KR102323299B1 - Aronia melanocarpa fermented ethanol extracts having anti-oxidative and antimicrobial activity and use thereof - Google Patents

Abstract

The present invention is an aronia fermented product or an alcohol extract thereof having antibacterial and antioxidant effects, and more specifically, a fermented product obtained by inoculating and fermenting aronia fruit with a Leuconostoc strain or an ethanol extract thereof as an active ingredient. It relates to a composition having antibacterial or antioxidant activity, a feed additive, and a method for preparing the same. The fermented Aronia fermented product or its alcohol extract according to the present invention has high antioxidant activity and antibacterial activity against various pathogens such as Listeria, Salmonella and Manjaimia bacteria, so that it can be used as a pharmaceutical material, feed / feed additive material, and It can be usefully used as various functional materials such as food materials. In particular, aronia fermented product or its alcohol extract has excellent antibacterial and antioxidant activity, so it can greatly help improve the productivity of livestock as a natural feed additive that can replace antibiotics. In addition, in the development of feed additives using aronia fruit, the physiological activity is greatly improved when using the alcohol extract of aronia after fermenting aronia using the leukonostok mesenteroid KCCM35046 or leukonostok mesenteroid KACC12315 strains as a starter strain. effect can be derived.

Description

Aronia fermented alcohol extract having antibacterial and antioxidant effects and uses thereof {Aronia melanocarpa fermented ethanol extracts having anti-oxidative and antimicrobial activity and use thereof}

The present invention is an aronia fermented product or an alcohol extract thereof having antibacterial and antioxidant effects, and more specifically, a fermented product obtained by inoculating and fermenting aronia fruit with a Leuconostoc strain or an ethanol extract thereof as an active ingredient. It relates to a composition having antibacterial or antioxidant activity, a feed additive, and a method for preparing the same.

In Korea's livestock industry, antibiotics have been used to promote productivity and growth of livestock, and ionophore antibiotics such as monensin, chloroform, and halogenated analogues have been used as representative antibiotics (Odongo et al., 2007 Journal of Dairy Science) (90: 1781-1788; McAllister and Newbold, 2008 Animal Production Science. 48: 7-13). 'Antibiotic (antibiotic)' is a soluble organic substance produced by microorganisms that inhibits the growth of other microorganisms or destroys their life. It was developed for medical and veterinary purposes to inhibit pathogens, but many researchers It has been used as a feed additive as it has also been recognized for its effects on animal growth promotion and feed efficiency improvement.

However, due to the recent increase in the types and usage of veterinary medicines due to the complex outbreak of diseases and increased resistance to pathogens, the misuse of antibiotics in animals and the excessive use of medicines for addition to compounded feed, the increasing demands of soybeans and consumers to secure the stability of livestock products. The use of antibiotics is limited. Therefore, the development of substances that can replace these antibiotics is a very important task in eco-friendly livestock farming.

Livestock are fed a highly concentrated, high-energy feed for the purpose of improving productivity. Therefore, the process of converting large amounts of nutrients into livestock products is continuously performed. During this process, active oxygen is generated in many livestock bodies, which promotes the weakening of cells and tissues, thereby exposing them to various diseases. Therefore, antioxidants with excellent activity are very helpful not only to improve livestock productivity but also to maintain the health of livestock.

Phenolic compounds contained in edible plants generally contain compounds of the benzene ring structure. These phenolic compounds are classified in various ways, and this classification is made according to the number of phenol rings, such as a benzene ring, and structural characteristics of molecules connected to the phenol ring (Manach et al. 2004: American Journal of Clinical Nutrition, 79: 727747). Until recently, these phenolic compounds were considered nutritionally undesirable. The reason can be found in the anti-nutrient function of phenolic compounds. In other words, phenolic compounds have been known to have a negative effect on nutrient digestion and interfere with the absorption of vitamins and minerals. However, recently, due to the excellent antioxidant ability of phenolic compounds, their value has been re-evaluated (Rodriguez et al. 2009: International Journal of Food Microbiology, 132: 7990). Antioxidant activity refers to the process of removing free radicals in the body. In the case of livestock, as mentioned above, the accumulation of free radicals in the body is unavoidable because they constantly decompose a large amount of nutrients. Superoxide radicals, hydroxy radicals, and hydrogen peroxide are typical types of active oxygen that accumulates in the body. If they are present in the body in small amounts, it is known that they are rather beneficial to livestock growth, but if the amount is large, the livestock is subjected to oxidative stress (Atmani et al. 2009: Food Chemistry, 112: 303309).

And this oxidative stress causes a decrease in the productivity of livestock. As an example, reactive oxygen species increase the activity of an enzyme called matrix metalloproteinase-2 (MMP2) in the muscle of beef cattle, and the increased enzyme activity inhibits collagen synthesis in the muscle. Eventually, oxidative stress leads to the production of tough meat by lowering the collagen synthesis of fibroblasts in the muscle (Archile-Contreras and Purslow, 2011: Food Research International, 44: 582588). Therefore, the development of phenolic compounds with excellent antioxidant activity derived from plants is very important for the development of organic livestock industry and for improving the quality of livestock products.

Phenolic compounds exhibit antibacterial activity as well as antioxidant activity. It is known that various lactic acid bacteria are involved in the natural fermentation of edible plants. It is known that lactic acid bacteria are involved in the natural fermentation of edible plants containing a large amount of phenolic compounds with antibacterial activity, and the lactic acid bacteria reduce the antibacterial activity by changing the structure of the phenolic compounds with antibacterial activity for their growth. . It is known that various enzymes are involved in the structural change of these phenolic compounds, and representatively, beta-glucosidase, tanninase, phenolic acid decarboxylase and esterase are known (Rodriguez, et al. 2009: International Journal). of Food Microbiology, 132: 7990). Therefore, changing the structure of the phenolic compounds contained in edible plants in the direction of improving their physiological activity can be obtained through the above-mentioned enzyme treatment or fermentation using lactic acid bacteria that produce the enzyme.

On the other hand, aronia (Aronia melanocarpa) is known as black chokeberry and is known to contain a large amount of phenolic compounds. In addition, the health effects of aronia extract have been reported to prevent cardiovascular disease, prevent diabetes, and protect the liver.

Accordingly, the present inventors tried to discover a material for a feed additive derived from nature that can replace antibiotics, and as a result, in the case of a fermented product obtained by inoculating and fermenting aronia fruit with Leukonostok strain, Listeria, Salmonella, and Manheimia bacteria. The present invention was completed by confirming that it has high antioxidant activity as well as antibacterial activity against various pathogens such as.

Korean Patent Publication No. 10-2011-0067308 Korean Patent No. 10-0889364

Accordingly, an object of the present invention is to provide a composition that can be used as a variety of functional materials such as pharmaceutical materials, feed/feed additive materials and food materials based on excellent antibacterial or antioxidant activity.

In addition, another object of the present invention is to provide an antibiotic-substitutable feed additive composition that can enhance the immunity of livestock through excellent antibacterial or antioxidant activity.

In addition, another object of the present invention is to provide a method for preparing a fermented aronia as a material having excellent antibacterial or antioxidant activity.

Another object of the present invention is to provide a method for preparing an aronia fermented alcohol extract as a material having excellent antibacterial or antioxidant activity.

In order to achieve the object of the present invention as described above, the present invention provides an antibacterial or antioxidant composition comprising aronia fermented product or aronia fermented alcohol extract as an active ingredient.

In one embodiment of the present invention, the aronia fermented product may be a fermented product obtained by inoculating and fermenting an aronia fruit with a Leuconostoc strain.

In one embodiment of the present invention, the aronia fermented alcohol extract may be an extract extracted by adding ethanol to a fermented product obtained by inoculating and fermenting aronia fruit with a Leuconostoc strain.

In one embodiment of the present invention, the Leuconostoc strain may be a Leuconostoc mesenteroides.

of the present invention In one embodiment, the Leuconostoc mesenteroides ) may be one selected from the group consisting of leukonostok mesenteroid KCCM35046, leukonostok mesenteroid KCTC3100, leukonostok mesenteroid KACC12312, leukonostok mesenteroid KACC12313 and leukonostok mesenteroid KACC12315 strains.

In one embodiment of the present invention, The composition may have antibacterial activity against one or more pathogens selected from the group consisting of Listeria, Salmonella, and Manheimia.

In addition, the present invention provides a livestock feed additive composition comprising aronia fermented product or aronia fermented alcohol extract as an active ingredient.

In one embodiment of the present invention, the livestock may be one kind of livestock selected from the group consisting of cattle, pigs, chickens, ducks, goats, sheep and horses.

In one embodiment of the present invention, the fermented product or extract can enhance the immunity of livestock through antibacterial and antioxidant activity.

In one embodiment of the present invention, the antibacterial activity may be the antibacterial activity against one or more pathogens selected from the group consisting of Listeria, Salmonella, and Manjaimia.

In addition, the present invention comprises the steps of (a) crushing the aronia fruit; (b) mixing the crushed aronia fruit juice with MRS medium solution; And (c) a base stock Pocono mesen steroid (Leuconostoc the mixture mesenteroides ) provides a method for producing fermented aronia having antibacterial and antioxidant activity, including the step of fermenting after inoculation of the strain.

In addition, the present invention comprises the steps of (i) mixing aronia fruit juice with MRS medium solution; (ii) Leuconostoc in the mixture mesenteroides ) inoculating the strain and then fermenting; and (iii) drying the fermented product and then extracting the fermented product by adding ethanol to it.

The fermented Aronia fermented product or its alcohol extract according to the present invention has high antioxidant activity and antibacterial activity against various pathogens such as Listeria, Salmonella and Manjaimia bacteria, so that it can be used as a pharmaceutical material, feed / feed additive material, and It can be usefully used as various functional materials such as food materials. In particular, aronia fermented product or its alcohol extract has excellent antibacterial and antioxidant activity, so it can greatly help improve the productivity of livestock as a natural feed additive that can replace antibiotics. In addition, in the development of feed additives using aronia fruit, when aronia is fermented using leukonostok mesenteroid KCCM35046 or leukonostok mesenteroid KACC12315 strains as starter strains and the alcohol extract is used, the physiological activity is greatly improved effect can be derived.

Figure 1 shows each test group fermented using leukonostok mesenteroid strains (KCCM35046, KCTC3100, KACC12312, KACC12313, KACC12315) (control group [untreated group]: CON, treatment group 1 [KCCM35046 strain inoculation fermentation]: Tr1, treatment group 2 [ KCTC3100 strain inoculation fermentation]: Tr2, treatment group 3 [KACC12312 strain inoculation fermentation]: Tr3, treatment group 4 [KACC12313 strain inoculation fermentation]: Tr4, treatment group 5 [KACC12315 strain inoculation fermentation]: Tr5, beta-glucosidase enzyme of aronia fermented broth It is the result of measuring the activity using the absorbance evaluation method.
2 is a result of measuring the concentration of lactic acid bacteria contained in the aronia fermentation broth for each test section fermented using the Leukonostok mesenteroid strain.
3 is a result of measuring the total polyphenol concentration contained in the aronia fermented alcohol extract for each test section fermented using the Leukonostok mesenteroid strain.
4 is a result of measuring the antioxidant activity of aronia fermented alcohol extract for each test section fermented using the Leukonostok mesenteroid strain.
5 is a result of separating the substances contained in the fermented alcohol extract of Aronia for each test section fermented using the Leukonostok mesenteroid strain using thin-layer chromatography. Plates A, B, and C are the results of evaluation of antioxidant-related substances using DPPH, and plates D, E, and F are the results of detection of substances through ultraviolet irradiation. Lane 1: Alcoholic extract of aronia fermented broth induced by natural fermentation without strain inoculation (control), Lane 2: Alcoholic extract of Aronia fermented broth fermented with Leukonostok mesenteroid KCCM35046 strain, Lane 3: Leukonostok mesenteroid KCTC3100 Alcoholic extract of Aronia fermented broth fermented with the strain, Lane 4: Alcoholic extract of Aronia fermented broth fermented with Leukonostok mesenteroid KACC12312 strain, Lane 5: Alcoholic extract of Aronia fermented broth fermented with Leukonostok mesenteroid KACC12313 strain , Lane 6: Alcoholic extract of Aronia fermentation broth fermented with Leukonostok mesenteroid KACC12315 strain.
6 is a result of calculating the area ratio of each material in the chromatogram using high-performance liquid chromatography on the change in total polyphenol substances contained in the aronia alcohol extract fermented using the Leukonostok mesenteroid strain.
7 shows the contents of caffeic acid, chlorogenic acid, and trans-ferulic acids contained in the aronia fermented alcohol extract for each test section fermented using the Leukonostok mesenteroid strain. (A: caffeic acid, B: chlorogenic acid, C: trans-ferulic acid).

In one aspect, the present invention relates to the antibacterial/antioxidant use of a fermented aronia or an alcoholic extract of fermented aronia.

In the present invention, 'aronia' means the fruit of the aronia tree, also called choke-berry.

This aronia contains a lot of anthocyanin pigments, so it is known that it not only prevents aging but also has a very good anticancer effect. It is also known to be of great help in the treatment of blood-related diseases, such as improving eyesight, heart and vascular diseases, and stroke.

Therefore, the present inventors conducted experiments focusing on aronia fruit while researching natural materials with various functionalities, and as a result, the fermented product inoculated and fermented with aronia leuconostoc strain has high antioxidant activity In addition, as it has antibacterial activity against various pathogens such as Listeria, Salmonella, and Manhaimia, the possibility of using it as a variety of functional materials such as pharmaceutical materials, feed/feed additive materials and food materials has been demonstrated.

In the present invention, a fermented product obtained by inoculating and fermenting aronia fruit with a Leuconostoc strain is simply called 'aronia fermented product', and the aronia fermented product has the same meaning as aronia fermented broth.

The fermented aronia of the present invention can be prepared by a general method applied in the art, but preferably, the MSR medium solution is mixed with the fruit juice obtained by crushing the aronia fruit, and then the Leuconostoc strain It can be prepared through the process of fermentation after inoculation using it as a starter.

In addition, the fermented aronia of the present invention may be additionally extracted by adding an ethanol extraction solvent to the dried fermented product after drying the fermented product in addition to the above process.

In the present invention, the material prepared through this additional process is simply called 'aronia fermented alcohol extract'.

Accordingly, the present invention features an antibacterial or antioxidant composition comprising fermented aronia or an aronia fermented alcohol extract as an active ingredient.

In one embodiment of the present invention, the Leuconostoc strain may be a Leuconostoc mesenteroides, preferably Leuconostoc mesenteroides KCCM35046, Leuconostoc mesenteroid KCTC3100, Leuconostoc mesenteroides At least one selected from the group consisting of mesenteroid KACC12312, leukonostok mesenteroid KACC12313 and leukonostok mesenteroid KACC12315 strain may be used.

In another aspect, the present invention relates to a livestock feed additive composition comprising aronia fermented product or aronia fermented alcohol extract as an active ingredient.

In the present invention, the term 'feed additive' refers to a substance added to feed for nutritional or specific purposes, and the specific purpose is to enhance immunity of livestock, promote growth, protect against bacteria or viruses, improve disease, relieve stress, and It means giving the same functionality.

The kind of livestock to be fed of the feed additive composition of the present invention may be exemplified by one kind of livestock selected from the group consisting of cattle, pigs, chickens, ducks, goats, sheep and horses, but is not particularly limited thereto .

As the feed additive composition of the present invention contains aronia fermented product or aronia fermented alcohol extract as an active ingredient, it has antibacterial activity (antibacterial activity against one or more pathogens selected from the group consisting of Listeria, Salmonella and Manheimia bacteria). ) and antioxidant activity to enhance the immunity of livestock.

In another aspect, the present invention comprises the steps of (a) crushing an aronia fruit; (b) mixing the crushed aronia fruit juice with MRS medium solution; and (c) inoculating the Leuconostoc mesenteroides strain into the mixed solution and then fermenting the aronia fermented product having antibacterial and antioxidant activity.

In another aspect, the present invention comprises the steps of (i) mixing aronia fruit juice with MRS medium solution; (ii) Leuconostoc in the mixture mesenteroides ) inoculating the strain and then fermenting; and (iii) drying the fermented product and then adding ethanol to the fermented product for extraction.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

< Example 1>

aronia Fermentation and alcohol extract manufacturing

<1-1> Preparation of materials

Aronia fruits used in the experiment were purchased from the Nonghyup in Sangju, Gyeongsangbuk-do. The Leukonostok mesenteroid strains used in the experiment were purchased from the Korea Microorganism Conservation Center (KCCM), the Microorganism Resource Center (KCTC), and the Agricultural Genetic Resources Information Center (KACC) (see Table 1 below). On the other hand, as pathogens for the evaluation of antibacterial activity, Staphylococcus aureus (wild type)), Listeria monocytogenes , KACC0550), Salmonella gallinarum ( Salmonella gallinarum , ATCC9184) and Mannheimia haemolytica (wild type)) strain was used.

Leukonostok mesenteroid strain used for aronia fermentation turn Leukonostok mesenteroid strain types One Leuconstoc mesenteroides KCCM35046 strain 2 Leuconstoc mesenteroides KCTC3100 strain 3 Leuconstoc mesenteroides KACC12312 strain 4 Leuconstoc mesenteroides KACC12313 strain 5 Leuconstoc mesenteroides KACC12315 strain

<1-2> aronia Fermented broth production

First, a fermentation broth was prepared by mixing aronia fruit ground with a grinder and MRS medium. Specifically, aronia fruit was crushed using a blade grinder, and 10 g of crushed fruit juice was mixed with 90 mL of sterilized 50% MRS medium to prepare an aronia fermented solution. The lactic acid bacteria culture solution for inoculation was prepared while stirring at a speed of 150 rpm at 30°C using MRS medium. Each of the leukonostok mesenteroid strains shown in Table 1 was finally inoculated into the aronia fermentation broth at a rate of 10% (v/v) as a starter inoculum, and fermented at 30° C. at a speed of 80 rpm for 48 hours. proceeded.

At this time, a total of 6 test spheres were set. The control was a test group that was not inoculated with any strain, and the natural fermentation of the aronia ferment was induced. Treatment group 1 was KCCM35046 strain, treatment group 2 was KCTC3100 strain, treatment group 3 was KACC12312 strain, treatment group 4 was KACC12313 strain, and treatment group 5 was fermented using the KACC12315 strain according to the leukonostok mesenteroid strains used. .

test set Kinds condition Abbreviation control no treatment CON treatment area 1 KCCM35046 strain fermentation Tr1 treatment area 2 KCTC3100 strain fermentation Tr2 treatment area 3 KACC12312 strain fermentation Tr3 treatment area 4 KACC12313 strain fermentation Tr4 treatment area 5 KACC12315 strain fermentation Tr5

<1-3> aronia Alcoholic extract production of fermentation broth

After fermentation was completed, a sample of 1 mL was collected from the fermentation broth for each test section and used for the measurement of the number of viable cells and the measurement of organic acids. Then, the remaining fermentation broth was transferred to a glass beaker and dried at 60° C. for one day. After drying was completed, the dried fermentation broth was pulverized using a mortar, and ethanol corresponding to 10 times the volume was added thereto and extracted at a speed of 80 rpm for one day. After the extraction was completed, the remaining solids were separated using filter paper (whatman no 1), and the remaining filtrate was concentrated with a vacuum concentrator. Finally, the concentrated samples were used for the following experimental analysis.

< Example 2>

aronia Analysis of physiological activity of fermented alcohol extract and development of strain for fermentation

<2-1> beta glucosidase Enzyme activity measurement

In this experiment, the beta-glucosidase enzyme activity of the aronia fermentation broth prepared in Example <1-2> was measured using the absorbance evaluation method. Enzyme activity was evaluated using each seed culture medium as an enzyme solution and a PNPG (p-nitrophenyl-β-D-glucoside) solution as a substrate solution. Enzyme activity was measured according to the method of Kim et al. (2007: Journal of Microbiology and Biotechnology. 17: 905912). The unit (U) representing the enzymatic activity was defined so that the enzyme corresponding to the turbidity of the culture solution represented 1 mM of p-nitrophenyl produced by decomposing the substrate for 1 minute. The concentration of p-nitrophenyl liberated from the substrate was evaluated by measuring the absorbance at 600 nm. For reference, the change of phenolic compounds contained in plants occurs by various microorganisms or enzymes secreted by them, and these enzymes include decarboxylase, tannase, and reductase. , benzyl alcohol dehydrogenase (benzyl alcohol dehydrogenase) and beta glucosidase (β-glycosidase) and the like are known.

As a result, as shown in FIG. 1, it was confirmed that beta-glucosidase enzyme activity was significantly high in treatment group 4 (L. mesenteroide KACC12313) and treatment group 5 ( L. mesenteroide KACC12315).

<2-2> Organic acid analysis

In this experiment, the organic acid content in the aronia fermentation broth prepared in Example <1-2> was measured using HPLC (Agilent 1100 series, CA, USA) equipped with a DAD detector and an automatic sample injector. Organic acids such as lactic acid, acetic acid, propionic acid, and butyric acid were separated and quantified using a column for organic acid analysis (Rezex TM ROA-Organic Acid H+). As the analysis conditions, a 0.005 N sulfuric acid solution was used for the mobile phase, and the mobile phase was flowed at a rate of 0.6 mL per minute. Detection of organic acids was performed at 220 nm.

The composition of organic acids produced during the fermentation of aronia fruit is shown in detail in Table 3 below.

In this test, lactic acid, acetic acid, propionic acid and butyric acid were investigated, and only lactic acid and acetic acid were detected in the analysis result. Significantly high lactic acid production was shown in control group and treatment group 1 (L. mesenteroide KCCM35046). Conversely, treatment group 2 (L. mesenteroide KCTC3100) showed the significantly lower lactic acid production. However, acetic acid production was significantly higher in treatment group 2. Even if lactic acid bacteria of the same genus were used, as shown in this result, the production amount of organic acid, a metabolite of lactic acid bacteria, was very different. That is, even lactic acid bacteria of the same genus may have different metabolic properties. Therefore, the selection of lactic acid bacteria with the most suitable characteristics according to the purpose of use has great significance and can be seen as important. In addition, it would not be desirable to apply one lactic acid bacterium to various applications just because it is a lactic acid bacterium. Incidentally, lactic acid is known as an organic acid involved in fermentation stabilization in plant fermentation, and acetic acid is known as an organic acid that helps to secure storage after fermentation because it has superior antibacterial activity compared to lactic acid (Keles, G., & Demirci, 2011: Animal Feed Science and Technology. 164:21-28). Therefore, information on organic acids produced by each lactic acid bacteria using the same substrate will be useful in selecting strains suitable for the purpose of use.

Analysis result of organic acid production in aronia fermented broth for each test group organic acid composition test piece ¹ SEM P-value control Tr 1 Tr 2 Tr 3 Tr 4 Tr 5 Lactic acid, mg/mL 27.45 ^c 28.06 ^c 21.39 ^a 22.76 ^b 27.39 ^c 23.44 ^b 0.64 <0.001 acetic acid,
mg/mL 4.38 ^b 4.13 ^a 5.14 ^d 5.20 ^d 5.24 ^d 4.93 ^c 0.11 <0.001 Lactic acid/acetic acid ratio 6.27 ^e 6.80 ^f 4.17 ^a 4.38 ^b 5.22 ^d 4.76 ^c 0.24 <0.001 ¹ Treatment: CON, control (non-inoculated Aronia); Tr 1, fermented by L. mesenteroides KCCM 35046; Tr 2, fermented by L. mesenteroides KCTC3100; Tr 3, fermented by L. mesenteroides KACC12312; Tr 4, fermented by L. mesenteroides KACC12313; Tr 5, fermented by L. mesenteroides KACC12315.
^abcdef Different superscripts in same row mean significantly different (P<0.05).

<2-3> cell growth measurement

In this experiment, the concentration of lactic acid bacteria in the aronia fermentation broth for each test section prepared in Example <1-2> was evaluated by the method of measuring the number of viable cells. The fermentation broth was serially diluted with sterilized 0.8% salt solution after fermentation was completed, and each diluted solution was spread on MRS plate medium. The MRS plate medium coated with the diluted solution was cultured for 24 hours in an incubator at 30° C., and the number of colonies formed was counted to evaluate the cell concentration.

As a result, as shown in FIG. 2, ^{cell growth of 10 8} CFU/mL or more was shown in all test groups. Significantly high cell growth was observed in treatment group 2 (L. mesenteroide KCTC3100). Interestingly, the control group (test group not inoculated with any strain) also showed high cell growth. As mentioned in the test method in the preceding text, sterilization was not performed on the crushed aronia fruit in this test. That is, the growth of lactic acid bacteria present in aronia fruit was not artificially excluded. Therefore, it is judged that the natural growth of lactic acid bacteria present in the aronia fruit was induced.

<2-4> Antibacterial activity measurement

In this experiment, the antibacterial activity of the fermented alcohol extract of Aronia for each test section prepared in Example <1-3> was measured by the growth inhibition ring evaluation method. The prepared pathogens were smeared on the LB plate medium. Thereafter, 50 μl of the aronia ferment extract was injected into a paper disk having a diameter of 6 mm. The paper disks prepared in Example <1-3> in which aronia fermented alcohol extracts were injected for each test section were placed on a plate medium smeared with pathogens, and then cultured at 37° C. for 24 hours. Thereafter, the diameter of the growth inhibition translucency formed on the plate medium was measured.

Detailed results are shown in Table 4 below.

Analysis of antibacterial activity of aronia fermented alcohol extract for each test group pathogen test piece ¹ CON Tr 1 Tr 2 Tr 3 Tr 4 Tr 5 Staphylococcus aureus
( Staphylococcus aureus ) ND ND ND ND ND ND Listeria
( Listeria monocytogenes ) ND 10 10 ND 11 12 man haimia haemoritica
( Mannheimia haemolytica ) 15 14 ND ND ND ND Salmonella Galinarum
( Salmonella gallinarum ) ND ND ND ND ND ND ¹ Treatment: CON, control (non-inoculated Aronia); Tr 1, fermented by L. mesenteroides KCCM 35046; Tr 2, fermented by L. mesenteroides KCTC3100; Tr 3, fermented by L. mesenteroides KACC12312; Tr 4, fermented by L. mesenteroides KACC12313; Tr 5, fermented by L. mesenteroides KACC12315.

As shown in Table 4 above, the antimicrobial activity of the aronia fermented alcohol extracts prepared in Example <1-3> was not observed in any of the test groups. On the other hand, antibacterial activity was observed in the case of Listeria and Manheimia. Antibacterial activity against Listeria was not observed in the control group, but was observed in treatment group 1 ( L. mesenteroides KCCM35046), treatment group 2 ( L. mesenteroides KCTC3100), treatment group 4 ( L. mesenteroides KACC12312) and treatment group 5 ( L. mesenteroides KACC12315). became In the case of Mannheimia haemolytica , antibacterial activity was observed only in the control group and treatment group 1. Through the above results, it was confirmed that the antibacterial activity and antibacterial activity spectrum were changed through fermentation.

<2-5> Analysis of total polyphenol content and measurement of antioxidant activity

In this experiment, the total polyphenol content contained in the aronia fermented alcohol extract for each test section prepared in Example <1-3> was evaluated according to the method of Juan and Chou (2010: Food Microbiology, 27: 586591). , gallic acid was used as a standard material. Antioxidant activity was measured by evaluating the concentration of free radicals removed by fermentation using DPPH (2,2-Diphenyl-1-picrylhydrazyl) reagent as a free radical. Antioxidant activity evaluation was performed according to the method of Brand-Williams et al. (1995: Food Science and Technology, 28: 2530), and the formula used for evaluation of antioxidant activity is as follows.

Free radical scavenging activity, % = (Black OD525 - Sample OD525)/Blank OD525×100

Changes in antioxidant activity-related substances were evaluated by thin layer chromatography and DPPH staining, and the test method was performed according to the method of Masoko and Eloff (2007: African Journal of Traditional, Complementary and Alternative Medicine, 4: 231239). In detail, a total of three mobile phase conditions were used. Each mobile phase solvent composition was EMW (polar/neutral), ethyl acetate/methanol/water = 40/5.4/5 (80 mL/10.8 mL/10 mL); CEF (intermediate polarity/acidic), chloroform/ethyl acetate/formic acid = 5/4/1; BEA (non-polar/basic): benzene/ethanol = 90/10/1. The material points of the fermented extracts separated by each mobile phase condition (aronia fermented alcohol extract for each test section prepared in Example <1-3>) were shown by staining with 0.2% DPPH solution.

As a result, as shown in Figure 3, significantly high polyphenol production was shown in treatment group 1 ( L. mesenteroide KCCM35046) and treatment group 5 ( L. mesenteroide KACC12315). The polyphenol content in aronia fruit is known to be about 7 mg/L (Kim et al. 2013: Nitric Oxide, 35: 5464; Malinowska et al., 2012: Nutrition, 28: 793798). In this experiment, about 10% of the aronia fruit crushed liquid was used in the fermentation broth, and as a result of the analysis, polyphenols at a level of 0.6 mg/L were found. Therefore, it was possible to obtain results consistent with the contents of previously reported studies.

On the other hand, the antioxidant activity results of each test group are as shown in FIG. The best antioxidant activity was shown in treatment group 5 ( L. mesenteroide KACC12315). Interestingly, in the case of treatment group 4 ( L. mesenteroide KACC12313), the total polyphenol content was low, but it was found that the antioxidant activity was very excellent. In addition, it was found that the total polyphenol content and antioxidant activity were significantly improved in some treatment groups compared to the control group. Through these results, it was found that the useful substances contained in Aronia were changed by the lactic acid bacteria used in the test. However, no significant material change was found in the thin-layer chromatography results stained with DPPH (see FIGS. 5A to 5C). However, the substances identified by UV showed different results compared to the control. In the case of the material separated into the EMW mobile phase, the results were shown to be new materials at Rf 0.4, 0.5 and 0.8 (see FIG. 5D ). The result considered to be such a novel material was also observed in the CEF mobile phase condition, and the result was found at Rf 0.25 (see FIG. 5E ).

<2-6> Phenolic compound profiling

In this experiment, the total polyphenolic substance change in the aronia fermented alcohol extract for each test section prepared in Example <1-3> was measured using high performance liquid chromatography (HPLC) for each substance on the chromatogram. Changes due to fermentation were investigated by calculating their area ratio. The analysis was performed according to the method reported by Ghasemzadeh and Jaafar (2013: BMC Complementary and Alternative Medicine, 13: 341350). Specifically, two different mobile phases were used. A 0.2% acetic acid solution was used for the mobile phase (A), and a 0.5% acetic acid solution and acetonitrile were mixed in a 1:1 ratio for the mobile phase (B). As the analysis conditions, each mobile phase was mixed at different ratios according to the analysis time, and the mixing conditions were as follows. 0 minutes 95(A): 5(B), 90:10 until 10 minutes, 60:40 until 40 minutes, 45:55 until 55 minutes, and 0:100 until 65 minutes.

As a result, as shown in FIG. 7, as a result of quantitative analysis on caffeic acid, chlorogenic acid, and trans-ferulic acids, in treatment group 1 ( L. mesenteroide KCCM35046) It showed a significantly high caffeic acid content. Chloronenic acid was significantly higher in treatment group 1 ( L. mesenteroide KCCM35046) and treatment group 5 ( L. mesenteroide KACC12315). Finally, in the case of ferulic acid, treatment group 5 ( L. mesenteroide KACC12315) was significantly higher.

<2-7> Relative performance index analysis

Various activities such as antibacterial activity, antioxidant activity, and total polyphenol content were analyzed as the physiological activity of the aronia fermented alcohol extract for each test section prepared in Example <1-3>. Therefore, it is necessary to draw an integrated conclusion by comprehensively calculating the different activities. Therefore, in this experiment, the relative performance index was calculated by applying the standardization method, and the best strain capable of improving the physiological activity of aronia was finally selected. Relative figure of merit was calculated using the following formula.

RPI (Relative figure of merit) = (A μ)/ σ + 3 (N ∼ 3,1)

In the above equation, A is the observed activity of the treatment group, μ is the average value of the activity, and σ is the standard deviation of the activity. The constant value of 3 is a constant value used to shift the standard normal distribution by a positive integer of 3 to prevent negative numbers from occurring.

As a result, as shown in Table 5 below, treatment group 1 ( L. mesenteroides KCCM35046) and treatment group 5 ( L. mesenteroides KACC12315) showed a significantly high relative performance index.

Relative performance index sum result for physiological activity of aronia fermented alcohol extract for each test group test piece ¹ SEM P value Tr 1 Tr 2 Tr 3 Tr 4 Tr 5 RPI ² 10.48 ^c 8.60 ^b 7.09 ^a 7.36 ^a 11.11 ^c 0.46 <0.001 ¹ Treatment: Tr 1, fermented by L. mesenteroides KCCM 35046; Tr 2, fermented by L. mesenteroides KCTC3100; Tr 3, fermented by L. mesenteroides KACC12312; Tr 4, fermented by L. mesenteroides KACC12313; Tr 5, fermented by L. mesenteroides KACC12315
² RPI: relative performance index was calculated with standardized values of antioxidant activity, antibacterial activity and total polyphenol content from each treatment.
^abc Different superscripts in same row mean significantly different (P<0.05).

<2-8> Statistical analysis

To analyze the effect of treatment, all observations were subjected to ANOVA according to the general linear model, and hypothesis testing was performed, and hypothesis testing was performed at the significance level of 95%. For the significance test between treatments, Duncan's multiple comparison analysis method was performed, and the significance level between treatments was tested at 95% significance level. All statistical analyzes were performed using the SPSS program (version 18, IBM, USA).

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (12)

In the antibacterial composition for Listeria monocytogene comprising an alcohol extract of Aronia fermented broth as an active ingredient,
The extract is an aronia fruit inoculated with any one of Leuconostoc mesenteroides KCCM35046, Leuconostoc mesenteroides KCTC3100, Leuconostoc mesenteroid KACC12313 and Leuconostoc mesenteroid KACC12315 strains and then fermented into alcohol Extracted, antibacterial composition. delete delete delete delete delete In a livestock feed additive composition having antibacterial activity against Listeria monocytogene , comprising an alcohol extract of Aronia fermented broth as an active ingredient,
The alcohol extract of the aronia fermented broth is aronia fruit by inoculating any one of Leuconostoc mesenteroides (Leuconostoc mesenteroides) KCCM35046, Leuconostoc mesenteroids KCTC3100, Leuconostoc mesenteroids KACC12313 and Leuconostoc mesenteroids KACC12315 strains. After fermentation, it is extracted with alcohol, livestock feed additive composition. 8. The method of claim 7,
The livestock is a livestock feed additive composition, characterized in that it is one kind of livestock selected from the group consisting of cattle, pigs, chickens, ducks, goats, sheep and horses. 8. The method of claim 7,
The composition is characterized in that for enhancing the immunity of livestock through antioxidant activity, livestock feed additive composition. delete delete delete

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