KR101161251B1 - Feed additive including resveratrol from mulberry stem as a antibiotic alternative and its feeding method - Google Patents

Abstract

The present invention relates to a growth-promoting antibiotic replacement feed additive using a mulberry branch-derived resveratrol having excellent physiological activity effect, a feed composition comprising the same, and a feeding method thereof. The resveratrol-containing feed additive according to the present invention increases the carcass rate of broilers when fed to feed, improves immune control function and antioxidant activity, and can be fed to the living body in a stable manner, which is essential for livestock replacement. Satisfy the physiological efficacy.

Broiler, Resveratrol, Productivity, Biostability, Blood Antioxidant Activity, Immune Regulation

Description

Feed additives, feed composition and feed method containing resveratrol from mulberry branch with antibiotic replacement effect {Feed additive including resveratrol from mulberry stem as a antibiotic alternative and its feeding method}

The present invention relates to a growth-promoting antibiotic replacement feed additive using a mulberry branch resveratrol excellent in physiological activity effect, a feed composition comprising the same, and a feeding method thereof.

In a paper published by Moore et al. In the 1940s, it was reported that the addition of the antibiotic streptomycin to animal feed improved the growth rate by 10 to 30%. Effective, its use has steadily increased worldwide. In 2000, the amount of antibiotics used in animals reached 12,000 tonnes for feed and 900 tonnes for disease treatment (Doyle, 2001). Here we need to note that more antibiotics are used to promote growth than to treat diseases.

The use of antibiotics works well, but there are also problems. This is a problem caused by antibiotic resistance (내성 性). In the 1960s, Swann raised the dangers of this problem and published the hazards that humanity may face. Due to these problems, the number of antibiotics that can be used as feed additives in the world has greatly decreased in recent years. The list of available antibiotics is expected to continue to decline in the future, and situations such as the total ban on antibiotic use cannot be excluded.

However, the use of antibiotics has been reported to increase the mortality rate by lowering livestock productivity and increasing disease rate. In Europe (especially Denmark), the use of antibiotics as growth promoters has been limited for the past few years. As a result, the number of antibiotics used to treat diseases was significantly increased, although it was greatly reduced for feed addition.

As a result, in the case of large-scale intensive livestock raising conditions, the high incidence of disease means that antibiotics added in small amounts to feed are very effective, and more aggressively, it is difficult to raise livestock economically without adding antibiotics. can do.

The use of growth-promoting antibiotics in livestock feed is aimed at maximizing livestock productivity, preventing high-density breeding and disease outbreaks due to poor breeding conditions. However, as problems such as antibiotic residue in livestock products and emergence of resistant bacteria have emerged, as the competitiveness of imported livestock products and consumer demand for safe livestock have increased, interest in non-antibiotic livestock products has increased. However, as described above, there is a problem of lowering livestock productivity and increasing disease occurrence during non-antibiotic breeding, and therefore countermeasures are urgently needed. In addition, the importance of the role of the livestock industry in the environmental aspects is emphasized not only for the sustainable operation of livestock, but also for the preservation of the environment and agricultural ecosystem. Fostering natural circulation agriculture and developing high value-added resources of by-products through the development of technology for utilizing by-products of agricultural and livestock products is one of the tasks to be preceded for green growth and eco-friendly livestock development.

The mulberry tree used for feeding silkworms in the sleep business contains various kinds of bioactive substances, but the remaining mulberry leaves and mulberry branches that have been used as food are discarded without being properly utilized. Mulberry leaves and mulberry branches, which are by-products of the latent business, have various useful substances such as 1-deoxynojirimycin, t-aminobutyric acid, and rutin, which are rich in their usefulness, but are mostly discarded. As a bioactive substance that shows efficacy such as anti-viral, nerve stabilization, and anti-aging, livestock can ingest it to promote health by promoting metabolism, strengthening immunity, etc., and improving productivity through improving appetite and improving digestion rate. You can. However, it is difficult to apply expensive resveratrol in actual livestock farms.

The purpose of the present invention is to establish the extraction and formulation technology of resveratrol from the mulberry leaves and mulberry branches, which are by-products of the latent business, and to achieve the effect of replacing the antibiotics for feed by adding and feeding the developed resveratrol-containing feed composition to broiler feed.

In order to achieve the above object, the present invention provides a method for preparing a feed additive comprising the following process.

According to one aspect of the invention,

Pressing and drying the mulberry branches;

Grinding the compressed mulberry leaves and mulberry branches to obtain solids;

Dissolving the obtained solid content in an 1: 1 weight ratio with an organic solvent, stirring and filtering to obtain an extract;

Removing the organic solvent from the extract and obtaining a solid; And

The method of preparing a feed additive comprising the step of completing the powder formulation so that the resveratrol content is 20 ~ 400ppm by adding silica, powder and soda ash to the obtained solid content.

In one embodiment, the organic solvent for dissolving the obtained extracted solid may be ethanol.

In one embodiment, the stirring may be performed at 200 to 400 rpm for 20 to 40 minutes at 35 to 45 ℃.

In one embodiment, the powder formulation may be added in a ratio of 0.7 ~ 1.0% by weight, mulberry extract from 35 to 45% by weight, 35 to 45% by weight of powder and 10 to 20% by weight of soda ash.

In addition, it is possible to provide a feed additive prepared according to the above method.

According to another aspect of the present invention, it is possible to provide a feed composition for replacing antibiotics with high immunity and productivity by adding the feed additive to 5 to 50% by weight of the total feed composition.

According to another aspect of the present invention, there can be provided a method of feeding a feed composition to enhance the immunity and productivity of the livestock, characterized in that to feed the antibiotic replacement feed composition excellent in the immune boosting and productivity.

The resveratrol-containing feed additive according to the present invention increases the carcass rate of broilers when fed to feed, improves immune control function and antioxidant activity, and can be fed to the living body in a stable manner, which is essential for livestock replacement. Satisfy the physiological efficacy.

Hereinafter, the content of the present invention will be described in more detail.

Feed additives used in the present invention means processed and formulated materials other than the material of the general feed for addition to the feed composition. In the present invention, it refers to a state in which the mulberry extract is processed and powdered by mixing silica, powder, soda ash and the like.

Feed composition refers to the final mixture that feed additives are added to a common feed to feed for livestock. In the present invention, it means a state in which the feed additive is mixed in a predetermined ratio to the general feed.

The present invention comprises the steps of pressing and drying the mulberry leaves and mulberry branches; Grinding the compressed mulberry leaves and mulberry branches to obtain solids; Dissolving the obtained solid content in an 1: 1 weight ratio with an organic solvent, stirring and filtering to obtain an extract; Removing the organic solvent from the extract and obtaining a solid; And it provides a method of producing a feed additive comprising the step of completing the powder formulation by adding silica and powder and soda ash to the obtained solid content.

Mulberry leaves and mulberry branches are crushed and immersed in ethanol in the state of blocking light and extracted at room temperature. Blocking the light is to suppress the photolysis of resveratrol contained in the mulberry leaves and mulberry branches to the maximum, and performing at room temperature is to prevent the degradation or precipitation of resveratrol that may occur in other high or low temperature conditions. The extract thus extracted may be concentrated by filtration under reduced pressure and then formulated through lyophilization.

At this time, it is preferable to stir at 200 to 400 rpm for 20 to 40 minutes at 35 to 45 ° C. for extraction of resveratrol. At temperatures higher than 45 ° C., the solvent may evaporate or the resveratrol may be unstable. At low temperatures, extraction of resveratrol may not occur sufficiently, which is undesirable. Stirring time and stirring speed is sufficient if the resveratrol can be extracted about 5mg per kg of mulberry leaves and mulberry branches in a stable state, if excessive stirring for longer than 40 minutes may cause problems in the stability of resveratrol, 400rpm When stirring at a high speed, the vibration of the solvent is too severe, the yield may be rather deteriorated, which is not preferable.

According to one embodiment of the present invention, the powder formulation may be prepared by adding a mulberry branch extract solid content of 0.7 to 1.0% by weight, silica 35 to 45% by weight, powder 35 to 45% by weight and soda ash 10 to 20% by weight. At this time, if the content of the mulberry eggplant extract solid content exceeds 1.0% by weight exceeds the content necessary for enhancing broiler productivity and immunity is not desirable in terms of economics, when added in less than 0.7% by weight is best in terms of productivity and immunity It is also undesirable to have no effect. Silica, powder, and soda ash are most preferably formulated at the above ratios. When the content of silica and powder exceeds 45% by weight, foreign substances increase in feed intake, which is undesirable, and in the case of soda ash, exceeding 20% by weight. It is not desirable to have a side effect of reducing feed intake due to off-flavor.

According to one embodiment of the present invention, a feed composition for improving antibiotics and an excellent antibiotic boosting agent having a feed additive added to 0.5 to 1.0% by weight of the total feed composition may be provided. As described above, the feed additive is 1.0 weight. If it exceeds the percentage, the resveratrol is contained more than necessary for productivity and immunity enhancement, which is not economically desirable.

The feed composition comprising the feed additive of the present invention can be used as an excellent antibiotic substitute in terms of immunological enhancement and productivity, and by administering a feed composition for replacing antibiotics with high immunity and productivity to livestock, it improves immunity and improves productivity of livestock. You can.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example  One. Resveratrol  Feed additive manufacture

1 kg of dried mulberry branches were crushed, and 1,000 ml of ethanol were added thereto, followed by light blocking for 72 hours, immersion, extraction at room temperature, and the extract was filtered under reduced pressure, and the residue was extracted twice more in the same manner. . At this time, the obtained filtrates were all concentrated under reduced pressure at 40 and lyophilized at -50 ° C to obtain an ethanol extract (21.15 g).

The ethanol extract was extracted three times with a mixture of hexane (n-hexane) and water (1: 1) for separation and extraction according to the polarity, and the hexane layer was removed, and the water layer was removed again with chloroform and water (1: 1). Extraction three times with. In addition, the water layer from which the chloroform layer was removed was extracted three times with a mixture of ethylacetate (ethylacetate) and water (1: 1), and each layer was concentrated under reduced pressure and lyophilized in the same manner as above, and the water layer from which ethyl acetate was removed was Concentration and freeze-drying under reduced pressure in the same manner as described above. Hexane fraction (7.64 g), chloroform fraction (1.65 g), ethyl acetate fraction (1.43 g) and water fraction (9.26 g) were obtained.

The resveratrol content of the fraction was analyzed using high performance liquid chromatography (HPLC). The instrument used was Agilent 1100 series, column Phenomenex Luna C18 (II) 5um 4.6 X 250mm, mobile phase dissolved in water (pH 3) And formic acid (pH 3) dissolved in acetonitrile were mixed and detected at 305 nm.

Resveratrol content of each fraction was 2.12 mg in ethanol fraction, 0.25 mg in hexane fraction, 1.64 mg in ethyl acetate fraction, and was not detected in chloroform fraction and water fraction.

Therefore, the content of resveratrol contained when the dried mulberry branch 1 was extracted with ethanol was found to be 4.01 mg, based on which the content of resveratrol to be included in the feed was calculated.

Mulberry branch extract was dried to obtain ethanol solids for feed formulation, wherein the resveratrol content ratio of solids was 57% by weight. In order to prepare a feed additive containing resveratrol at a concentration of about 400 ppm for feeding broilers, it was mixed at a ratio of 0.7 wt% solids, 40 wt% silica, 45 wt% powder, and 14.3 wt% soda ash.

Example  2. For broilers Resveratrol  Feeding Effect of Containing Feed Composition

In order to investigate the effect of the additive composition of the resveratrol-containing feed composition prepared in Example 1, a 1-day-old broiler cockerel was subjected to a specification test for 5 weeks. Comparative Example 1 was an antibiotic-free addition, Comparative Example 2 was an antibiotic (avilamycin 10ppm + salinomycin 60ppm) addition, Comparative Example 3 was a vitamin E (a-tocopherol) 200IU addition well known as an antioxidant. In the case of Example, the developed resveratrol-containing feed composition was added to the feed level of 20ppm) and 200ppm, thereby leaving Example 1 and Example 2. Table 1 shows the contents of the treatment for broiler specification test.

division Antibiotic addition
Processing content Comparative Example 1
× - Comparative Example 2
○ Avilamycin 10ppm +
Salinomycin 50ppm  Comparative Example 3 ○
 Vitamin E (a-tocopherol) 200 IU  Example 1 ○
Resveratrol 20ppm  Example 2 ○
Resveratrol 200ppm

Table 2 compares broiler productivity. All of the examples showed an improvement in weight gain compared to Comparative Example 1 (antibiotic), especially in Example 2 (200 ppm resveratrol) body weight and weight gain of 1,905g and 1,857g showed better results than Comparative Example 2, the antibiotic addition group It was. In addition, the feed requirement of the Example was improved compared to Comparative Example 1. In terms of productivity, resveratrol was superior to vitamin E, which is known as a natural antioxidant, and it was effective to add 200 ppm more than 20 ppm in the level of resveratrol.

Comparative Example 1
(Antibiotic) Comparative Example 2
(Antibiotic) Comparative Example 3
(Vitamin E) Example 1
(Resveratrol)
20 ppm Example 2
(Resveratrol)
200 ppm  Onset weight (g) 48.3 48.3 48.4 48.3 48.2  5 weeks body weight (g) 1,812 (100) 1,890 (104) 1,858 (103) 1,865 (103) 1,905 (105)  Weight gain (g) 1,764 (100) 1,841 (104) 1,810 (103) 1,817 (103) 1,857 (105)  Feed Intake (g) 2,844 (100) 2,937 (103) 2,858 (100) 2898 (102) 2,898 (102)  Feed rate 1.61 (100) 1.60 (99) 1.60 (99) 1.60 (99) 1.56 (97)

Figures in parentheses compared to Comparative Example 1

Table 3 shows the carcass rate and the relative weight of the tissue, and the result of measuring the carcass rate and the weight of liver, spleen, and abdominal fat after supplementation for 5 weeks was compared to Comparative Example 1 (Antibiotics). It was confirmed that the carcass rate of (resveratrol) increased and the abdominal fat decreased, and the relative weight of liver and spleen did not show a big difference.

Comparative Example 1
(Antibiotic) Comparative Example 2
(Antibiotic) Comparative Example 3
(Vitamin E) Example 1
(Resveratrol)
20 ppm Example 2
(Resveratrol)
200 ppm Conductor Rate (%)
68.3 (100) 71.1 (104) 69.9 (102) 70.9 (104) 71.1 (104)  Liver (g / 100g body weight) 2.52 (100) 2.57 (102) 2.58 (102) 2.33 (102) 2.54 (101)  Spleen (g / 100g body weight) 0.10 (100) 0.09 (90) 0.10 (100) 0.10 (100) 0.10 (100)  Abdominal fat (g / 100g body weight) 1.52 (100) 1.65 (109) 1.27 (84) 1.44 (95) 1.31 (86)

Figures in parentheses compared to Comparative Example 1

Table 4 shows the contents of metabolites in the blood and compares the biochemical compositions.

Comparative Example 1
(Antibiotic) Comparative Example 2
(Antibiotic) Comparative Example 3
(Vitamin E) Example 1
(Resveratrol)
20 ppm Example 2
(Resveratrol)
200 ppm Creatinine
(mg / dL) 0.26 (100) 0.21 (81) 0.16 (62) 0.19 (73) 0.18 (69)
BUN (mg / dL) 2.00 (100) 2.20 (110) 2.80 (140) 2.60 (130) 2.40 (120) Total protein
(g / dL) 3.24 (100) 3.22 (99) 3.28 (101) 2.98 (92) 2.92 (90) Albumin (g / dL)
(Albumin) 1.50 (100) 1.72 (115) 1.70 (113) 1.54 (103) 1.70 (113) Globulin (g / dL)
(Globulin) 1.74 (100) 1.50 (86) 1.58 (91) 1.44 (83) 1.22 (70) albumin/
globulin 0.89 (100) 1.15 (129) 1.08 (121) 1.08 (121) 1.44 (162) AST (U / L)
176.8 (100) 165.6 (94) 170.8 (97) 168.0 (95) 160.0 (90) ALT (U / L)
6.40 (100) 6.20 (97) 6.20 (97) 6.20 (97) 6.60 (103)

() Shows the index compared with the comparative example 1.

The biochemical composition of blood components described in the table has the following meanings.

Creatinine: A product of muscle metabolism. Increased levels are associated with renal failure or dehydration, and are used as effective indicators of renal failure determination along with blood urea nitrogen concentrations.

2. Urea Nitrogen (BUN, blood urea nitrogen): It shows kidney function and increases in case of disorder.

3. Total protein: There are more than 500 kinds of proteins in the blood, and they are responsible for the movement of various substances, iron metabolism, immunity, and blood coagulation. The total protein is an indicator of such a living state, and if it is lower or higher than the normal value, the synthesis or degradation of the protein may be suspected or the protein absorption disorder may be suspected.

4. Albumin (Albumin): Albumin is a protein that accounts for two-thirds of the total protein in the blood, plays an important role in maintaining blood pressure, and is responsible for transporting various nutrients and hormones.

5. Globulin: Determination of immune system abnormalities by measuring the amount of proteins responsible for immune function in the blood.

6. Albumin / globulin ratio: As a criterion for determining the abnormality of blood proteins, if decreased, it may indicate chronic inflammation, digestive disease, kidney disease, and the like.

7. AST (aspartate aminotransferase): An enzyme in the liver that increases when the liver cells are damaged.

8. ALT (alanine aminotransferase): ALT is an enzyme in the liver that increases when hepatocytes are damaged by hepatitis.

Creatinine, BUN, total protein, albumin, globulin, AST and ALT are indicators of liver and kidney damage and are used as indicators to determine the toxicity of new feed additives. As a result, creatinine, total protein, globulin, AST and ALT were decreased in the Examples compared to the Comparative Examples. Therefore, resveratrol was found to reduce the damage of cells and tissues through antioxidant and anti-inflammatory action, but did not have a negative effect.

Table 5 and Figure 1 compare IgA, IgG, and IgM, which are immunoglobulins present in the blood. Compared with Comparative Example 1, IgA, IgG and IgM of Comparative Examples 2 and 3 and Examples 1 and 2 were reduced. In broiler breeding stages, various types of harmful factors and stresses can act and cause abnormalities in the immune response. This increase in immune response can negatively affect growth by increasing basal metabolic rate, changing nutrient uptake, as well as allowing nutrients to be used for skeletal and muscle accumulation in the immune response. Resveratrol, like antibiotics, regulates the broiler's immune system to reduce the development of immune response abnormalities in normal breeding conditions. Table 5 shows the blood immunoglobulin content (unit: μg / dL).

Table 5

Comparative Example 1
(Antibiotic) Comparative Example 2
(Antibiotic) Comparative Example 3
(Vitamin E) Example 1
(Resveratrol)
20 ppm Example 2
(Resveratrol)
200 ppm ------ μg / dL ------ IgA
3.05 (100) 1.45 (48) 2.49 (82) 2.25 (74) 1.69 (55) IgG
49.07 (100) 30.82 (63) 47.50 (97) 37.83 (77) 39.17 (80) IgM
0.82 (100) 0.66 (80) 0.82 (100) 0.79 (96) 0.59 (72)

Figures in parentheses compared to Comparative Example 1

Table 6 and Figure 2 compare the antioxidant factors in the blood. Antioxidant enzyme-like activity (SOD like activity) showing blood antioxidant activity was superior to Examples 1 and 2 compared to Comparative Examples 1 and 2, Example 2 supplemented with 200 ppm of resveratrol vitamin E 200IU known as a natural antioxidant It showed higher activity than the comparative example 3 which added the supplementation. MDA (malondialdehyde) formed by the lipid peroxidation reaction also showed lower results than Comparative Examples 1 and 2 in Examples 1 and 2. Investigation of blood-related antioxidant factors revealed that resveratrol exerts strong antioxidant activity, thereby increasing blood antioxidant activity and reducing lipid peroxidation. Table 6 shows the contents of blood antioxidant related factors.

Comparative Example 1
(Antibiotic) Comparative Example 2
(Antibiotic) Comparative Example 3
(Vitamin E) Example 1
(Resveratrol)
20 ppm Example 2
(Resveratrol)
200 ppm  SOD like activity (%) 21.58 (100) 21.42 (99) 23.61 (109) 23.10 (107) 25.98 (120) MDA content (Malondialdehyde) (μg / mL) 0.206 (100) 0.206 (100) 0.189 (92) 0.204 (99) 0.194 (94)

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

1 is a schematic diagram showing a process of obtaining resveratrol extract from the mulberry leaves and mulberry branches.

Figure 2 is a formulated form of the resveratrol extract extracted from mulberry leaves and mulberry branches.

Figure 3 shows the immunoglobulin content in the blood of each treatment.

Figure 4 shows the content of antioxidant related factor MDA (Malondialdehyde) in the blood of each treatment.

Claims (7)

Method for producing an antibiotic replacement feed additive comprising the following steps: Pressing and drying the mulberry leaves and the mulberry branches; Grinding the compressed mulberry leaves and mulberry branches to obtain solids; Dissolving the obtained solid content with ethanol in a weight ratio of 1: 1 to 200 to 400 rpm at 35 to 45 ° C. for 20 to 40 minutes, and filtering to obtain an extract; Removing ethanol from the extract to obtain a solid; And Comprising 0.7 to 1.0% by weight of the obtained mulberry leaf and mulberry branch extract solids 35 to 45% by weight and powder 35 to 45% by weight and soda ash 10 to 20% by weight to complete the powder formulation. delete delete delete Antibiotic substitute feed additive prepared according to the method of claim 1. delete delete

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