ANTICOCCIDIAL COMPOSITION COMPRISING VIOLACEIN, AND USE THEREOF

Abstract

The present application relates to: an anticoccidial composition comprising violacein, a violacein derivative and/or a salt thereof; and a use thereof. A composition comprising violacein, according to one embodiment, has excellent effects of direct killing of protozoa that can induce coccidiosis, inhibiting the cell penetration of the protozoa and/or inhibiting the intracellular proliferation of the protozoa, and preventing, alleviating, and treating in vivo coccidiosis.

Claims

1-9. (canceled)

10. A method for preventing, alleviating or treating coccidiosis, comprising a step of administering a composition comprising at least one selected from the group consisting of violacein, violacein derivative and pharmaceutically acceptable salt thereof as an active ingredient, to an animal.

11. The method according to claim 10, wherein the violacein derivative is at least one selected from the group consisting of deoxyviolacein, proviolacein, and oxyviolacein.

12. The method according to claim 10, wherein the coccidiosis is induced by an Eimeria sp. protozoan.

13. The method according to claim 10, wherein the preventing, alleviating, or treating coccidiosis is at least one selected from the group consisting of the following (1) to (4): (1) reduction of at least one selected from the group consisting of lesion score, fecal oocyst excretion amount and mortality; (2) inhibition of weight loss due to coccidiosis; (3) increase of an anticoccidial index (ACI); and (4) reduction of cell invasion of an Eimeria sp. protozoan, propagation of the protozoan in cells, or both of them.

14. The method according to claim 10, wherein the composition is a feed composition comprising the active ingredient at a concentration of 1%(w/w) or less based on the total weight.

15. The composition according to claim 10, wherein the composition is a feed additive.

16. A method for killing Eimeria sp. protozoan, or inhibiting cell invasion or propagation of Eimeria sp. protozoan, comprising a step of administering at least one selected from the group consisting of violacein, violacein derivative and salt thereof as an active ingredient, to an animal.

17. The method according to claim 16, wherein the violacein derivative is at least one selected from the group consisting of deoxyviolacein, proviolacein, and oxyviolacein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0090] FIG. 1 shows the anticoccidial index (ACI) of violacein upon single inoculation with protozoa.

[0091] FIG. 2 shows the anticoccidial index (ACI) of violacein upon complex inoculation of 3 kinds of protozoa.

[0092] FIG. 3 shows the protozoan cell invasion rate inhibitory effect and propagation inhibitory effect of the protozoa in cells of violacein.

[0093] FIG. 4 shows the result of measuring acid resistance of violacein.

[0094] FIG. 5 shows the result of measuring heat resistance of violacein.

MODE FOR INVENTION

[0095] The present invention will be described in more detail by the following examples, but it is not intended to limit the scope by the following examples.

Example 1

In vivo Anticoccidial Activity of Violacein Upon Single Inoculation

Example 1-1

Experimental Facility and Research Design

[0096] An in vivo anticoccidial efficacy evaluation test was performed in an animal experiment facility in Gyeongsangnam-do, South Korea. One-day-old female Ross broilers were individually weighed and randomly divided into groups to use in an experiment. Matters and conditions for experimental design were described in Table 2.

TABLE-US-00002 TABLE 2 Category Experiment variable Breeding type Cage Broiler stocking age 1-day-old Total experiment period 22 days Gender Female Number of broilers per cage 15 Number of repetitions per treatment group 2 repetitions Number of treatment groups 6 Total number of broilers 165 broilers Kind of challenge inoculation protozoa Eimeria tenella Number of challenge inoculation oocysts 10,000 oocysts oral inoculation/broiler

[0097] A breeding farm was managed according to the Korean poultry breeding management guidelines. Cages and the breeding farm were cleaned and disinfected before starting the test. The temperature of 40 to 41° C. and the humidity of 40 to 50% of the breeding farm were maintained, and it was continuously monitored.

Example 1-2

Experimental Design

[0098] For the feed, Korea Feed Al-choi product was used, and each material (diclazuril (Yuhan Corporation YUHAN DICLA product), salinomycin (Cheil Bio Cheilsalino-60 product), violacein (CJ Cheiljedang)) was added to the feed, respectively, at a concentration described in Table 3 below to be self-mixed. Antibiotics and supplements were not used in general feed and mixed feed, and no anticoccidial agent other than each material was added. Broilers were fed ad libitum throughout the experiment period. After putting 15 1-day-age broilers in cages randomly placed for each of the control group or test group and breeding, general feed (Al-choi product) was fed for 7 days, and then the mixed feed prepared above was ingested by dividing by control group or test group. The non-infected negative control group used lcage, and the infected negative control group and experimental group used 2cage to progress the experiment.

[0099] The feed formulation administered to the control group (negative control group or positive control group) and test group and whether coccidiosis was induced by Eimeria tenella were described in Table 3 below.

[0100] 14-day-old broilers were orally inoculated with 10,000 oocysts (90% sporulated Eimeria tenella oocysts) per individual to induce coccidiosis.

TABLE-US-00003 TABLE 3 Group Treatment Non-infected negative General feed control group Infected negative control Eimeria tenella infection + general feed group Positive control group 1 Eimeria tenella infection + formulated feed comprising diclazuril 1 ppm Positive control group 2 Eimeria tenella infection + formulated feed comprising salinomycin 60 ppm Violacein treated group 1 Eimeria tenella infection + formulated feed comprising violacein 10 ppm Violacein treated group 2 Eimeria tenella infection + formulated feed comprising violacein 40 ppm

Example 1-3

Measurement of Anticoccidial Activity of Violacein

[0101] The anticoccidial efficacy by test group designed in Example 1-2 above was shown as an anticoccidial index (ACI), and the anticoccidial index was calculated by Equation 2 below. The ACI score is out of 200 points, and the higher the ACI score, the more excellent the anticoccidial ability, and when it is 120 points or more to less than 140 points, it is determined that it is effective as an anticoccidial material, and when it is 140 points or more to less than 160 points, it is determined that it is excellent as an anticoccidial material, and when it is 160 points or more, it is determined that the anticoccidial effect is very excellent (Luis Miguel De Pablos et al., Anticoccidial activity of maslinic acid against infection with Eimeria tenella in chickens, Parasitol Res, 2010).

Anticoccidial index (ACI)=(survival rate after challenge inoculation (%))+(daily weight gain compared to negative control group (RWG, %))−(lesion score×10)−(fecal oocyst excretion amount index)  (Equation 2)

[0102] 1) Survival rate: the number of dead individuals was recorded daily, and post mortem autopsy was performed to determine a cause of mortality, and the number of individuals who died due to causes other than coccidiosis was excluded. The survival rate (%) up to the 8th day after challenge inoculation was used for anticoccidial index calculation. The measured survival rate was described in Table 4 below.

[0103] 2) Daily weight gain: body weight was measured for each cage before challenge inoculation with a protozoan into an individual and at the 7th day after challenge inoculation, and the difference was divided by the number of days to calculate the daily weight gain (ADG, g/d). ‘Daily weight gain compared to the negative control group (RWG, %)’ which was calculated by dividing the daily weight gain (ADG, average daily gain; g/d) of each experimental group by the weight gain (ADG, g/d) of the non-infected negative control group and multiplying by 100 was used for anticoccidial index calculation. The daily weight gain (ADG, g/d) measured in each control group and test group and the daily weight gain compared to the negative control group (RWG, %) were shown in Table 4 below.

[0104] 3) Lesion scoring: On the 8th day after challenge inoculation, an autopsy was conducted for 4 broilers per cage, and the intestines were incised and opened. Scoring was performed for each coccidial lesion in the appendix region of the broilers. The lesion scoring method was performed by referring to Johnson J K & Reid W M (1970) document (Joyce Johnson, W. Malcolm Reid, Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens, Experimental parasitology, 1970). The lesion score is on a scale of 0-4, and 0 point corresponds to normal appendix, and 1 point is a mild infection symptom, and 2 points is a moderate infection symptom, and 3 points is a severe infection symptom and 4 points is a case of showing a very severe infection symptom or causing death. A lesion index was calculated by multiplying the measured appendix lesion score by 10, and this was used for anticoccidial index calculation. The measured lesion score and lesion index in each control group and test group were described in Table 4 below.

[0105] 4) Fecal oocyst excretion amount: the entire feces on the 6˜8th day of challenge inoculation were collected by cage, mixed evenly, and then randomly sampled 3 times in total by lg each. After floating the oocysts in lg feces using salt water, the oocyst excretion amount was measured using McMaster chamber, and the result was described in Table 4 below. The oocyst excretion amount (%) compared to the infected negative control group was calculated by dividing the oocyst excretion amount by each group into the oocyst excretion amount of the infected negative control group and multiplying by 100. The oocyst excretion amount index was calculated as 0 when the calculated oocyst excretion amount compared to the infected negative control group was at a level of 0% to less than 1%, 5 when it was 1% or more to less than 26%, 10 when it was 26% or more to less than 51%, 20 when it was 51% or more to less than 76%, and 40 when it was 76% or more to 100% or less, and the result was described in Table 4 below, and this was used for anticoccidial index calculation.

TABLE-US-00004 TABLE 4 Group Control group Test group Compounds Non-infected Infected negative negative control group control group Salinomycin Diclazuril Violacein Conc (ppm) 60 1 40 10 Number of broilers 15 30 30 30 30 30 ADG(g/d) 64 54 56 61 58 60 after challenge inoculation RWG(%) 100 85 88 96 91 94 Survival rate 93 97 87 100 90 100 after challenge inoculation (%) Lesion score 0.4 3.1 2.6 1.7 2.7 2.1 Lesion index 4 31 26 17 27 21 Oocyst 0 2.9E+08 1.3E+08 1.3E+08 9.0E+07 1.3E+08 excretion amount/g Oocyst index 0 40 10 10 10 10 ACI 190 111 139 169 145 162

[0106] As aforementioned, the anticoccidial index by each test group measured by Equation 2 above was shown in Table 4 and FIG. 1 below. As shown in Table 4 and FIG. 1, in the Eimeria tenella-infected negative control group, the weight gain was reduced and the lesion score and oocyst excretion amount were increased, compared to the non-infected negative control group. In addition, in all the violacein treated groups, compared to the infected negative control group, the weight gain and survival rate after challenge inoculation were increased, and the lesion score and oocyst excretion amount were reduced, and the anticoccidial index was excellent. In particular, in the treated group of the formulated feed comprising violacein of 40 ppm, the egg excretion amount was significantly reduced, compared to the positive control group ingesting salinomycin or diclazuril, so it could be confirmed that violacein could reduce the rate of the live stock contamination and secondary infection by coccidiosis. Furthermore, as the result of Example 7 below, violacein is not remained in organs and blood differently from the positive control group, salinomycin or diclazuril, so it does not need a withdrawal period, therefore it can be utilized as a more excellent anticoccidial agent

Example 2

In Vivo Anticoccidial Activity of Violacein Upon Complex Inoculation

Example 2-1

Experimental Facility and Research Design

[0107] An in vivo anticoccidial efficacy evaluation test was performed in an animal experiment facility in Chungcheongnam-do, South Korea. One-day-old male Ross broilers were individually weighed and randomly divided into groups to use in an experiment. Matters and conditions for experimental design were described in Table 5.

TABLE-US-00005 TABLE 5 Category Experiment variable Breeding type Cage Broiler stocking age 1-day-old Total experiment period 22 days Gender Male Number of broilers per cage 8 Number of repetitions per 7 repetitions treatment group Number of treatment groups 6 Total number of broilers 336 broilers Kind of challenge inoculation Eimeria tenella, acervulina, maxima protozoa Number of challenge inoculation Eimeria tenella: 10000/broiler oral oocysts inoculation Eimeria acervulina: 50000/broiler oral inoculation Eimeria maxima: 50000/broiler oral inoculation

[0108] A breeding farm was managed according to the Korean poultry breeding management guidelines. Cages and the breeding farm were cleaned and disinfected before starting the test. The temperature of 40 to 41° C. and the humidity of 40 to 50% of the breeding farm were maintained, and it was continuously monitored.

Example 2-2

Experimental Design

[0109] The feed was mixed in Neobase, and each material (salinomycin (Cheilbio Cheilsalino-60 product), violacein (CJ Cheiljedang)) was added to the feed, respectively, at a concentration described in Table 6 below to be self-mixed. Antibiotics and supplements were not used in general feed and mixed feed, and no anticoccidial agent other than each material was added. Broilers were fed ad libitum throughout the experiment period. After putting 8 1-day-age broilers in cages randomly placed for each of the control group or test group and breeding, general feed (Al-choi product) was fed for 7 days, and then the mixed feed prepared above was ingested by dividing by control group or test group.

[0110] The feed formulation administered to the control group (negative control group or positive control group) and test group and whether coccidiosis was induced by three kinds of protozoa were described in Table 6 below.

[0111] Coccidiosis induction was performed by oral inoculation (challenge inoculation) in an oral tube type of 10,000, 50,000, 50,000 oocysts (eggs) with over 90% mature (sporulation) Eimeria tenella, Eimeria acervulina, and Eimeria maxima per one individual to 14-day-old broilers.

TABLE-US-00006 TABLE 6 Group Treatment Non-infected negative General feed control group Infected negative control Eimeria tenella, acervulina, maxima (3 kinds) group infection + general feed Positive control group 1 Eimeria tenella, acervulina, maxima (3 kinds) infection + formulated feed comprising salinomycin 60 ppm Violacein treated group 1 Eimeria tenella, acervulina, maxima (3 kinds) infection + formulated feed comprising violacein 10 ppm Violacein treated group 2 Eimeria tenella, acervulina, maxima (3 kinds) infection + formulated feed comprising violacein 15 ppm Violacein treated group 3 Eimeria tenella, acervulina, maxima (3 kinds) infection + formulated feed comprising violacein 20 ppm

Example 2-3

Measurement of Anticoccidial Activity of Violacein

[0112] The anticoccidial efficacy by test group designed in Example 2-2 above was shown as an anticoccidial index (ACI), and the anticoccidial index was calculated by the similar method to Example 1-3 above. However, in the present example, the protozoa of Eimeria tenella, Eimeria acervulina, and Eimeria maxima were challenge inoculated, so the lesion score was calculated by summing the scores for each of the duodenum, jejunum and appendix, respectively, differently from Example 1-3.

TABLE-US-00007 TABLE 7 Group Control group Test group Compounds Non-infected Infected negative negative control group control group Salinomycin Violacein Conc (ppm) 60 10 15 20 Number of broilers 56 56 56 56 56 56 ADG(g/d) 44.3 43.7 46.6 40.4 45.2 42.1 after challenge inoculation RWG(%) 100 98.6 105.1 91.1 102.0 95 Survival rate 100 100 100 100 100 100 after challenge inoculation (%) Lesion score 1.3 8.4 5.5 5.1 3.1 4.0 Lesion index 13 84 55 51 31 40 Oocyst 0.0 8.6E+05 5.4E+05 6.8E+05 3.8E+05 15.6E+05 excretion amount/g Oocyst index 0 34.3 20.0 31.4 15.7 22.9 ACI 187 80 130 109 155 132

[0113] As aforementioned, the anticoccidial index by each test group measured by Equation 2 above was shown in Table 7 and FIG. 2 below. As shown in Table 7 and FIG. 2, in the negative control group to which 3 kinds of the protozoa were complex infected, the weight gain was reduced and the lesion score and oocyst excretion amount were increased, compared to the non-infected negative control group. In addition, in all the violacein treated groups, compared to the infected negative control group, the lesion score and oocyst excretion amount were reduced, and the anticoccidial index was excellent. In particular, in the treated group of the formulated feed comprising violacein of 15 ppm, compared to the salinomycin-ingested positive control group, the lesion score and oocyst excretion amount were significantly reduced, so it could be confirmed that violacein could reduce the rate of the livestock contamination and secondary infection by coccidiosis.

Example 3

Measurement of Protozoan Direct Killing Effect of Violacein

[0114] In the present example, protozoan (sporozoite) direct killing ability evaluation was conducted against 3 representative Eimeria kinds known to be infected in most farms (E. tenella, E. acervulina, E. maxima).

[0115] A certain amount of oocysts of each coccidial protozoan was put in a tube containing glass beads and pulverized, and then to remove the crushed oocyst cell wall and other debris, internal sporocysts were purified using the percoll density gradient, and washed with PBS solution. A reagent comprising sodium taurocholic acid (Sigma aldrich, USA) and trypsin (Gibco, USA), respectively, was treated to sporocysts of Eimeria tenella, Eimeria acervulina and Eimeria maxima for excystation of the internal sporozoites, and they were incubated, and then they were washed with PBS solution once and the protozoa were obtained.

[0116] After reacting the violacein and anticoccidial agents, salinomycin, diclazuril and gallic acid (hereinafter, material) with 3 kinds of Eimeria protozoa at various concentrations of 1 to 500 ppm, respectively, only alive protozoa (sporozoites) were counted through microscopic observation. Then, the death rate (%) of the protozoa when each material was treated compared to the PBS-treated negative control group was measured, and the minimum concentration which directly killed the protozoa by 100% was shown in Table 8 below.

TABLE-US-00008 TABLE 8 Minimum concentration for killing 50% protozoa (sporozoites) (ppm) Material Eimeria tenella Eimeria acervulina Eimeria maxima Negative control — — — group Salinomycin 500 250 500 Diclazuril >500 500 >500 Gallic acid >500 >500 >500 Violacein 0.63 3.9 5

[0117] As the result of confirming the in vitro direct killing effect against the coccidial protozoan species (3 kinds), as shown in Table 8, in the gallic acid treatment group known as an anticoccidial agent, the 100% killing effect against the protozoa up to 500 ppm was not shown, and also in the diclazuril treatment group, 100% sporozoite killing effect was not shown up to 500 ppm against Eimeria tenella and Eimeria maxima. In the violacein treatment group, even at a significantly lower concentration than other groups, Eimeria tenella, Eimeria acervulina and Eimeria maxima of 100% could be killed, so it could be confirmed that the protozoan (sporozoite) killing effect was significantly excellent.

Example 4

Cell Invasion and Propagation Inhibitory Effect Against Eimeria protozoa of violacein

[0118] In the present example, using MDBK cell line which is a representative animal cell known to cause Eimeria infection and propagation, the inhibition ability of intracellular protozoan invasion and intracellular protozoal propagation of the violacein was investigated.

[0119] 100,000 MDBK cells (purchased from ATCC) were aliquoted in a 24-well plate, and then incubated at a temperature of 37° C. for 12 hours. The protozoan of Eimeria tenella was obtained similar to the method of Example 3 above. 200,000 protozoa per one well were added to wells in which cells were aliquoted, and each material (violacein and anticoccidial agents, salinomycin, diclazuril and gallic acid) was treated to the cells by concentration, and cultured at a temperature of 41° C. for 24 hours. A negative control group is a MDBK cell infected by protozoa of Eimeria tenella, and a positive control group means a group in which salinomycin, diclazuril or gallic acid solution was incubated with the Eimeria tenella protozoan. Thereafter, in order to remove the protozoa that did not invade cells, the cells were washed twice using PBS solution. After removing the cells and protozoa inside the cells through pipetting, DNA was extracted from the cells, and PCR was performed using E. tenella ITS-1 (Internal transcribed spacer-1) gene-specific primers. The sequences of the used primers were described in Table 9 below.

TABLE-US-00009 TABLE 9 Primer Nucleotide sequence (5′.fwdarw.3′) SEQ ID NO E.tenella ITS-1 Forward TGGAGGGGATTATGAGAG SEQ ID NO: 1 GA Reverse CAAGCAGCATGTAACGGA SEQ ID NO: 2 GA

[0120] Ct values before/after washing for each material were compared and corrected with the ΔCt value of the negative control group to calculate the cell invasion inhibition rate (%) of the protozoa through treatment of each material, and the result was shown in FIG. 3 and Table 10 below.

[0121] 100,000 MDBK cells (purchased from ATCC) were aliquoted in a 24-well plate, and then incubated at a temperature of 37° C. for 12 hours. The protozoan of Eimeria tenella was obtained similar to the method of Example 3 above. 200,000 protozoa per one well were added to wells in which cells were aliquoted, and cultured at a temperature of 41° C. for 24 hours, and then the cells were washed using PBS solution twice to remove protozoa which were not attached to the cells. Each material (violacein and anticoccidial agents, salinomycin, diclazuril and gallic acid) was treated to the cells by concentration, and further cultured at a temperature of 41° C. for 24 hours. A negative control group is a MDBK cell infected by protozoa, and a positive control group means a group in which salinomycin and diclazuril solution was incubated with the Eimeria tenella protozoan. After removing the cells and protozoa inside the cells through pipetting, DNA was extracted from the cells, and PCR was performed using E. tenella ITS-1 (Internal transcribed spacer-1) gene-specific primers. The sequences of the used primers were described in Table 9 above.

[0122] The intracellular protozoan propagation inhibition rate (%) through treatment of the material was calculated by comparing Ct values in the material treatment groups compared to the negative control group, and the result was shown in FIG. 3 and Table 10.

[0123] As shown in FIG. 3 and Table 10 above, diclazuril showed an effect only in inhibition of the intracellular protozoan propagation, and gallic acid showed an effect in cell invasion of protozoa, but on the other hand, violacein inhibited both cell invasion and intracellular protozoan propagation of the Eimeria tenella protozoan, and degree thereof was more excellent than the positive control group. In the violacein 1 ppm and 0.5 ppm administration groups, the experimental result related to the intracellular protozoan propagation inhibitory effect did not show a statistically significant difference.

TABLE-US-00010 TABLE 10 Cell invasion Intracellular protozoan inhibitory effect propagation inhibitory Material of protozoa (%) effect (%) Negative control group 0.0 0.0 Diclazuril   1 ppm 6.3 52.9 0.5 ppm 0.0 45.9 Salinomycin  10 ppm 56.4 34.8   5 ppm 50.1 32.2 Gallic acid 10 ppm 90.1 0.0   5 ppm 76.0 0.0 Violacein   1 ppm 78.6 45.8 0.5 ppm 59.5 53.7

Example 5

Evaluation of Acid Resistance of Violacein

[0124] In the present example, the acid resistance of the violacein was to be evaluated. Hydrochloric acid (HCl) solution was added to the violacein solution of 200 ppm, to adjust to be pH 2, 3 and 5.5, and then it was left at a temperature of 40° C. for 1 hour. Then, it was neutralized (pH 7.0) by adding sodium hydroxide (NaOH) solution, and 200,000 Eimeria maxima (E. maxima) protozoa (sporozoites) were exposed to the violacein at various concentrations of 0.1 to 100 ppm diluted using PBS solution, and they were reacted at a temperature of 41° C. for 4 hours. Thereafter, the protozoal death rate (%) was measured by the similar method to Example 3 above, and this was shown in FIG. 4 and Table 11. In Table 11, the control group means a violacein treatment group without treating the acidic condition, and the protozoa alone means a group to which violacein was not treated.

[0125] As shown in FIG. 4 and Table 11, it could be confirmed that the violacein did not lose the killing efficacy against protozoa inducing coccidiosis even under a strong acidic condition.

TABLE-US-00011 TABLE 11 Addition Protozoa Death rate according to violacein treatment condition level alone Control group pH 2, 1 h pH 2.5, 1 h pH 3, 1 h 100 ppm 0 ± 2.39 100.00 ± 0 100.00 ± 0 100.00 ± 0 100.00 ± 0  10 ppm 100 ± 0.62  99 ± 1.08  99 ± 1.24 99 ± 0.62    1 ppm 85 ± 3.79 84 ± 7.55 83 ± 4.94 86 ± 7.34  0.1 ppm 64 ± 7.95 64 ± 3.79 61 ± 11.66  63 ± 4.49

[0126] As shown in FIG. 4 and Table 11, it could be confirmed that in the control group and all the experimental groups, the protozoal killing effect was excellent on a concentration-dependent manner of violacein, and even if violacein was left under the acidic condition of pH 2 to 3 for 1 hour and then recovered to neutrality, the protozoal killing efficacy of acidic violacein of pH 2 to 3 did not appear.

Example 6

Evaluation of Heat Resistance of Violacein

[0127] In the present example, the heat resistance of the violacein was to be evaluated. The violacein was exposed at a temperature of 85 to 95° C. for 10 minutes, and then heat was cooled and it was diluted with PBS solution at a concentration of 0.1 to 100 ppm. 200,000 Eimeria maxima (E. maxima) protozoa were reacted to the violacein at various concentrations of 0.1 to 100 ppm at a temperature of 41° C. for 4 hours, and the protozoal death rate (%) was measured by the similar method to Example 3 above, and this was shown in FIG. 5 and Table 12. In Table 12 below, the control group means a violacein solution without treating the high temperature condition, and the protozoa alone means a group to which violacein was not treated.

TABLE-US-00012 TABLE 12 Addition Protozoa Death rate according to violacein treatment condition level alone Control group 85° C., 10 min 90° C., 10 min 95° C., 10 min 100 ppm 0 ± 2.39 100 ± 0 100 ± 0 100 ± 0 100 ± 0  10 ppm 100 ± 0.62 99 ± 0.62 99 ± 1.24 99 ± 0.62   1 ppm 85 ± 3.79 89 ± 4.49 88 ± 2.85 90 ± 3.47  0.1 ppm 64 ± 7.95 62 ± 8.98 65 ± 4.49 69 ± 5.43

[0128] As shown in Table 12 and FIG. 5, it could be confirmed that in the control group and all the experimental groups, the protozoal killing effect was excellent on a concentration-dependent manner of violacein, and even if violacein was left at a high temperature (80 to 95° C.), the protozoal killing efficacy of violacein did not appear at all the concentrations.

Example 7

In Vivo Safety Evaluation

[0129] The safety evaluation of violacein was conducted in a breeding facility under the condition described in Example 1. At 7 days of age, broilers were fed a feed comprising 50 ppm violacein for 7 days, and an autopsy was conducted at 14 days of age, and the feed, appendix luminal intestinal contents, blood, spleen, liver and kidney were collected. For the feed used in formulation with the violacein, Daehanfeed Al-Choi product was used.

[0130] Each sample of the collected tissue was tested for the content of violacein through the HPLC method. For feed pretreatment, lg of each treatment group was weighed and diluted by adding 10 of MTBE solution, and then sonication was progressed for 1 hour, and filtration was performed using a 0.45 μM syringe filter. Then, after evaporating the filtered sample for 1 hour, 1 , of 100% methanol was added. In addition, sonication was conducted for 1 minute, and it not dissolved, the supernatant was filtered once more using a 0.45 μM syringe filter, and then placed in a vial for analysis. For HPCL pretreatment of blood and organs, samples for each treatment group were pooled and homogenized 3 times for 10 seconds at 4000bpm. Then, the supernatant was collected in a 1.5 microtube and frozen at −70° C. After that, after lyophilizing the sample, 50 mg of the sample was weighed and placed in a microspin tube. 3000 of MTBE solution was added to the weighed sample, and the microspin tube itself was placed in a 1.5 canonical tube, and vortexed for 60 seconds. Sonication was further performed for 1 hour, and centrifugation was performed twice at 5000 rpm. 200-300 solution collected in the 1.5 tube was confirmed and the MTBE solution was evaporated. 100 of 100% methanol was added and dissolved, and if not dissolved, sonication was performed for 10 minutes and then 50 of the supernatant was collected and placed in a vial for analysis. Then, the samples in which pretreatment was completed were analyzed using a set LC/MS instrument to measure the amount of violacein remaining in a body after feeding, and the result was shown in Table 13 below. In Table 13, ND (not detected) means not detected.

TABLE-US-00013 TABLE 13 Violacein content (mg/kg) Treatment Cecum luminal group Feed intestinal contents Blood Liver Kidney Spleen NC ND ND ND ND ND ND Violacein 13 2.1 ND ND ND ND

[0131] As shown in Table 13, as the result of analyzing the violacein content in the feed and the cecum luminal intestinal contents, blood, spleen, liver and kidney of the broilers fed with the feed, the violacein was orally ingested by the broilers and then passed through the stomach and reached the appendix. In addition, violacein was not detected in blood, liver, kidney and spleen, so it was confirmed that it did not remain in a body, and it was confirmed that violacein could inhibit coccidium generation while staying only in the intestine.