KR20240049533A - 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 containing fermented chives or extracts thereof fermented using Lactobacillus plantarum SK4719 strain (KACC 92270P).

Description

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

The present invention relates to a feed additive composition containing chive fermentation product fermented using Lactobacillus plantarum SK4719 strain (KACC 92270P).

Recently, economic losses due to avian influenza, a contagious respiratory disease, have continued to occur in Korea, and disease outbreaks and damage caused by other pathogenic microorganisms continue, so there is a need to develop product production technologies that can commonly respond to these.

Chives are perennial plants that can be harvested eight times a year, have very high farmland use efficiency, are inexpensive, and are especially useful as an eco-friendly antibacterial/antiviral agent as they contain various functional substances. However, there are no products developed, commercialized, or sold using chives, such as feed additives. Currently, research is being conducted on feeding livestock using fermented and non-fermented natural products of chives, and the results are reported to be effective in improving livestock productivity, antioxidants, blood, and immune activity.

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 fermented chives or extracts thereof using the leek-derived Lactobacillus plantarum SK4719 strain.

Therefore, the purpose of the present invention is to provide a feed additive composition containing fermented chives fermented using Lactobacillus plantarum SK4719 strain (KACC 92270P) and a method for producing the same.

To achieve the above object, the present invention provides a feed additive composition comprising fermented chives or extracts thereof.

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

Additionally, the present invention provides a composition for inhibiting poultry pathogenic bacteria and avian influenza virus, comprising fermented chives or extracts thereof.

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

Figure 1 is a graph showing (A) cell viability and (B) pH of fermented chives fermented in MRS in an embodiment of the present invention.
Figure 2 is a graph showing (A) cell survival rate and (B) pH of fermented chives fermented in water in an embodiment of the present invention.
Figure 3 is a graph showing the cell survival rate over time of (A) MRS and (B) fermented chives fermented in water in an embodiment of the present invention.
Figure 4 shows the DPPH radical scavenger activity of fermented chives in one embodiment of the present invention. In the graph, a and b indicate a statistically significant difference at p<0.05 using Students' t-test.
Figure 5 shows the antibacterial activity of fermented chives against poultry pathogens in one embodiment of the present invention. WFCC: water-fermented CC juice extract; MFCC: MRS-fermented CC juice extract.
Figure 6 shows LC-MS/MS chromatograms of methanol extract of fermented chives according to an example 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 attached drawings. In the following description, detailed descriptions of techniques well known to those skilled in the art may be omitted. Additionally, when describing the present invention, if 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 terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of 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 is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

The present invention relates to a feed additive composition comprising fermented chives or extracts thereof and a method for producing the same.

Additionally, the present invention relates to a composition for inhibiting poultry pathogenic bacteria and avian influenza virus, comprising fermented chives or extracts thereof.

The fermented chive product may be fermented using the leek-derived Lactobacillus plantarum SK4719 strain, but is not limited thereto.

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

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

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

The fermented chives or extracts thereof have antibacterial activity against poultry pathogenic bacteria, for example, Salmonella Gallinarum, S. pullorum , Salmonella typhi , and Salmonella typhi. Murium ( S. typhimurium ), Salmonella paratyphi ( S. paratyphi ), Salmonella enteritidis ( S. enteritidis ), Salmonella anatum ( S. anatum ), Enterococcus faecalis, Escherichia coli ( E coli ), Staphylococcus aureus , and Clostridium perfringens, but are not limited to this.

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

The water or MRS and the chive juice may be mixed at a volume ratio of 95 to 85:5 to 15, for example, may be mixed at a volume ratio of 90:10, but are not limited thereto.

The extract of the fermented chives contains kaempferol, kaempferol 3- O -β-sophoroside, and kaempferol-3- O -rutinoside . - O -rutinoside), kaempferol 3- O -glucoside, oxodihydroxy-octadecenoic acid, (Z)-5,8,11 - trihydride 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)-octadecatetraenoate ((6Z,9Z,12Z,15Z)-octadecatetraenoate), 9-OxoOTrE (9-oxo-10e,12z,15z-octadecatrienoic acid), 12(13)-epoxy-9Z-octadecenoic acid, and 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 for illustrating the content of the present invention and are not intended to limit the present invention.

<Example 1> Determination of optimal concentration of fermented chives

The chives used in the experiment were green belt chives sold at a general supermarket (Gwangjin-gu, Seoul), and were juiced using a green juicer (Angel-7700, Korea). Lactobacillus plantarum SK4719 (KACC 92270P) isolated from chives was used as a fermentation microorganism.

Chive juice was fermented in MRS medium (MFCC) and sterilized water (WFCC). To find the optimal concentration for fermentation of Lactobacillus plantarum SK4719, MRS and chive juice in water were prepared at concentrations of 0, 5, 10, 20, 25, 30, 35, and 40% (v/v). 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 pH was measured. The pH was measured using a pH meter (ISTEC 735P, Korea), and the number of viable cells was confirmed using the 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 was measured at certain 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.

Freeze-dried non-fermented and fermented chive juice (20 g) was mixed with 200 ml 80% (v/v) methanol, shaken in a shaking incubator (30℃, 100 rpm) for 30 minutes, and sonicated for 15 minutes at 30℃ or lower. Processed. The mixture was centrifuged at 13,500 rpm (4°C, 10 min) to obtain the supernatant. This process was repeated 5 times, and the obtained supernatants were mixed, rotary evaporated at 45°C, freeze-dried, freeze-dried, and kept at -80°C until analysis.

As a result, as can be seen in Figure 1, when MRS and leek juice were fermented together for 0 hours, the pH ranged from 5.5 at 0% to 5.8 at 35%. After 18 hours of fermentation, the pH was 3.9 at 0% and 3.8 at 35%. 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 Figure 2, when water and leek juice were fermented together, the pH decreased rapidly at a concentration between -5% and maintained the decrease up to a concentration of 10% during 18 hours of fermentation. At 0 hours of fermentation, pH increased from 6.6 to 7.1, but after 18 hours of fermentation, pH decreased to 3.27 at 10% concentration. Lactobacillus Plantarum SK4719 viable cells increased when 0-35% chive juice and MRS were fermented for 18 hours. Non-lactic acid bacteria grew better as the concentration of chive juice increased, while lactic acid bacteria decreased. When fermented with water and 10% chive juice, the lowest pH and highest cell survival rate were observed. Similarly, high survival rate and low survival rate of Lactobacillus plantarum SK4719 when co-fermented with MRS and 10% chive juice. pH was shown (Figure 1). Therefore, 10% (v/v) of chive juice was suitable for fermentation of Lactobacillus plantarum SK4719.

As shown in Figure 3, time profiling confirmed that 24 hours was optimal for fermentation of leek juice. When water and chive juice were fermented using Lactobacillus Plantarum SK4719, the highest CFU of 8.1 was seen at 24 hours, and the pH also rapidly 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 is initially When fermented for 15 hours at 7.6 CFU/ml, it increased to 9.5. In a previous study, Lactobacillus plantarum SK4719 was fermented with MRS, showing a viable count of 9 CFU/ml within 12 hours (Niu et al., 2019).

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

Total phenol and flavonoid content

The total phenolic content of the samples was measured using the Folin-Ciocalteu method (Dudonne et al., 2009). 20 μl of sample (10 mg/ml in 80% (v/v) MeOH) and 100 μl of 0.2 N Fc reagent (Sigma f-9252) were mixed and incubated for 5 minutes in the dark. 80 μl of 7.5% Na ₂ CO ₃ was mixed and reacted for 60 minutes in the dark at room temperature. Gallic acid was used as a standard solution. Absorbance was measured at 750 nm using an ELISA reader (Synergy 2, BioTek Instruments Inc.). Total phenols were expressed as mg of equivalent gallic acid 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 incubated in the dark at room temperature. It 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 meiolysis of flavonoid compounds by enzymes released by probiotics. Previous studies have reported that the initial metabolism of flavonoids is advantageous because they are poorly absorbed into the intestines (Lotito et al., 2011). When Graptopetalum paraguayense E. Walther was fermented with Lactobacillus plantarum SK4719 BCRC 10357, flavonoid 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 measured 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 for 10 minutes at room temperature. . Absorbance was measured at 412 nm, and thiol content was expressed as μM of cysteine equivalent per g of plant extract (μM CE/g).

To measure allicin content, cysteine was diluted to 10, 20, 30, 40, and 50 μM using 50 mM HEPES buffer. 100 μl of 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 allicin concentration was calculated using the following formula. Allicin concentration was expressed as μM of cysteine equivalent 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, allicin content was reduced due to its volatile nature and the use of Lactobacillus plantarum SK4719. In the previous literature (Yang et al., 2014), chives were treated with Lactobacillus plantarum SK4719. It was reported that when fermented with LK8 for 48 hours, allicin decreased by 85.45% and thiol increased by 39.37%. However, no significant changes were observed in the case of fermentation in MRS medium, which is likely due to the dark color of the extract containing MRS medium.

<Example 3> Biological activity of fermented chive juice extract

antioxidant activity

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

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, the antioxidant activity of leek juice was significantly reduced to 24% and 9%, respectively, after fermentation in water and MRS medium for 24 hours (p<0.05). This is due to the reduction of flavonoids and organosulfur compounds, allicin and thiol, after fermentation of chive juice.

Antibacterial and antiviral activity of chive juice

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

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

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

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

Fermented chive 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 kaempferol and its glycosides contained in the ethanol extract of WFCC are believed to exhibit anti-influenza activity.

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

Metabolite profiling of water-fermented chive 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 using a Phenomenex KINETEX C18 column (100 mm × 2.1 mm, 1.7 μm particle size; Torrance, CA, USA) while maintaining the column temperature at 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 over 1 minute, step up to 100% solvent B over 9 minutes, hold for 1 minute, then decrease to 5% solvent B over 3 minutes. MS data were collected using an Orbitrap Velos ProTM system equipped with an ion-trap mass spectrometer and HESI-II probe. Mass spectra were acquired in the 100-2000 m/z range. The probe heater and capillary temperatures were set at 300°C and 350°C, respectively. The capillary voltage was set at 2.5 KV and 3.7 KV in negative and positive modes, 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) . The MW, RT, and mass fragmentation patterns (MSn) data of metabolites evaluated by UHPLCLTQ-Orbitrap-MS/MS were confirmed by comparing them with data obtained from the literature and public online databases.

Chromatograms of WFCC measured in positive and negative modes are shown in Figure 6. Overall, 12 different secondary metabolites belonging to flavonols and fatty acids were identified. The identified compounds are kaempferol, kaempferol 3- O -β-sophoroside, and kaempferol-3- O -rutinoside . O - rutinoside), kaempferol 3- O -glucoside, oxodihydroxy-octadecenoic acid, (Z)-5,8,11 - trihydride 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)-octadecatetraenoate ((6Z,9Z,12Z,15Z)-octade catetraenoate), 9-OxoOTrE (9-oxo-10e,12z,15z -octadecatrienoic acid), 12(13)-epoxy-9Z-octadecenoic acid (12(13)-epoxy-9Z-octadecenoic acid) (Table 3).

<Example 5> Chive feed additive

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

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 123132-50 0L, Korea), and then fed to broilers.

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

<Example 6> Animal experiment

Experimental feed and experimental design

For each treatment group, 40 seedlings were stocked in 5 repetitions, and they were placed completely randomly. The basic experimental feed used in this experiment was mainly corn and soybean meal, and the ME of the early broiler feed was 3,100 kcal/kg and the ME of the latter was 3,150 kcal/kg. From the start date, the first phase feed was fed until the 3rd week, and the second phase feed was fed from the 3rd to the 5th week.

Public animal and specification management

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

The experimental cage was a no-window cage with a concrete floor, and rice husk was used to spread it to a thickness of 5 cm. The temperature inside the house was 27.8±1.6℃ and humidity 74.8±8. The experiment was conducted in a 1% environment, and the room temperature was set at 32 ± 1℃ for the first week and then decreased by 2℃ every week to maintain 25 ± 1℃ at the end of the experiment. The lights were on for 24 hours from the start of the experiment to the end of the experiment, and feed and water were fed ad libitum.

Body weight, feed intake and feed efficiency

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

sample collection

At the end of the feeding experiment, samples were collected by randomly selecting 2 animals per pen and slaughtering 10 animals per treatment group. Samples collected included blood, breast meat, leg meat, organs (liver, spleen, F-sac), small intestine (duodenum, jejunum, ileum), and cecum.

Blood was slaughtered using carbon dioxide and collected using a heart blood collection method, injected into an EDTA-treated blood collection tube, and stored at 4°C. Afterwards, the serum was separated by centrifugation at 1500 rpm * g for 10 minutes, and the serum was stored at -20°C.

meat quality characteristics

The relative weight of chicken meat was measured by measuring the weight of selected broiler meat for each treatment group and the weight of 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 shaped into a circular shape, placed in a poly ethylene bag, and heated in a 75°C water bath (C-WBE, Chang Shin co., Korea) for 30 minutes. Afterwards, it was left to cool at room temperature for 10 minutes, and the weight loss was calculated by comparing the weight difference before and after heating.

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

Meat color is measured using a colorimeter (Chromameter, CR210, Minolta, Japan) on the surface of the sample. L* value indicates lightness, a* value indicates redness, and b* value indicates yellowness. was measured. The standard color at this time was a calibration plate with an L* value of 97.69, a* value of -0.43, and b* value of +1.98.

pH was calculated as the value displayed 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, F-cyst)

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

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

The length and weight of the small intestine and cecum were measured according to the addition of chive feed additives and antibiotics to the feed. The small intestine was measured by dividing it into three parts: duodenum, jejunum, and ileum, and the weight was measured after removing the contents of the small intestine and cecum.

Testing the number of viable bacteria in the small intestine (duodenum, jejunum, ileum) and cecum

The number of viable bacteria in the small intestine and cecum was measured using standard agar culture using MRS, NA, Macconkey, ST (Streptococcus thermophilus), and SS (Salmonella shigella) (Difco, USA) plate media. The contents of the intestines were 8 times per treatment group, and 1 g of sample was added to 9 ml of sterilized distill water and sequentially diluted through a homogenization process.

After that, 10㎕ was dispensed into each medium, spotted, and cultured in an incubator at 37°C for 24 hours. The number of effective colonies was measured, CFU/g was calculated, and converted to log10 value. All experiments were conducted in 3 repetitions.

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

To measure blood composition, blood was collected from 10 randomly selected broiler chickens per treatment group using the heart blood collection method at the end of the experiment. 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 LDL+VLDL value was calculated by subtracting the HDL content from the total cholesterol content.

The results of the above analysis are shown in Table 6.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled 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 should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

National Institute of Agricultural Sciences KACC92270P 20190709

Claims (12)

A feed additive composition comprising fermented chives or extracts thereof,
The extract of the fermented leek product is an alcohol extract,
The extract of the fermented chives contains kaempferol, kaempferol 3- O -β-sophoroside, and kaempferol-3- O -rutinoside . - O -rutinoside), kaempferol 3- O -glucoside, oxodihydroxy-octadecenoic acid, (Z)-5,8,11 - trihydride 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)-octadecatetraenoate ((6Z,9Z,12Z,15Z)-octadecatetraenoate), 9-OxoOTrE (9-oxo-10e,12z,15z-octadecatrienoic A feed additive composition comprising one or more compounds selected from the group consisting of acid), 12(13)-epoxy-9Z-octadecenoic acid, and combinations thereof. According to paragraph 1,
The feed additive composition wherein the chive fermented product is fermented using Lactobacillus plantarum SK4719 strain. According to paragraph 2,
The feed additive composition wherein the fermented chive is fermented by mixing water or MRS and chive juice. According to paragraph 3,
A feed additive composition wherein the water or MRS and the chive juice are mixed in a volume ratio of 95 to 85:5 to 15. 1) Squeezing chives to produce chive juice;
2) mixing the chive juice prepared in step 1) with water or MRS to prepare a mixture; and
3) Fermenting the mixture prepared in step 2) using Lactobacillus plantarum SK4719 strain. A method of producing a feed additive composition comprising a. According to clause 5,
In step 2), water or MRS and the chive juice are mixed in a volume ratio of 95 to 85: 5 to 15. According to clause 5,
A manufacturing method in which the fermentation step of step 3) is performed for 12 to 24 hours. According to clause 5,
A production method further comprising the step of extracting the fermented chive product with alcohol to produce an extract of the fermented chive product. A composition for inhibiting poultry pathogenic bacteria and avian influenza virus, comprising fermented chives or extracts thereof,
The extract of the fermented chives contains kaempferol, kaempferol 3- O -β-sophoroside, and kaempferol-3- O -rutinoside . - O -rutinoside), kaempferol 3- O -glucoside, oxodihydroxy-octadecenoic acid, (Z)-5,8,11 - trihydride 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)-octadecatetraenoate ((6Z,9Z,12Z,15Z)-octadecatetraenoate), 9-OxoOTrE (9-oxo-10e,12z,15z-octadecatrienoic A composition for inhibiting a virus, comprising at least one compound selected from the group consisting of 12(13)-epoxy-9Z-octadecenoic acid, 12(13)-epoxy-9Z-octadecenoic acid, and combinations thereof. . According to clause 9,
A composition for inhibiting poultry pathogenic bacteria and avian influenza virus, wherein the chive fermented product is fermented using Lactobacillus plantarum SK4719 strain. According to clause 10,
The chive fermented product is a composition for inhibiting poultry pathogenic bacteria and avian influenza virus, which is fermented by mixing water or MRS and chive juice. 1) Squeezing chives to produce chive juice;
2) mixing the chive juice prepared in step 1) with water or MRS to prepare a mixture; and
3) Fermenting the mixture prepared in step 2) using Lactobacillus plantarum SK4719 strain; A method of increasing the polyphenol content in chives, including the step.