KR20220109196A - Composition for feed additive comprising fermentation product of allium tuberosum for inhibiting poultry pathogenic microorganism and avian influenza viruses - Google Patents

Abstract

The present invention relates to a feed additive composition comprising fermented leek, which is obtained by fermenting the leek by using Lactobacillus plantarum SK4719 strain (KACC 92270P) or an extract thereof. The fermented leek obtained by fermenting the leek by using Lactobacillus plantarum SK4719 strain derived from the leek or the extract thereof exhibits poultry disease controlling efficacy.

Description

Fermented leek feed additive composition for suppressing poultry pathogenic bacteria and avian influenza virus and its efficacy

The present invention relates to a feed additive composition comprising a fermented leek fermented using Lactobacillus plantarum SK4719 strain (KACC 92270P).

Recently, economic losses due to bird flu, an infectious respiratory disease, have occurred continuously in Korea, and disease outbreaks and damage due to other pathogenic microorganisms continue to occur, so it is necessary to develop product production technology that can respond in common.

Leek is a perennial plant that can be harvested 8 times a year, has very high farmland utilization efficiency, is inexpensive, and contains various functional substances, so it is useful as an eco-friendly antibacterial/antiviral agent. However, there is no development, commercialization and sale of products such as feed additives using leek. Currently, research on feeding livestock using fermented and non-fermented natural products of leek is in progress, and as a result, it is reported that there is an effect of improving the productivity, antioxidant, blood and immune activity of livestock.

Republic of Korea Patent Publication No. 10-2019-0033987

Accordingly, the inventor of the present invention completed the present invention by confirming the poultry disease control efficacy of leek-derived Lactobacillus plantarum ( Lactobacillus plantarum ) fermented leek fermented product or extract thereof using SK4719 strain.

Accordingly, an object of the present invention is to provide a feed additive composition comprising a fermented leek fermented using Lactobacillus plantarum SK4719 strain (KACC 92270P) and a method for preparing the same.

In order to achieve the above object, the present invention provides a feed additive composition comprising a leek fermented product or an extract thereof.

In addition, the present invention provides a method for producing a feed additive composition, comprising the step of mixing water or MRS and leek juice, and then inoculating and fermenting the Lactobacillus plantarum SK4719 strain.

In addition, the present invention provides a composition for inhibiting poultry pathogenic bacteria and avian influenza virus, comprising a fermented leek or an extract thereof.

The feed additive composition of the present invention is effective in controlling diseases of poultry because it exhibits excellent antibacterial activity.

1 is a graph showing (A) cell viability and (B) pH of leek fermented in MRS in an embodiment of the present invention.
Figure 2 is a graph showing (A) cell viability and (B) pH of the fermented leek in water according to an embodiment of the present invention.
Figure 3 is a graph showing the cell viability according to time of the fermented leek fermented in (A) MRS and (B) water in one embodiment of the present invention.
Figure 4 shows the DPPH radical scavenger activity of the leek fermentation in an embodiment of the present invention. In the graph, a and b mean that there is a statistically significant difference at p<0.05 using Students' t-test.
Figure 5 shows the antibacterial activity against poultry pathogens of the leek fermentation in an embodiment of the present invention. WFCC: water-fermented CC juice extract; MFCC: MRS-fermented CC juice extract.
6 shows an LC-MS/MS chromatogram of a methanol extract of a leek fermented product according to an embodiment of the present invention: (A) positive mode and (B) negative mode.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of well-known techniques known to those skilled in the art may be omitted. In addition, in describing the present invention, when it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, the terms used in this specification are terms used to properly express preferred embodiments of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs.

Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

The present invention relates to a feed additive composition comprising a fermented leek or an extract thereof, and a method for preparing the same.

In addition, the present invention relates to a composition for inhibiting poultry pathogenic bacteria and avian influenza virus, comprising a fermented leek or an extract thereof.

The leek fermented product may be fermented using a Lactobacillus plantarum SK4719 strain derived from leek, but is not limited thereto.

The fermented leek may be fermented by mixing water or MRS and leek juice, but is not limited thereto.

The extract of the leek fermented product may be an alcohol extract, for example, may be a methanol extract, but is not limited thereto.

The leek fermented product may be prepared by a method comprising the step of mixing water or MRS and leek juice, and then inoculating and fermenting the Lactobacillus plantarum SK4719 strain, but is not limited thereto. In addition, the method may further include the step of extracting the leek fermented product with alcohol to prepare an extract of the leek fermented product, but is not limited thereto.

The leek ferment or its extract has antibacterial activity against poultry pathogenic bacteria, for example, Salmonella Gallinarum , Salmonella pullorum ( S. pullorum ), Salmonella typhi ( S. typhi ), Salmonella typhi ) Murium ( S. typhimurium ), Salmonella paratyphi ( S. paratyphi ), Salmonella enteritidis ( S. enteritidis ), Salmonella anatum ( S. anatum ), Enterococcus faecalis ( Enterococcus faecalis ), Escherichia coli ( E . _ _

The leek fermented product or extract thereof may inhibit avian influenza virus, but is not limited thereto.

The water or MRS and the leek juice may be mixed in a volume ratio of 95-85: 5-15, for example, may be mixed in a volume ratio of 90: 10, but is not limited thereto.

The extract of the leek ferment is campterol (kaempferol), campferol 3- O -β-sophoroside (kaempferol 3- O -β-sophoroside), campferol-3- O -lutinoside (kaempferol-3) -O -rutinoside), camperol 3- O -glucoside (kaempferol 3- O -glucoside), oxodihydroxy-octadecenoic acid, (Z)-5,8,11-trihydro Roxyoctadec-9-enoic acid ((Z)-5,8,11-trihydroxyoctadec-9-enoic acid), 9(S)-HpOTrE, fatty acid derivative, 9S-HOTrE (9S-hydroxy-10E,12Z,15Z) -octadecatrienoic acid), (6Z,9Z,12Z,15Z)-octadecatrienoate ((6Z,9Z,12Z,15Z)-octadecatetraenoate), 9-OxoOTrE (9-oxo-10e,12z,15z-octadecatrienoic acid), 12(13)-epoxy-9Z-octadecenoic acid (12(13)-epoxy-9Z-octadecenoic acid), and at least one compound selected from the group consisting of combinations thereof, but is not limited thereto. .

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

<Example 1> Determination of optimum concentration of leek fermentation

Greenbelt leek sold at a general mart (Gwangjin-gu, Seoul) was used as the leek used in the experiment, and it was juiced using a green juicer (Angel-7700, Korea). Lactobacillus plantarum SK4719 (KACC 92270P) isolated from leek was used as a fermenting microorganism.

Leek juice was fermented in MRS medium (MFCC) and sterile water (WFCC). To find the optimal concentration for the fermentation of Lactobacillus plantarum SK4719, MRS and leek juice in water were prepared at concentrations of 0, 5, 10, 20, 25, 30, 35, 40% (v/v) and Lactobacillus plantarum SK4719 (107 log10 CFU/ml; 1%, w/v) was inoculated. After incubation at 30° C. and 100 rpm for 18 hours, the number of viable cells was counted and the pH was measured. The pH was measured using a pH meter (ISTEC 735P, Korea) and the number of viable cells was confirmed by a drop plate method on MRS agar (Difco, USA). To determine the optimal time for fermentation of leek juice using Lactobacillus plantarum SK4719 at 30°C, the number of viable cells at regular time intervals (0, 6, 12, 18, 24, 30, 36, 42, 48 h) were counted and the pH was measured. All experiments were repeated three times.

After mixing the freeze-dried non-fermented and fermented leek juice (20 g) with 200 ml 80% (v/v) methanol, it was shaken for 30 minutes in a shaking incubator (30 ° C, 100 rpm), and ultrasonicated for 15 minutes at 30 ° C or less. processed. The mixture was centrifuged at 13,500 rpm (4° C., 10 min) to obtain a supernatant. This process was repeated 5 times, the obtained supernatant was mixed, and after rotary evaporation at 45°C, freeze-dried, and stored at -80°C and freeze-dried until analysis.

As a result, as can be seen in FIG. 1 , when MRS and leek juice were fermented together for 0 hours, the pH was from 0% to 5.5 to 35% to 5.8. After 18 hours of fermentation, the pH was from 0% to 3.9 and from 35% to 3.8, the pH decreased by more than 1.3 after 18 hours of fermentation. The decrease in pH is due to the production of organic acids.

In addition, as can be seen in FIG. 2 , when water and leek juice were fermented together, the pH decreased sharply at a concentration between -5% during fermentation for 18 hours and maintained a decrease up to a concentration of 10%. At 0 hours of fermentation, the pH increased from 6.6 to 7.1, but after 18 hours of fermentation, the pH decreased to 3.27 at 10% concentration. Lactobacillus plantarum SK4719 viable cells were increased when 0-35% leek juice and MRS were fermented for 18 hours. Non-lactic acid bacteria grew well as the concentration of leek juice increased, whereas lactic acid bacteria decreased. When water and 10% leek juice were fermented together, the lowest pH and highest cell viability were obtained. Similarly, high viability and low survival rates of Lactobacillus plantarum SK4719 when MRS and 10% leek juice were fermented together. pH was shown (FIG. 1). Therefore, 10% (v/v) leek juice was suitable for fermentation of Lactobacillus plantarum SK4719.

As shown in FIG. 3 , it was confirmed by time profiling that 24 hours was optimal for fermentation of leek juice. When water and leek juice were fermented using Lactobacillus plantarum SK4719, it showed the highest CFU of 8.1 at 24 hours, and the pH was also sharply decreased from 7.3 to 4.4. When MRS and leek juice were fermented, the pH decreased from 6.2 to 3.9. Lactobacillus plantarum SK4719 was the initial It increased to 9.5 after 15 hours of fermentation at 7.6 CFU/ml. In a previous study, when Lactobacillus plantarum SK4719 was fermented with MRS, it showed a viability of 9 CFU/ml within 12 hours (Niu et al., 2019).

<Example 2> Analysis of bioactive compounds of leek juice extract

Total phenolic and flavonoid content

The total phenol content of the samples was determined by the Folin-Ciocalteu method (Dudonne et al., 2009). 20 μl of the sample (10 mg/ml in 80% (v/v) MeOH) and 100 μl of 0.2 N Fc reagent (Sigma f-9252) were mixed and reacted in the dark for 5 minutes. After mixing 80 μl of 7.5% Na ₂ CO ₃ , it was reacted for 60 minutes in the dark at room temperature. Gallic acid was used as standard solution. Absorbance at 750 nm was measured using an ELISA reader (Synergy 2, BioTek Instruments Inc.). Total phenols were expressed in mg of gallic acid equivalent per gram of extract (mg GAE/g).

For total flavonoid content (TFC) determination, 20 μl of sample (10 mg/ml in 80% MeOH), 180 μl of 90% diethylene glycol and 20 μl of 1 N NaOH were mixed in a 96 well plate and mixed at room temperature in the dark. was reacted for 60 minutes. Quercetin was used as a standard solution. Absorbance was measured at 415 nm using an ELISA reader. TFC was expressed as mg of equivalent quercetin per gram of extract (mg QE/g).

As a result, as shown in Table 1, in the case of water fermentation (p<0.05), the polyphenol content increased by 24% and the flavonoid content decreased by 27% after fermentation. The decrease in TFC is due to the meiosis of flavonoid compounds by enzymes released by probiotics. In previous studies, it has been reported that the initial metabolism of flavonoids is not well absorbed into the intestine, so it is advantageous (Lotito et al., 2011). When Graptopetalum paraguayense E. Walther was fermented with Lactobacillus plantarum SK4719 BCRC 10357, flavonoids and phenolic compounds increased from 17.2 and 92.2 μg/mg to 22.9 and 111 μg/mg, respectively (Wu et al., 2011).

Allicin and thiol concentrations

Thiol concentration was determined by adding 100 μl of diluted sample (50 mg/ml) and 100 μl of 1.5 mM 5,5'-dithiobis (2-nitro-benzoic acid) (DTNB) in a 96-well plate at room temperature for 10 minutes. . Absorbance was measured at 412 nm, and thiol content was expressed as μM of cysteine equivalents per g of plant extract (μM CE/g).

To determine allicin content, cysteine was diluted to 10, 20, 30, 40, 50 μM using 50 mM HEPES buffer. 100 μl of the sample (50 mg/ml) and 100 μl of L-cysteine (250 μM) were reacted in a 96-well plate for 10 minutes. 100 μl of the mixture was reacted with 100 μl of 1.5 mM (DTNB) for 10 minutes. Absorbance was measured at 412 nm, and the allicin concentration was calculated by the following formula. Allicin concentrations were expressed in μM of cysteine equivalents per gram of plant extract (μM CE/g).

In the above formula, a is the thiol in the sample, b is the thiol after reaction with L-cysteine, and c is the added L-cysteine.

As a result, in the case of water fermentation, the contents of thiol and allicin decreased by 52% (p<0.05) and 17% (p>0.05), respectively, after 24 hours of fermentation. During fermentation, the allicin content decreased due to its volatilization properties and the use of Lactobacillus plantarum SK4719. In the prior literature (Yang et al., 2014), leek Lactobacillus plantarum SK4719 It was reported that allicin decreased by 85.45% and thiol increased by 39.37% when fermented with LK8 for 48 hours. However, no significant change was observed in the case of fermentation in MRS medium, probably due to the dark color of the extracts containing MRS medium.

<Example 3> Biological activity of fermented leek juice extract

antioxidant activity

In a 96 well plate, mix 180 μl of 2,2-diphenyl-1-picrylhydrazyl (DPPH) stock solution (0.2 mM in 80% (v/v) methanol) and 20 μl of sample (5 mg/ml) at room temperature. It was placed in the dark for 30 minutes. Trolox was used as a standard at concentrations of 100, 200, 400, 600, and 800 μM diluted with 80% MeOH. Absorbance was measured at 517 nm using an ELISA reader. DPPH scavenger activity was calculated by the following formula.

In the above equation, A ₀ is the absorbance of the control at 517 nm, and A ₁ is the absorbance of the sample at 517 nm.

As a result, as shown in Figure 4, after 24 hours of fermentation in water and MRS medium, the antioxidant activity of leek juice was significantly reduced to 24% and 9%, respectively (p<0.05). This is due to the reduction of allicin and thiol, which are flavonoids and organosulfur compounds, after fermentation of leek juice.

Antibacterial and antiviral activity of leek juice

The activity of the fermented leek juice extract prepared as in Example 1 was confirmed by agar well dispersion assay. After mixing 100 mg/ml of fermented leek juice extract with water (WFCC) and MRS (MFCC), 100 μl was added to the wells of NA agar plates containing pathogens and incubated at 37° C. for 24 hours.

In addition, by performing an antiviral test by a cytopathic effect (CPE) reduction assay, the virus-induced lytic protective effect of MDCK (Madin-Darby Canine Kidney) cells by methanol extracts of WFCC and MFCC was confirmed. MDCK cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRI, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) at 37°C, 5% CO ₂ wet incubator. . Avian influenza, A H1N1 virus (A/Puerto Rico/8/34) was cultured in virus growth medium (VGM). VGM was prepared in DMEM containing 0.3% bovine serum albumin (BSA) and 1% P/S.

First, Lactobacillus plantarum SK4719 in 50 mg/ml water (WFCC) and MRS (MFCC) A sample solution (10 mg/ml) was prepared by diluting MeOH extract of fermented leek juice (10% v/v) in VGM 5 times. MDCK cells were seeded in 96-well plates and cultured overnight at 37° C., 5% CO ₂ wet incubator. Then, MDCK cells were infected with influenza A H1N1 virus of 50% tissue culture infective dose (TCID50) and incubated in a wet incubator at 37° C., 5% CO ₂ . Then, the cells were washed with PBS (pH 7.0), and 3-fold serially diluted methanol extract from 10 mg/ml was added. After incubation for 48 hours, the anti-influenza activity of the extract was confirmed by direct microscopic observation of CPE reduction and crystal violet uptake method.

S. Typhi, S. Typhimurium, S. Paratyphi, S. Enteritidis, S. Anatum, E. faecalis , E. coli , St. aureus , Lactobacillus plantarum SK4719 in water (WFCC) and MRS medium (MFCC) against pathogens such as C. perfringens As a result of confirming the antibacterial activity of the fermented leek juice extract (100 mg/ml), as shown in FIG. 5, both extracts showed high antibacterial activity against poultry pathogens, and the antibacterial activity of the fermented leek juice extract was It appears to be due to the flavonoid content.

Fermented leek juice extracts WFCC and MFCC in water and MRS) showed antiviral activity as shown in Table 2. The WFCC extract showed antiviral activity at a concentration of 1.1 mg/ml and the MFCC extract showed antiviral activity at a concentration of 3.3 mg/ml. This is because WFCC contains more bioactive compounds than MFCC, and it is believed that kaempferol and its glycosides contained in the ethanol extract of WFCC show anti-influenza activity.

<Example 4> UHPLC-LTQ-Orbitrap-MS/MS analysis

Metabolite profiling of water-fermented leek juice extract (10 mg/mL) was performed using ultra-high-performance liquid chromatography-linear trap quadrupole-orbitrap-tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS/MS). did. Chloramphenicol (2.5 mg/mL) was used as an internal standard (IS). A sample injection volume of 5 μL was injected into a UHPLC system equipped with a Vanquish binary pump H system (Thermo Fisher Scientific, Waltham, MA, USA), auto-sampler, and column compartment at a flow rate of 0.3 mL/min. Chromatographic separation was performed with a Phenomenex KINETEX® C18 column (100 mm × 2.1 mm, 1.7 μm particle size; Torrance, CA, USA) while maintaining the column temperature of 40°C. The mobile phase consisted of 0.1% formic acid in water (v/v, solvent A) and 0.1% formic acid in acetonitrile (v/v, solvent B). The mobile phase solvent gradient was programmed as follows: 5% solvent B for 1 minute, stepwise increase to 100% solvent B for 9 minutes, and hold for 1 minute and then decrease to 5% solvent B for 3 minutes. MS data were collected using an Orbitrap Velos Pro™ system equipped with an ion-trap mass spectrometer and HESI-II probe. Mass spectra were acquired in the range of 100-2000 m/z. The probe heater and capillary temperature were set to 300°C and 350°C, respectively. The capillary voltage was set at 2.5 KV and 3.7 KV in negative and positive mode, respectively. UHPLC-LTQ-Orbitrap-MS/MS data were acquired with Xcalibur software (version 2.00, ThermoFisher Scientific) and converted to netCDF format (*.cdf) using Xcalibur software. Peak detection, retention time collection, and alignment were performed using the MetAlign software package ( http://www.metalign.nl) . MW, RT, and mass fragmentation patterns (MSn) data of metabolites evaluated by UHPLCLTQ-Orbitrap-MS/MS were verified by comparison with data obtained from literature and published online databases.

Chromatograms of WFCC measured in positive and negative modes are shown in FIG. 6 . Collectively, 12 other secondary metabolites belonging to flavonols and fatty acids were identified. Identified compounds are campterol (kaempferol), campferol 3- O -β-sophoroside (kaempferol 3- O -β-sophoroside), campferol-3- O -lutinoside (kaempferol-3- O) -rutinoside), camperol 3- O -glucoside (kaempferol 3- O -glucoside), oxodihydroxy-octadecenoic acid, (Z)-5,8,11-trihydroxyoctane Dec-9-enoic acid ((Z)-5,8,11-trihydroxyoctadec-9-enoic acid), 9(S)-HpOTrE, fatty acid derivative, 9S-HOTrE (9S-hydroxy-10E,12Z,15Z-octadecatrienoic acid) acid), (6Z,9Z,12Z,15Z)-octadecatrienoic acid , 12(13)-epoxy-9Z-octadecenoic acid (12(13)-epoxy-9Z-octadecenoic acid) (Table 3).

<Example 5> Leek feed additive

Greenbelt leek sold at a general mart (Gwangjin-gu, Seoul) was used as the leek used in the experiment, and it was juiced using a green juicer (Angel-7700, Korea). After squeezing, 90% water and 10% leek juice were mixed and used to prepare non-fermented, fermented leek feed additive, and the fermented leek feed additive was cultured in the above mixture at 30° C., 100 rpm for 18 hours. The fermentation was carried out using Lactobacillus plantarum SK4719 (KACC 92270P) isolated from leek as described in Example 1 as a fermenting microorganism.

Non-fermented and fermented leek feed additives were added at a level of 3% of the total feed ingredients, stirred for 10 minutes using a stirrer (HC 123132-500L, Korea), and then fed to broilers.

As an antibiotic, Enramycin-10 product was used, 0.01% of the total broiler feed amount was added, mixed with a stirrer for 10 minutes, and then fed to the broilers. The composition of raw materials for broiler early and late broilers are as described in Table 4 below.

<Example 6> Animal experiment

Experimental feed and experimental design

At 5 repetitions per treatment group, 40 new juveniles were stocked per repetition, and complete random arrangement was carried out. The basic experimental feeds used in this experiment were mainly corn and soybean meal, and ME was prepared at 3,100 kcal/kg in the first half of the broiler feed and 3,150 kcal/kg in the second half. The first feed was fed from the start date to the 3rd week, and the late feed was fed from the 3rd to 5th weeks, respectively.

Management of published animals and breeding

As a test animal, 800 male chicks (starting weight: 134.5±0.2 g) of broiler Ross308 breed were used, and the experiment was repeated 5 times by dividing it into 4 treatment groups as shown in Table 5 above. Each pen (repeat) was stocked by 40 trees, and the experiment was conducted for 4 weeks from August 26 to September 23, 2019.

The experimental cage is a cage-free cage with a concrete floor installed, and rice husks were used to spread it to a thickness of 5 cm. The temperature inside the house was tested in an environment of 27.8±1.6℃ and humidity of 74.8±8.1%, and the indoor temperature was set to 32±1℃ for the first week and then decreased by 2℃ every week to 25±1℃ in the last week of the experiment. was to be maintained. The lighting was turned on for 24 hours from the start of the experiment to the end of the experiment, and feed and water were fed unlimitedly (ad-libitum), respectively.

Body weight, feed intake and feed efficiency

Daily feed intake (ADFI), daily gain (ADG), and feed efficiency (Feed/Gain) were measured. Body weight and feed intake were measured weekly from the start date to the end of the experiment, and body weight was obtained as the average body weight per animal by measuring all stocked sorghum in one pen at once. The daily feed intake was calculated by dividing the total feed amount by excluding the remaining feed amount and the loss amount by the number of days consumed. Feed efficiency was calculated as the ratio of weight gain and feed intake for each period.

sampling

On the day of the end of the breeding experiment, 2 numbers per pen were randomly selected, and 10 pieces per treatment group were slaughtered to collect samples. Blood, breast meat, leg meat, organs (liver, spleen, cyst F), small intestine (duodenum, jejunum, ileum) and cecum were collected.

Blood was collected using the slaughter and cardiac blood sampling method using carbon dioxide, and was stored at 4°C after being injected into an EDTA-treated blood collection tube. Thereafter, the serum was separated by centrifugation at 1500 rpm * g for 10 minutes, and the serum was stored at -20°C.

meat quality

The relative weight of chicken meat was measured by measuring the weight of the selected chickens for each treatment group and the weight of the collected chicken meat (breast meat, leg meat), and expressed as a ratio of the weight of chicken meat per 100 g of live weight.

For cooking loss, the sample was molded into a circular shape, placed in a polyethylene bag, and heated in a 75℃ water bath (C-WBE, Chang Shin co., Korea) for 30 minutes. Then, it was allowed to cool at room temperature for 10 minutes, and the weight difference before and after heating was compared to calculate the reduced amount.

* Cooking loss(%) = Sample weight before cooking - Sample weight after cooking / Sample weight before cooking × 100

For meat color, measure the surface of the sample using a colorimeter (Chromameter, CR210, minolta, Japan) to indicate L* value indicating lightness, a* value indicating redness, and b* value indicating yellowness. was measured. At this time, a calibration plate with an L* value of 97.69, a* value of -0.43, and b* value of +1.98 was used as the standard color.

The pH was calculated as the value shown when a pH meter (Hanna Instruments, Nusfalau, Romania) was placed at a depth of 1 cm of breast meat and leg meat, and was measured three times per sample.

Weighing organs (liver, spleen, sac)

The weight of the excised organs (liver, spleen, F sac) was measured using a scale (EL4002, Mettlertoledo) after removing foreign substances such as fat, and calculated as the ratio of weight per 100 g of live weight.

Length and weight of small intestine (duodenum, jejunum, ileum) and cecum

The length and weight of the small intestine and caecum according to the addition of leek feed additives and antibiotics to the feed were measured. The small intestine was divided into three parts: the duodenum, the jejunum and the ileum, and the weight was measured after the contents in the small intestine and cecum were removed.

Examination of live bacteria in the small intestine (duodenum, jejunum, ileum) and cecum

The number of viable cells in the small intestine and caecum was measured using a standard agar culture method using plate plates of MRS, NA, Macconkey, ST (Streptococcus thermophilus), SS (Salmonella shigella) (Difco, USA). For intestinal contents, 8 contents per treatment group were used, and 1 g of the sample was placed in 9 ml of sterilized distill water and then diluted sequentially through a homogenization process.

After spotting, 10 μl of each medium was dispensed and cultured in an incubator at 37° C. for 24 hours. By measuring the number of effective colonies that appeared, CFU/g was calculated and converted into a log10 value. All experiments were performed in 3 repetitions.

Analysis of general components in broiler blood according to leek feed additive feeding

To measure the composition of blood, blood was collected from broilers randomly selected at the end of the experiment by 10 numbers per treatment group using the cardiac blood sampling method. The collected blood was used for analysis with serum separated using a centrifuge (HA-12, Korea) at 3,000 rpm for 15 minutes.

A total of 15 blood components were measured, including GGT, GOT, GPT, LDH, ALP, Glucose, BUN, Creatine, Uric acid, Total cholesterol, HDL, LDL, Triglyceride, Total protein, and Albumin.

HDL-cholesterol (%) was calculated by subtracting the HDL content from the total cholesterol content and the ratio of HDL to the total cholesterol content, and the value of LDL+VLDL was calculated.

The analysis results are shown in Table 6.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may 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.

National Academy of Agricultural Sciences KACC92270P 20190709

Claims (13)

A feed additive composition comprising a leek ferment or an extract thereof. The feed additive composition according to claim 1, wherein the leek fermented product is fermented using a Lactobacillus plantarum SK4719 strain. The feed additive composition according to claim 2, wherein the leek fermented product is fermented by mixing water or MRS and leek juice. The feed additive composition according to claim 3, wherein the water or MRS and the leek juice are mixed in a volume ratio of 95-85: 5-15. The feed additive composition of claim 1, wherein the extract of the leek fermented product is an alcohol extract. The method of claim 5, wherein the extract of the fermented leek is campterol (kaempferol), campferol 3- O -β-sophoroside (kaempferol 3- O -β-sophoroside), campferol-3- O -ru Tinoside (kaempferol-3- O -rutinoside), camperol 3- O -glucoside (kaempferol 3- O -glucoside), oxodihydroxy-octadecenoic acid, (Z)-5, 8,11-trihydroxyoctadec-9-enoic acid ((Z)-5,8,11-trihydroxyoctadec-9-enoic acid), 9(S)-HpOTrE, fatty acid derivative, 9S-HOTrE (9S-hydroxy) -10E,12Z,15Z-octadecatrienoic acid), (6Z,9Z,12Z,15Z)-octadecatetraenoate ((6Z,9Z,12Z,15Z)-octadecatetraenoate), 9-OxoOTrE (9-oxo-10e) ,12z,15z-octadecatrienoic acid), 12(13)-epoxy-9Z-octadecenoic acid (12(13)-epoxy-9Z-octadecenoic acid), and combinations thereof comprising at least one compound selected from the group consisting of Phosphorus, a feed additive composition. After mixing water or MRS and leek juice, inoculating and fermenting the Lactobacillus plantarum SK4719 strain, a method for producing a feed additive composition. The method according to claim 7, wherein the water or MRS and the leek juice are mixed in a volume ratio of 95-85: 5-15. The method according to claim 7, wherein the fermentation is carried out for 12 to 24 hours. The method according to claim 7, further comprising the step of extracting the leek fermented product with alcohol to prepare an extract of the leek fermented product. A composition for inhibiting poultry pathogenic bacteria and avian influenza virus, comprising a fermented leek or an extract thereof. The composition for inhibiting pathogenic poultry bacteria and avian influenza virus according to claim 10, wherein the leek fermented product is fermented using Lactobacillus plantarum SK4719 strain. The composition for inhibiting poultry pathogenic bacteria and avian influenza virus according to claim 12, wherein the leek fermented product is fermented by mixing water or MRS and leek juice.

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