Novel isolated strain of leuconostoc mesenteroides, and use thereof

Abstract

The present invention relates to a novel isolated strain of Leuconostoc mesenteroides and a use thereof. According to the present invention, in order to derive optimal conditions for preventing crustacean discoloration, the optimum treatment conditions of the novel Leuconostoc mesenteroides strain or a culture medium thereof, of the present invention, have been established using response surface methodology (RSM). Therefore, palatability and freshness are maintained so that productivity can be improved, and a high-nutrition marine product with maintained taste, nutrition, and texture can be provided.

Description

New isolate of Leuconostoc mesenteroides and its use

[0001] The present invention relates to a novel isolate of Leuconostoc mesenteroides and its use.

[0002] Recently, seafood has been gaining attention as a healthy protein source, leading to an increase in supply and consumption. Among these, crustaceans such as shrimp and crab are highly preferred fish in the domestic consumer market, and their supply and consumption are continuously increasing. However, in the case of shrimp, a popular food among modern consumers, discoloration, or melanosis, occurs frequently in areas such as the head and thorax, tail, and joints due to melanin, a water-soluble substance (tyrosine, dihydroxy phenylalanine), which is a protein breakdown product by bacteria, caused by temperature changes during storage and distribution. This melanin is produced by tyrosinase, an enzyme that oxidizes melanin in the presence of oxygen and ultraviolet rays. While melanosis does not directly harm consumers, it reduces commercial value and consumer desire due to its appearance.

[0003] As a preservative to prevent the decline in commercial value of these crustaceans, sulfite compounds containing sulfur dioxide have been confirmed to be used for shrimp and crabs. Sulfite compounds have antioxidant properties and can suppress deterioration such as blackening of the shrimp's head, whiskers, or shell, or whitening and loss of gloss of the shell. Therefore, it is reported that most frozen or refrigerated shrimp sold in the market have been treated with preservatives containing sulfurite compounds such as sulfites or pyrosulfites to prevent deterioration.

[0004] However, sulfite compounds are powerful reducing agents, and are used not only as food additives for preservatives but also as bleaching agents and antioxidants. Therefore, caution is required when using them for food. Furthermore, shrimp treated with preservatives containing sulfite compounds can experience discoloration of the shell and muscle, loss of luster, and off-flavors, as well as unpleasant or bitter flavors.

[0005] Furthermore, prior research on preventing blackening in shrimp has utilized natural antioxidants. However, natural antioxidants typically require high concentrations to be effective in preventing blackening when applied to food, which can cause off-flavors and odors. Furthermore, synthetic additives have been used to prevent blackening in crustaceans, but these are strictly prohibited due to side effects.

[0006] Therefore, it is necessary to develop a method using effective natural materials that can overcome these problems and prevent or prevent discoloration by suppressing melanin production that occurs during the distribution process of crustaceans.

[0007] 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.

[0008] 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.

[0009] Therefore, if these lactic acid bacteria and / or their culture solution are used as natural materials that can prevent discoloration (blackening) of crustaceans, it is expected that the quality of crustaceans during processing and distribution will be improved, the sales period will be extended, and high marketability will be achieved.

[0010] Under these circumstances, the present inventors sought to develop a method for suppressing discoloration of crustaceans during distribution. As a result, a novel lactic acid bacteria strain, Leuconostoc mesenteroides M10, with excellent antioxidant activity and tyrosinase inhibition was isolated and identified from aquatic products. Through RSM analysis, the optimal mixing conditions for the strain culture and rosemary extract for preventing discoloration of crustaceans were determined. Furthermore, statistical significance was demonstrated to demonstrate that this method not only satisfies consumer sensory and taste preferences but also maintains freshness, thereby completing the present invention.

[0011] Accordingly, one object of the present invention is to provide a novel Leuconostoc mesenteroides strain or a culture medium thereof having an effect of preventing discoloration of crustaceans, deposited under the accession number KCTC15455BP.

[0012] In addition, another object of the present invention is to provide a composition for preventing discoloration of crustaceans, comprising rosemary extract and the strain or a culture solution thereof as active ingredients.

[0013] In addition, another object of the present invention is to provide a food preservative composition comprising a rosemary extract and the strain or a culture solution thereof as active ingredients.

[0014] In addition, another object of the present invention is to provide a method for preventing discoloration of crustaceans.

[0015]

[0016] Other objects and advantages of the present invention will become more apparent from the detailed description of the invention and the claims below.

[0017] 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.

[0018] 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.

[0019]

[0020] Hereinafter, the present invention will be described in detail.

[0021]

[0022] According to one aspect of the present invention, the present invention provides a strain of Leuconostoc mesenteroides (L. mesenteroides) or a culture solution thereof having an effect of preventing discoloration of crustaceans, deposited under the accession number KCTC15455BP.

[0023] The Leuconostoc mesenteroides (L. mesenteroides) strain of the present invention is a novel Leuconostoc mesenteroides strain isolated from salted hairtail squid, and was identified by the inventors as a lactic acid bacteria strain. It was named M10 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 KCTC15455BP.

[0024] Isolation and cultivation of the above Leuconostoc mesenteroides strain can be performed by any method known in the art.

[0025] Additionally, the Leuconostoc mesenteroides strain has a 16S rDNA comprising the base sequence of sequence number 1.

[0026] The novel lactic acid bacteria strain of the present invention, Leuconostoc mesenteroides (L. mesenteroides), exhibits excellent mycological characteristics, including the ability to produce organic acids, and excellent antioxidant and tyrosinase inhibitory abilities.

[0027] 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.

[0028] 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.

[0029] 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.

[0030]

[0031] In addition, according to another aspect of the present invention, the present invention provides a composition for preventing discoloration of crustaceans, comprising as active ingredients a Leuconostoc mesenteroides (L. mesenteroides) strain or a culture solution thereof having a discoloration preventing effect on crustaceans, deposited under accession number KCTC15455BP; and a rosemary extract.

[0032] Additionally, in the present invention, the effective ingredient means a mixture of the Leuconostoc mesenteroides strain or a culture solution thereof and the rosemary extract.

[0033] The mixing ratio of the above Leuconostoc mesenteroides strain or its culture medium and the above rosemary extract is mixed in a ratio of 1:10 to 10:1 (v / v), preferably 1:1.9 (v / v), but is not limited thereto as long as the purpose of the present invention can be achieved.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] The alcohol having 1 to 4 carbon atoms may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, and isobutanol.

[0038] 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.

[0039] 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.

[0040] The culture solution of the Leuconostoc mesenteroides M10 strain and the rosemary extract not only effectively prevent or suppress discoloration of crustaceans 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 mesenteroides M10 strain and the rosemary extract can be applied to various purposes requiring prevention of food discoloration.

[0041] The above composition includes a culture medium of a strain of Leuconostoc mesenteroides (L. mesenteroides) 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 medium; a dried product of the culture medium and a fraction of the culture medium may be appropriately employed and used.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] The effective ingredients of the composition of the present invention are a lactic acid bacteria strain isolated from fermented anchovies or a culture solution 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.

[0046] In addition, as long as the purpose of the present invention can be achieved, any crustacean known in the art can be used.

[0047] Preferably, the crustacean is at least one species selected from the group consisting of shrimp, crayfish, lobster, and krill, and in one embodiment of the present invention, shrimp is used, but is not limited thereto.

[0048]

[0049] In addition, according to another aspect of the present invention, the present invention provides a food preservative composition comprising, as active ingredients, a Leuconostoc mesenteroides (L. mesenteroides) strain or a culture solution thereof, having a discoloration-preventing effect on crustaceans, deposited under the accession number KCTC15455BP; and a rosemary extract.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055]

[0056] In addition, according to another aspect of the present invention, the present invention provides a method for preventing discoloration of crustaceans, comprising the following steps:

[0057] (a) a step of washing crustaceans and removing moisture; and

[0058] (b) a step of immersing the crustacean in a composition for preventing discoloration of crustaceans, comprising a Leuconostoc mesenteroides (L. mesenteroides) strain or a culture solution thereof having a discoloration preventing effect of crustaceans, deposited under the accession number KCTC15455BP, and a rosemary extract.

[0059] 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.

[0060] The above crustaceans can be de-veined prior to washing, and can be soaked and dried immediately after washing. However, in order for the anti-discoloration 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 crustaceans 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.

[0061] The above drying can be appropriately adjusted by a person skilled in the art using any drying method known in the art.

[0062] Preferably, the immersion time in step (b) is 1 minute to 100 minutes, more preferably 1 minute to 50 minutes, even more preferably 5 minutes to 10 minutes, and most preferably 7 minutes 38 seconds. The immersion of the crustacean in the composition can be repeated 1 to 10 times at intervals of 3 to 10 hours from the time of immersion until completion. In addition, if the immersion is performed for less than 1 minute or for more than 100 minutes, discoloration may not be effectively prevented.

[0063] In the above step (b), the crustacean and the discoloration prevention composition are immersed in a ratio of 1:10 to 10:1 (w / w).

[0064] The processed material, crustaceans, can be immersed in the discoloration prevention composition at a ratio of 1:10 to 10:1 (w / v), but is not limited thereto.

[0065] In one embodiment of the present invention, in order to maximize the discoloration prevention effect, the mixing ratio of the crustacean and the discoloration prevention composition was explored, and it was found that it was preferable to mix the crustacean and the discoloration prevention composition at a ratio of 1:3.3 (w / v).

[0066] If the mixing ratio of the crustacean and the discoloration prevention composition is less than 1:10 (w / v) or exceeds 10:1 (w / v), discoloration is not effectively prevented.

[0067] In the step (b), the Leuconostoc mesenteroides 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 mesenteroides 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.

[0068] In one embodiment, the culture solution of the Leuconostoc mesenteroides 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).

[0069] 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.

[0070] In one embodiment of the present invention, the present invention has established for the first time optimal conditions for preventing discoloration and maintaining freshness when treating a novel lactic acid bacteria strain, Leuconostoc mesenteroides (L. mesenteroides) or a culture solution thereof and a rosemary extract to a target crustacean.

[0071] 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.

[0072] Since the method of the present invention utilizes the composition for preventing discoloration of crustaceans of the present invention described above, description of common contents between the two is omitted to avoid excessive complexity of the present specification.

[0073]

[0074] In conclusion, the present invention is expected to present a new method for preventing discoloration of various crustaceans in aquatic products and for applying lactic acid bacteria to the aquatic food industry by optimizing a bio-treatment technology that can effectively suppress discoloration of crustaceans while maintaining their freshness.

[0075] The present invention uses response surface methodology (RSM) to establish optimal treatment conditions for a novel Leuconostoc mesenteroides (L. mesenteroides) strain or a culture medium thereof in order to derive optimal conditions for preventing discoloration of crustaceans, thereby maintaining palatability and freshness and thus improving productivity, and providing highly nutritious aquatic products with maintained taste, nutrition, and texture.

[0076] Figure 1 shows a 3D response surface model of Lightness according to the immersion time and immersion ratio of the method of the present invention.

[0077] Figure 2 shows a 3D response surface model of Lightness according to the immersion time and the ratio of lactic acid bacteria and rosemary in the method of the present invention.

[0078] Figure 3 shows a 3D response surface model of Lightness according to the immersion time and rosemary concentration of the method of the present invention.

[0079] Figure 4 shows a 3D response surface model of Lightness according to the immersion ratio and the ratio of lactic acid bacteria and rosemary of the method of the present invention.

[0080] Figure 5 shows a 3D response surface model of Lightness according to the immersion ratio and rosemary concentration of the method of the present invention.

[0081] Figure 6 shows a 3D response surface model of Lightness according to the ratio of lactic acid bacteria and rosemary and rosemary concentration of the method of the present invention.

[0082] Figure 7 shows a 3D response surface model of blackening evaluation according to the immersion time and immersion ratio of the method of the present invention.

[0083] Figure 8 shows a 3D response surface model of the blackening evaluation according to the immersion time and the ratio of lactic acid bacteria and rosemary of the method of the present invention.

[0084] Figure 9 shows a 3D response surface model of blackening evaluation according to the immersion time and rosemary concentration of the method of the present invention.

[0085] Figure 10 shows a 3D response surface model of blackening evaluation according to the immersion ratio and the ratio of lactic acid bacteria and rosemary of the method of the present invention.

[0086] Figure 11 shows a 3D response surface model of blackening evaluation according to the immersion ratio and rosemary concentration of the method of the present invention.

[0087] Figure 12 shows a 3D response surface model of the blackening evaluation according to the ratio of lactic acid bacteria and rosemary and rosemary concentration of the method of the present invention.

[0088] Figures 13a to 13c show the general bacterial count (a), VBN (b), and lightness value (c) levels of shrimp treated with the optimal conditions derived from the present invention during the 4℃ storage period.

[0089] 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.

[0090]

[0091] Example 1. Isolation and selection of a novel lactic acid bacteria strain (Leuconostoc mesenteroides M10) of the present invention

[0092] The present inventors isolated a novel lactic acid bacteria strain, Leuconostoc mesenteroides M10, from a traditional domestic fermented seafood food (fermented hairtail fish) as follows:

[0093] The fermented anchovy sauce was diluted and inoculated onto Bromo cresol purple agar (plate count agar with 0.006% bromo cresol purple). After incubation at 30°C for 48 hours, colonies that turned yellow were selected. A total of 12 colonies were isolated, and the strain with the highest antioxidant and tyrosinase inhibitory activities was selected.

[0094] Additionally, to identify the finally selected strain, the 16S rRNA base sequence of the isolated strain was determined and compared with known strains.

[0095] 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').

[0096] 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 mesenteroides.

[0097] In addition, the lactic acid bacteria according to this specification were identified as strains closely related to the public strain Leuconostoc mesenteroides (Genbank accession No. NR_118557.1), but were confirmed to be different strains with sequence differences from the aforementioned strains.

[0098] Accordingly, the inventors of the present invention named the isolated Leuconostoc mesenteroides strain as M10 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 KCTC15455BP.

[0099] Accordingly, the strain of the present invention, Leuconostoc mesenteroides M10, is a novel strain that has been found to have differences in base sequence compared to the test strain, and the strain exhibits mycological characteristics such as excellent production of organic acids and high antioxidant activity and excellent tyrosinase inhibition ability, so that it can achieve effects not only as the strain itself but also as its culture metabolites.

[0100]

[0101] Example 2. Derivation of optimal treatment conditions for lactic acid bacteria culture solution (cell-free supernatant) and rosemary extract using RSM.

[0102] The present inventors analyzed the discoloration prevention ability of a mixture of a culture medium of Leuconostoc mesenteroides (L. mesenteroides) M10, a novel strain of the present invention isolated in Example 1, and a rosemary extract against crustaceans, and applied RSM (Response surface methodology) as a statistical experimental method to derive optimization of immersion conditions.

[0103] At this time, in order to derive an optimized model that can prevent or prevent discoloration of crustaceans 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), the immersion ratio of shrimp and treatment solution (w / v), the immersion time (min), and the rosemary extract concentration (mg / mL). In addition, Box-Behnken Design (BBD) among RSMs was used to analyze the optimal conditions for discoloration inhibition through treatment with lactic acid bacteria and rosemary extract.

[0104] More specifically, Leuconostoc mesenteroides M10 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. Frozen shrimp were thawed, marinated according to each experimental condition, dehydrated for an additional 3 minutes, stored at 10℃ for 24 hours, and changes in lightness were checked and an evaluation of blackening was conducted.

[0105] As a result, the experiment was designed according to BBD among RSM to derive the optimal treatment conditions that can effectively prevent discoloration of shrimp by treating shrimp with cell-free culture medium and rosemary extract, and the independent variable (X i ) dependent variable (Y) according to the immersion time (X1), immersion ratio (X2), ratio of lactic acid bacteria cell-free supernatant and rosemary extract (X3), and rosemary concentration (X4) i ) The results of Lightness (Y1) and Blackness Evaluation (Y2) were analyzed.

[0106] 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.

[0107] In Table 1, to optimize the immersion conditions of shrimp, the values ​​were coded into three levels of -1, 0, and +1, and the ranges of independent variables were set as immersion times of 5, 7, and 10 minutes, immersion ratios of 1:2, 1:3, and 1:4 (w / v), lactic acid bacteria and rosemary ratios of 2:1, 1:1, and 1:2 (v / v), and rosemary concentrations of 0.15, 0.2, and 0.25 (mg / mL).

[0108]

[0109] [Table 1]

[0110]

[0111]

[0112] 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.

[0113]

[0114] [Table 2]

[0115]

[0116]

[0117] 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.

[0118]

[0119] [Table 3]

[0120]

[0121]

[0122] The optimal conditions for treatment of a mixture of M10 (cell-free) culture medium (supernatant) and rosemary extract using RSM are shown in Table 4 below.

[0123]

[0124] [Table 4]

[0125]

[0126]

[0127] In addition, to verify the optimal conditions predicted through modeling using RSM, the results of a verification test on the prevention of discoloration of shrimp 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.

[0128] 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).

[0129]

[0130] [Table 5]

[0131]

[0132]

[0133] As a result, the optimal conditions modeled according to the present invention were derived as an immersion time of 7 minutes and 38 seconds, an immersion ratio of a mixture of cell-free culture medium and rosemary extract to shrimp of 1:3.3 (w / v), a ratio of cell-free culture medium and rosemary extract of 1:1.9 (v / v), and a concentration of rosemary extract of 0.2185 (mg / mL), and it was confirmed that this was a statistically significant model.

[0134] Therefore, this proves that the optimal treatment conditions of the Leuconostoc mesenteroides (L. mesenteroides) M10 culture solution (L. mesenteroidesM10 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 crustaceans.

[0135]

[0136] Example 3. Confirmation of quality changes during the storage period of shrimp using the optimal conditions of the present invention.

[0137] The inventors of the present invention measured changes in the general bacterial count, volatile basic nitrogen (VBN), and surface L* (lightness) value during storage at 4°C to determine whether the lactic acid bacteria strain and rosemary extract derived in the present invention affect the quality when treated on shrimp according to the optimal treatment conditions.

[0138] As a result, as shown in Figures 13a and 13b, compared to the untreated control group, the optimal condition treatment group of the present invention with Leuconostoc mesenteroides M10 and rosemary extract showed a significantly lower increase in the number of general bacteria and VBN content during the storage period. In other words, this proves that the optimal condition treatment group of the present invention not only exhibits antibacterial activity but also maintains freshness without affecting the quality of crustaceans.

[0139] In addition, as shown in Fig. 13c, compared to the control group, the optimal condition treatment group of the present invention with Leuconostoc mesenteroides M10 and rosemary extract showed a significantly lower decrease in the Lightness value during the storage period. In other words, this proves that the optimal condition treatment group of the present invention can prevent blackening of crustaceans.

[0140]

[0141] In summary, in the present invention, when the lactic acid bacteria strain Leuconostoc mesenteroides (L. mesenteroides) M10 culture solution and rosemary extract of the present invention are treated under optimal conditions for crustaceans, the level of blackening is low through Lightness analysis, which has a great influence on consumer preference, and it is possible to maintain freshness through a freshness judgment method. Therefore, the method for preventing discoloration using the lactic acid bacteria strain culture solution and rosemary extract of the present invention not only maintains the quality of crustaceans to satisfy consumer preference and sensory needs, but also extends the distribution period and edible period, and thus can have high marketability in the aquatic products processing industry and the environmental industry.

[0142]

[0143] 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.

[0144]

[0145] [Accession number]

[0146] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0147] Accession number: KCTC15455BP

[0148] Date of acceptance: 20230525

[0149] [Correction pursuant to Rule 91, November 15, 2024]

Claims

1. A strain of Leuconostoc mesenteroides or a culture solution thereof having an effect of preventing discoloration of crustaceans, deposited under the deposit number KCTC15455BP.

2. In paragraph 1, The above Leuconostoc mesenteroides strain is characterized by having 16S rDNA comprising the base sequence of sequence number 1. A strain of Leuconostoc mesenteroides (L. mesenterodies) or a culture solution thereof, deposited under the accession number KCTC15455BP, having an effect of preventing discoloration of crustaceans.

3. A composition for preventing discoloration of crustaceans, comprising as active ingredients a strain of Leuconostoc mesenteroides (L. mesenterodies) or a culture solution thereof, deposited under the deposit number KCTC15455BP, having an effect of preventing discoloration of crustaceans; and a rosemary extract.

4. In paragraph 3, The above Leuconostoc mesenteroides 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 crustaceans.

5. In paragraph 3, The above culture solution is characterized by being a culture solution in which the strain is cultured or a culture filtrate obtained by physically filtering the culture solution. A composition for preventing discoloration of crustaceans.

6. In paragraph 3, 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, alcohol having 1 to 4 carbon atoms, and mixed solvents thereof. A composition for preventing discoloration of crustaceans.

7. In paragraph 3, The concentration of the rosemary extract is characterized by being 0.01 to 10 mg / mL. A composition for preventing discoloration of crustaceans.

8. In paragraph 3, The above crustaceans are characterized in that they are selected from the group consisting of shrimp, crayfish, lobster and krill. A composition for preventing discoloration of crustaceans.

9. A food preservative composition comprising, as active ingredients, a strain of Leuconostoc mesenteroides (L. mesenteroides) or a culture solution thereof, deposited under the deposit number KCTC15455BP, having an effect of preventing discoloration of crustaceans; and a rosemary extract.

10. A method for preventing discoloration of crustaceans, comprising the following steps: (a) a step of washing crustaceans and removing moisture; and (b) a step of immersing the crustacean in a composition for preventing discoloration of crustaceans, comprising a Leuconostoc mesenteroides (L. mesenteroides) strain having an effect of preventing discoloration of crustaceans, deposited under the deposit number KCTC15455BP, or a culture solution thereof, and a rosemary extract.

11. In paragraph 10, In the step (b), the immersion time is characterized by being 1 to 100 minutes. Method for preventing discoloration of crustaceans.

12. In paragraph 10, In the step (b), the crustacean and the composition for preventing discoloration of the crustacean are immersed in a ratio of 1:10 to 10:1 (w / v). Method for preventing discoloration of crustaceans.

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