KR20220030376A - New Leuconostoc mesenteroides Wikim0120 and complex fermented Abeliophyllum distichum extract using the same - Google Patents

Abstract

The present invention relates to a novel Leuconostoc mecenteroides Wikim0120 strain and a complex fermented Abeliophyllum distichum extract using the same, and more specifically, to a novel Leuconostoc mecenteroides Wikim0120 strain and a complex fermented Abeliophyllum distichum extract using the same, which are obtained by applying a complex fermented Abeliophyllum distichum extract using a novel Leuconostoc mesenteroides Wikim0120 strain isolated from kimchi and having antibacterial activity and oligosaccharide production ability (including maltotriose and isomaltose) to a pickled cabbage processing process, thereby improving the quality of pickled cabbage, but also storing and distributing pickled cabbage in a fresh state for a long time.

Description

New Leuconostoc mesenteroides Wikim0120 strain and complex fermented extract using the same {New Leuconostoc mesenteroides Wikim0120 and complex fermented Abeliophyllum distichum extract using the same}

The present invention relates to a Leukonostok mecenteroides Wikim0120 strain separated from kimchi and having antibacterial activity and oligosaccharides [maltotriose and isomaltose] producing ability and a complex fermented extract of Miseon tree fermented using the strain. it's about

Kimchi is a traditional vegetable fermented food unique to Korea. Recently, as the excellent functionality of kimchi has been known, 'Health', an American health magazine, has selected kimchi as one of the world's top 5 health foods. There is an increasing trend of interest. It has been reported that kimchi contains various lactic acid bacteria and contains a large amount of organic acids, dietary fiber, and nutrients. Recently, studies on the functionality of kimchi are being actively conducted. The physiological functions of kimchi include antioxidant, anti-aging, anti-cancer effect, immune improvement effect, cardiovascular disease prevention and constipation prevention through bowel action, appetite enhancement, diet effect, etc. This has been reported

Recently, in addition to probiotics, the term prebiotics is often used, and prebiotics refer to nutrients that serve as food for beneficial intestinal probiotic lactic acid bacteria. say material. Recently, in the health functional food industry, interest and demand for prebiotics materials along with probiotics having various functions such as immune activity and skin improvement as well as improving healthy intestinal function are increasing at the same time. Prebiotics are oligosaccharides that reach the intestine without being absorbed because they are not decomposed by enzymes in the body. As oligosaccharides mainly used as prebiotics, six types of oligosaccharides are listed in the Food Code: fructooligosaccharide, isomaltooligosaccharide, galactooligosaccharide, maltooligosaccharide, xylooligosaccharide, and genchioligosaccharide. Isomaltooligosaccharide and maltooligosaccharide have been reported to improve taste due to imparting umami taste. As a physiological function, they have functions such as selective increase in the number of beneficial intestinal bacteria, Bifidobacterium. Functionality is approved. In addition, it has been reported that the selective proliferation effect of Bifidobacterium is almost the same as that of raffinose or fructooligosaccharide, and it selectively activates only Bifidobacterium because the magnetization is poor for bacteria other than Bifidobacterium compared to other sugars.

Kimchi production starts with salting cabbage. The purpose of pickling is to prevent the growth of harmful microorganisms or bacteria in the cabbage so that the cabbage does not decay and salt-resistant microorganisms dominate, so it is the most basic process for fermenting and fermenting kimchi. Osmosis occurs between salt and Chinese cabbage during pickling, which affects the taste of kimchi and is an important process that affects overall kimchi quality. Therefore, in kimchi lacking in pickling, the flavor deteriorates due to excessive production of exudate during the fermentation process, and claims may occur due to excessive salt content during over-picking.

Recently, due to busy social activities, rather than making kimchi at home, it is changing to eat it by purchasing it from the market or by purchasing pickled cabbage and seasoning and tossing them together. Therefore, pickled cabbage is commercialized and sold, and the proportion of purchase of such pickled cabbage is increasing more and more than that of purchasing fresh cabbage.

Although pickled cabbage is distributed in the market, there are many cases where consumers experience browning or softness when they receive the pickled cabbage, so complaints about this are increasing. According to the press release of the Korea Consumer Federation, among the consumer complaints related to cabbage in 2014, advice on pickled cabbage accounted for 75% of the total. When looking at the types of complaints related to the quality of the pickled cabbage in detail, the most common complaints about the quality of the pickled cabbage were brittleness (softening phenomenon), followed by deterioration (browning), degree of pickling, odor (medicine, strange odor, chemical, hoarse smell, smell of disinfectant), It can be seen that there are many problems in the distribution and storage of pickled cabbage such as foreign substances (sand, larvae, etc.) and others.

Korean Patent Registration No. 10-1761186 Korean Patent Registration No. 10-2033439 Korean Patent Registration No. 10-1933711 Korean Patent Registration No. 10-1531349

Accordingly, the present invention has been devised to solve the above problems, and the problem to be solved by the present invention is to be separated from kimchi and have antibacterial activity and oligosaccharides [maltotriose and isomaltose] producing ability. An object of the present invention is to provide a novel Leukonostok mecenteroides Wikim0120 strain.

In addition, the present invention, using the novel Leukonostok mecenteroides Wikim0120 strain, applied to the processing process of pickled cabbage to provide a complex fermented extract of Miseon tree effective for extending the quality and shelf life of pickled cabbage. Another object There is this.

However, the technical problems to be achieved in the present invention are not limited to the technical problems mentioned above, and other technical problems that are not mentioned are clearly to those of ordinary skill in the art to which the present invention belongs from the description below. can be understood

In order to achieve the above technical problem, the present invention provides a Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120, KCCM12658P) strain having antibacterial activity and oligosaccharide production ability.

In addition, the present invention provides a Leukonostok mecenteroides Wikim0120 strain having antibacterial activity against food poisoning bacteria

In addition, the present invention provides a Leukonostok mecenteroides Wikim0120 strain having antibacterial activity against coliform.

In addition, the present invention provides a Leukonostok mecenteroides Wikim0120 strain, characterized in that the oligosaccharide is an oligosaccharide comprising maltotriose and isomaltose

In addition, the present invention is a Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120, KCCM12658P) strain or a culture thereof, a concentrate of the culture and a dried product of the culture. It provides a complex Miseon tree fermented extract, characterized in that.

In addition, the present invention provides a complex fermented Miseon tree extract, characterized in that the complex Miseon tree ferment extract has antibacterial activity against food poisoning bacteria.

In addition, the present invention provides a complex Miseon tree fermented extract, characterized in that the food poisoning bacteria is Listeria monocytogenes ( Listeria monocytogenes )

In addition, the present invention provides a complex Miseon tree ferment extract, characterized in that the complex Miseon tree ferment extract has the ability to produce oligosaccharides containing maltotriose and isomaltose

In addition, the present invention comprises the steps of preparing a liquid medium by mixing Miseon tree extract powder and yeast extract, Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120, KCCM12658P) strain or its culture, the culture on the liquid medium It provides a method for producing a complex Miseon tree ferment extract comprising the step of fermenting by mixing one or more components selected from the group consisting of a concentrate of water and a dried product of the culture.

In addition, the present invention is a Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120, KCCM12658P) strain or a culture thereof, a concentrate of the culture and a dried product of the culture. It provides a food composition comprising the complex Miseon tree ferment extract as an active ingredient.

In addition, the present invention provides a food composition, characterized in that the food is a functional beverage, health functional food, kimchi inoculum or fermented food.

In addition, the present invention provides a food composition, characterized in that the food is pickled cabbage

In addition, the present invention provides a food composition, characterized in that the pickled cabbage containing the fermented extract of the complex Miseon tree as an active ingredient has an increased glucose content compared to the pickled cabbage.

The novel strain according to the present invention has an advantage in that it has excellent antimicrobial activity against food poisoning microorganisms and oligosaccharide production ability including oligosaccharides, particularly maltotriose and isomaltose.

In addition, the fermented complex Miseon tree ferment extract using the novel strain according to the present invention contains various functional substances such as oligosaccharides and glucose including maltotriose and isomaltose when applied to the processing process of pickled cabbage. This has the advantage of not only improving the quality of pickled cabbage, but also preventing browning or softening of the pickled cabbage so that it can be stored and distributed in a fresh state for a long time.

However, the effects obtainable in the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description. will be able

1 shows a list of 16S rRNA nucleotide sequences of Leukonostok mecenteroides Wikim0120 according to the present invention.
2 shows the results of microbial identification through 16S rRNA sequencing of Leukonostok mecenteroides Wikim0120 according to the present invention.
Figure 3 shows the results of antibacterial activity against E. coli and E. coli group of Leukonostok mecenteroides Wikim0120 according to the present invention.
Figure 4 shows the glucose standard curve for detecting the sugar content of the pickled cabbage with the addition of the complex Miseon tree ferment extract according to the present invention.
5 shows the results of the qualitative analysis of monosaccharides and oligosaccharides of pickled cabbage with the addition of the complex Miseon tree ferment extract according to the present invention.
6 to 9 show the results of changes in the microbiological properties of the fermented extract of Miseon tree complex according to the present invention.

Hereinafter, with reference to the accompanying drawings, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, since the description of the present invention is merely an embodiment for structural or functional description, the scope of the present invention should not be construed as being limited by the embodiment described in the text. That is, since the embodiment may have various changes and may have various forms, it should be understood that the scope of the present invention includes equivalents capable of realizing the technical idea. In addition, since the object or effect presented in the present invention does not mean that a specific embodiment should include all of them or only such effects, it should not be understood that the scope of the present invention is limited thereby.

The meaning of the terms described in the present invention should be understood as follows.

Terms such as “first” and “second” are for distinguishing one component from another, and the scope of rights should not be limited by these terms. For example, a first component may be termed a second component, and similarly, a second component may also be termed a first component. When a component is referred to as being “connected to” another component, it may be directly connected to the other component, but it should be understood that other components may exist in between. On the other hand, when it is mentioned that a certain element is "directly connected" to another element, it should be understood that the other element does not exist in the middle. Meanwhile, other expressions describing the relationship between elements, that is, "between" and "between" or "neighboring to" and "directly adjacent to", etc., should be interpreted similarly.

The singular expression is to be understood to include the plural expression unless the context clearly dictates otherwise, and terms such as "comprise" or "have" are not intended to refer to the specified feature, number, step, action, component, part or any of them. It is intended to indicate that a combination exists, and it should be understood that it does not preclude the possibility of the existence or addition of one or more other features or numbers, steps, operations, components, parts, or combinations thereof.

All terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs, unless otherwise defined. Terms defined in the dictionary should be interpreted as being consistent with the meaning of the context of the related art, and cannot be interpreted as having an ideal or excessively formal meaning unless explicitly defined in the present invention.

The present invention is derived from kimchi and relates to a Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120, KCCM12658P) strain having antibacterial activity and oligosaccharide production ability.

The Leukonostok mecenteroides strain is a gram-positive cocci and is facultative anaerobic. This strain is a representative kimchi lactic acid bacteria that grows predominantly in the early stage of kimchi fermentation, and increases the preservation of kimchi by producing bacteriocin, an antibacterial substance. This strain plays a very important role in the taste of kimchi by producing some organic acids and ethanol as well as lactic acid through heterofermentation during fermentation. In addition, it can be applied as dietary fiber while making monosaccharides into high molecular weight carbohydrates called hard-to-decompose dextrin.

As used herein, the term "antibacterial" refers to the action of inhibiting or controlling the growth and proliferation of microorganisms such as bacteria or fungi, and the Leukonostok mecenteroides Wikim0120 of the present invention is not limited thereto, but coliform (coliform) ) may be food quality indicator microorganisms such as

It was confirmed that the Leukonostok mecenteroides Wikim0120 strain according to the present invention has an oligosaccharide production ability, and is an oligosaccharide including, in particular, maltotriose and isomaltose.

In addition, the present invention is a Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120, KCCM12658P) strain or a culture thereof, a concentrate of the culture and a dried product of the culture. It provides a complex Miseon tree fermented extract, characterized in that.

Miseon tree is a special plant that grows only in Korea, and belongs to the genus Ash > Miseon tree, and it is a very rare plant with only one genus and one species in the world. The Miseon tree is known as the tree that expresses the most Korean beauty, and the name of the Miseon tree was given because the shape of the fruit is reminiscent of the round fan used by our ancestors in the past, the Miseon (尾扇). The value of Miseon tree lies in its rarity, and research is being conducted by confirming the possibility of its use as a food material to create high added value and applying it to various food industries.

Antioxidant, anti-inflammatory, whitening, and anti-wrinkle effects are known as related functional studies on Miseon tree extract, and Miseon tree leaves and stems contain polyphenols (leaf 50.6 mg/g, stem 13.5 mg/g) exhibiting physiological activity. ) and flavonoids (leaf 96.5 mg/g, stem 18.5 mg/g) are reported to have excellent antioxidant activity. It is known that various types of polyphenols and flavonoids with excellent physiological effects exist in the leaves of Miseon tree as well. According to Kwon et al. (2014), as a result of analyzing the components of Miseon tree leaves, fructose (32.1 mg/g), glucose (56.2 mg/g), and fatty acid content were palmitic acid (31.8%), stearic acid (14.8%), water-soluble vitamins It is known to contain phosphorus ascobic acid (1.32 mg/g), calcium (Ca) (166.2 mg/100 g), and iron (Fe) (3.6 mg/100 g) in mineral content. In addition, as a result of the safety evaluation of Miseon tree, its safety has been proven through genotoxic effects and toxicity tests, and it is judged that it has a high potential for use as a material for functional foods and pharmaceuticals.

In particular, the fermented extract of Miseon tree complex has antibacterial activity against food poisoning bacteria such as Listeria monocytogenes, and has the ability to produce oligosaccharides including maltotriose and isomaltose.

In addition, the present invention comprises the steps of preparing a liquid medium by mixing Miseon tree extract powder and yeast extract, Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120, KCCM12658P) strain or its culture, the culture on the liquid medium It provides a method for producing a complex Miseon tree ferment extract comprising the step of fermenting by mixing one or more components selected from the group consisting of a concentrate of water and a dried product of the culture.

The Miseon tree extract may be obtained by extracting the roots, stems or leaves of Miseon tree with water, C1 to C4 lower alcohol, or a mixture thereof. Specifically, the extraction is preferably performed by adding water, alcohol, or a mixture thereof to 2 to 20 times the volume of the Miseon tree after chopping the dried Miseon tree, and extracting by adding 3 to 10 times the volume of the Miseon tree. More preferably, it is not limited thereto. The extraction temperature is preferably 30°C to 100°C, more preferably 70°C to 100°C, but is not limited thereto. The extraction time is preferably 1 hour to 20 hours, more preferably 10 hours to 15 hours, but is not limited thereto. As the extraction method, cold extraction, ultrasonic extraction, or reflux cooling extraction method are all available, but the present invention is not limited thereto. The number of extractions is preferably 1 to 5 times, more preferably 2 to 3 times repeated extraction, but is not limited thereto. Additionally, the Miseon tree extract may be diluted, concentrated, or purified and used after dilution or concentration.

The method for preparing the liquid medium is not particularly limited as it is generally used in the art, and specifically, distilled water is added to the Miseon tree extract powder and yeast extract to prepare a Miseon tree extract culture solution, and it is sterilized and used. At this time, the yeast extract is preferably used in an amount of 5 to 25 parts by weight based on 100 parts by weight of the Miseon tree extract powder. .

Fermentation is performed using the liquid medium, and the fermentation is preferably performed for 36 to 60 hours at a fermentation (culture) temperature generally used in the art, specifically 25 to 35°C.

In addition, the present invention is a Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120, KCCM12658P) strain or a culture thereof, a concentrate of the culture and a dried product of the culture. It provides a food composition comprising the complex Miseon tree ferment extract as an active ingredient.

The food composition may be used as food, food additive, beverage, beverage additive, fermented milk and/or health functional food, but is not limited thereto. The food includes meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and/or vitamin complexes, etc. may be, but is not limited thereto. The food composition of the present invention may further include a food pharmaceutically acceptable carrier.

Specifically, the food composition may be a functional beverage, health functional food, kimchi inoculant or fermented food.

In the present invention, the "functional beverage" may include various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. As the natural carbohydrate, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame may be used. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 1 g per 100 ml of the composition of the present invention, but is not limited thereto.

In the present invention, "health functional food" refers to food with excellent medical and medical effects processed to efficiently exhibit bioregulatory functions in addition to nutritional supply. The food may be prepared in any one formulation selected from the group including powders, granules, tablets, capsules, liquids, and pills, but any formulation commonly used in the art may be used without limitation.

In the present invention, "fermented food" may be yogurt, curd, cheese, fermented milk, powdered milk, milk-based fermented food, ice cream and/or fermented cereal based product, but is not limited thereto.

In particular, the present invention is effective in improving the quality and extending the shelf life of the pickled cabbage when the complex fermented extract of Miseon tree is applied to the pickled cabbage processing process. The characteristics of the pickled cabbage containing the fermented extract of the complex Miseon tree as an active ingredient were confirmed through pickling rate, texture, pH and titratable acidity, salinity, reducing sugar analysis, saccharide qualitative analysis, and microbiological characteristics.

In particular, the pickled cabbage containing the complex Miseon tree ferment extract as an active ingredient has an increased glucose content compared to the untreated pickled cabbage, and contains oligosaccharides including maltotriose and isomaltose. Thus, it was confirmed that there is an effect of improving the quality of taste.

These results were similar to the research results reported by Cho et al. (2014) that isomaltooligosaccharide (IMO) was synthesized by the dextransucrase enzyme produced by Leuconostoc citreum KACC91035, a starter for kimchi fermentation, reported by Cho et al. (Cho et al. 2014. An improved process of isomaltooligosaccharide) production in kimchi involving the addition of a Leuconostoc starter and sugars. International Journal of Food Microbiology. 170: 61-64.).

Isomaltooligosaccharide is contained in traditional fermented foods such as kimchi, cheongju, and soybean paste, and is known as a sugar that not only sweetens but also has a savory taste. Isomaltooligosaccharide has a low water activity compared to sugar and other sugars, so it has the effect of inhibiting the growth of various microorganisms. In terms of physical properties, IMO is widely used in food because it has superior moisturizing properties compared to sugar or maltose, and is effective in improving sweetness and preventing starch aging. Its main use is mainly for confectionery, beverages, dairy products, and candy, and the effect of improving taste by imparting umami has been recognized, and its use is expanding to high-quality vinegar, confectionery, baking, and beverages. In addition, as a physiological function, it has functions such as a selective increase in the number of Bifidobacterium, a beneficial bacteria in the intestine, so it has been approved for the intestinal function of certain health food by the Ministry of Health and Welfare of Japan. The selective growth effect of Bifidobacterium is almost the same as that of raffinose or fructooligosaccharide, and it is known that only Bifidobacterium is selectively activated because the magnetization is poor for bacteria other than Bifidobacterium compared to other sugars.

In addition, it was confirmed that pickled cabbage can be stored and distributed in a fresh state for a long period of time due to excellent microbiological properties against various bacteria.

Hereinafter, preferred examples are presented to help the understanding of the present invention, but the following examples are merely illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention. , it is natural that such variations and modifications fall within the scope of the appended claims.

Example 1: Novel Leuconostoc mesenteroides Wikim0120 strain

A) Isolation and culture of Kimchi Lactobacillus

10 g of various kimchi samples (cabbage kimchi, kkakdugi, dongchimi, cabbage kimchi, cucumber kimchi, radish kimchi) are taken and diluted decimal with sterile physiological saline, spread on solid starch agar and corn meal agar medium, and anaerobic system gaspack (BBL, Becton) Dickinson, USA) in anaerobic culture conditions, 30 ℃, after culturing for 48 hours, different microorganisms were isolated.

B) Identification of isolated microorganisms

The selected kimchi lactic acid bacteria were identified through 16S ribosomal DNA gene sequencing analysis. After taking the cells of the selected microorganisms and washing the cells twice in sterile physiological saline, DNA was extracted using a DNeasy tissue kit (Qiagen, Valecia, CA, USA). Universal primer for 16S ribosomal DNA gene amplification of extracted DNA; 27F (5'-AGAGTTTGAT CATGGCTCAG-3') and 1492R (5'-GGATACCTTGTTACGA CTT-3') primers were used. For PCR reaction, 1 μl of DNA template (20 μg/ml), 1.0 μM forward primer and 1.0 μM reverse in 10 μl of Takara Perfect Premix (Takara, Japan) containing 0.4 mM dNTP, 0.5 units Taq polymerase, and 4 mM Mg ²⁺ 1 μl of each primer was added, and distilled water was added to the remainder to make a total volume of 20 μl. PCR amplification was performed with a Mastercycler gradient (Eppendorf, Hamburg, Germany). The PCR reaction was carried out for 30 cycles of 5 minutes at 95°C (initial denaturation), 94°C for 45 seconds (denaturation), 52°C for 45 seconds (annealing), and 1 minute at 72°C (extension), and finally at 72°C for 5 minutes. extension was performed.

After binding the amplified fragment of about 1,473bp to T vector (Invitrogen, Carlsbad, CA, USA), it was transformed. The nucleotide sequence was determined using T vector sequencing primer, and the result was ribosomal DNA of GenBank (NCBI, Bethesda, MD, USA) using BLAST search (http://www.ncbi.nlm.nih.gov) program. It was identified by comparison with gene sequencing.

As a result of microbial identification through 16S rRNA sequencing of the Wikim0120 strain isolated from kimchi as shown in FIG. 2, it was confirmed that it is 100% identical to the Leuconostoc mesenteroides strain, and Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120). At this time, the 16S rRNA nucleotide sequence of the Leukonostok mecenteroides Wikim0120 is as shown in FIG. 1 .

The strain Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120) has been deposited with the Korea Microorganism Conservation Center, and is being preserved after being given an accession number KCCM12658P as of January 16, 2020.

Experimental Example 1: Antibacterial activity of the strain

Antibacterial activity was measured for E. coli and coliform derived from pickled cabbage using the paper disc diffusion method.

50 μl of the Leukonostok mecenteroides Wikim0120 culture solution was added dropwise to the paper disc, incubated at 30° C. for 24 hours, and the formation of a transparent ring was confirmed. In this case, Kimchi Lactobacillus CM2 and CM3, which have excellent extracellular polysaccharide production ability, were used as controls.

3 shows the antibacterial activity results, the strain of Example 1 exhibited similar antibacterial activity to CM2 and CM3, and in particular, the antibacterial activity (47 mm) against the coliform group was very excellent.

Experimental Example 2: Extracellular polysaccharide production

Sucrose solid medium for extracellular polysaccharide production among isolated Kimchi lactic acid bacteria [4% sucrose, 1.5% agar, tryptone (10 g/L), meat extract (10 g/L), yeast extract (5 g/L) , sodium acetate (5 g/L), disodium phosphate (2 g/L), tri-ammonium citrate (2 g/L), MgSO ₄ (0.1 g/L), MnSO ₄ (0.05 g/L), pH 6.5 ], and then cultured at 30°C for 48 hours to first select slime bacteria representing mucoid colonies. The primary selection strain was inoculated in sucrose broth, and then cultured at 30° C. for 48 hours to generate extracellular polysaccharide (EPS). The culture solution was centrifuged at 4° C. (10,000 × g, 25 min, 4° C.) to remove cells, and 95% ethanol cooled twice in the recovered supernatant was added and precipitated at 4° C. for 15 hours. The precipitate was recovered by centrifugation (10,000 × g, 25 min, 4℃) again, and the remaining ethanol was dried and then freeze-dried to obtain crude EPS. To purify crude EPS, trihloloacetic acid was added to the culture medium to a final concentration of 4% (w/v), treated at 4°C for 2 hours, and centrifuged (10,000 × g, 25 min, 4°C) to precipitate the cells and Protein was removed. After the supernatant was recovered, the remaining protein was removed by filtration with a 0.2 μm filter, and a double amount of 95% ethanol was added to the recovered supernatant, followed by precipitation at 4° C. for 15 hours. The precipitate was recovered by centrifugation (10,000 × g, 25 min, 4 ℃), the remaining ethanol was dried, dissolved in tertiary distilled water, put in a dialysis sack, dialyzed at 4 ℃ for 24 hours, and then freeze-dried. The production amount of EPS with respect to the culture medium was expressed as mg/mL.

The amount of EPS (crude exopolysaccharide) produced with respect to the culture medium was 810 mg/50 mL.

Example 2: Complex Miseon tree fermented extract

Distilled water (1L) was added to 20 g of hot air-dried Miseon extract (leaf, stem) powder and 5 g of yeast extract to prepare a 2% (w/v) Miseon extract culture solution, sterilized at 121°C for 15 minutes, and then fermented with kimchi lactic acid bacteria. It was used as a liquid medium. The Leukonostok mecenteroides Wikim0120 strain (10 ⁶ CFU/ml) selected in Example 1 was inoculated at 0.5% (v/v) each and cultured alone or co-cultured at 30° C. for 48 hours to obtain a complex Miseon tree ferment extract. prepared.

The prepared complex Miseon tree ferment extract was confirmed to have a pH of 4.0, an acidity of 0.22, and a number of live cells of 4.0×10 ⁵ CFU/mL.

Experimental Example 3: Antibacterial activity of the fermented extract of Miseon tree complex

After 0.5 wt% of Listeria monocytogenes food poisoning microorganisms were inoculated (10 ⁵ CFU/ml) in the heat-treated cabbage juice, 0.1 wt% of the fermented extract of Example 2 was added to confirm the antibacterial effect did

Food poisoning microorganism detection was confirmed by using solid media such as Listeria monocytogenes selective media, eosin methylene blue (EMB), baird parker agar (BPA), oxford agar, etc. The number of viable cells was confirmed by using phenyl ethyl alcohol sucrose (PES) agar, a selective medium.

In the case of the complex fermented extract of Miseon tree of Example 2, the antibacterial effect was confirmed against Listeria monocytogenes food poisoning microorganisms, and the number of viable cells in the cabbage juice was confirmed at 10 ⁶ CFU/ml.

Example 3: Preparation of pickled cabbage

The raw cabbage variety was purchased from Haenam, and the trimmed raw cabbage was cut in two and immersed in pickled water. 7.4 kg) to prepare an initial 10% brine. The pickling conditions were marinated at the temperature of tap water (based on 18 ° C) for 16 hours, washed twice with tap water, and 3 times the extract of Miseon tree (leaf, stem) and the complex Miseon tree ferment extract of Example 2 (concentration 50 mg/10 kg) After leaving it for 10 minutes in the washing water containing it, it was naturally dehydrated at room temperature for 2-3 hours. While storing the pickled cabbage in 2% brine for up to 14 days (10°C), samples were taken at intervals of 1, 3, 7, and 14 days to analyze the physicochemical properties of the pickled cabbage.

Experimental Example 4: Characteristic evaluation

1. Pickle rate

The pickling rate was calculated by the formula (1) below considering the change of raw materials through the process of pickling, washing, and dehydration to sea salt in the main raw material.

[Formula 1]

Abandoned Cabbage control
(untreated group) Miseon Tree Extract fermented rice tree Cabbage Pumon 6.1±0.3 6.4±0.1 6.0±0.1 Raw cabbage weight (kg) 8.5 9.3 8.4 Cabbage weight after trimming (kg) 6.0 5.9 6.1 10% brine used (kg) 7.2 7.1 7.4 Amount of salt used (kg) 0.9 0.9 1.0 Weight after dehydration (kg) 5.1 5.1 5.3 Pickle rate (%) 85.0 86.4 86.8 Initial saline salinity (%) 10.0 10.0 10.0 Salt brine after pickling (%) 9.5 9.3 9.2 Salt water temperature (℃) after pickling 13.0 13.0 13.0

As shown in Table 1 above, the pickling rate of pickled cabbage was 86.4% and 86.8% when the Miseon tree extract and the complex Miseon tree ferment extract of Example 2 were added, respectively, which increased to 1.4% and 1.8% compared to the control pickled cabbage.

2. Texture (hardness)

Hardness was expressed as the magnitude of the force passing 10 mm through a 2 mm diameter probe using a texture analyzer (TA.XT plus, Stable Micro Systems, Ltd., England). At this time, the operating conditions of the texture analyzer are pretest speed 2 mm/s, test speed 10 mm/s, post test speed 10 mm/s, rupture test speed 2 mm/s, distance 15 mm. Texture is evaluated as a force representing strength.

Texture (kg) Control group (untreated group) Miseon Tree Extract Fermented Miseon Tree Save Day 1 0.6±0.1 0.8±0.1 0.7±0.1 Save Day 3 0.7±0.0 0.8±0.1 0.7±0.1 Save Day 7 0.7±0.0 0.8±0.0 0.8±0.1 Save Day 14 0.5±0.0 0.6±0.0 0.7±0.0

As shown in Table 2, the texture (force, kg) of the pickled cabbage was increased to 0.7±0.0 ~ 0.8±0.1 force (kg) when the Miseon tree extract and the fermented Miseon tree extract were added. It is expected that this will have an effect with the calcium contained in the Miseon tree extract.

Various types of polyphenols and flavonoids, which are currently known to have excellent physiological effects, exist in the leaves of Miseon tree, and they contain inorganic nutrients such as calcium (166.2 mg/100g). It is reported that if the tissue is partially damaged during pickling, it is somewhat recovered by treatment with calcium, and it is reported that the hardness is maintained higher than that of pickling in 10% brine, so that damage to the tissue is minimized during pickling by mixing calcium chloride.

3. pH and titratable acidity

After squeezing the kneaded sample with a blender using gauze, 20 mL of the juice was taken and measured with a pH meter (Corning Incorporated, USA). After titration to obtain the consumption of 0.1N NaOH solution was calculated by the following formula (2).

[Formula 2]

pH Control group (untreated group) Miseon Tree Extract Fermented Miseon Tree Save Day 1 6.0±0.0 6.0±0.0 6.0±0.0 Save Day 3 5.9±0.0 6.2±0.0 6.2±0.0 Save Day 7 6.0±0.0 6.2±0.0 6.5±0.0 Save Day 14 6.1±0.0 6.2±0.1 6.2±0.2

Acidity (%) Control group (untreated group) Miseon Tree Extract Fermented Miseon Tree Save Day 1 0.2±0.0 0.2±0.0 0.2±0.0 Save Day 3 0.2±0.0 0.2±0.0 0.2±0.0 Save Day 7 0.1±0.0 0.1±0.0 0.1±0.0 Save Day 14 0.1±0.0 0.2±0.0 0.2±0.0

As shown in Tables 3 and 4, there was no significant difference between the treated sample groups in the range of pH 5.9 to 6.5 and acidity 0.1 to 0.2%.

4. Salinity

Weigh about 1 g of the kneaded sample with a blender, dilute (about 100 times), take 10 mL of the filtered (ADVANTEC, No. 1) filtrate, add 1 mL of 2% potassium chromate, and titrate with 0.02 N AgNO ₃ It was calculated using the formula (3). In this case, the unit is % (w/v).

[Formula 3]

5. Reducing sugar (DNS method) analysis

Accurately measure 1 g of the kneaded sample with a blender, dilute (50 mL), add 3 mL of DNS reagent to 1 mL of the filtered (ADVANTEC, No. 1) filtrate, immediately mix with a vortex mixer, and bathe in boiling water for 5 minutes. . After cooling at room temperature, it was diluted with 16 mL of distilled water and absorbance was measured at 510 nm using a UV-VIS spectrophotometer (SHIMADZU, UV1800, Japan), and the reducing sugar content was calculated by a glucose standard curve (Fig. 4).

Unprocessed Pickled Cabbage Pickled Cabbage with Complex Miseon Tree Fermented Extract Pickled cabbage with Miseon tree extract Abs. at 510 nm 0.382 0.435 0.377 dilution rate 100 100 100 Glucose (mg/mL) 15.554 17.728 15.349

As shown in Table 5, untreated pickled cabbage, Leuconostoc mesenteroides Wikim0120, fermented with Leuconostoc mesenteroides Wikim0120. Analysis was performed. As a result, it was confirmed that the glucose content (17.728 mg/mL) of the pickled cabbage to which the complex Miseon tree ferment extract was added was the highest.

6. Qualitative analysis of sugars in pickled cabbage (TLC method)

Thin layer chromatography is a method of separation using the difference in the movement speed of the substances constituting the mixture when the mixture moves by the mobile phase on a stationary phase such as silica gel. Fermentation with untreated pickled cabbage (Control) and Leuconostoc mesenteroides Wikim0120 The types of monosaccharides and oligosaccharides of pickled cabbages stored in 2% brine for up to 14 days (10°C) by adding a complex fermented misun and Misun tree extracts were identified.

At this time, the sugars used are all sugars with reducing ends, and the monosaccharides are glucose, fructose, mannose, and arabinose, the disaccharides are maltose, isomaltose, sucrose, cellobiose, and panose, and the oligosaccharides are maltooligosaccharides [glucose (G1) to maltooctane ose (G8)] and polysaccharides were cellodextrin. For TLC, a silica gel TLC plate (Merck Co., Darmstadt, Germany) was used. For other reagents, general grade reagents were used.

After centrifugation (8,500g, 20 minutes, 4℃) to remove the precipitate, 5% TCA 10mL is added to 10mL of the supernatant and refrigerated (4℃) for 2 hours to prevent protein precipitation. proceeded. After centrifugation (8,500g, 20 minutes, 4℃), it was dripped onto a TLC plate (20 × 20cm), and sugars (monosaccharides, disaccharides and oligosaccharides) were made at a concentration of 7.5 μg/μl and dropped onto the TLC plate. It was developed under a solvent composition of isopropanol : ethyl acetate : H ₂ O (3 : 1 : 1, v/v/v). After development, the TLC plate was dried at room temperature. After spraying the urea-metaphosphoric acid solution, it was left in an oven at 110°C for 5 minutes to develop color. Then, the spot position and color development level of the TLC plate were checked.

Sugars (monosaccharides, disaccharides and oligosaccharides) were dripped using a silica gel TLC plate and developed with a solvent of isopropanol : ethyl acetate : H ₂ O (3 : 1 : 1, v/v/v). As a result, it was confirmed that glucose(G1) or sucrose had the highest content in the result values of all pickled cabbage samples. A trace amount of maltose spot was also shown in the sample, and it was confirmed that a trace amount of maltotriose or isomaltose spot was also present. In particular, maltotriose or isomaltose spots were most intensely formed only on pickled cabbage to which the complex fermented extract of Miseon tree was added.

7. Microbiological Characteristics

The sample was taken aseptically and ground using a blender, put in a sterile filter bag, diluted with 0.85% NaCl, and stomached for 1 minute. After homogenization, the suspension was diluted decimal with sterile physiological saline, and then smeared on the following solid medium for separation to measure the number of viable cells.

Among lactic acid bacteria 1) Lactobacillus spp. was counted by inoculating a sample diluted step by step using LBS (Lactobacillus Selection) agar by the spreading culture method, and then culturing at 30° C. for 48 hours.

2) Leuconostoc spp. was counted by inoculating a sample diluted step by step using PES (phenyl ethylalcohol sucrose) agar by the spreading culture method and then culturing at 30° C. for 48 hours.

3) In the case of the total number of bacteria, samples diluted step by step using PCA (Plate count agar, Difco) were inoculated by the spreading culture method, and then cultured at 30° C. for 24 hours and counted.

4) In the case of Bacillus, samples diluted step by step using TSA (Tryptic soy agar) were inoculated by the spreading culture method and then cultured at 30° C. for 24 hours and counted.

5) In the case of basophils, samples diluted step by step using MA (Marine agar) were inoculated by the spreading culture method and then cultured at 30° C. for 48 hours and counted.

6) In the case of yeast and mold bacteria, YPD (yeast potato dextrose agar) was used, and for PDA (potato dextrose agar), 1.4 mL/100 mL of 10% tartaric acid was added after sterilization and inoculated with a sample diluted step by step. It was counted after 48-72 hours incubation at 25 °C by the spreading culture method.

7) E. coli/E. coli group was inoculated with EMB (eosin methylene blue) agar with step-by-step dilution solution, and then cultured at 36°C for 24 hours. did

The results of the change in the microbiological properties are shown in FIGS. 6 to 9 below.

Figure 6 shows the number of Lactobacillus lactic acid bacteria and the number of leukonostok lactic acid bacteria. As shown in Figure 6, 1) The number of Lactobacillus lactobacilli is in the range of 1.6×10 ⁴ to 2.3×10 ⁵ CFU/mL on the first day of storage, and it gradually decreases from the 7th day of storage, and on the 14th day of storage, pickled cabbage with the addition of fermented extract Only in Lactobacillus was confirmed as 5.0×10 ² CFU/mL viable cells. 2) On the first day of storage, the number of lactic acid bacteria in Leukonostok ranges from 1.5×10 ⁴ to 1.2×10 ⁵ CFU/mL, and gradually decreases from the 7th day of storage. It was confirmed that the number of lactic acid bacteria maintained 1.1×10 ⁴ CFU/mL viable bacteria.

7 shows the total number of bacteria (the number of normal bacteria) and the number of basophils (the number of resistant bacteria). As shown in Figure 7, 3) The total number of bacteria (normal bacteria) is in the range of 1.5×10 ⁵ to 2.8×10 ⁵ CFU/mL on the first day of storage, and gradually decreases from the 3rd day of storage in the pickled cabbage with the addition of the fermented extract of Miseon tree. On the 14th day of storage, the number of general bacteria was confirmed as 7.0×10 ² CFU/mL viable cells. As a control, the number of normal bacteria in the untreated pickled cabbage was confirmed as the number of viable cells of 2.1× ^{10 4} CFU/mL. 5) The number of basophilic bacteria (the number of salt-resistant bacteria) is in the range of 7.6×10 ⁴ to 4.7×10 ⁵ CFU/mL on the first day of storage. The number of resistant bacteria was confirmed as the number of viable bacteria at 6.8×10 ³ CFU/mL. As a control, the number of salt-resistant bacteria in the untreated pickled cabbage was 3.4× ^{10 4} CFU/mL viable.

Figure 8 shows the number of 6) yeast and mold bacteria. As shown in Figure 8, the yeast gradually decreased from the 3rd day of storage in the range of 2.6×10 ⁴ to 2.3×10 ⁵ CFU/mL of storage on the 1st day of storage in the pickled cabbage with the addition of the fermented extract of Miseon tree ferment, and the number of yeast on the 7th and 14th days of storage was not detected. On the 1st day of storage, the mold was 3.3×10 ⁴ ~ 2.5×10 ⁵ CFU/mL, and in the pickled cabbage with the addition of the fermented extract of P. fermented extract, it gradually decreased from the 3rd day of storage and was confirmed to be 7.4×10 ² CFU/mL on the 14th day of storage. did However, based on the colony form, it was judged to be yeast or bacterial growth, not mold.

9 shows Bacillus and E. coli/E. coli group. As shown in FIG. 9, 4) The number of Bacillus bacteria decreased gradually from the 7th day of storage in the range of 1.7×10 ⁵ to 4.9×10 ⁵ CFU/mL in the range of 1.7×10 5 to 4.9×10 5 CFU/mL of storage on the first day of storage, and Bacillus on the 14th day of storage The number of bacteria was confirmed as 9.8×10 ³ CFU/mL viable cells. As a control, the number of Bacillus bacteria in untreated pickled cabbage was 4.5×10 ⁴ CFU/mL viable cells, and the effect of reducing the number of Bacillus bacteria (10 ¹ CFU/mL) was confirmed when the compound fermented extract of Miseon tree was added. Bacillus, which is widely distributed in nature including soil, produces endospores and has the characteristic of surviving even after heat treatment, so it is a microorganism that is difficult to control during fermentation of kimchi. It was considered necessary to check. 7) The number of coliform bacteria was in the range of 7.6×10 ⁴ to 3.2×10 ⁵ CFU/mL on the first day of storage. 5.7×10 ³ CFU/mL was confirmed as the number of viable cells. In the untreated pickled cabbage sample, which is a control, not only the coliform group (2.5×10 ⁴ CFU/mL) but also E. coli forming a green metallic luster were confirmed as the number of viable cells of 8.0×10 ² CFU/mL. The effect of reducing the number of coliform bacteria (10 ¹ CFU/mL) and the non-detection of E. coli was confirmed when the complex fermented extract of Miseon tree was added.

Korea Microorganism Conservation Center KCCM12658P 20200116

<110> Korea Food Research Institute <120> New Leuconostoc mesenteroides Wikim0120 and complex fermented Abeliophyllum distichum extract using the same <130> A20-167 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1473 <212> DNA <213> Lactobacillus plantarum <400> 1 gatgaacgct ggcggcgtgc ctaatacatg caagtcgaac gcacagcgaa aggtgcttgc 60 acctttcaag tgagtggcga acgggtgagt aacacgtgga caacctgcct caaggctggg 120 gataacattt ggaaacagat gctaataccg aataaaactt agtgtcgcat gacaaaaagt 180 taaaaggcgc ttcggcgtca cctagagatg gatccgcggt gcattagtta gttggtgggg 240 taaaggccta ccaagacaat gatgcatagc cgagttgaga gactgatcgg ccacattggg 300 actgagacac ggcccaaact cctacgggag gctgcagtag ggaatcttcc acaatgggcg 360 aaagcctgat ggagcaacgc cgcgtgtgtg atgaaggctt tcgggtcgta aagcactgtt 420 gtatgggaag aacagctaga ataggaaatg attttagttt gacggtacca taccagaaag 480 ggacggctaa atacgtgcca gcagccgcgg taatacgtat gtcccgagcg ttatccggat 540 ttattgggcg taaagcgagc gcagacggtt tattaagtct gatgtgaaag cccggagctc 600 aactccggaa tggcattgga aactggttaa cttgagtgca gtagaggtaa gtggaactcc 660 atgtgtagcg gtggaatgcg tagatatatg gaagaacacc agtggcgaag gcggcttact 720 ggactgcaac tgacgttgag gctcgaaagt gtgggtagca aacaggatta gataccctgg 780 tagtccacac cgtaaacgat gaacactagg tgttaggagg tttccgcctc ttagtgccga 840 agctaacgca ttaagtgttc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaggaa 900 ttgacgggga cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960 cttaccaggt cttgacatcc tttgaagctt ttagagatag aagtgttctc ttcggagaca 1020 aagtgacagg tggtgcatgg tcgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080 gcaacgagcg caacccttat tgttagttgc cagcattcag atgggcactc tagcgagact 1140 gccggtgaca aaccggagga aggcggggac gacgtcagat catcatgccc cttatgacct 1200 gggctacaca cgtgctacaa tggcgtatac aacgagttgc caacccgcga gggtgagcta 1260 atctcttaaa gtacgtctca gttcggattg tagtctgcaa ctcgactaca tgaagtcgga 1320 atcgctagta atcgcggatc agcacgccgc ggtgaatacg ttcccgggtc ttgtacacac 1380 cgcccgtcac accatgggag tttgtaatgc ccaaagccgg tggcctaacc ttttaggaag 1440 gagccgtcta aggcaggaca gatgactggg gtg 1473

Claims (13)

Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120, KCCM12658P) strain having antibacterial activity and oligosaccharide production ability.
The method according to claim 1, wherein the antibacterial activity is Leukonostok mecenteroides Wikim0120 strain, characterized in that the antibacterial activity against food poisoning bacteria.
The method according to claim 2, wherein the food poisoning bacteria is a Leukonostok mecenteroides Wikim0120 strain, characterized in that the coliform (coliform).
The method according to claim 1, wherein the oligosaccharide is Leukonostok mecenteroides Wikim0120 strain, characterized in that the oligosaccharide comprising maltotriose and isomaltose.
Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120, KCCM12658P) strain or a culture thereof, a concentrate of the culture, and a dried product of the culture A complex characterized in that the extract is fermented by at least one component selected from the group consisting of Miseon tree fermented extract.
The method according to claim 5, The complex fermented Miseon tree ferment extract, characterized in that it has antibacterial activity against food poisoning bacteria.
The method according to claim 6, wherein the food poisoning bacteria is Listeria monocytogenes ( Listeria monocytogenes ) Complex Miseon tree ferment extract, characterized in that.
The method according to claim 5, The complex Miseon tree ferment extract is a complex Miseon tree fermented extract, characterized in that it has the ability to produce oligosaccharides containing maltotriose (maltotriose) and isomaltose (isomaltose).
Preparing a liquid medium by mixing Miseon tree extract powder and yeast extract;
Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120, KCCM12658P) strain or a culture thereof, the concentrate of the culture on the liquid medium, and the step of fermenting by mixing one or more components selected from the group consisting of a dry material of the culture A method for producing a complex Miseon tree fermented extract, characterized in that
Leuconostoc mesenteroides Wikim0120 ( Leuconostoc mesenteroides Wikim0120, KCCM12658P) strain or a culture thereof, a concentrate of the culture, and a complex fermented extract of fermented Miseon tree extract by at least one component selected from the group consisting of a dried product of the culture A food composition comprising as an active ingredient.
The food composition according to claim 10, wherein the food is a functional beverage, a health functional food, a kimchi inoculant, or a fermented food.
The food composition according to claim 10, wherein the food is pickled cabbage.
The food composition according to claim 12, wherein the pickled cabbage containing the fermented extract of the compound Miseon tree as an active ingredient has an increased glucose content compared to the pickled cabbage.

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