KR20160133120A - Feed or feed additive with IgY for prevention of edema disease of swine - Google Patents

Abstract

The present invention relates to a feed or a feed additive comprising IgY which is a yolk antibody separated from the yolk of an egg laid by a haying hen inoculated with Escherichia coli (E. coli) F18 (KCTC 12787BP). The feed or feed additive comprising IgY exhibits excellent efficacy of preventing edema disease of swine.

Description

[0002] Feed or feed additive with IgY for prevention of edema disease of swine [

The present invention relates to a feed or feed additive for preventing swine edema disease, and more particularly to a feed or feed additive for preventing swine edema disease containing IgY which is an egg yolk antibody.

Edema disease of swine (swine edema disease) is one of the E. coli, 8 to 12 weeks of well-nourished piglets with good mortality, the mortality rate is up to 80% acute disease. After the induction, various serotypes of Escherichia coli (0-138, 0-139, 0-141, etc.) enter the small intestine and multiply rapidly due to various stresses. Various components such as endotoxins are absorbed in the small intestine. It is reported to develop in the form of hypersensitivity.

On the other hand, immunoglobulin antibody (IgY) has already been proved to be effective in the prevention and treatment of diseases by various studies. It has been reported that when used at a high concentration, the therapeutic effect can be expected to be almost the same as that of antibiotics. In addition, IgY is derived from a complete food egg, so there is no concern about safety.

The egg yolk antibody is a form of passive immunization using the avian immune system. The immune antibody obtained by the active immunization of the mother chicken is transferred to the egg yolk, and the immune system is transmitted to the offspring. The IgY is the most abundant of the antibodies in the egg yolk. IgY has a molecular weight of about 160 to 180 kDa, which is larger than that of mammalian IgG, and has a wide range of use because it has various advantages that mammalian antibodies do not have, as described above.

Korean Patent Publication No. 10-2010-0044813 (published on Apr. 30, 2010) discloses a technique for producing a swine swine vial vaccine at low cost and high efficiency. The gene of the toxin protein (Stx2e protein) Technology for producing plant vaccines of swine swine disease by efficiently expressing them in cells and at low cost.

The present invention relates to a method for preventing pig swine disease that causes large economic damage in the domestic swine industry, and is a method different from the conventional method for preventing vaccination, which comprises adding IgY, a specific egg yolk antibody produced in a chicken egg, The purpose of this research is to develop and provide a method for demonstrating the defense efficacy against swine edema disease.

The present invention provides an egg yolk antibody IgY obtained by inoculating E. coli F18 (KCTC 12787BP) into a laying hens and then separating the yolk from the eggs produced by the laying hens.

In the present invention, E. coli F18 (KCTC 12787BP) was used as an antigen among several E. coli strains. When Escherichia coli is used as an antigen to vaccinate the laying hens, IgY may be produced. The effectiveness of IgY produced varies depending on the type and condition of the antigen. In the present invention, E. coli F18 (KCTC 12787BP) was selected, and it was confirmed that the IgY production ability was excellent. In addition, it was confirmed that the produced IgY exerts excellent pig swine disease prevention ability.

Meanwhile, the egg yolk antibody IgY against E. coli F18 (KCTC 12787BP) can be produced through a method known in the art. For example, the egg yolk antibody IgY can be produced by the following method. First, a Sadox vaccine is prepared by mixing antigen E. coli F18 (KCTC 12787BP) and ISA70. Then, inject the muscle into the chest of the laying hens and perform boosting. Thereafter, the egg is collected and the egg yolk is separated, whereby IgY in egg yolk can be separated.

On the other hand, the present invention provides a pig feed or feed additive characterized by containing an egg yolk antibody IgY obtained by inoculating E. coli F18 (KCTC 12787BP) into a laying hens and then separating the egg yolk from the eggs produced by the laying hens . At this time, the pig may be, for example, a weaning pig. In addition, the pig feed or feed additive may preferably be one for prevention of swine edema disease.

According to the present invention, when IgY was added to the feed of the weaned pig diets, even though there was a hardness-moderate edema biopsy, at least NC (negative control) and PC (positive control) As shown in Fig. In addition, it was confirmed that the daily gain of body weight during the whole test period and the feed efficiency of 0 ~ 2 weeks were significantly increased compared to the control group. In addition, it was confirmed that the IgY supplementation was significantly lower in the 1st, 2nd and 3rd digits of fecal index than NC and PC. These results suggest that the addition of IgY in weaned pig diets improves the productivity of weanling pigs and inhibits swelling diseases in pigs.

On the other hand, the present invention provides a pig breeding method characterized in that the egg yolk antibody IgY obtained by inoculating E. coli F18 (KCTC 12787BP) into a laying hens and separated from egg yolk of an egg laying hens is fed to pigs . At this time, the pig may be, for example, weanling pig.

The egg yolk antibody IgY isolated from the egg yolk of the egg laying hens after inoculation of the E. coli F18 (KCTC 12787BP) of the present invention on the laying hens was significantly higher than that of the IgY produced by using other E. coli strains as the antigen, , And demonstrate the efficacy of preventing swine swine disease.

FIG. 1 shows the results of SDS-PAGE of a specific egg yolk antibody protein according to the concentration of ammonium sulfate in the separation of IgY antibody using the ammonium sulfate method.
Figure 2 shows the result of SDS-PAGE of the E. E. coli F18-IgY isolated using 35% ammonium sulfate.
Figure 3 shows the SDS-PAGE and Western blot results showing the binding affinity of the anti- E. coli F18-IgY to the antifungal.
FIG. 4 shows the HPLC analysis (FIG. 4A) and standard curve creation results (FIG. 4B) of IgY standard solution concentration.
Figure 5 shows the suitability of the established IgY assay conditions in the yolk powder.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following embodiments and experimental examples, and includes modifications of equivalent technical ideas.

[ Example  1: Through inoculation of laying hens High station  Specific egg yolk antibody production]

1. Production and vaccination of laying hens

 end. Swelling disease  Causative antigen production

For the production of the antigen, E. coli F18 (KCTC 12787BP) strain was plated on platinum, plated on a blood agar, and cultured in a 37 ° C incubator for 24 hours. Thereafter, the colonies were confirmed, inoculated into BHI broth, and cultured at 37 ° C in a shaking incubator at 110 rpm for 24 hours. Cultured E. coli was identified by plating on selective medium, and incubated in broth containing 0.1% formaldehyde for 24 hours. After centrifugation at 7,000 rpm at 4 ° C for 10 min, the cells were collected as an antigen. After 0.1% formaldehyde was added, it was inactivated for 24 hours and then used with an UV spectrophotometer to adjust the antigen concentration to 0.5 at OD 600nm.

 I. Production of high-concentration vaccine using produced antigen

For the preparation of the vaccine, Sadox vaccine was prepared using a homogenizer after mixing the above-cultured antigen with ISA70 at a ratio of about 3: 7. Then, aseptic test and inactivation confirmation test were conducted to prepare a vaccine for the production of specific egg yolk antibody.

 All. Laying immunity

The prepared vaccine was injected intramuscularly into the chest with 0.8 ml of Hy-Line Brown laying hens at 22 weeks of age. Boosting was performed two times after the first inoculation at intervals of two weeks.

 la. Yolk  Sample collection

Egg yolk samples were collected every day from the day before the inoculation to the eggs collected every day at 7 days intervals. Five eggs were randomly collected from each egg yolk, and the sterilized DWs, which were 10 times the weight of the yolk, were mixed and stored in a 50 ml tube for 24 hours. After 24 hours, the mixture was dissolved in a bath at 37 ° C, and the mixture was centrifuged at 4 ° C for 30 minutes at 3,500 rpm.

 hemp. singularity Egg yolk antibody Potent  Measure

  (1) Separation of OMP (Outer membrane protein) for antibody titration

The produced antigen E. coli F18 was centrifuged at 7,000 rpm for 50 minutes. After centrifugation, the supernatant was discarded, the pellet was suspended in 10 mM HEPES buffer, and then the microbes were lysed by ultrasonication. The supernatant was harvested by centrifugation at 7,000 rpm for 30 minutes at 4 ° C. 1% N-Lauroly sarcosine (SIGMA, L-9150) was added to the harvested supernatant to a final concentration of 0.01% and treated at room temperature for 10 minutes. After the treatment, N-lauroyl sarcosine was removed by centrifugation at 15,000 rpm for 50 minutes at 4 ° C, and then suspended again in 50 ml of 10 mM HEPES buffer. The OMP was recovered by centrifugation at 15,000 rpm for 50 minutes at 4 ° C. The collected antigen was quantitated by BCA method, and then set to 100 μg / ml, and coated on an ELISA plate for antibody titer measurement.

  (2) Measurement of antibody titer by ELISA

The specificity of egg yolk antibodies in egg yolk was measured using the 'Indirect ELISA method'. The activity of egg yolk antibodies was determined by Mine (Y., 1997. Seperation of Salmonella enteritidis from experimentally enclosed liquid eggs using hen IgY immobilized immunomanetic separation system. J. Agric. Food Chem., 45, 3723-3727). First, the antigen was diluted in a coating buffer at a concentration of 100 μg / ml, coated on a 96-well polystyrene plate in an amount of 100 μl per well, and incubated at 4 ° C. , And allowed to stand at 37 DEG C for 1 hour. After washing three times with PBS-T (phosphate buffer saline, 0.05% Tween 20, pH 7.4), the cells were blocked with PBS buffer containing 3% BSA for 1 hour at 37 ° C and washed in the same manner as described above . Sample preparation was prepared by placing 200 μl of PBS buffer in 20 μl of negative samples in U-plate and 5 wells of negative control.

The egg yolk samples were dispensed in 110 wells of PBS in 11 wells except the first well for binary dilution. 110 μl of the egg yolk sample was added to 110 μl of PBS buffer, and the resultant was subjected to binary dilution using an 8-well multi-pipette. After the blocking step, the sample was diluted with U-plate and transferred to a 100 μl aliquot. Respectively. (Anti-chicken: Sigma, USA) diluted in 'HRP Conjugate Stabilizer (SURMODICS, SZ02-1000)' was diluted to a proper volume in PBS, and 100 μl of each antibody was dispensed into each well. Lt; 0 > C for 1 hour. After washing three times, 100 μl of 'TMB 2-Component Microwell Peroxidase Substrate Kit (KPL, 50-76-03)' was mixed at 1: 1 and each well was dispensed at 100 μl. The reaction was stopped by dispensing 100 μl of the stop solution, and the absorbance of each well was measured with an ELISA reader at a wavelength of 450 nm and expressed as an ELISA value. The OD value of the voice was doubled to the measured result, and the dilution ratio of the treated egg yolk was substituted.

  (3) Measurement result of antibody by ELISA

Generation of specific yolk antibodies was confirmed by ELISA on the egg yolk samples obtained from the laying hens. The test was carried out by the above ELISA method. As shown in the results of Table 2, the activity of IgY against the pathogenic bacterium E. coli F18, which is a cause of edema of the weaned piglets, was confirmed by collecting the yolk samples collected.

The IgG titer of the antigen was measured using egg yolk obtained after immunization and E. coli F18 vaccine was prepared from the laying hens. As a result, the titers of the antigens were measured from the 2nd week of the first inoculation to 6,400 ~ 25,600 were found to be active.

Results of weekly yolk sac antibody
Inoculation Parking
Antigen ( E. coli F18): Emulsifier = 3: 7 Primary 2 weeks 12,800 Secondary 1 week 12,800 2 weeks 12,800 Third 1 week 25,600 2 weeks 25,600 Fourth 1 week 12,800 2 weeks 12,800 5th 1 week 12,800 2 weeks 6,400 3 weeks 6,400 4 weeks 6,400 5 weeks 6,400 6 weeks 6,400

[ Example  2: Produced peculiarities Of the immune protein  Confirm]

1. Establishment of methods for separation and purification of antibodies and separation of antibodies using Ammonium sulfate method

 end. Ammonium sulfate treatment concentration setting

  (1) Fractionation experiment by concentration

Separation and purification were carried out using the ammonium sulfate treatment method to purely separate the produced specific egg yolk antibody protein. Ammonium sulfate was tested at each concentration to determine the best concentration for separation and purification.

In the laying hens, E. coli The F18 vaccine was inoculated to collect high-frequency eggs, and egg white and arsenic were removed using an egg yolk separator. After removing the egg yolk with Whatman No.1 filter paper, only yolk was removed from the beaker. The amount of egg yolk isolated was measured, and the amount of primary distilled water was adjusted to 4 times by weight. After stirring for 1 hour, the pH was adjusted to 5 using 1M HCl. This egg yolk solution was stored in a -70 ° C ultra-cold refrigerator for one day.

The egg yolk solution was taken out at room temperature, dissolved for about one day, and centrifuged (9,000 rpm, 4) for 10 minutes to obtain supernatant, which was then filtered on Whatman No.1 filter paper using a vacuum pump. The volume of the filtered supernatant was measured and 25% ammonia sulfate was added in small portions under ice conditions to sufficiently dissolve it. The ammonium sulfate-treated solution was allowed to stand overnight at 4 ° C., and the pellet was collected by centrifugation (9,000 rpm, 4 ° C.) for 30 minutes and resuspended in 1 × PBS buffer to collect the sample. The supernatant was measured for volume, and the pellet and the supernatant were collected by addition of 30% of ammonium sulfate. The pellet and supernatant samples were collected by treating the same with 35% and 50% of ammonium sulfate, and the purity of the specific egg yolk antibody was determined by SDS-PAGE.

(2) Fractionation test results by concentration

When ammonium sulfate was treated, 10 μg of each sample was loaded on a 12% SDS-PGAE gel to confirm the best separation at which concentration, and SDS-PAGE results as shown in FIG. 1 were confirmed. For the control general IgY, a heavy chain of 70 kDa and a light chain band of 21 kDa were identified. From the results of samples 1 to 8, it was found that 4 to 6 of the two chains were best separated and other proteins were not identified. Through this experiment, it was confirmed that when the ammonium sulfate was treated at a concentration of 30 to 35%, the specific egg yolk antibody was well separated (FIG. 1). Figure 1 shows the results of protein SDS-PGE with different concentrations of ammonium sulfate (C, normal chicken IgY; 1, 25% ammonia sulfate, and 2, 25% pellet; 4, 30% pellet after treatment, 5, 35% after treatment, 6, 35% pellet after treatment, 7, 50% after treatment, pellet after 8, 50% treatment).

 I. Isolation and purification of edible egg specific egg yolk antibody protein

(1) 35% ammonium sulfate treatment method

To isolate the Anti- E. coli F18-IgY protein, E. coli The F18 vaccine was inoculated and the second round of inoculation was collected. The prepared egg was removed with egg yolk separator, egg yolk was removed with Whatman No.1 filter paper, and egg yolk was separated from the beaker. The amount of egg yolk isolated was measured, and the amount of primary distilled water was adjusted to 4 times by weight, and the mixture was stirred for 1 hour and adjusted to pH 5 with 1M HCl. This egg yolk solution was stored in a -70 ° C ultra-cold refrigerator for one day. The egg yolk solution was taken out at room temperature and dissolved for one day. The supernatant was obtained by centrifugation (9,000 rpm, 4 ° C) for 10 minutes and filtered through a Whatman No.1 filter paper using a vacuum pump. The volume of the filtered supernatant was measured, and 35% ammonia sulfate was added little by little at 4 ° C and dissolved by stirring. At this time, the treated concentration was set through experiments for confirming the concentration of gastric ammonium sulfate. The ammonium sulfate-treated solution was allowed to stand at 4 占 폚 for one day, and the supernatant was discarded by centrifugation (9,000 rpm, 4 占 폚) for 30 minutes. The pellet was collected and resuspended in 1x PBS buffer to collect the IgY sample. The collected samples were subjected to protein quantification using the BCA protein assay kit (Pierce, 23227) and their purity was confirmed by SDS-PAGE.

  (2) Results of 35% ammonium sulfate treatment

(1) Results of protein quantification and purity determination of E. coli F18-IgY (SDS-PAGE)

The amount of Anti- E. coli F18-IgY isolated by ammonium sulfate treatment was quantified using the BCA protein assay kit and was found to be 30.2 mg / ml. As a result of loading 5 μg of Anti- E. coli F18-IgY in a 12% SDS-PAGE gel, the two bands were confirmed at about 30 kDa and 70 kDa (FIG. 2). Figure 2 shows the result of SDS-PAGE analysis of the anti- E. coli IgY antibody. The band of similar size to that of the normal chicken IgY, which is sold in the market, was confirmed. Therefore, it is considered that the egg yolk antibody protein is easily separated by the treatment with the ammonium sulfate of the produced Anti- E. coli F18-IgY.

(2) The binding affinity result (Western-blot) of anti- E. coli F18-IgY against antifungal agent

SDS-PAGE and Western blotting were carried out to confirm the binding affinity of the above-produced Anti- E. coli F18-IgY to E. coli F18 strain. SDS-PAGE was confirmed by loading 5 μg of E. coli F18 strain using 12% gel, and the band for the strain was confirmed. The confirmed gel was transferred to NC membrane at 100 V for 1 hour and blocked in '5% skim milk in TBST' for 1 hour. After washing three times with 1 × TBST buffer, 2 μg / ml of anti- E. coli F18-IgY isolated as the primary antibody was treated for 1 hour and washed three times. Anti-Chicken-IgY-HRP was treated with a secondary antibody at 1: 30,000 for 1 hour, washed three times, and band was confirmed using 'Chemiluminescent HRP Substrate (Millipore, WBKLS0100)'. As a result, some of the bands confirmed by the results of E. coli F18 SDS-PAGE were confirmed in the Western blot results and confirmed to be bound to the F18 antigen protein and E. coli F18-IgY, and the produced E. coli F18 -IgY and E. coli F18 antigen (Fig. 3). Figure 3 shows the results of SDS-PAGE and Western blotting.

2. Study on criteria and test methods for setting the indicator material content

 end. Establishment of analytical method of IgY content by HPLC

  (1) HPLC analysis conditions

In order to measure the total IgY content in the egg yolk powder of spontaneus, the HPLC system as shown in Table 4 was used. The column temperature was 80 ° C, the detector was UV 280 nm, the sample injection amount was 2 μl and the mobile phase was acetonitrile (AcN) and water in a gradient profile and the flow rate was 0.8 ml / min.

HPLC system for IgY qualitative and quantitative analysis HPLC system

Shimadzu HPLC  Pump LC-20AD  Detecter SPD-20A UV / VIS Analysis program Lab solutions v5.3, Shimadzu corporation Column  Waters XBridge BEH300 C4 3.5 μm 4.6 × 250 Solvent
_{A: 0.1% TFA in H 2} O B: 0.1% TFA in AcN

HPLC Analysis Gradient Profile Time
(min) Flow
(ml / min) Mobile phase A B 0 0.8 70 30 3 0.8 70 30 10 0.8 60 40 17 0.8 55 45 22 0.8 0 100 27 0.8 0 100 28 0.8 70 30 40 0.8 70 30

  (2) Preparation of standard liquid and measurement of linearity

The standard reagents were prepared by using IgY protein (from egg yolk 21 mg / ml, abcam, ad119138) and using H ₂ O (HPLC grade) at concentrations of 0.0625, 0.125, 0.25, 0.5 and 1 mg / ml The standard solution was used. Standard curves were prepared using HPLC analysis program (Lab solution v5.3, Shimadzu).

  (3) HPLC system suitability measurement

IgY content To determine the suitability of the HPLC analysis conditions, the measurement was repeated 9 times using 0.5 mg / ml IgY standard solution. The retention time (RT), peak area and peak height at 50% (RSD,%) of 50% width satisfy the value of 1.0% or less and whether the value of the lowest theoretical plate number satisfies 2,000 or more.

  (4) Precision measurement

To determine the daily and intra-day variability of the standard solutions, the five standard concentrations (RSD,%) of the peak area ratios were determined by repeating the above 5 concentrations for 3 days.

[Equation 1]

Relative standard deviation (RSD,%) = (standard deviation / average) x 100

  (5) Accuracy

To evaluate the accuracy of the HPLC analysis conditions, the accuracy was evaluated based on the results obtained by substituting the results obtained by injecting each standard solution three times at each concentration into the calibration curve and the degree of error (recovery rate,%) of true values.

&Quot; (2) "

Recovery (%) = (measured concentration / theoretical concentration) × 100

I. IgY analysis in samples

  (1) Sample treatment

The egg yolk powder was mixed with 10 times of water (H ₂ O, HPLC grade), adjusted to pH 5 (± 0.2) with 1N HCl and homogenized for 1 minute. The homogenate was centrifuged at 4 to 4,000 rpm for 30 minutes, filtered through Whatman No.1 filter paper, and the filtrate was filtered through a 0.2 μm syringe filter and used as a sample for HPLC analysis.

  (2) Establishment conditions for analyzing IgY content in the sample

To confirm the reliability of the HPLC assay conditions for the IgY content in the samples prepared from the egg yolk powder, the relative standard deviation of the retention time and the peak area was determined by repeating the measurement of one analytical sample six times. The analytical samples were repeatedly measured for 3 days and the precision was evaluated by the relative standard deviation of the peak area.

 All. Results of Establishment of Conditions for Analysis of IgY Content by HPLC

  (1) HPLC analysis Linearity measurement

As a result of HPLC analysis of the concentration of the IgY standard solution, it was confirmed that the peak analyzed at the same retention time (RT) of 14.5 min as in FIG. 4 (A). The correlation coefficient (R ² ) of the linearity was calculated from the standard curve prepared for each standard solution, and as a result, the correlation was 1.000 as shown in FIG. 4 (B), and the linearity was recognized. Figure 4 shows the results of HPLC analysis and standard curve creation for IgY standard solution concentration. The detection limit (LOD) and the limit of quantification (LOQ) of the standard solution were 0.001 mg / ml and 0.012 mg / ml, respectively (Table 4).

IgY std. Standard curve equation, detection limit (LOD) and quantitation limit (LOQ) Sample Equations R ² LOD (mg / ml) LOQ (mg / ml) IgY protein y = 6.886e-006 * x + 0.033747 0.999 0.001 0.012

  (2) HPLC analysis system suiability measurement

The relative standard deviation (RSD,%) of retention time (RT), peak area and peak height 50% width (50% width) was determined by repeatedly measuring one standard solution. ) Were all less than 1.0%, and the minimum number of plates was more than 2000, thus satisfying all of the conformity criteria (Table 5).

HPLC analysis system suitability measurement RT area 50% width Plate nunmber One 14.542 67568 0.597 3330 2 14.522 68122 0.611 2942 3 14.544 67395 0.6 3286 4 14.514 68631 0.599 3021 5 14.557 67880 0.602 3245 6 14.573 67999 0.598 3151 7 14.514 67996 0.602 3214 8 14.568 68014 0.602 3146 9 14.547 68402 0.603 3272 mean 14.540 68001 0.602 3178 % RSD ^{a )} 0.14 0.52 0.642 Criteria              RSD (%) Max 1.0% Min 2000

^a) RSD (relative standard deviation,%) = (standard deviation / average) x 100

  (3) Precision measurement

The daily and intra-day variations of each HPLC analysis were examined. As a result, the relative standard deviation of the reference standard value was 1.0% or less (Table 6).

IgY analysis precision measurement IgY Conc. Precision RSD (%) ^a) (mg / ml) intra day inter day 1st 2nd 3rd 0.0625 0.29 0.25 0.32 0.44 0.125 0.11 0.21 0.11 0.43 0.25 0.58 0.68 0.64 0.78 0.5 0.31 0.89 0.66 0.70 One 0.54 0.79 0.31 0.71 Criteria RSD (%) Max 1.0%

^a) RSD (relative standard deviation,%) = (standard deviation / average) x 100

 (4) Accuracy

As a result of the accuracy of the HPLC method, the results were found to correspond to the reference recovery of 100 ± 3% at all concentrations (Table 7).

Measuring IgY assay accuracy IgY Conc. (mg / ml) Area RSD (%) ^a) Real data IgY Conc. (mg / ml) Recovery (%) ^b) 0.0625  4197 ± 12.2 0.29 0.063 ± 0.001 101.5 0.125 12974 ± 14.3 0.11 0.123 + - 0.001 98.1 0.25  31090 ± 180.3 0.58 0.247 ± 0.004 99.0 0.5  68647 ± 212.8 0.31 0.506 ± 0.002 101.2 One  139961 ± 881.8 0.63 0.998 0.005 99.8 Criteria - RSD (%)
Max 1.0% - 100 ± 3%

^a) RSD (relative standard deviation,%) = (standard deviation / average) x 100

^b) Recovery (recovery rate,%) = (measured concentration / theoretical concentration) × 100

From the above results, it was found that the conditions of IgY assay using HPLC system were established, and the range of analysis under the established analytical conditions was set to 0.0625 ~ 1 mg / ml.

la. Yolk  In the powder sample IgY  analysis

 (1) Conditions for analysis of IgY content in egg yolk spray dried powder samples

As a result of examining the suitability of IgY assay conditions using the HPLC system for the IgY analysis in the sample, it was confirmed that the peak of resolution factor (R = 2.5) was observed at the same retention time (14.52 min) The suitability of FIG. 5 shows the results of confirming the suitability of the IgY assay conditions in the established egg yolk powder. Also, as a result of measurement of precision, the relative standard deviation (RSD,%) of retention time (RT) and peak area were all below 1.0%.

Measurement of IgY content in egg yolk powder Precision measurement 1st 2nd 3rd 4th 5th 6th mean SD % RSD ^{a )} Criteria RT 14.55 14.49 14.51 14.50 14.52 14.52 14.52 0.02 0.14 RSD (%)
Max1.0% Peak
area 56813 57016 57207 56475 56947 56749 56867 251 0.44

^a) RSD (relative standard deviation,%) = (standard deviation / average) x 100

The accuracy of the IgY assay in the samples was analyzed by the relative standard deviation (RSD,%) within 1% of the measured IgY content within the day and within the day, and the results were appropriate for the accuracy criterion (Table 9). As a result, the method of analyzing IgY in the sample was established.

Measurement of IgY content in egg yolk powder Accuracy measurement intra day inter day 1st 2nd 3rd Precision RSD (%) ^a) 0.093 0.491 0.408 0.342 Criteria RSD (%) Max 1.0%

^a) RSD (relative standard deviation,%) = (standard deviation / average) x 100

[ Example  3: Identification of efficacy of specific immune protein]

One. Weaned pigeon  In feed IgY  Additive Feed Specification Test (Domestic)

 end. Research Purpose

To investigate the effect of the anti - subunit IgY yolk powder in feed on the productivity and edema disease of weaned piglets.

 I. Materials and methods

  (1) Test animals and test design

(Landrace × Yorkshire × Duroc) Weighed 27 dogs were weighed and weighed 5.22 ± 1.21 kg at the start of the test. The treatments were 1) IgY (99.8% basal diet + 0.2% IgY), 2) NC (100% basal diet) and 3) PC (99.8% basal diet + 0.2% % AIJI guard ^®, add Biotech production) the 3 treatment, three replications, 3 rats per repeating unit to a random arrangement seed ^{10 7, 10 8, 10 9} cfu / 1ml per repetition were each administered orally.

  (2) Management of test feeds and specifications

The test was conducted at Dankook University test farm. Feeds were fed infinitely to corn-soybean meal based on NRC (2012) requirement and water was adjusted to be freely fed using an automatic water dispenser.

  (3) Survey items

   ① daily gain, daily dietary intake and feed efficiency

Body weight and feed intake were measured at 1 week before, 1 week after, and 3 weeks after the inoculation, and daily body weight gain, daily feed intake and feed efficiency were calculated.

   ② Fecal index

Fecal index was measured weekly and scored as a score. 2 = hard, formed stool that remains firm and soft; 3 = soft, formed, and moist stool that retains its shape; 4 = soft, unformed stool that assumes the 5 = watery, liquid stool that can be poured.

   ③ edema

The presence or absence of edema was assessed by a professional veterinarian (n = 0: normal, 1: mild, 2: severe) by observing edema and neurological symptoms in various parts of eyelid, ear, face, ).

   ④ Autopsy

On the 22nd day after the initiation of the test, the veterinarian autopsied and quantified (0: normal, 1: mild, 2: severe) by observing multiple reservoirs, enlargement of the jejunal membrane, size of the edema, thickening of the intestinal wall, Respectively.

  (4) Statistical processing

All data were analyzed using the 'General Linear Model Procedure' of SAS (2001), and the significance of the mean between the treatment averages was tested with Duncan's multiple range test (Duncan, 1955).

 All. Specification Test Result

  (1) Productivity

The effects of IgY supplementation on the productivity of weaned piglets are shown in Table 10. The IgY treatment group showed significantly higher IgE treatment groups than the NC treatments ( p <0.05). In addition, IgY treatment group was significantly higher ( p > 0.05) than the NC treatment group at 2 ~ 4th day of gestation. During the whole test period, IgY treatment was significantly higher ( p <0.05) in the daily gain than the NC treatment.

Effect of IgY supplementation on productivity of weaned piglets^One Items IgY NC PC SE ² Body weight, kg   d-7  5.54  5.38  5.60 0.01  d 7  8.87  8.19  8.52 0.08   d 14 14.59 13.25 13.80 0.19 Phase 1 (-7d - 7d)    ADG, g 248 ^a 201 ^b 227 ^ab 10     ADFI, g 293 283 287 12 G / F 0.849 ^a 0.709 ^b 0.790 ^a 0.020 Phase 2 (7-21d)    ADG, g 409 ^a 348 ^b 377 ^ab 14     ADFI, g 501 481 494 9 G / F 0.815 0.725 0.765 0.040 Overall (-7d - 21d)    ADG, g 329 ^a 274 ^b 302 ^ab 12     ADFI, g 350 344 340 6 G / F 0.827 0.718 0.773 0.029

¹⁾ Abbreviation: IgY, NC + IgY 0.2%; NC, Basal diet (No antibiotics); PC, NC + additive 0.2%

²⁾ Standard error.

^{a, b} Means in the same row with different superscripts differ ( P <0.05).

  (2) Fecal index

The effects of IgY supplementation on fecal indices of weaned piglets are shown in Table 11. IgY treatment was significantly lower ( P <0.05) in the 1, 2, and 3 feces indexes after challenge than NC and PC treatments.

Effect of IgY supplementation on fecal indices of weaned piglets ¹ Items IgY NC PC SE ² Fecal score ³   d-7 3.67 3.67 3.67 0.14  d 7 4.00 ^b 4.33 ^a 4.33 ^a 0.08   d 14 3.33 ^b 4.33 ^a 4.00 ^a 0.16   d 21 3.70 ^b 4.10 ^a 4.00 ^a 0.90

¹⁾ Abbreviation: IgY, NC + IgY 0.2%; NC, Basal diet (No antibiotics); PC, NC + additive 0.2%

²⁾ Standard error.

³⁾ Fecal scores: 1 hard, dry pellet; 2 firm, formed stool; 3 soft, moist stool that retains shape; 4 soft, unformed stool that assumes shape of container; 5 watery liquid that can be poured.

^{a, b} Means in the same row with different superscripts differ ( P <0.05).

  (3) presence or absence of edema disease

IgY treatment We detected both hardness and moderate edema at autopsy, but IgY treated weaned piglets showed milder symptoms than NC and PC treatment.

  (4) Effect of edema disease concentration

The effects of IgY supplementation on the productivity of weaned piglets according to edema germ concentration are shown in Table 12.

Effect of IgY supplementation on the productivity of weaned piglets according to edema germ concentration ¹ Items IgY NC PC SE ² 10 ⁷ 10 ⁸ 10 ⁹ 10 ⁷ 10 ⁸ 10 ⁹ 10 ⁷ 10 ⁸ 10 ⁹ Body Weight, kg d-7 5.60 5.28 5.30 5.52 5.38 5.25 5.42 5.45 5.14 9.73 d7 9.03 ^a 8.81 ^a 8.76 ^a 8.50 ^ab 8.28 ^ab 7.80 ^b 8.26 ^ab 8.90 ^a 8.39 ^ab 29.30 d14 14.33 ^ab 14.79 ^a 14.66 ^a 13.32 ^abc 13.48 ^abc 12.39 ^c 12.76 ^bc 14.67 ^a 13.97 ^abc 16.55 Phase 1 (-7d - 7d) ADG, g 246 ^a 253 ^a 247 ^a 213 ^bc 207 ^bc 182 ^c 203 ^bc 246 ^a 232 ^ab 10 ADFI, g 285 307 287 306 275 269 265 300 296 - G / F 0.863 ^a 0.822 ^a 0.862 ^a 0.685 ^b 0.754 ^ab 0.677 ^b 0.766 ^ab 0.822 ^a 0.784 ^ab 0.034 Phase 2 (7d - 21d) ADG, g 378 ^ab 427 ^a 421 ^a 345 ^ab 372 ^ab 328 ^ab 321 ^b 4112 ^ab 398 ^ab 29 ADFI, g 492 495 517 463 484 496 508 489 486 - G / F 0.768 ^ab 0.863 ^a 0.815 ^ab 0.745 ^ab 0.767 ^ab 0.662 ^ab 0.632 ^b 0.843 ^a 0.820 ^ab 0.060 Overall (-7d - 21d) ADG, g 312 ^abc 340 ^a 334 ^a 279 ^bcd 289 ^abcd 255 ^d 262 ^cd 329 ^ab 315 ^abc 17 ADFI, g 389 401 402 384 379 382 386 394 391 - G / F 0.802 ^ab 0.847 ^a 0.832 ^a 0.725 ^ab 0.763 ^ab 0.667 ^b 0.678 ^b 0.835 ^a 0.806 ^ab 0.043

¹⁾ Abbreviation: IgY, NC + IgY 0.2%; NC, Basal diet (No antibiotics); PC, NC + additive 0.2%

²⁾ Standard error.

^{a- d Means in the same row with} different superscripts differ (P <0.05).

In the piglet weight, swelling of the after swelling germs administration treatment groups at 7 primary IgY germs 10 administration of ^{7 cfu / 1ml, 10 8 cfu} / 1ml, 10 9 cfu / administration of 1ml group and PC treatment groups of 10 ⁸ cfu / 1ml One treatment group was significantly higher than the treatment group treated with 10 ⁹ cfu / 1 ml of NC treatment ( P <0.05). On the 14th day, 10 ⁸ cfu / 1 ml, 10 ⁹ cfu / 1 ml of IgE in the treated group and 10 ⁸ cfu / 1 ml of PC treated group were the highest ( P <0.05) The treatment group treated with 10 ⁹ cfu / 1 ml of NC treatment showed the lowest ( P <0.05).

In the 0th and 2nd weeks of the experiment, the daily weight gain was significantly higher in all IgY treatments (10 ⁷ cfu / 1 ml, 10 ⁸ cfu / 1 ml, 10 ⁹ cfu / 1 ml of swelling germ) and 10 ⁸ cfu / ( P <0.05). The treatment group treated with 10 ⁹ cfu / 1 ml of NC treatment showed the lowest value ( P <0.05).

Feed efficiency was significantly higher in all IgY treatments (10 ⁷ cfu / 1 ml, 10 ⁸ cfu / 1 ml, 10 ⁹ cfu / 1 ml of edema germs) and 10 ⁸ cfu / ml of edema germ in PC treatments ( P <0.05). The treatment group treated with 10 ⁷ cfu / 1 ml and 10 ⁹ cfu / 1 ml of NC treatment showed the lowest ( P <0.05).

In the 2 nd and 4 th weeks of gestation, the IgE treatment group showed the highest ( p <0.05) the treatment groups with 10 ⁸ cfu / 1 ml and 10 ⁹ cfu / 1 ml edema germs, 10 ⁷ cfu / 1 ml was significantly lower than the control group ( P <0.05).

Edema of it IgY treatment during the whole experiment period, daily gain germs ^{10 8 cfu / 1ml, 10 9} cfu / 1ml one treatment a was the highest significantly with administration of (P <0.05), NC edema germs 10 ⁹ cfu of treatment / 1 ml was the lowest ( P <0.05).

  (5) Autopsy

   ① IgY treatment: There was a small amount of multiple retention findings and showed mild mesenteric, lymphatic hemorrhage and mild variceal thickening.

   ② NC treatment: Large amount of multiple retention and hardness - Midterm mesentery, lymph node hemorrhage and moderate intimal thickening.

③ PC treatment: Small amount of multiple retention findings, showing mild mesentery, lymphatic hemorrhage and mild thickening of the wall.

In conclusion, the addition of IgY in weanling diets significantly increased the daily gain and the 0 - to - 2 - feed efficiency during the whole test period compared to the NC treatment. In the fecundity index, the IgY additions were significantly lower in the 1, 2, and 3-week fecal indices than NC and PC. In the case of edema disease, all IgY tumors showed mild to moderate edema, but mild symptoms were found in the NC and PC treatments. In the autopsy results, a small amount of multiple retention was seen in the IgY appendages and showed mild mesenteric, lymph node hemorrhage, and mild thickening of the wall. However, NC treatments showed large amounts of multiple reservoirs and mild-to-moderate mesentery, lymph node hemorrhage and moderate thickening of the wall hydronephrosis, PC treatment showed a small amount of multiple retention and mild mesentery, lymphatic hemorrhage and hardness barrier And bilateral hypertrophy.

As a result, it was concluded that the addition of IgY in weaned diets resulted in improved productivity, decreased diarrhea index, and suppressed edema of the pigs.

Korea Biotechnology Research Institute KCTC12787BP 20150403

Claims (6)

Egg yolk antibody IgY isolated from egg yolk of laying hens after inoculation of E. coli F18 (KCTC 12787BP) on the laying hens.
Characterized in that it comprises an egg yolk antibody IgY obtained by inoculating E. coli F18 (KCTC 12787BP) on a laying hens and then separated from the yolk of an egg laid by the laying hens.
3. The method of claim 2,
The pig feed or feed additive may contain,
A pig feed or feed additive characterized by the prevention of swine edema.
3. The method of claim 2,
The pig,
A pig feed or feed additive characterized by being a progeny pig.
An egg yolk antibody IgY obtained by inoculating E. coli F18 (KCTC 12787BP) into a laying hens and then separated from yolk of eggs produced by the laying hens is fed to a pig.
6. The method of claim 5,
The pig,
A method for breeding pigs characterized in that they are weaned pigs.

Images