KR102265268B1 - Feed composition for increasing immunity having grub as active component - Google Patents

Abstract

The present invention is slugs dry powder 30% by weight; 30% by weight of sweet potato net dry powder; 30% by weight of garlic peel dry powder; And it provides a livestock immunity enhancement feed composition comprising 10% by weight of tangerine peel dry powder. The feed composition according to the present invention as described above has an effect of improving immunity when ingested by pets or livestock, so that the ingested pet or livestock has strong resistance to various diseases such as bacterial diseases and viral diseases.

Description

Immunity-improving feed composition containing slugs as an active ingredient {Feed composition for increasing immunity having grub as active component}

The present invention relates to an immunity-improving feed composition containing slugs as an active ingredient to improve immunity of pets such as dogs and cats, or livestock such as chickens and pigs, thereby enabling strong breeding against bacterial and viral diseases.

Coronavirus (corona virus) is a virus belonging to the coronaviridae family, and diseases caused by coronavirus infection are very diverse. Coronaviruses are RNA viruses that cause respiratory, digestive, liver, and brain diseases in mammals and birds (Thomas M. et al. irology, 279(2): 371-374; Wege et al. (1982) Curr). Top. Microbiol. Immunol. 99: 165-200) It is a virus that mainly infects animals such as dogs, pigs, and cattle.

Coronavirus causes serious economic losses in the livestock industry. Another type of coronavirus, porcine epidemic diarrhea virus (PEDV), is a highly contagious viral disease that invades the gastrointestinal tract and causes dehydration due to vomiting and diarrhea. It is a virus that causes considerable economic loss due to its high mortality rate and high fever (Duarte M, Laude H (1994) Sequence of the spike protein of the porcine epidemic diarrhea virus. J Gen Virol. 75 (Pt 5): 1195-200) .

The swine transmissible gastroenteritis virus (TGEV), which has a high mortality rate in pigs, also belongs to coronaviruses. In addition, mouse coronavirus causes hepatitis, rat coronavirus causes severe respiratory diseases, and bovine coronavirus causes neurological diseases. It causes a variety of diseases in animals (Brian DA, Baric RS Curr. Top. Microbiol. Immunol. (2005). 287: 1-30).

As such, there is an urgent need to develop a feed composition capable of improving immunity against viruses that cause various diseases.

In addition, the overseas pet-related market continues to grow at an annual rate of 13% to a total of $60 billion (about KRW 60 trillion) in the United States, Japan, Europe, etc., and the domestic pet (companion) animal-related industry rapidly developed in the late 1990s. It has developed and is currently estimated to reach 1 trillion in the pet dog market, 5 million dogs, and 3 million dogs (including companion animals). Not only these companion animals, but also carnivorous insects such as arowanas, scorpions, tarantulas, beards, monitors, and geckos, as well as reptiles, rodents, birds and ornamental fish The population of breeders raising at home is increasing, and interest in improving the health of these animals is increasing. As the demand increases, the development of premium functional animal feed is required.

Accordingly, the present inventors can enhance immunity to pathogens while continuing research to develop a feed that can promote growth and improve immunity to pathogens during animal breeding, and can increase weight gain as well as pathogens The present invention was completed by discovering the surprising fact that mortality due to infection can be greatly reduced.

1. Republic of Korea Patent Publication No. 10-2002-0086814

Therefore, to solve the above problems, it contains to improve the immunity of pets such as dogs and cats, chickens, and livestock such as pigs, thereby providing an immunity-improving feed composition that enables strong breeding against bacterial and viral diseases.

In order to achieve the above object, the present invention
30% by weight of slug dry powder;
30% by weight of sweet potato net dry powder;
Garlic peel dry powder 30% by weight; and

It provides a livestock immunity enhancement feed composition comprising 10% by weight of tangerine peel dry powder.

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The feed composition according to the present invention as described above has an effect of improving immunity when ingested by pets or livestock, so that the ingested pet or livestock has strong resistance to various diseases such as bacterial diseases and viral diseases.

Hereinafter, the present invention will be described in detail.

the present invention

10 to 90% by weight of any one or more selected from the group consisting of slug dry powder, slug blood lymph, and slug fermented broth; and

It provides a livestock immunity-improving feed composition comprising 10 to 90% by weight of sweet potato net dry powder.

In another embodiment, the present invention

Garlic peel dry powder further comprising,

20 to 60% by weight of any one or more selected from the group consisting of the slug dry powder, slug blood lymph and slug fermented broth,

20 to 60% by weight of the sweet potato net dry powder,

It provides a livestock immunity enhancement feed composition comprising 20 to 60% by weight of the garlic peel dry powder.

In another embodiment, the present invention

The feed composition according to the present invention is

Eoseongcho dry powder, honeysuckle dry powder, Yeongyo dry powder, Centella asiatica dry powder, purslane dry powder, Pogongyeong dry powder, tangerine peel dry powder, and local herb dry powder further comprising any one or more herbal powders selected from the group consisting of,

In this case, 20 to 50% by weight of any one or more selected from the group consisting of the slug dry powder, slug blood lymph and slug fermented broth, 20 to 50% by weight of the sweet potato dry powder, 20 to 50% by weight of the garlic peel dry powder, the It provides a livestock immunity enhancement feed composition comprising 10 to 40% by weight of the herbal powder powder.

The powder in the present invention includes all of the powder prepared by drying itself (hot air drying, freeze-drying) and pulverizing the powder or the extract extracted using various extraction solvents to concentration and drying, as the extraction solvent, purified water or ethanol It is preferable to use

The slug used in the present invention has been used as a medicine for various diseases from oriental medicine for a long time, and is now widely bred in farms and used in oriental medicine or folk remedies, and is an insect of the order Coleopteran and Etonian, Protaetia. brevitarsis) larvae.

The general component of such slugs is very high about 58% of protein, and also contains a large amount of unsaturated fatty acids. In particular, oleic acid, which is known for its blood pressure lowering effect, accounts for 60% of the total fatty acids, and among minerals, phosphorus, potassium, and vitamins. Silver contains B3 and B9, which has the effect of removing toxins and smoothing blood circulation. Histidine, an essential amino acid for infancy that relieves severe allergy and helps blood formation, helps improve cognitive ability and improves memory. It contains a large amount of glutamic acid and proline, which helps to form cartilage by helping to form collagen.

In addition, slugs are a herbal medicine used as a treatment for liver disease in the private sector, and the liver protection effect of slugs has been scientifically verified in Korea. GOT, GPT, bile acid, and bilirubin levels were investigated for the hepatoprotective effect of Slugfish extract in acute hepatitis induced by carbon tetrachloride. It has been shown to inhibit liver fibrosis in mice with liver cirrhosis, and is expected to be effective as a therapeutic agent.

Such slugs can be used in the composition according to the present invention in powder form, and the powder manufacturing method can be used without any particular limitation as long as it is widely known in the art to which the present invention pertains. For example, a preferred method for manufacturing slug dry powder is Fasting the slugs before manufacturing, preferably for about 4 days to completely remove the wastes remaining in the intestines of the slugs, then boil water and blanch for about 5 to 20 seconds to remove the sterilization effect and bad smell on the surface of the slugs. It is manufactured by a method of drying at 60° C. (hot air drying, drying by a heater, freeze drying are all possible) and manufacturing a powder.

In the present invention, slug blood lymph collected from slugs can be used, and the blood lymph collecting method can be used without particular limitation as long as it is widely known in the art to which the present invention pertains. After freezing and thawing, it is installed in a centrifuge, and after centrifugation, the supernatant is taken to collect the blood lymph from the slugs.

In addition, in the present invention, liquid hemolymph can be prepared and used as a powder. In this case, there are various methods, but the present invention includes a hemolymph powder manufacturing step of lyophilizing the collected hemolymph to produce a powder, which is freeze-dried. This is because when a porous amorphous powder is prepared and dissolved again in a liquid phase, it can be easily dissolved, and it can be used for various purposes by being prepared as a powder having an appropriate volume. Here, the freeze-drying method can be used without particular limitation as long as it is widely known in the art to which the present invention pertains.

Another use form of slugs applicable in the present invention is to put slugs or pulverized slugs in water and Bacillus subtilis var. natto strain, Bacillus subtilis strain and Aspergillus kawachii strain is to use any one or more strains selected from the group consisting of inoculated and fermented fermentation broth.

The above-mentioned fermentation broth consists of an oil phase in the upper layer containing fatty acid oil and an aqueous phase in the lower layer containing water-soluble peptides, which are used together in the present invention, and may be used separately or by adjusting the amount if necessary. .

The slugs used in the fermentation can be used without any particular limitation, whether they are live, dead or dry (the use of water is to sufficiently moisten the slugs so that the fermentation can be performed efficiently, and the amount of water is sufficient for the slugs to You can put it so that it can be locked, and there is no special limitation.

The fermentation temperature for the fermentation may be any suitable temperature to proceed with the fermentation, preferably, the fermentation is performed at room temperature, and the fermentation time is not particularly limited, but is about 5 to 50 hours. Since the fermentation in the present invention can be over-fermented, the inoculation amount of the strain may be inoculated with a sufficient amount to be fermented, but in consideration of efficiency, it is preferable to set it to about 2 to 15% by weight relative to the weight of the used slugs.

When the fermentation according to the present invention proceeds, the oil phase gradually elutes and separates toward the upper layer as the fermentation proceeds, and the lower layer undergoes fermentation of the slugs, disappears and dissolves in the aqueous phase. Therefore, the end of fermentation can be done until the time when the shape of the slugs disappears. In the present invention, as a fermentation strain, Bacillus subtilis var. One or more strains selected from the group consisting of natto strains, Bacillus subtilis strains and Aspergillus kawachii strains were selected. As a result of testing with various strains, the strain most suitable for the purpose of the present invention was selected.

The composition according to the present invention comprises sweet potato net dry powder and garlic peel dry powder. The drying method, extraction method, and concentration method for producing the pure sweet potato pure dry powder and garlic peel dry powder can be used without particular limitation as long as they are widely known in the art.

The sweet potato has a scientific name of Ipomoea batatas, and is a herbaceous perennial plant belonging to the genus Convolvulaceae and sweet potato, and sweet potato shoots are commonly used for food as stems of sweet potatoes. The origin of sweet potato is Central America, and it spread to Europe and spread all over the world due to Columbus's arrival in the Americas. According to the data reported so far, sweet potatoes are one of the representative alkaline foods that contain carbohydrates, crude fiber, calcium, potassium, phosphorus, beta-carotene, a precursor of vitamin A, and vitamin C, and contain a small amount of fat, vitamin B2 and antioxidant activity. It is known to contain chlorogenic acid, which is a polyphenol compound shown. Studies on the pharmacological effects of sweet potatoes have mainly focused on anticancer, antioxidant, and blood cholesterol lowering effects.

Garlic (Garlic; Allium sativum L) is a perennial herbaceous plant belonging to the Liliaceae family. It has a unique and strong taste that improves the taste of food, removes fishy smell, and improves appetite, so it is a very important spice raw material. Agricultural products are part of our daily life. Garlic contains polyphenols and flavonoids, and is known to have several useful effects on the human body when ingested. Korea has the second highest garlic production after China and India, and the area and production of garlic have been continuously increasing since 2010. According to the Food Supply and Demand Table (2013) of the Korea Rural Economic Research Institute, garlic tends to be consumed after the primary processing process, and 45,000 tons of garlic processing by-products such as stems, skins and roots are generated annually. Only a small part of it is used as compost and most of it is discarded as it is. Garlic peel contains about 7 times more phenolic components and flavonoid aglycone than garlic meat, which is an edible part. In particular, the phenol component is known to have high antioxidant and antibacterial effects.

In addition, the feed composition according to the present invention may further include various herbal powders exhibiting functionality by being used as an active ingredient in feed, and preferred examples thereof include, for example, dried sagebrush dry powder, dry honeysuckle powder, dry liana dry powder, and dried Centella asiatica powder. , purslane dry powder, pogongyeong dry powder, tangerine peel dry powder, and local herb dry powder may be mentioned at least one herbal powder selected from the group, and more preferably, tangerine peel powder.

The tangerine peel (橘皮, citrus peel) is a ripe fruit peel of a citrus tree belonging to the citrus (genus Citrus) of the subfamily of citrus fruits and dicotyledonous plants. The dried tangerine peel is called dermis and is used as a herbal medicine. The dermis has a beneficial effect in treating nausea and flatulence, indigestion, loss of appetite, and vomiting. It is also used as an antiperspirant, antibacterial agent, and expectorant, in addition to being an air freshener and a digestive agent (健胃劑). In modern medicine, it is used for gastritis and indigestion as an aromatic phlegm, and is used as a cough or sputum medicine. Tangerine peel contains abundant citric acid, vitamin C and vitamin A, and rutine, deosmine, hesperidin, naringin, which strengthen capillaries, antioxidant and free radical scavenging, and anti-aging, prevent cancer cell invasion and metastasis It contains a number of functional substances, such as tangeretin, nobiletin, coumarin, which detoxifies carcinogens, carotenoid, which acts as an antioxidant, and limonene, which is a kind of terpene that acts as an excitatory and sedative of the central nervous system. is known As for the research report on tangerine peel, ingredients, functionality and bread using it (Kwon Soo-kyung et al. 2002, quality characteristics of bread with tangerine peel water homogenate added. Journal of the East Asian Dietetic Society, 12, 397; Lee Eun-jin et al. 2012 tangerine peel powder added There is something about the quality characteristics of bread, Journal of the Korean Culinary Society, 18, 27).

When such herbal powder is further included, the immune-enhancing effect is strengthened, and thus an excellent effect can be achieved for chronic inflammatory diseases of pets or livestock.

The content range of the above-described components is determined in an optimal form through various tests.

Hereinafter, the present invention will be described in detail through Examples and Test Examples. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more clearly explain the present invention to those of ordinary skill in the art.

<Example 1: Preparation of composition>

Each component according to Table 1 was mixed to prepare a composition according to the present invention.

composition 1
(weight%) composition 2
(weight%) composition 3
(weight%) slug dry powder 50 slug fermentation broth 50 slug blood lymph 50 Sweet Potato Net Dry Powder 50 50 50

The dried powders were dried by hot air to obtain a moisture content of about 5% by weight and then pulverized powder was used.

The slug fermented broth is a dried slug, and as a fermentation strain, Bacillus subtilis var. The natto was added in an amount of about 3% by weight based on the weight of the dried slugs, and the fermentation was carried out for 20 hours while maintaining at room temperature, and the liquid obtained after the slugs disappeared.

For the slug blood lymph, the slugs are thoroughly washed with purified water, then the surface is dehydrated, and a hole is made in the slug using a needle, and then 30 g of the slug is put in a tube for centrifugation with a diameter of 10 mm. After putting it back in a 15 mm centrifuge tube, it was created in a centrifuge and centrifuged at about 1000 rpm for 1 minute, and then the supernatant was used.

< test example 1: Confirmation of the safety of the composition>

In order to confirm the stability of the composition as an additive in the weaning feed after weaning, 70 piglets (3 weeks old) immediately after weaning were classified into 7 groups of 10 each. Seven groups were determined as a control group without the composition added, compositions 1, 2, and 3 added by 7% by weight, respectively, and the result of measuring the mortality rate by conducting a test with free feeding for 3 weeks was the composition. After 1 death was recorded in the first week of the test in the control group without addition, no additional mortality occurred in all groups.

< test example 2: Measurement of absorption rate when administered in combination with feed and additives>

Digestive absorption was measured for the group administered with the composition of the present invention of Test Example 1.

As a result, the digestibility and absorption rate of the general feed group showed a high absorption rate close to about 72%, and the average of about 81% in the feed group containing the composition according to the present invention showed a better absorption rate than that of the general feed group. observed.

< test example 3: Composition of phagocytes (macrophage) in gluttony Impact>

Using the composition of Example 1, the change in the number of phagocytic cells and the phagocytic ability of the phagocytic cells were measured. The amount of the composition used in the test was 50 mg, and only PBS was administered as a control. The mice used in the test were grouped with 10 mice each, and 5-week-old magnetic ICR mice (purchased from Orient Co., Ltd.) were used.

① Change in the number of phagocytic cells according to the composition treatment

After oral administration for 10 days for Compositions 1, 2, 3 and the control group, the number of phagocytic cells was collected and the number of phagocytic cells was measured. As a result, in the group treated with Compositions 1, 2, and 3, the number of phagocytic cells was the control group over time. showed an increase of about 15% compared to

When the composition was orally administered, it could be confirmed that the composition had a positive effect on the primary immune response from the results showing a tendency to increase the number of phagocytic cells mediating the primary immune response.

② of phagocytic cells gluttony ( phagocytosis Effect of composition on efficiency)

^{Phagocytosis of 1×10 5} phagocytic cells isolated from each treatment group was measured using a phagocytosis assay kit manufactured by molecular probe.

First, the separated phagocytic cells are laid out on a plate and sonicated E coli marked with fluorescence that enables the phagocytic ability to be checked under a microscope is prepared. After culturing the phagocytic cells in a 96-well plate, the E coli solution Put 100 μl, incubate for 2 hours, discard all the culture medium, add 100 μl of standard cell counting solution (trypan blue), incubate for 1 minute, remove the standard cell counting solution, and measure absorbance at a wavelength of 495 nm .

As a result, all composition-treated groups showed about 13% higher phagocytic activity than the control group.

These results mean that when the composition is orally administered, not only the quantitative increase of phagocytic cells but also the qualitative phagocytic ability is improved.

< test example 4: Analysis of the effect of the composition on the blood immunoglobulin G secretion>

Compositions 1, 2, and 3 were orally administered to each of 10 5-week-old magnetic ICR mice (purchased from Orient Co., Ltd.) for 21 days, and as a control, 10 untreated mice were used as a control to obtain blood immunoglobulin (immunoglobulin) was compared and measured.

As a measurement method, first put the mouse blood into a heparin tube, centrifuge at 1600 rpm for 10 minutes to separate the serum. After coating an IgG protein in a 96-well plate, block with blocking buffer and wash 3 times, The separated serum is added and incubated for 1 hour.

Again, after washing 5 times, add an antibody conjugated with Horse Radish Peroxide (HRP) and react for 1 hour. Add a substrate that reacts with HRP, react in the dark for 30 minutes, dissolve with 2M sulfuric acid, and absorb absorbance at a wavelength of 450 nm measure

The significance of all result values obtained from the test was verified using the 'one way ANOVA' statistical method for the result values of each treatment group.

In the case of the control group, the concentration of IgG in blood did not change significantly over time, however, the composition-treated group showed a sharp increase from the 4th day, and thereafter, the increased amount was maintained.

When the composition was administered, it was confirmed that the administered composition directly stimulates the secretion of immunoglobulin or promotes the secretion of a factor that promotes the secretion of immunoglobulin by showing a rapid rise in blood IgG, and it was confirmed that the immune system could be improved by proper administration of the composition. could

< test example 5: Evaluation of survival rate according to Salmonella infection>

A 5-week-old magnetic ICR mouse (purchased from Orient Co., Ltd.) was used. After a one-week adaptation period, group separation was performed by randomization.

During the adaptation period and the test period, the breeding environment was set at a temperature of 23±2℃, relative humidity of 55±5%, illumination time of 12 hours, and illuminance of 150~300Lux, and the supplied sterilized solid feed for test animals (Orient Bio Co., Ltd.) (purchased from) and water were provided as free intake conditions.

After completion of the adaptation period, Salmonella (S typhimurium) was orally administered as shown in Table 2 below after ^{intraperitoneal injection (1×10 7 CFUm/ice).} composition was included.

group
treatment head number
Salmonella (S typhimurium) matter I 1×10 ⁷ CFUm/ice solid feed 10 II 1×10 ⁷ CFUm/ice Solid feed + composition 1 10 Ⅲ 1×10 ⁷ CFUm/ice Solid feed + composition 2 10 IV 1×10 ⁷ CFUm/ice Solid feed + composition 3 10

As a result of confirming the survival rate of the test animals, in group I, 5 animals died on the 1st day of infection, 4 animals on the 3rd day, and on the 7th day on the end of the test, all died. On the other hand, in the group administered with the composition according to the present invention, 2 to 3 animals died on the second day of infection, and all survived thereafter. The results are shown in Table 3.

group
Dead (number of horses) 1 day 2 days 3 days 7 days I 5 4 One II 0 3 0 0 Ⅲ 0 2 0 0 IV 0 2 0 0

As can be seen from Table 3, it was confirmed that the composition according to the present invention is very useful for enhancing immunity.

< test example 6: Dog breeding>

As test animals, 32 healthy pet dogs aged 2 to 3 years were divided into 4 groups using 700±20 g of Paknaise, respectively, and 8 animals in group I were allowed to ingest the existing feed (ANF holistic) as a control group, and the rest of the group Each of the 8 animals was mixed with the feed prepared in Example 1 and the conventional feeding practice with a content of 7% by weight. In addition, it was bred in a cage measuring 50cm in width, 50cm in length, and 30cm in height, and did not exercise. The feeding method was 50g±50g free feeding twice a day for 30 days, and the results are shown in Table 4.

group feed intake
(%< days) weight of steam
(%) Cough (cold) (%) diarrhea and excretion
(%) aspect of hair excrement
odor level I (control) 92±4% 54±2% 12±2% 52±2% usually strong Ⅱ (Composition 1) 93±3% 56±2% 0 0 burnish weakness Ⅲ (Composition 2) 93±3% 56±2% 0 0 burnish weakness Ⅳ (Composition 3) 93±3% 56±2% 0 0 burnish weakness

Looking at the results of Table 4, the feed according to the present invention was smoothly fed, and no specific pathogenic symptoms appeared during feeding, in particular, there was no death of young test animals, there was no dog with diarrhea (feces), and there was no feces. It was found that the odor was significantly reduced.

< test example 7: porcine epidemic diarrhea virus (PEDV)>

One-week-old pigs were purchased from a place where there was no PEDV vaccine vaccination and no recent viral diarrhea. Pigs without disease and seemingly healthy were classified as having similar weight averages, and infant formula was fed. In addition, in the PED virus culture, PEDV was cultured as Vero cells ^{and inoculated 3 ml each at a titer of 10 5} TCID ₅₀ /ml, and then orally administered to the animals of the test group as shown in Table 5 below. , The control group included only powdered milk, and the test group included a composition of 7% by weight of the total weight of the powdered milk.

group
treatment head number
PEDV matter I 3ml Powdered milk 3 II 3ml Milk powder + composition 1 3 Ⅲ 3ml Milk powder + composition 2 3 IV 3ml Milk powder + composition 3 3

During the course of the test, the status of feces was observed every day, the fecal score of each individual was recorded, and the mean and standard deviation of each group were calculated. Fecal score was "3; normal, 2; soft stool, 1; diarrhea, 0; death". Table 5 shows the mean and standard deviation of the fecal score of each group after PEDV inoculation.

After PEDV inoculation, subjects in group I showed severe diarrhea. On the other hand, the fecal score of the group administered with the composition according to the present invention on the 7th day after PEDV inoculation was significantly higher than that of group I (p<0.05). Therefore, it was confirmed that the extract of the present invention relieves diarrhea caused by PEDV.

group
evaluation time 1 day 2 days 3 days 4 days 5 days 6 days 7 days I 2.0 1.3 1.5 1.6 1.6 1.3 1.0 II 2.0 1.6 2.0 2.3 2.3 2.5 2.6 Ⅲ 2.0 1.6 2.1 2.2 2.3 2.5 2.7 IV 2.0 1.6 2.0 2.3 2.3 2.5 2.6

In addition, after euthanizing each individual, the small intestine is collected and dissected, fixed in 10% formalin, undergoes the usual process for pathological tissue production, and each tissue has a hematoxylin & eosin stained slide. Histopathological examination was performed to determine whether the small intestine villi were atrophy.

The atrophy of the small intestine villi was compared by obtaining the vili:crypt ratio. As a result, the ratio of the villi:crypt ratio of the group administered with the extract according to the present invention was significantly higher than that of group I.

Therefore, it was confirmed that the composition of the present invention alleviates small intestine damage caused by PEDV.

< Example 2: Preparation of composition>

Each component according to Table 7 was mixed to prepare a composition according to the present invention.

composition 4
(weight%) composition 5
(weight%) slug dry powder 35 30 Sweet Potato Net Dry Powder 35 30 Garlic Peel Dry Powder 30 30 Tangerine Peel Dry Powder 10

The dried powders were dried by hot air to obtain a moisture content of about 5% by weight and then pulverized powder was used.

< test example 8: Confirmation of the safety of the composition>

In order to confirm the stability of the composition as an additive to weaning feed after weaning, 50 piglets (3 weeks old) immediately after weaning were grouped into 5 groups of 10 each. Five groups were determined as a control group to which no composition was added, and a group to which 7 wt% of each of compositions 4 and 5 were added, and the result of measuring the mortality rate by conducting a test with free feeding for 3 weeks was that the composition was not added. After 1 death was recorded in the control group at the first week of the trial, no additional mortality occurred in any group.

< test example 9: Measurement of absorption rate when administered in combination with feed and additives>

Digestive absorption was measured for the administration group of the composition of the present invention of Test Example 9.

As a result, the digestibility and absorption rate of the general feed group showed a high absorption rate close to about 72%, and the average of about 88% in the feed group containing the composition according to the present invention, showing a better absorption rate than that of the general feed group It was observed that this was better than the composition of Example 1.

< test example 10: composition of phagocytes (macrophage) in gluttony Impact>

Using the composition of Example 2, the change in the number of phagocytic cells and the phagocytic ability of the phagocytic cells were measured. The amount of the composition used in the test was 50 mg, and only PBS was administered as a control. The mice used in the test were grouped with 10 mice each, and 5-week-old magnetic ICR mice (purchased from Orient Co., Ltd.) were used.

① Change in the number of phagocytic cells according to the composition treatment

After oral administration for 10 days to Compositions 4 and 5 and the control group, the number of phagocytic cells was collected and the number of phagocytic cells was measured. As a result, the number of phagocytic cells in the group treated with Compositions 4 and 5 was about less than that of the control group over time. showed a 20% increase.

When the composition was orally administered, it was confirmed that the composition had a positive effect on the primary immune response from the results showing the tendency to increase the number of phagocytic cells mediating the primary immune response, which is more This is an improved result.

② of phagocytic cells gluttony ( phagocytosis Effect of composition on efficiency)

^{Phagocytosis of 1×10 5} phagocytic cells isolated from each treatment group was measured using a phagocytosis assay kit manufactured by molecular probe.

First, the separated phagocytic cells are laid out on a plate and sonicated E coli marked with fluorescence that enables the phagocytic ability to be checked under a microscope is prepared. After culturing the phagocytic cells in a 96-well plate, the E coli solution Put 100 μl, incubate for 2 hours, discard all the culture medium, add 100 μl of standard cell counting solution (trypan blue), incubate for 1 minute, remove the standard cell counting solution, and measure absorbance at a wavelength of 495 nm .

As a result, all composition treatment groups showed about 18% higher phagocytic activity than the control group.

These results mean that when the composition is orally administered, not only the quantitative increase of phagocytic cells but also the qualitative phagocytic ability is improved.

< test example 11: Analysis of the effect of the composition on blood immunoglobulin G secretion>

Compositions 4 and 5 were orally administered to each of 10 5-week-old magnetic ICR mice (purchased from Orient Co., Ltd.) for 21 days, and 10 mice that were not treated with anything as a control group were used as a blood immunoglobulin (immunoglobulin). ) was compared and measured.

As a measurement method, first put the mouse blood into a heparin tube, centrifuge at 1600 rpm for 10 minutes to separate the serum. After coating an IgG protein on a 96-well plate, block with blocking buffer and wash 3 times, The separated serum is added and incubated for 1 hour.

Again, after washing 5 times, add an antibody conjugated with Horse Radish Peroxide (HRP) and react for 1 hour. Add a substrate that reacts with HRP, react in the dark for 30 minutes, dissolve with 2M sulfuric acid, and absorb absorbance at a wavelength of 450 nm measure

The significance of all result values obtained from the test was verified using the 'one way ANOVA' statistical method for the result values of each treatment group.

In the case of the control group, the concentration of IgG in blood did not change significantly over time, but the composition-treated group showed a sharp increase from the second day, and the increased amount was maintained thereafter.

When the composition was administered, it was confirmed that the administered composition directly stimulates the secretion of immunoglobulin or promotes the secretion of a factor that promotes the secretion of immunoglobulin by showing a rapid rise in blood IgG, and it was confirmed that the immune system could be improved by proper administration of the composition. It was determined that when the composition of Example 2 was used, a rapid effect could be exerted because the time of rise of IgG in the blood was faster than the composition of Example 2 compared to the composition of Example 1.

< test example 12: Evaluation of survival rate according to Salmonella infection>

A 5-week-old magnetic ICR mouse (purchased from Orient Co., Ltd.) was used. After a one-week adaptation period, group separation was performed by randomization.

During the adaptation period and the test period, the breeding environment was set at a temperature of 23±2℃, relative humidity of 55±5%, illumination time of 12 hours, and illuminance of 150~300Lux, and the supplied sterilized solid feed for test animals (Orient Bio Co., Ltd.) (purchased from) and water were provided as free intake conditions.

After completion of the adaptation period, Salmonella (S typhimurium) was orally administered as shown in Table 8 after ^{intraperitoneal injection (1×10 7 CFUm/ice).} composition was included.

group
treatment head number
Salmonella (S typhimurium) matter I 1×10 ⁷ CFUm/ice solid feed 10 II 1×10 ⁷ CFUm/ice Solid feed + composition 4 10 Ⅲ 1×10 ⁷ CFUm/ice Solid feed + composition 5 10

As a result of confirming the survival rate of the test animals, in group I, 5 animals died on the 1st day of infection, 4 animals on the 3rd day, and on the 7th day on the end of the test, all died. On the other hand, in the group administered with the composition according to the present invention, one animal died on the second day of infection, and all survived thereafter. The results are shown in Table 9.

group
Dead (number of horses) 1 day 2 days 3 days 7 days I 5 4 One II 0 One 0 0 Ⅲ 0 One 0 0

As can be seen from Table 9, the composition of Example 2 was found to be very useful for enhancing immunity, which was a more synergistic effect than the composition of Example 1.

< test example 13: Dog breeding>

As test animals, 16 healthy pet dogs aged 2-3 years were divided into 3 groups using 700±20 g of Paknaise, respectively, and 8 animals in group I were allowed to consume the existing feed (ANF holistic) as a control, and the rest of the group Each of the 8 animals was mixed with the feed prepared in Example 2 and the conventional feeding practice with a content of 7% by weight. In addition, it was bred in a cage measuring 50cm in width, 50cm in length, and 30cm in height, and did not exercise. The feeding method was 50g±50g daily for 30 days, twice a day, and the results are shown in Table 10.

group feed intake
(%< days) weight of steam
(%) Cough (cold) (%) diarrhea and excretion
(%) aspect of hair excrement
odor level I (control) 92±4% 54±2% 12±2% 52±2% usually strong Ⅱ (Composition 4) 94±3% 57±2% 0 0 burnish weakness Ⅲ (Composition 5) 94±3% 57±2% 0 0 burnish weakness

Looking at the results of Table 10, the feed according to the present invention was smoothly fed, and no pathogenic symptoms appeared during feeding, there was no death of young test animals in particular, there was no dog with diarrhea (feces), and there was no feces. It was found that the odor was significantly reduced. In addition, it showed an improved effect than the composition of Example 1.

< test example 14: porcine epidemic diarrhea virus (PEDV)>

One-week-old pigs were purchased from a place where there was no PEDV vaccine vaccination and no recent viral diarrhea. Pigs without disease and seemingly healthy were classified as having similar weight averages, and infant formula was fed. In addition, for PED virus culture, PEDV was cultured as Vero cells ^{and inoculated 3 ml each at a titer of 10 5} TCID ₅₀ /ml, and then orally administered to the animals of the test group as shown in Table 11 below. , The control group included only powdered milk, and the test group included a composition of 7% by weight of the total weight of the powdered milk.

group
treatment head number
PEDV matter I 3ml Powdered milk 3 II 3ml Milk powder + composition 4 3 Ⅲ 3ml Milk powder + composition 5 3

During the course of the test, the status of feces was observed every day, the fecal score of each individual was recorded, and the mean and standard deviation of each group were calculated. Fecal score was "3; normal, 2; soft stool, 1; diarrhea, 0; death". Table 10 shows the mean and standard deviation of the fecal score of each group after PEDV inoculation. After PEDV inoculation, subjects in group I showed severe diarrhea. On the other hand, the fecal score of the group administered with the composition according to the present invention on the 7th day after PEDV inoculation was significantly higher than that of group I (p<0.05). Therefore, it was confirmed that the extract of the present invention relieves diarrhea caused by PEDV.

group
evaluation time 1 day 2 days 3 days 4 days 5 days 6 days 7 days I 2.0 1.3 1.5 1.6 1.6 1.3 1.0 II 2.0 1.9 2.4 2.7 2.8 2.9 3.0 Ⅲ 2.0 1.9 2.4 2.8 2.8 2.9 3.0

In addition, after euthanizing each individual, the small intestine is collected and dissected, fixed in 10% formalin, undergoes the usual process for pathological tissue production, and each tissue has a hematoxylin & eosin stained slide. Histopathological examination was performed to determine whether the small intestine villi were atrophy.

The atrophy of the small intestine villi was compared by obtaining the vili:crypt ratio. As a result, the ratio of the villi:crypt ratio of the group administered with the extract according to the present invention was significantly higher than that of group I.

Therefore, it was confirmed that the composition of the present invention alleviates small intestine damage caused by PEDV.

The feed composition according to the present invention as described above has an effect of improving immunity when ingested by pets or livestock, so that the ingested pet or livestock has strong resistance to various diseases such as bacterial diseases and viral diseases.

Claims (5)

30% by weight of slug dry powder;
30% by weight of sweet potato net dry powder;
30% by weight of garlic peel dry powder; and
Livestock immunity enhancement feed composition, characterized in that it consists of 10% by weight of tangerine peel dry powder.


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