085] Figure 13 shows a 3D response surface model of the concentration of rosemary extract and lactic acid bacteria supernatant of the present invention: oxy-Mb content according to rosemary extract.

[0086] Figure 14 shows a 3D response surface model of oxy-Mb content according to the concentration and immersion time of the rosemary extract of the present invention.

[0087] Figure 15 shows a 3D response surface model of oxy-Mb content according to the concentration and immersion ratio of the rosemary extract of the present invention.

[0088] Figure 16 shows a 3D response surface model of the lactic acid bacteria supernatant of the present invention: the rosemary extract and the oxy-Mb content according to the immersion time.

[0089] Figure 17 shows a 3D response surface model of the lactic acid bacteria supernatant of the present invention: oxy-Mb content according to rosemary extract and immersion ratio.

[0090] Figure 18 shows a 3D response surface model of oxy-Mb content according to the immersion time and immersion ratio of the present invention.

[0091] Figure 19 shows a 3D response surface model of the concentration of rosemary extract and the lactic acid bacteria supernatant of the present invention: the degree of redness of the sensory evaluation according to the rosemary extract.

[0092] Figure 20 shows a 3D response surface model of the sensory evaluation redness according to the concentration and immersion time of the rosemary extract of the present invention.

[0093] Figure 21 shows a 3D response surface model of the sensory evaluation redness according to the concentration and immersion ratio of the rosemary extract of the present invention.

[0094] Figure 22 shows a 3D response surface model of the sensory evaluation redness according to the lactic acid bacteria supernatant of the present invention: rosemary extract and immersion time.

[0095] Figure 23 shows a 3D response surface model of the sensory evaluation redness according to the lactic acid bacteria supernatant of the present invention: rosemary extract and immersion ratio.

[0096] Figure 24 shows a 3D response surface model of the sensory evaluation redness according to the immersion time and immersion ratio of the present invention.

[0097] Figure 25 shows a 3D response surface model of the overall preference of the sensory evaluation according to the concentration of the rosemary extract and the lactic acid bacteria supernatant of the present invention: rosemary extract.

[0098] Figure 26 shows a 3D response surface model of the overall preference of the sensory evaluation according to the concentration and immersion time of the rosemary extract of the present invention.

[0099] Figure 27 shows a 3D response surface model of the overall preference of the sensory evaluation according to the concentration and immersion ratio of the rosemary extract of the present invention.

[0100] Figure 28 shows a 3D response surface model of the overall preference of the sensory evaluation according to the lactic acid bacteria supernatant of the present invention: rosemary extract and immersion time.

[0101] Figure 29 shows a 3D response surface model of the overall preference of the sensory evaluation according to the lactic acid bacteria supernatant of the present invention: rosemary extract and immersion ratio.

[0102] Figure 30 shows a 3D response surface model of the overall preference of the sensory evaluation according to the immersion time and immersion ratio of the present invention.

[0103] Figure 31 shows the general bacterial count (Figure 31a) and VBN (Figure 31b) levels of tuna meat treated with the optimal conditions derived from the present invention during the 10℃ storage period.

[0104] The following examples are provided solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

[0105]

[0106] Example 1. Isolation and selection of the lactic acid bacteria strain (Leuconostoc citreumM8) of the present invention

[0107] The present inventors isolated Leuconostoc citreum M8, a lactic acid bacteria strain, from a traditional domestic fermented seafood food (fermented hairtail fish) as follows:

[0108] The diluted salted anchovy was inoculated onto Bromo cresol purple agar (plate count agar with 0.006% bromo cresol purple), incubated at 30°C for 48 hours, and colonies that turned yellow were selected. A total of 12 colonies were isolated, and the strain with the highest antioxidant activity was selected.

[0109] Additionally, to identify the finally selected strain, the 16S rRNA base sequence of the isolated strain was determined and compared with known strains.

[0110] 16S rRNA sequencing of the isolate was performed by isolating the chromosomal DNA of the isolate and amplifying it by PCR using primers 27F (SEQ ID NO: 2; 5'-AGAGTTTGATCCTGGCTCAG -3') and 1492R (SEQ ID NO: 3; 5'-TACGGYTACCTTGTTACGTACTT-3').

[0111] The DNA base sequence encoding 16S rRNA of the above strain (16S rDNA; sequence number 1) was analyzed for phylogenetic relationships by examining the homology with the 16S rRNA base sequences of strains registered in the GeneBank database using the Basic local alignment search tool (Blast, NCBI), and as a result, it was confirmed that the selected isolated strain of the present invention is a lactic acid bacteria strain belonging to Leuconostoc citreum.

[0112] In addition, the lactic acid bacteria according to this specification were identified as strains closely related to the public strain Leuconostoc citrium (Genbank accession No. LC09622.1), but were confirmed to be different strains with sequence differences from the aforementioned strains.

[0113] Accordingly, the inventors of the present invention named the isolated Leuconostoc citreum strain as M8 and deposited it with the Korea Research Institute of Bioscience and Biotechnology (KCTC) Biological Resource Center on May 25, 2023, and were assigned the accession number KCTC15454BP.

[0114] Accordingly, the strain of the present invention, Leuconostoc citreum M8, is a novel strain that has been found to have differences in base sequence when compared to the test strain, and since the strain exhibits mycological characteristics of producing excellent organic acids and exhibiting high antioxidant capacity, it can achieve effects not only through the strain itself but also through its culture metabolites.

[0115]

[0116] Example 2. Derivation of optimal treatment conditions for lactic acid bacteria culture solution (cell-free supernatant) and rosemary extract using RSM.

[0117] The present inventors analyzed the discoloration prevention ability of red fish meat by a mixture of Leuconostoc citreum (L. citreum) M8 culture solution, a strain isolated in Example 1, and rosemary extract, and applied RSM (Response surface methodology) as a statistical experimental method to derive the optimization of immersion conditions.

[0118] In addition, tuna was used as a representative red meat fish, and the discoloration prevention effect was measured through the redness value of the tuna meat surface, the redness value of the crude extract, the oxy-myoglobin content, the met-myoglobin content, the degree of redness in the sensory evaluation, and the overall preference.

[0119] At this time, in order to derive an optimized model that can prevent or suppress discoloration of red fish meat using RSM, a single factor experiment was performed on the conditions of the mixing ratio of lactic acid bacteria culture solution and rosemary extract (v / v), immersion ratio of tuna and treatment solution (w / v), immersion time (min), and rosemary extract concentration (mg / mL), and Box-Behnken Design (BBD) among RSM was used to analyze the optimal conditions for discoloration suppression through lactic acid bacteria and rosemary extract treatment.

[0120] More specifically, Leuconostoc citreum M8 was inoculated into MRS broth adjusted to 10% glucose and cultured at 30℃ for 24 hours under conditions of 9 log CFU / mL cell count, centrifuged (10,000Хg, 20 min, 4℃), and only the supernatant was collected and filtered using a 0.22 μm membrane filter to prepare a cell-free culture medium and used in the experiment. Tuna was thawed from frozen tuna, marinated according to each experimental condition, dehydrated for an additional 1 minute, and stored at 10℃ for 1 hour to check changes in redness and myoglobin content, and then sensory evaluation was conducted.

[0121] As a result, in order to derive the environmentally friendly optimal treatment conditions that can effectively prevent discoloration of tuna by treating tuna with cell-free culture medium and rosemary extract, the experiment was designed according to BBD among RSM and the independent variable (X i ) dependent variable (Y) according to the concentration of rosemary extract (X1), the ratio of lactic acid bacteria cell-free supernatant and rosemary extract (X2), the steeping time (X3) and the steeping ratio (X4) i ) the flesh surface a * value (Y1), crude extract a * The results of the sensory evaluation items, such as value (Y2), oxy-myoglobin content (Y3), redness (Y4), and overall preference (Y5), were analyzed.

[0122] More specifically, the experimental levels and ranges of independent variables designed for model optimization are shown in Table 1, the second-order polynomial model equation is shown in Table 2, and the ANOVA for variance and adequacy analysis of the second-order model is shown in Table 3.

[0123] In Table 1, to optimize the immersion conditions, they were coded into three levels of -1, 0, and +1, and the ranges of independent variables were set as immersion times of 10, 15, and 25 seconds, immersion ratios of 1:2, 1:3, and 1:4 (w / v), lactic acid bacteria supernatant and rosemary extract ratios of 2:1, 1:1, and 1:2 (v / v), and rosemary extract concentrations of 0.01, 0.03, and 0.05 (mg / mL).

[0124]

[0125] [Table 1]

[0126]

[0127]

[0128] The results of the experiment according to the conditions presented in RSM and the quadratic equation derived for each dependent variable are as shown in Table 2.

[0129]

[0130] [Table 2]

[0131]

[0132]

[0133] To verify the significance of the model presented in this invention, ANOVA analysis was conducted, and the results are shown in Table 3. The models for all dependent variables developed in this study were found to be significant.

[0134]

[0135] [Table 3]

[0136]

[0137]

[0138] The optimal conditions for treatment of a mixture of M8 (cell-free) culture medium (supernatant) and rosemary extract using RSM are shown in Table 4 below.

[0139]

[0140] [Table 4]

[0141]

[0142]

[0143] In addition, to verify the optimal conditions predicted through modeling using RSM, the results of a verification test for preventing discoloration of tuna immersed in a mixture of lactic acid bacteria cell-free culture medium and rosemary extract according to the suggested optimal conditions are shown in Table 5 below.

[0144] This indicates that the model optimized using RSM is appropriate, as the deviation between the actual and predicted values ​​in all variables is less than 5% (p< 0.05).

[0145]

[0146] [Table 5]

[0147]

[0148]

[0149] As a result, the optimal conditions modeled according to the present invention were derived as an immersion time of 13 seconds, an immersion ratio of a mixture of cell-free culture solution and rosemary extract for tuna of 1:3.31 (w / v), a ratio of cell-free culture solution and rosemary extract (LCFS: RE) of 1.2:1 (v / v), and a concentration of rosemary extract of 0.027 (mg / mL), and it was confirmed that this was a statistically significant model.

[0150] Therefore, this proves that the optimal treatment conditions of the Leuconostoc citreum (L. citreum) M8 culture solution (L. citreumM8 cell-free culture) and rosemary extract of the present invention, derived based on the RSM analysis of the present invention, can excellently prevent discoloration of red-fleshed fish.

[0151]

[0152] Example 3. Confirmation of quality changes during storage of tuna meat using the optimal conditions of the present invention.

[0153] The inventors of the present invention measured the changes in the number of general bacteria and volatile basic nitrogen (VBN) during the storage period at 10°C to determine whether the quality of tuna meat was affected when treated according to the optimal treatment conditions of the mixture of lactic acid bacteria strain and rosemary extract derived from the present invention.

[0154] As a result, as shown in Fig. 31, compared to the untreated control group, the group treated with the optimal conditions of the mixture of Leuconostoc citreum M8 of the present invention and rosemary extract showed a significantly lower increase in the number of general bacteria and VBB content during the storage period. In other words, this means that the group treated with the optimal conditions of the mixture of the lactic acid bacteria strain of the present invention and rosemary extract not only exhibits antibacterial activity without affecting the quality of red fish meat, but also maintains freshness.

[0155]

[0156] In summary, the present invention has revealed that when the lactic acid bacteria strain Leuconostoc citreum (L. citreum) M8 culture solution and rosemary extract of the present invention are treated under optimal conditions to red-fleshed fish, the overall preference can be improved through color difference analysis and sensory evaluation, which have a significant impact on consumer preference. Therefore, the method for preventing discoloration using the lactic acid bacteria strain culture solution and rosemary extract of the present invention can maintain the quality of red-fleshed fish and satisfy consumer preference and sensory needs, and thus can have high marketability in the aquatic products processing industry and the environmental industry.

[0157]

[0158] Although the present invention has been described with reference to the preferred embodiments mentioned above, various modifications and variations are possible without departing from the spirit and scope of the invention. Furthermore, the appended claims encompass such modifications and variations as fall within the spirit of the invention.

[0159]

[0160] [Accession number]

[0161] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0162] Accession number: KCTC15454BP

[0163] Date of acceptance: 20230525

[0164]

[0165] [Correction pursuant to Rule 91, November 15, 2024]

Claims

1. A composition for preventing discoloration of red-fleshed fish, comprising as active ingredients a Leuconostoc citreum (L. citreum) strain or a culture solution thereof deposited under the deposit number KCTC15454BP; and a rosemary extract.

2. In paragraph 1, The above Leuconostoc citrium strain is characterized by having 16S rDNA containing the base sequence of sequence number 1. A composition for preventing discoloration of red-fleshed fish.

3. In paragraph 1, The above Leuconostoc citrium strain or its culture solution and the rosemary extract are characterized in that they are mixed in a ratio of 1:10 to 10:1 (v / v). A composition for preventing discoloration of red-fleshed fish.

4. In paragraph 1, The above culture medium is characterized in that it is a culture medium in which a strain is cultured or a culture filtrate obtained by physically filtering the culture medium. A composition for preventing discoloration of red-fleshed fish.

5. In paragraph 1, The above rosemary extract is characterized in that it is an extract extracted with at least one solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof. A composition for preventing discoloration of red-fleshed fish.

6. In paragraph 5, The concentration of the rosemary extract is characterized by being 0.01 to 10 mg / mL. A composition for preventing discoloration of red-fleshed fish.

7. In paragraph 1, The above red meat fish is characterized in that it is a fish selected from the group consisting of tuna, sardine, mackerel, yellowtail, herring and saury. A composition for preventing discoloration of red-fleshed fish.

8. A food preservative composition comprising Leuconostoc citreum (L. citreum) strain or a culture solution thereof deposited under the deposit number KCTC15454BP; and rosemary extract; as active ingredients.

9. Method for preventing discoloration of red-fleshed fish, comprising the following steps: (a) a step of washing red meat fish and removing moisture; and (b) A step of immersing the red-fleshed fish in a composition for preventing discoloration of red-fleshed fish, comprising a Leuconostoc citreum (L. citreum) strain deposited under accession number KCTC15454BP or a culture solution thereof and a rosemary extract.

10. In paragraph 9, In the above step (b), the immersion time is characterized by being 1 second to 100 seconds. Method for preventing discoloration of red meat fish.

11. In paragraph 9, In the step (b), the red meat fish and the composition for preventing discoloration of the red meat fish are immersed in a ratio of 1:10 to 10:1 (w / v). Method for preventing discoloration of red meat fish.

Images