KR101479619B1 - A method for preparation of propolis by-product powder and feed additives comprising it - Google Patents

Abstract

The present invention relates to a process for preparing powder of propolis byproduct which powder is highly viscous and water-insoluble propolis byproduct, and a functional feed additive having a natural antibiotic effect comprising the powder propolis by-product. In the present invention, there is provided a process for the production of (a) Starch; dextrin; Lactose; glucose; (B) obtaining a dried material by vacuum drying or lyophilizing the mixture obtained in the step (c); and (c) pulverizing the dried material obtained in the above step with a pulverizer And pulverizing the pulverized powder while cooling the pulverized product so as to prevent entanglement of the pulverized particles from occurring, thereby obtaining a powder having a particle size of 50 to 1,500 mesh; and a method for producing powder of propolis byproduct, By-products are provided. The powdered propolis by-product obtained according to the present invention can be used in various fields because it can be supplied at a low cost while having the functionality of refined propolis such as excellent antibacterial action. In particular, it can be used for natural antibiotic Can be used as a functional feed additive.

Description

[0001] The present invention relates to a process for preparing propolis by-product powder and a feed additive comprising the propolis by-

The present invention relates to a process for preparing powders for pulverizing propolis byproducts and functional feed additives with natural antibiotic effect comprising the powder propolis byproducts.

Propolis is a substance made by mixing beeswax with wax and saliva secreted by a plant. It is composed of vegetable resin, wax, essential oil, pollen, flavonoid, other organic matter and minerals. Propolis is a natural substance based on antimicrobial and antioxidant effects. It has various physiological activities such as antibacterial and anti-inflammatory action, analgesic and narcotic action, immune action, antioxidative action, antiviral action, . Propolis, based on antimicrobial and antioxidant properties, is one of the natural candidate substances that can replace antibiotics whose use for livestock is currently limited due to resistance problems.

Propolis is well known for its efficacy, but propolis has many difficulties in its use due to its unique viscosity and water insolubility. Conventional propolis extraction methods include, but are not limited to, a method using a spoon, a method using water, and a method using glycerin. However, when a propolis is used, the extracted propolis has low digestibility and water absorption, And when water is used, the content of the active ingredient, flavonoid, is as low as about 0.2%, so that the antibacterial and antioxidative effects of propolis are difficult to be exerted. Improved extraction methods that overcome these problems have been proposed in Korean Patent Laid-Open Nos. 10-1999-75441, 10-2002-76979, and 10-0550165.

Although a considerable amount of propolis remains in the residual propolis byproduct (hereinafter referred to as " propolis by-product ") after extracting propolis from the propolis raw material, it is hardly used due to the nature of the propolis byproduct, It is a fact that I can not. If the propolis byproduct can be pulverized, the propolis can be utilized for a variety of purposes because it can utilize the antibacterial and antioxidant properties of propolis at low cost. However, unlike propolis extracts, there are no successful cases of powdered propolis byproducts that are highly viscous and non-aqueous.

Korean Patent Publication No. 10-1999-75441 Korean Patent Publication No. 10-2002-76979 Korean Patent Publication No. 10-0550165

It is an object of the present invention to provide a process for producing propolis by-product powder which is high in tackiness and powdery by-product of propolis which has not been utilized so far.

Propolis is a highly viscous dendritic material made by mixing a resin collected from a plant with a saliva secreted by a bee. The propolis by-product is extracted from the ingested material and the propolis by-product is water-insoluble and has a higher viscosity do. This propolis by-product contains a large amount of propolis, but is difficult to use due to its high viscosity. The object of the present invention is to provide a powdered propolis by-product which can be obtained by powdering the propolis by-product to utilize the antibacterial and antioxidant-effective functions of propolis at low cost.

The present invention also aims at using this powdered propolis by-product, which has propolis functionalities such as excellent antibacterial activity, as a functional feed additive capable of replacing antibiotics.

In the present invention,

delete

delete

(a) Wheat flour on propolis byproducts; Starch; dextrin; Lactose; glucose; And at least one excipient selected from silicon dioxide to obtain a combination,
(b) vacuum drying or lyophilizing the compound obtained in the above step to obtain a dried product, and
(c) pulverizing the dried material obtained in the above step with a pulverizer while pulverizing the pulverized material while cooling the pulverized material using a pulverizer equipped with a cooling device to obtain a powder of 50 to 1,500 mesh, A powder production method and a powdered propolis by-product are provided.

In addition, the present invention provides a functional feed additive comprising the powdered propolis by-product as an active ingredient having antibacterial activity. The livestock is particularly any one of cattle, pigs, chickens, ducks, and fish.

The term " propolis by-product " or " byproduct by-product " or " by-product " in the present invention means a by-product obtained by extracting propolis from a propolis raw material. Unless otherwise specified, propolis is firstly extracted It means to include all the remaining by-products.

&Quot; Powder " in the present invention means both powder and particle state.

The present invention can provide a propolis by-product in the form of powder in which the propolis by-product which is highly viscous and is non-aqueous is powdered. The powdered propolis by-products obtained according to the present invention can be supplied at a low cost while having the functionality of refined propolis such as excellent antibacterial and antioxidant functions. The powdered propolis byproducts of the present invention can be utilized in various fields, and can be used as a functional feed additive for natural antibiotic use, which replaces existing antibiotics in livestock feeds.

Figs. 1 to 3 show a crusher used in an embodiment of the present invention. Fig. 1 is an external view of the crusher, Fig. 2 is a front view of the crusher, and Fig. 3 is a side view of the crusher.
FIGS. 4A and 4B show the results of an experiment on resistance to fish microbes and viruses by an attack experiment on flounder, FIG. 4A shows the result of an attack test on VHS virus, FIG. 4B shows the results of Streptococcus This is the result of an attack on iniae .

The method for producing powders of propolis by-products of the present invention includes the following steps (a) to (c).

(a) blending an excipient with the propolis by-product to obtain a blend,

(b) vacuum drying or lyophilizing the compound obtained in the above step to obtain a dried product,

(c) pulverizing the dried material obtained in the above step with a pulverizer to obtain pulverized powders while cooling the heat generated in the pulverizing process so that the pulverized particles are not re-entangled.

Each step will be described in detail below.

Step of compounding the excipient with propolis byproduct

Add an appropriate amount of excipient to the propolis byproduct. Propolis byproduct is a byproduct byproduct of propolis extracted from propolis, which is water-insoluble and very viscous. Regardless of the method of extracting propolis from the nodule, all of the remaining nodule byproducts after extraction of propolis can be used as propolis byproducts in the present invention.

The excipients to be mixed with the propolis byproduct should be highly viscous and mix well with non-aqueous byproducts. Among the excipients that can be used in the pharmaceutical, food and feed fields, those suitable are selected. In view of the mixing properties and the physical properties and costs which prevent agglomeration after crushing, it is preferable to use wheat flour; Starch; dextrin; Lactose; glucose; And at least one excipient selected from silicon dioxide is used. More preferably, wheat flour or starch or both are used together as an excipient.

In the present mixing step, additives may optionally be further added as needed for nutritional fortification, propolis-specific odor masking, taste improvement, physical properties and the like. As an additive, preferably a fruit by-product, a sweetener or a property improving agent is formulated. The fruit by-products are not limited to specific fruits, and any fruit by-products available as feed can be used. The fruit by-products are added for nutritional fortification, odor masking, taste improvement, etc. The fruit by-products are preferably selected from the group consisting of grape, persimmon, apple, pear, peach, kiwi, And the stem on which the stem was placed. The sweetener may be any sweetener which can be used in medicine, food or feed, without limitation. As a sweetening agent, for example, fruit concentrates such as aspartame, a concentrate of macellum or a concentrate of a bait, stevioside, citric acid, nutrient enhancer and glucose serving as a sweetener may be used. The physical property improving agent is used for improving physical properties and tactile properties of the composition, for example, gellan gum may be used. The kind of additives which can be mixed with the excipient is not limited, and can be selectively carried out according to need by a person skilled in the art.

The mixing ratio of propolis by-product and excipient is suitably 5 to 50% by weight of propolis by-product and 50 to 95% by weight of excipient. If the proportion of excipients is lower than that of the excipients, the overall viscosity of the excipients is high, which is difficult to be powdered in the next step. If the ratio of excipients is higher than that, the effective amount of propolis byproducts in the final powder is lowered. However, depending on the purpose of use, it is also possible to increase the proportion of the excipient. More preferably, 60 to 90% by weight of an excipient is added to 10 to 40% by weight of the propolis by-product.

The compound  Vacuum drying or lyophilizing step

The compound obtained in the above step is subjected to vacuum drying or lyophilization. This step can be carried out by using a vacuum dryer or a freeze dryer which is usually used in the field of food. Preferably at 10 to 100 DEG C, at -0.5 to 3 atm, for 2 to 36 hours. In one embodiment of the present invention, vacuum drying was performed under conditions of 65 ° C and -1 atm for 12 hours.

Crushing step

The vacuum (or freeze) dried material obtained in the above step is pulverized by a pulverizer. In this case, heat is generated in the pulverization process by the pulverizer, and heat generated in the pulverization process increases the adhesion of the pulverized propolis by-product, so that the pulverized particles are re-entangled. In order to prevent such coagulation phenomenon, the present invention grinds while cooling the heat generated in the grinding process. In a preferred embodiment of the present invention, a grinder equipped with a cooling device as shown in Figs. 1 to 3 is used.

1 to 3, the mill 100 used in the preferred embodiment has a hopper 110, a feed screw for feeding a predetermined amount of the propellant by-product introduced into the hopper 110, A pulverizing mill 130 for pulverizing the raw material charged in the charging part 120 and a recovery box 140 for recovering powders of propolis by-product pulverized in the pulverizing mill 130, And a cooling water line is provided at a rear portion of the mill mill 130 so that the cooling water generated in the cooler 150 is circulated and the heat generated in the milling process by the mill mill 130 can be reduced. In the figure, reference numeral 160 denotes a lift for the lifting operation of the collection box 140, and 170 denotes an operation panel for controlling the driving of the pulverized mill and the rotation of the inlet part screw. The pulverizer 100 shown in FIGS. 1 to 3 is only one preferred embodiment of the present invention, but the pulverizer used in the present invention is not limited thereto. The pulverizing step of the present invention is not limited to the form of the cooling device or the bonding structure with the pulverizer so long as the heat generated in the pulverization process can be cooled so that no entanglement phenomenon occurs between the pulverized particles.

In this grinding step, preferably, the dried material is pulverized to 50 mesh or more, preferably 50 to 1,500 mesh, more preferably 100 to 1,000 mesh.

The propolis by-product in powder form is obtained through the above steps. The powder propolis by-product obtained by the above method preferably contains 0.3% by weight or more of total flavonoids. This powdered propolis by-product has functionalities such as antibacterial and antioxidative effects of propolis, and can be applied to various fields to utilize such functionality. Particularly, the powdered propolis by-product of the present invention can be suitably utilized in the field where the functionality of propolis can be utilized at low cost. In particular, it may suitably be used as a functional feed additive that can replace antibiotics in feed for livestock. The target livestock is not particularly limited and can be used for all kinds of mammals such as mammals, birds, and fish, and is particularly applicable to the species including cattle, pigs, chickens, ducks, and fishes.

[ Example ]

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, these embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these embodiments, and the present invention can be implemented in various forms within the scope of the claims.

Example  1 to 7

Powder production of propolis by-products

(1) The propolis was extracted from the propolis raw material, and the excipient was blended with the residual byproduct of the raw material according to the compounding ratio shown in Table 1 below.

(2) The compound obtained in (1) was vacuum dried using a vacuum drier at 65 ° C and -1 atmospheric pressure for 12 hours.

(3) The vacuum dried material obtained in (2) was pulverized to a size of 100 mesh or more while cooling the heat generated in the pulverization process using the pulverizer shown in Fig.

Test Example  One

Total flavonoid content of powdered propolis by-products

This test is performed according to the total flavonoid content analysis method (Health Functional Food Code). The flavonoid substance is extracted from the sample using ethanol and analyzed using a spectrophotometer. The absorbance is measured at 415 nm, which is the maximum absorption wavelength of flavonoid .

(3,000 rpm, 10 min), and the supernatant was taken. The residue was extracted three times with 8 ml of 80% ethanol, and the extract was washed with water And the total volume was adjusted to 50 ml with 80% ethanol to prepare a test solution. Take 0.5 ml of this test solution into a test tube, add 1.5 ml of ethanol, 0.1 ml of 10% aluminum nitrate solution, 0.1 ml of 1 M potassium acetate solution and 2.8 ml of water, and sufficiently stir. After standing at room temperature for 40 minutes, the absorbance of the liquid layer is measured at 415 nm using a UV / VIS Spectrophotometer (Varian, Inc. USA) using a 10 mm cell as a reference solution. Separately from the absorbance of the sample, the total flavonoid content is calculated by the following equation using the absorbance difference obtained by subtracting the absorbance of 0.1 ml of water instead of the aluminum nitrate solution during the above operation. The results are shown in Table 2.

       Total flavonoid content (w / w%, or w / v%) =

                                    50 ml

        a ㎎ / ㎖ × ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ × 100

The amount of sample (g or ml) × 1,000 (mg)

Test Example  2

Experiments to measure the antibacterial activity against livestock pathogens and harmful bacteria of powdered propolis byproducts

(One) Used strain

To determine the antimicrobial activity of the powdered propolis by-products prepared in the above examples, 10 kinds of harmful species were selected. Staphylococcus aureus , one of the important strains of bovine mastitis subp . aureus ( KCTC 1928) is the causative agent of gangrenous mastitis. In addition, it exists in the mouth and skin of animals and is harmless in the normal state. When the antibiotic substance is used for a long time or the immunity is weakened, the body propagates abnormally in the body (mucous membranes such as the mouth and the vagina) Candida , a fungus that causes fingernails, scaly patches (athlete's foot) albicans were selected. Most of the bacteria in the Gram negative group are known to be the most known. They are found in human or animal fields, have pathogenic properties such as diarrhea and sometimes have cystitis, cholecystitis, pyelonephritis, Escherichia that can cause meningitis coli And Bacillus subtilis , one of the most studied bacteria among Gram positive group, was selected as the cause of food spoilage. In addition, food and including the world-renowned food poisoning microorganisms of Salmonella typhimurium food poisoning it is spread by water, causing various infectious diseases the most important strains of Staphylococcus aureus (Staphylococcus aureus ) were selected. The antimicrobial activity of the selected strains was measured.

The strains used in this experiment were Eshcherichia coli (KCTC 1924), Staphylococcus aures subp . aureus (KCTC 1928), Staphylococcus epidermidis (KCTC 1917), Steptococcus pyogenes (KCTC 3096), Proteus vulgaris (KCTC 2433), Clostridium perfringens (KCTC 3269), Klebesiella pneumoniae subsp . pneumoniae (KCTC 1726), Candida albicans (KCTC 7965), Salmonella enterica subsp . enterica (KCTC 1925), Bacillus subtilis subsp . and subtilis (KCTC 1021) were distributed from KTCT (Korea Research Institute of Bioscience and Biotechnology) (Table 3).

(2) Antibacterial activity experiment

The antibacterial activity was tested in a medium containing 100 mg / ml of powdered propolis by-product ('PBP') obtained in Example 5. As a comparative example, a mixture of RP and PBP (RP + PBP) at a ratio of 1: 1 was used as a refined propolis (RP) (Seoul Propolis Co.). Purified propolis (RP) was purified by WEEP method from Seoul Propolis Co., Ltd. and used commercially.

The strains were incubated overnight in an incubator and used as inoculum. Antimicrobial activity was measured by Disc diffusion method (paper disc: ø 6mm, Whatman). First, 0.1 mL of the preculture medium was transferred to 15 mL of the sterilized medium prepared at 45 ° C. by the pour-plate method, mixed well, and placed in a Petri dish having a diameter of 8.8 cm. After the paper disc was placed on the paper disc, the sample was absorbed and then cultured in an incubator to measure the antimicrobial activity to the size of the clear zone around the paper disc.

(3) Results

Table 4 shows the results of the agar well diffusion assay at a concentration of 100 mg / ml of the powdered propolis by-products (PBP) obtained in the examples and the comparative examples 'RP' and 'RP + PBP'. As can be seen in Table 4, the antimicrobial activity against 10 strains was exhibited, but the activity against Bacillus subtilis ( KCTC 1021) was the highest overall, while that of Candida albicans ( KCTC 7965), Staphylococcus epidermidis ( KCTC 1917), Klebesiella pneumoniae subsp . pneumoniae ( KCTC 1726) .

Test Example  3

Antioxidant and anti-inflammatory effects of powdered propolis by-products

The antioxidant and anti-inflammatory effects of the powdered propolis by-products of the present invention were evaluated. In particular, it has been verified by comparison with refined propolis (RP) that it can be used to develop economical antibiotic products for powdered propolis by-products.

(1) Experimental method

(A) Sample

The powder propolis byproduct ('PBP') obtained in Examples 5 to 7 was used as a sample, and a refined propolis (RP ') (Seoul Propolis Co., Ltd.) was used as a comparative example. Purified propolis (RP) was purified by WEEP method from Seoul Propolis Co., Ltd. and used commercially. PBP 12.5 ',' PBP 25 ', and' PBP 50 ', which contained 12.5 mg, 25 mg and 50 mg, respectively, in 100 ml of propolis byproduct, Refined propolis ('RP') was also labeled 'RP 12.5', 'RP 25', 'RP 50' depending on the content in 100 ml.

(I) In vitro  Antioxidant activity evaluation

① DPPH Radical  Measurement of scavenging activity

In general, DPPH (2,2-diphenyl-picryl-hydrazyl) method is used to measure antioxidative activity. DPPH is dark purple and consists of radicals of nitrogen center power. The antioxidant activity of the samples was calculated by scaling the percent of radical scavenging by the value of the DPPH absorbance change after the addition of the sample. In other words, 198 μl of 0.15 mM DPPH methanol solution was dispensed into a 96-well plate, and 2 μl of a different concentration of the sample was added. After reacting at room temperature for 30 minutes, absorbance at 517 nm was measured using a microplate reader (Tecan Infinite M200) Were measured. The concentration of the sample to which 50% of the DPPH radical was erased was expressed as IC50. The results obtained by repeated experiments were averaged and expressed as values.

DPPH radical scavenging activity (%) =

A _control : absorbance of control without addition of sample

A _sample : absorbance of the reaction solution to which the sample is added

② Superoxide anion radical  Measurement of scavenging activity

Superoxide radicals (O ₂ ^- ) act as precursors of other harmful free radicals, so the search for substances capable of directly eliminating these radicals has been used as an effective method for the development of antioxidants. In this experiment, the degree of inhibition of the reaction of Xanthine / Xanthine oxidase by superoxide generation system and WST-1 reduced by formazan production was measured. 15 μl of the sample, 150 μl of 50 mM sodium phosphate buffer (pH 7.4) containing 1 mM EDTA, and 15 μl of 0.5 mU xanthine oxidase and WST-8 solution were added to a 96-well plate and stabilized at room temperature for 1 minute. 120 [mu] l of 2.5 mM xanthine was added. The final concentrations of the samples used were 500, 250, 125 and 62.5 μg / ㎖, respectively. The final concentrations of the control group Epigallocatechin gallate (EGCG) were 100, 50, 25 and 12.5 ㎍ / ㎖. After incubation at room temperature for 30 min, absorbance was measured at 450 nm wavelength using a microplate reader (Tecan Infinite M200). The superoxide anion radical scavenging activity was expressed as a percentage of the absorbance of the control.

Superoxide anion radical scavenging activity (%) =

A _blank : absorbance of blank without sample and Xanthine oxidase

A _control : absorbance of control without addition of sample

A _sample : absorbance of the reaction solution to which the sample is added

(All) In mice  Evaluation of antioxidative effect

① Test animal and sample treatment

8 weeks old ICR mouse females were purchased from Orient and used in the experiment in the animal room of SPF condition for about one week. Breeding environment was adjusted to 26 ℃, humidity 60%, indoor lighting was changed to 12 hours cycle (08: 00 ~ 20: 00). Experimental groups were divided into 8 groups of 7 animals each. Group 1 was set as a control group, and propylene glycol was orally administered at 100 쨉 l per 30 g of body weight. Groups 2 and 3 were administered with carbon tetrachloride (CCl ₄ ), and propylene glycol was orally administered at a dose of 100 μl per 30 g of body weight. Groups 3 and 4 (RP 50 mg / kg, 100 mg / kg) PBP 50 mg / kg, 100 mg / kg) and Group 7, 8 (RP 50% + PBP 50% 50 mg / kg, 100 mg / kg). Each sample was intubated at 9 o'clock every morning for 7 days and fed normal diet and drinking water. Groups 2 to 8 were intraperitoneally administered with 1.0 mg / kg of a mixture solution of CCl ₄ and an equivalent amount of olive oil on the seventh day. All mice were sacrificed after fasting for 21 hours and liver and plasma were collected to measure lipid peroxidation and glutathione (GSH) content.

② Measurement of lipid peroxidation ( TBARS assay )

Lipid peroxidation was analyzed using OxiSelect ^™ TBARSAssayKit using thiobarbituric acid (TBA). Blood collected from the abdominal vein of mice was placed in a tube containing anticoagulant ACD and centrifuged at 3000 rpm for 15 minutes. The separated plasma was stored at -70 ° C and used for TBARS analysis the next day. Immediately after the blood was collected, the liver tissue was perfused with cold physiological saline to remove the blood, and the blood was removed. The blood was taken out, washed with 1 × BHT in cold physiological saline, washed with filter paper, and stored at -70 ° C. Respectively. About 0.1 g of liver was homogenized in cold physiological saline containing 1X BHT at a concentration of 100 mg / mL, and the homogenate was centrifuged at 10,000 g for 5 minutes to quantify the protein, Of TBARS were measured. Detailed experiments were carried out in accordance with the method described in the kit. Absorbance was measured at 532 nm using a microplate reader (Tecan Infinite M200).

(la) In vitro  Evaluation of anti-inflammatory effect

① Cell culture

RAW 264.7 cells were cultured in a 5% CO2 incubator at 37 ° C in a DMEM medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin in a Korean Cell Line Bank (KCLB, Seoul, Korea) Respectively. RAW 264.7 cells were plated on a 24-well plate at a concentration of 4 × 10 ⁵ cells / ml and cultured for 24 hours. After 4 hours, RP, PBP 12.5, 25, 50 ㎍ / Ml) and cultured for 24 hours and 48 hours. The vehicle group was treated with DMSO as a control and incubated for the same time.

② Nitric oxide  Measurement of secretion inhibitory activity

The NO assay was performed to investigate the effect of propolis processed raw material (RP) and processing byproduct (PBP) on nitric oxide (NO) production. Raw 264.7 cells were cultured in 24 well plates, and RP and PBP were pretreated for 12 hours at 12.5, 25, and 50 ㎍ / ㎖, respectively. LPS (0.1 μg / ml) was then treated for 48 hours and the NO concentration was measured with the cell culture supernatant thus treated. NO concentrations were measured using the Griess reaction and compared with the nitrate standard curve. 100 μl of the supernatant and the same amount of Griess reagent were mixed in 96 wells and absorbance was measured at 540 nm using a microplate reader (Tecan Infinite M200).

③ inflammatory cytokine ( TNF -α, IL -6) secretion inhibitory activity measurement

TNF-α and IL-6 were measured in the medium of cultured RAW 264.7 cells. Anti-mouse TNF-α and IL-6 capture monoclonal antibodies were coated on a 96-well plate at 0.2 μg / ml and reacted at 4 ° C for 12 hours. To prevent nonspecific reactions after coating, the cells were incubated with assay diluent buffer for 1 hour, washed 3 times with PBS containing 0.05% tween 20, and incubated with appropriate concentrations of TNF-α and IL-6 Diluted and prepared. Each culture supernatant was counted, and 100 쨉 l of each supernatant was added to each well, followed by reaction for 3 hours. After washing 5 times with washing buffer, 100 μl of biotinylated anti-mouse TNF-α and IL-6 (0.2 μg / ml) were added to each well for 2 hours. After washing with washing buffer, streptavidin-horseradish peroxidase conjugate enzyme (SAV-HRP) was treated at 2.5 μg / ml for 1 hour. After washing 7 times, 50 μl of TMB substrate solution was added to each well to induce color development for 10 min. After stopping the reaction using 2N sulfuric acid, TNF-α was detected at 450-570 nm using an ELISA reader The amount of IL-6 protein was measured.

(hemp) In mice  Evaluation of anti-inflammatory effect Cotton pellet granuloma  Formation test)

8 weeks old ICR mouse females were purchased from Orient and used in the experiment in the animal room of SPF condition for about one week. Breeding environment was adjusted to 26 ℃, humidity 60%, indoor lighting was changed to 12 hours cycle (08: 00 ~ 20: 00). The experimental group was divided into 7 groups of 7 animals each. Groups 1 and 2 were treated with propylene glycol at a dose of 100 μl per 30 g of body weight and group 2 and 3 (RP 50 ㎎ / ㎏, 100 ㎎ / ㎏), Group 4 and 5 (PBP 50 ㎎ / Mg / kg), group 6, 7 (RP 50% + PBP 50% 50 mg / kg, 100 mg / kg). On the first day of the experiment, each sample was incubated, and an autoclaved cotton pellet (10 mg) was anesthetized with diethyl ether and inserted aseptically into the subcutaneous area of the right underarm. Each sample was intubated at 9 o'clock every morning for 7 days and sacrificed at 9 o'clock on the eighth day to collect cotton pellets surgically. The collected cotton pellets were dried in 60 o C dry oven for 18 hours and weighed to determine the weight of granuloma tissue.

(2) Results

One) RAW  264. 7 cells nitric oxide  And inflammation cytokine  Evaluation of secretion inhibition

RAW 264.7 cells secrete nitric oxide (NO) and inflammatory cytokines (TNF-α, IL-6, etc.) known as inflammation-promoting substances by LPS stimulation. In this experiment, the effects of propolis processed raw material (RP) and processing byproduct (PBP) on the secretion of inflammation-related substances in RAW 264.7 cells were observed.

First, the effect of RP and PBP on NO production is shown in the graph 1 below. As can be seen, increasing the concentration of each sample in the concentration range of 12.5-50 μg / ml without cytotoxicity showed that the production of NO induced by LPS was decreased, and RP was the most effective . In the control group, nitrite production was 1.83 ± 0.94 μM, whereas in the LPS alone treatment group, the concentration was increased to 53.28 ± 4.98 μM. 3.32 ± 1.28 μM in the RP 50 μg / ㎖ treatment group and 7.39 ± 1.85 μM in the PBP 50 μg / ml treatment group inhibited LPS induced nitrite production. These results showed that RP and PBP had a high inhibitory effect on NO production in RAW 264.7 cells. PBP, which was powdered by-product, had a lower activity than purified propolis but had a considerable effect.

[Graph 1]

The values of independent experiments are expressed as means ± SEM (n = 4).

We next examined the effect of TNF-α and IL-6 on the inflammatory cytokines secretion. RP, and PBP at concentrations of 2.5, 25, and 50 were pretreated with RAW 264.7 cells at a concentration of 1 mg / ml, treated with LPS at a concentration of 0.1 μg / ml, and then secreted into the culture medium after 24 hours and 48 hours TNF-a and IL-6 levels were measured. As a result, LPS significantly increased TNF-α and IL-6 production, which are proinflammatory cytokines, and TNF-α induced by LPS in RP and PBP-treated groups at 25 and 50 mg / RP decreased 29.73%, 37.69%, 14.10% and 21.36% at 24 hours, 40.99% and 44.p6% at 48 hours, and 35.68% and 45.49% for PBP, respectively Graph 2). In the same way, IL-6 was reduced to 15.13% and 35.14% in the RP group and 31.07% and 42.88% in the RP and PBP 25 and 50 mg / 20.50%, and 30.62%, respectively. The decrease rate of PBP group was 36.26% and 45.64% (Graph 3). These results suggest that both purified propolis (RP) and powdered propolis byproduct (PBP) inhibit the production of inflammatory cytokines induced by LPS, although PBP has lower activity than RP but has a significant activity .

[Graph 2]

Graph 2. Inhibitory effect of RP , PBP on the LPS - induced production of TNF -a. Values represent mean ± _SD (n = 4).

[Graph 3]

Values represent mean ± _SD (n = 4).

2) In mice  Evaluation of anti-inflammatory effect Cotton pellet On by granuloma  Evaluation of formation inhibition effect

In the anti - inflammatory test using mice or rats, granuloma formation test by cotton pellet is widely used as a model for evaluating the inhibitory effect on chronic inflammation. When a cotton pellet is implanted subcutaneously, an inflammatory reaction takes place around the cotton pellet and inflammatory cells gather to form a granuloma, an inflammatory tissue. The in vivo anti-inflammatory effects of propolis processed raw materials (RP) and processed by-products (PBP) were evaluated using this test model in the graph 4 below. Graph 4 shows that the RP, PBP, RP 50% and PBP 50% mixture decreased granuloma tissue proliferation compared to the control group. The weight of granuloma was increased by 50 ± 4.47 ㎎ in the untreated group after 10 ㎎ of cotton pellet. In the group treated with 100 ㎎ / ㎏ of RP, it was increased by 16.27 ± 2.07 ㎎. In the group treated with 100 ㎎ / ㎏ of PBP, 12.34 ± 0.94 ㎎ was increased by 15.35 ± 0.93 ㎎ in the group treated with 50% . 100 ㎎ / ㎏ treatment group showed significant anti - inflammatory effects in RP, PBP and RP + PBP mixed 50% of RP 50 and PBP 50, and 50 ㎎ / ㎏ treatment group , RP showed the best anti-inflammatory effect.

[Graph 4]

Data are means ± SEM (n = 7)

As a result of the in vivo activity evaluation, both the processed and the processed by-products showed high antioxidative and anti-inflammatory effects. In particular, in vivo activity was observed when oral administration of 50 mg / kg or 100 mg / kg was used in the evaluation of antioxidative and antiinflammatory efficacy using mice. In particular, it was observed that the activity of PBP was slightly lower than that of RP, but still exhibited a significant physiological activity such as exhibiting an activity of about 50% or less of that of RP. Thus, the inventors of the present invention Of powdered propolis byproducts could be used as natural antibiotics for livestock feeds.

Test Example  4

Weaned pigeon  And broiler specimen test

The efficacy of the powdered propolis byproducts of the present invention as an antibiotic substitute was confirmed through a test of broodstock and broiler chickens.

(1) Experimental method

(end) Weaned pigeon  Specification Test

1) Test animals and test design

Landrace × Yorkshire × Duroc Weighed 125 dogs were weighed and weighed 6.68 ± 1.03 kg at the start of the test. The powder propolis by-product prepared in Example 1 was used as the powder propolis by-product (PBP) of the present invention. The treatments were 1) NC (basal diet, no antibiotics), 2) PC (NC + tiamulin 500ppm), 3) PRO1 (NC + 0.05% PBP) + 0.20% PBP), 5 repetitions per treatment, and 5 repetitions per arrangement.

2) Test feed and specification management

The test was carried out at Dankook University Experimental Research Station. The test diets were fed with corn - soybean meal according to the requirement of NRC (1998), and the water was freely fed using an automatic water dispenser.

3) Survey items

① Productivity

Body weight and feed intake were measured at the start of the test, at 2 weeks and at the end (5 weeks), and daily gain, daily feed intake and feed efficiency were calculated. Mortality was calculated by dividing the number of deaths by the percentage of the total number of deaths by treatment.

② Nutrient digestibility

Nutrient digestibility was obtained by adding 0.2% of chromium oxide (Cr2O3) as a labeling substance at the end of 2 weeks and at the end (5 weeks). The samples were dried in a dryer at 60 ° C for 72 hours, and then pulverized with a Willey mill. Cr mixed with the general components of the feed and labeling was analyzed according to the method of AOAC (2000).

③ Blood characteristics

Blood samples were collected at 2 weeks and at the 5th week of treatment (8 weeks), and blood was collected from the jugular vein using a K ₃ EDTA Vacuum tube (Becton Dickson Vacutainer Systems, Franklin Lakes, NJ) mL, and WBC (white blood cell) and lymphocyte were examined using an automatic blood analyzer (ADVID 120, Bayer, USA). The serum biochemical test was carried out at the start of the test and at 2 weeks using a vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA) to collect 5 mL of blood, centrifuging at 3,000 rpm for 15 minutes at 4 ° C, Were used to investigate IgG and TNF-a.

④ Microbial bacteria

Eight specimens were selected for treatment at the 2nd week and at the end of the test (5 weeks), and the specimens were collected by an anal massage method. The specimens were stored frozen at -20 ° C until the experiment, and then suspended in sterilized physiological saline and homogenized And diluted 103-107 steps, and used as a sample for measuring viable cell count. MRS agar for Lactobacillus and MacConkey agar (Difco, USA) for E. coli were used to measure the numbers of Lactobacillus and E. coli in the pork by experimental treatment.

⑤ Fecal index

Fecal index was measured weekly and scored. 4 = soft, unformed stool that assumes (2 = hard, formed stool that remains firm and soft; 3 = soft, formed, and moist; 4 = soft, dry pellets in a small, hard mass; 5 = watery, liquid stool that can be poured).

⑥ morphological characteristics of intestines

After the end of the test, 5 samples were collected from the duodenum, the plant and the spermatozoa after slaughtering. Samples were stored in formalin solution, and then villi length and depth were measured.

⑦ Statistical processing

All data were analyzed using the General Linear Model Procedure of SAS (1999) and Duncan's multiple range test (Duncan, 1955) was used to determine the difference between the mean values.

 (B) Broiler Specification Test

1) Test animals and test design

This test was performed on 720 rats 308 (♀, female) at 1 day, and the weight of the test was 40.14 ± 0.14g. The test was conducted for 29 days. The powder propolis by-product prepared in Example 1 was used as the powder propolis by-product (PBP) of the present invention. The experimental designs were 1) NC (Basal diet, No antibiotics) 2) PC (Antibiotics diet) 3) PRO1 (NC + 0.05% PBP) 4) PRO2 (NC + 0.10% PBP) 0.20% PBP), and 9 replicates per treatment, 16 replicates per replicate.

2) Test feed and specification management

ROSS 308 chicks were raised in a three - stage cage, and the position of the treatments was adjusted, and the feed and water were free - veganized.

3) Survey items and methods

① Productivity

Body weight was measured at the initiation, 14 days and 2 end (29 days) by treatments. The feed intake was calculated by subtracting the remaining amount from the feed amount during body weight measurement. The feed conversion ratio was calculated by dividing the feed intake by the body weight gain. The mortality rate was calculated by dividing the number of deaths by the total number of deaths per treatment.

② Blood characteristics

Blood samples were collected at the end of the study (29 days), randomly selected by the number of 10, and 2 mL of blood was collected from the subclavian vein using a K ₃ EDTA Vacuum tube (Becton Dickinson Vacutainer Systems, Fraklin Lakes, NJ) WBC (white blood cell), red blood cell (RBC), and lymphocyte were examined using a Bayer, USA. Serum biochemical tests were performed by using a Vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) in the subclavian vein at the end of the study (29 days) and collecting 5 ㎖ of blood and centrifuging at 4,000 rpm for 15 minutes Were used for analysis. The IgG content of isolated serum was analyzed by nephelometry using a nephelometer (Behring, Germany) analyzer.

③ Long-term weight

At the end of the test, the treatment groups were randomly selected and slaughtered by tongue withdrawal method. The weight of the liver, spleen, proximal part, F sac, chest and abdominal fat were measured and calculated as the ratio to the body weight.

④ Meat quality

The meat color was measured by repeating a colorimetric method (Model CR-210, Minolta Co., Japan) twice per each breast meat sample. At this time, the standard color plates were L = 89.2, a = 0.921, and b = 0.783. The water holding capacity was measured by the method of Hofmann et al. (1982) by measuring the ratio of the total area to the meat area. The pH was measured using a pH meter (77P, Istek, Korea) Respectively. The drop loss was determined by measuring the amount of loss after 1 day, 3 days, 5 days and 7 days after storing the sample in a constant shape of 2 cm thickness and storing it in a polyethylene bag for 7 days at 4 ℃ refrigerated room.

⑤ Observation of field shape

At the end of the study (4 weeks), long - term weight analysis was performed to observe intestinal morphology, to identify intestinal diseases, to identify necrotizing enterocolitis, and to take pictures.

⑥ Statistical processing

All data were analyzed using the Duncan's multiple range test (Duncan, 1955) using the General Linear Model procedure of SAS (1999).

(2) Experimental results

(end) Weaned pigeon  Specification Test Result

1) Productivity

The effects of dietary propolis addition on the productivity of weaned piglets are shown in Table 5. PRO1, PRO2 and PRO3 treatments were significantly higher (p <0.05) than those of NC treatments at 0-2 weekly gestational weight gain (P <0.05), but there was no significant difference in daily feed intake and feed efficiency 0.05). There was no statistically significant difference between two treatments in daily gain, daily feed intake and feed efficiency (P> 0.05). PRO2 and PRO3 were significantly higher (P <0.05) than control (P <0.05), and PRO3 was significantly higher than control (P <0.05) . There was no statistically significant difference in dietary intakes between treatments (P> 0.05). The mortality rate did not show significant difference (P> 0.05).

2) Nutrient digestibility

The effects of dietary propolis on the nutrient digestibility of weaned piglets are shown in Table 6. The digestibility of PRO1, PRO2 and PRO3 with PC and propolis was significantly higher than that of non - antibiotic NC treatment (P <0.05), and the digestibility of PRO1, PRO2 and PRO3 were significantly higher than those of non-antibiotic NC treatment (P <0.05). No significant differences in digestibility of building, nitrogen and energy were found at 5th week (P> 0.05).

3) Blood characteristics

The effects of the addition of propolis in the feed on the blood profile of the weaned piglets are shown in Table 7. There was no significant difference between the two treatment groups in IgG, WBC, lymphocyte and TNF-a (P> 0.05). At the end of the experiment, PRO2 treatment was significantly higher in IgG (P <0.05) than NC treatment (p <0.05), but WBC, lymphocyte and TNF-a showed no significant difference in treatment interval (P> 0.05).

4) Intrauterine microorganisms Lichen

The effects of the addition of propolis in the feed on microbial microflora of the herbal infant are shown in Table 8. There was no significant difference between the treatments (P> 0.05) for E. coli and Lactobacillus at 2 weeks and at the end of the test.

5) Feces  Indices

The effects of the addition of propolis in the feed on the fecal indices of the weaned piglets are shown in Table 9. PRO2 treatment was significantly lower (P <0.05) than NC and PC treatments at 1 week, but there was no significant difference between treatments at initiation, 2 weeks, 3 weeks, 4 weeks, > 0.05).

6) Intestinal morphological characteristics

The effects of the addition of propolis in the feed on intestinal morphological characteristics of the weaned piglets are shown in Table 10. There was no statistically significant difference (P> 0.05) in the ratio of villi, jungwa and villus / jungwa within the small intestine.

(B) Broiler Specimen Test Result

1) Productivity

The effects of the addition of propolis in the feed on the productivity of broiler chickens are shown in Table 11. Feeding rate of PRO2 was lower than that of NC treatment (P <0.05). Feeding rate of PRO2 was lower than that of NC treatment (P <0.05). Feed intake, however, did not show any difference (P> 0.05). There was no statistically significant difference between the treatments (P> 0.05) for weight gain, feed intake and feed conversion rate during 15-29 days. During the whole test period, the PRO2 treatment group showed higher (P <0.05) than the NC treatment group (P <0.05) and the PRO2 treatment group showed lower than the NC treatment group. However, there was no significant difference in feed intake between treatments (P> 0.05). There was no significant difference in the mortality between the treatments (P> 0.05).

2) Blood characteristics

The effects of the addition of propolis in the feed on the blood characteristics of the broiler are shown in Table 12. The WBC, RBC and Lymphocyte contents showed no significant difference in the feed intake (P> 0.05) but the IgG level was higher (P <0.05).

3) Long term weight ratio

The effects of the addition of propolis in the feed on the long term weight ratios of broilers are shown in Table 13. There was no significant difference between the treatment groups in the spleen, F sac, chest and proximal weights (P> 0.05).

4) Meat quality characteristics

The effects of the addition of propolis in the feed on the productivity of broiler chickens are shown in Table 14. There was no significant difference in pH, chest color, water holding capacity and storage loss (P> 0.05).

5) Field observation

The effects of the addition of propolis in the feed on the morphology of broiler chickens are shown in Table 15. There was no specific disease or disease in the examination of intestinal morphology and intestinal necrotizing enterocolitis, and no specific disease was found because there was no case of death or severe diarrhea especially during the specimen test period.

Test Example  5

halibut( Paralichthys olivaceus ) On growth, feed efficiency, nonspecific immunity and disease resistance

(1) Materials and methods

1) Experimental feed

Experimental diets were prepared by mixing five feeds and liquid propolis ('L') ('P') obtained by adding 0 (Control), 0.25, 0.50, 0.75 and 1.0% (Con, P0%, P0.25, P 0.25%, P0.5, P 0.50%) were prepared with three feeds containing 0.25, 0.5, and 1.0% L0.25, L0.25%, L0.5, L0.50%, L1.0, L1.0%). All experimental diets were prepared using the practical feed source, with 46% crude protein, 15% crude fat, and 17 MJ / kg diet. The difference of available energy according to the amount of propolis in the experimental diets was controlled by the addition of cellulose. Table 16 shows the basic feed composition table. The experimental diets were prepared by uniformly mixing the feed materials in a mixer, adding fish oil, adding distilled water equivalent to 30% of the total amount of the feed material, and kneading with a feed mixer. Mixed dough water was sized to the appropriate size of the experimental fish using a small-sized digging machine (SMC-12, Kuposlice, Busan, Korea). Molded experimental diets were naturally dried for 24 h and stored at -20 ° C until feeding.

2) Experimental language  And breeding management

The fish used in the experiment were purchased from the nursery culture facility located in Awol - eup, Cheju - do, and transferred to the Institute of Oceanography and Environment, Cheju National University. Experimental fish were adapted to the experimental environment while feeding the commercial feed for two weeks. After preliminary breeding, the flounder (initial average weight: 8.94 ± 0.02 g) was randomly selected and placed in a total of 24 150 L circular plastic tanks with 45 fish per tank. Breeding water was regulated so as to supply 2 - 3 L / min flow rate using sand filtration seawater and air stones were installed in all experimental tanks to maintain dissolved oxygen. The temperature of the rearing water was dependent on the natural water temperature (18-23 ℃) and the light intensity was maintained at 12L: 12D using fluorescent lamps. Experimental feeding was performed twice a day (08:00 hr and 18:00 hr) and the experiment was conducted for a total of 8 weeks.

3) Sample Collection

Fish weighing was measured every 2 weeks and all experimental fishes were fasted to reduce the stress of the experimental fish 24 hours before the measurement. After final weight measurement, eight fish were randomly selected per animal and anesthetized with 2-phenoxyethanol (200ppm) solution. Three out of seven animals were collected from the tail vein using a heparinized syringe. Blood collected was used for hematocrit, hemoglobin and nitroblue-tetrazolium (NBT) analysis. The blood of the remaining four fish was collected using a syringe without heparin treatment. The whole blood was left at room temperature for 60 minutes and centrifuged (5,000 × g) to separate the serum and used for nonspecific immunological analysis.

4) Survey items

① Growth rate measurement

Survey items related to growth rate are as follows. Growth rate (WG,%) = 100 × (final mean body weight) / initial mean body weight; Feed Conversion Efficiency (FCR) = dry feed fed / wet weight gain; Daily growth rate (SGR,%) = [(loge initial body weight) / days] x 100; Feed intake (FI) = dry feed consumed (g) / fish; Protein conversion efficiency (PER) = wet weight gain / total protein given.

② Analysis of general components of experimental diets

The general components of the experimental diets were analyzed by the AOAC (1995) method using the normal pressure heating drying method (125 ° C, 3 hr), the direct filtration method (550 ° C, 6 hr) , Sweden) and the fat was analyzed by Folch et al. (Soxhlet heater system C-SH6, Korea) according to the method of Sanghoon et al. (1959).

③ Blood analysis

Hematocrit and hemoglobin analyzes were performed to determine the general health of the experimental fish. Hematocrit was analyzed by microhematocrit technique and hemoglobin content was measured by automatic biochemical analyzer (Express plus system, Bayer, USA).

④ Immunological analysis

Macrophage is a phagocytic cell that engages in pathogens that enter the body and performs predation, and is involved in both innate and adaptive immunity. Enhancing macrophage function is called macrophage activation, which promotes the hydrolysis of captured pathogens and increases the production of reactive oxygen species (ROS). Therefore, the macrophage activity, ie innate immune response, can be confirmed indirectly through the measurement of ROS. Macrophage activity assay was analyzed as follows (Kumari and Sahoo, 2005). First, 50 μl of blood and NBT solution (0.2%) were mixed and reacted at 25 ° C for 30 minutes. 50 μl of the reaction mixture was transferred to a glass tube and 1 ml of dimethyl formamide was added thereto. After centrifugation at 2000 rpm for 5 minutes, the absorbance was analyzed at 540 nm using an upper spectrophotometer (Genesys, Series 10 UV, Rochester, NY, USA). At this time, the blank was made of dimethyl formamide.

Myeloperoxidase (MPO) is a peroxidase with antimicrobial activity in neutrophils, basophils, and eosinophils. MPO produces hypochlorous acid and tyrosyl radical with cytotoxicity through hydrogen peroxide and chloride anion produced during the respiratory burst process, thereby killing the pathogen. Therefore, the high content of MPO in vivo increases the nonspecific immune response through amplification of the antimicrobial effect. Serum MPO activity was analyzed based on the method of Kumari and Sahoo (2005). First, 80 μl of HBSS (Hanks balanced salt solution) is dispensed into 96-well plates and 20 μl of serum is added. Then, add 20 mM TMB (3,3'5,5'-tetramethyllbenzidine hydrochloride) solution and 5 mM H 2 O 2 solution. After reacting for 2 minutes, add 35 μl of 4 M H2SO4 solution and measure the absorbance at 450 nm in a microplate reader (Thermo, USA).

Lysozyme is one of the antimicrobial enzymes involved in the immune response. It is an enzyme that exhibits antimicrobial activity against various bacteria nonspecifically, not specific antibacterial activity against specific bacteria. The antimicrobial mechanism against pathogenic bacteria is the antibacterial mechanism by hydrolyzing β-1,4-glucoside bond between N-acetylmuramic acid and N-acetyl-D-glucosamine residues of peptidoglycan which is a constituent of bacterial cell wall, . Serum lysozyme analysis was analyzed by Sankaran and Shanto (1972). A suspension of 0.2 mg / ml was prepared by adding lyophilized Micrococcus lysodeikticus (Sigma, USA) to sodium citrate buffer (0.02 M, pH 5.52), and the suspension and serum were mixed at a ratio of 10: Absorbance values were measured at 450 nm. After reacting at 24 ° C for one hour, the final absorbance value was measured. Lyophilized hen egg white lysozyme (HEWL; Sigma, USA) was used to calculate the standard curve and expressed as ugHEWL / ㎖.

Superoxide dismutase (SOD) is a typical antioxidant enzyme that catalyzes the conversion of peroxide ions into oxygen and hydrogen peroxide. When the amount of active oxygen in vivo is increased, the SOD level also increases, thereby improving the immunity of the host organism by eliminating the free toxic group generated in the aerobic metabolic process. Serum SOD (Superoxide dismutase) activity was analyzed using the superoxide dismutase assay kit (Sigma, 19160). Add 20 μl of radical detector to 96-well plates and add 10 μl of serum from the experimental fish. After incubation for 20 minutes with 20 μl xanthine oxidase, the absorbance was measured at 450 nm using a microplate reader (Themo, USA).

Serum antiprotease activity was analyzed by the method of Ellis (1990). 20 μl of trypsin solution (Sigma, USA) was added and 20 μl of serum was reacted at 22 ° C for 10 minutes. Then, 200 μl of phosphate buffer (0.1M, pH 7.0) and 250 μl of 2% azocasein The reaction is carried out at 22 ° C for 60 minutes. After the reaction, 500 μl of 10% trichloroacetic acid was added and reacted at 22 ° C for 10 minutes. The reaction mixture was centrifuged (6000 × g) for 5 min and 100 μl of the supernatant was dispensed into 96-well plates containing 100 μl of NaOH (2N). The absorbance at 430 nm was measured using a microplate reader Were measured.

5) Disease resistance test for fish microbes and viruses through attack experiment

① Hospital strain

The bacterium and virus used in the offensive experiment are Streptococcus iniae bacteria, which is mainly developed in the flounder farms of Jeju Island, and viral hemorrhagic septicemia virus (VHS) virus, which has been suffering the most damage in recent flounder. Infectious symptoms of S. iniae can be seen in the cases of protrusion of the eye, clouding and redness, redness of the head and upper jaw, hemorrhage in the lower part of the gill lid, and mucus secretion in the gills and body surface. VSH virus is caused by pigmentation blackening, Hernias and other symptoms of the disease.

② Attack test method

After the end of the growth experiment, the experiment was carried out on blood samples and remaining fish in order to investigate the effect of Propolis on the flounder flounder. The VSH virus was prepared in the following manner. The CPE was confirmed by inoculating the EPC cell line with the filter cake of the spermatozoa and culturing at 15 ° C for 3 days. Eagle's minimum essential medium (EMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotic-antimycotic (Gibco BRL) was used for cell culture. The passage of the virus was carried out with low multiplicity of infection (MOI, <0.01), and for all experiments, the viral solution was used for less than 3 times. S. iniae The strain was cultured in TSA medium at 25 ° C for 24 hours and then suspended in PBS to 1 × 10 ⁸ cfu / ml. Infection method was performed by injecting 10 μl of intraperitoneal virus and strain of experimental fish using a syringe.

6) Statistical analysis

Completely randomized design was used for the placement of experimental diets, and growth and analysis results were analyzed by one-way ANOVA using SPSS (Version 12.0) program. The significant difference in the data values was compared with Turkey's test (P? 0.05). Data are expressed as means ± standard deviation (mean ± SD). Percentage data were calculated by arcsine strain value and analyzed statistically.

(3) Results

 Table 17 shows the results of growth investigated at the end of the breeding experiment. As a result, no significant difference was observed between 'P' and 'L' additions in all experimental groups. However, the final weights of the P0.5 and L0.25 and L0.5 experimental groups tended to be higher than those of the control, and the feed conversion efficiency, protein utilization efficiency and daily growth rate were similar. Survival rates were not significantly different in all experimental groups.

The hematological analysis results are shown in Table 18. Hemoglobin analysis showed that L0.25, L0.5 and L1.0 were significantly higher than those of control. Hematocrit was not significantly different among all experimental groups.

The nonspecific immunological analysis results are shown in Table 19. As a result, there was no significant difference in all experimental items. However, the L0.25 and L0.5 experimental groups showed a higher tendency to macrophage activity than the control, and the MPO activity was higher in the P0.75 experimental group than in the other experimental groups. Lysozyme and SOD activities were higher in the P0.5, P0.75 and L0.5 experimental groups than in the control, and the antiprotease activity was slightly higher in the propolis supplemented groups than the P0.5 experimental group Respectively.

4A, 4B, and 5 show the results of the attack test performed after the external symptoms and the breeding experiment of the experimental fish in the course of the attack experiment. As a result of the attack test on VHS virus, the lungs were observed from the 2nd day after the inoculation of the virus, and the survival rate of the Con group after 26 days was 48.9%. On the other hand, the survival rate of the other groups except the Con group was over 70%, among which the P0.75 group was the highest at 93.3%, followed by the P1.0 and L0.5 groups at 86.7% . As a result of the attack on Streptococcus iniae , the lungs were observed from the 2nd day after inoculation, and the survival rate of Con group was 24.4% after 24 days. On the other hand, the survival rate of the other groups except the Con group was over 60%, among which the P0.75 group was the highest at 80.0%, followed by the P0.5 and L0.5 groups at 73.3% and 68.9% Respectively. In both experiments, the P0.75 group with 0.75% of powder propolis showed the highest survival rate.

[Graph 5]

Graph 5. Cumulative mortality of olive flounder fed the eight experimental diets after challenge with VHS (A) and Streptococcus iniae (B) by injection.

All the results suggest that the addition of 0.5-0.75% propolis in the feed can improve the nonspecific immune response of the flounder and the resistance of the flounder to the virus and bacteria. Certain results on growth effects should be reevaluated based on long - term breeding experiments.

4) Review and Conclusion

As a result of all the analyzes, the addition of the powdered propolis byproduct or the purified propolis of the present invention in the feed did not significantly affect the growth and nonspecific immune activity of the flounder. However, as a result of the growth-related analysis, the addition of the appropriate content seems to have some growth effect. Growth was slightly decreased in the experimental group added with 1.0% high concentration of propolis byproduct and liquid propolis. These results suggest that when the breeding experiment is carried out for a long time, the low growth rate of the high concentration additive experimental group is predicted and it should be added below 1% in the flounder feed. Nonspecific immunological analysis showed that the immunoreactivity of P0.5, P0.75 and L0.5 was higher than that of the control. Especially, the survival rates of P0.5, P0.75 and L0.5 were higher than those of control.

The powdered propolis by-product obtained according to the present invention can be used in various fields because it can be supplied at a low cost while having the functionality of refined propolis such as excellent antibacterial and antioxidative action. Especially, It can be used as a functional additive for antibiotic applications.

Claims (12)

(a) Wheat flour on propolis byproducts; Starch; dextrin; Lactose; glucose; And at least one excipient selected from silicon dioxide to obtain a combination,
(b) vacuum drying or lyophilizing the compound obtained in the above step to obtain a dried product, and
(c) pulverizing the dried material obtained in the above step with a pulverizer while pulverizing the pulverized material while cooling the pulverized material using a pulverizer equipped with a cooling device to obtain a powder of 50 to 1,500 mesh, Powder. The method of claim 1, wherein 5 to 50% by weight of the propolis by-product is mixed with 50 to 95% by weight of an excipient. 2. The method of claim 1, wherein the cooling device comprises a cooling water line in contact with the crusher. The method of claim 1, wherein the powder has a size of 100-1,000 mesh. The method of claim 1, wherein the powder has a total flavonoid content of at least 0.3 wt%. The method according to any one of claims 1 to 5, further comprising adding an additive for at least one of nutritional fortification, odor masking, taste improvement, and physical properties with the excipient in the step of obtaining the combination Process for the production of powders of by-products. 7. The method of claim 6, wherein the additive is a fruit by-product or a sweetener. The method according to claim 7, wherein the fruit byproduct includes at least one of a shell, a seed, and a stem bearing a fruit selected from the group consisting of grape, persimmon, apple, pear, peach, kiwi and mandarin orange By weight of propolis by-products. The method of claim 7, wherein the sweetener comprises at least one of aspartame, fruit concentrate, stevioside, citric acid, and glucose. A powdered propolis by-product prepared by the method of any one of claims 1 to 5. A feed additive comprising the powdered propolis by-product of claim 10 as an active ingredient having antibacterial activity. 12. The feed additive of claim 11, wherein the animal is any one of cattle, pigs, chickens, ducks, and fish.

Images