KR20170132475A - A feed additive for improving meat quality and feed composition using the same - Google Patents

Abstract

The present invention relates to a feed additive for improving meat quality of a livestock and a feed composition using the same. More specifically, the present invention relates to a feed additive for improving meat quality of a livestock and a feed composition using the same, which include any one ingredient of a licochalcone A, a licochalcone B, and a mixture thereof isolated from licorice as an active ingredient, suppress oxidative stress, and increase livestock growth ability, so that meat quality can be improved.

Description

[0001] The present invention relates to a feed additive for improving meat quality of a livestock and a feed composition using the feed additive,

The present invention relates to a feed additive for improving meat quality of livestock and a feed composition using the feed additive. More particularly, the present invention relates to a feed additive for improving the meat quality of livestock, comprising livestock, lycopene, And a feed composition using the feed additive.

As consumers become more and more diverse in their diet, awareness and sales of high quality meats with good health as well as good taste are increasing. Therefore, according to the demand of these consumers, producers are carrying out many tests and researches to improve the quality of meat products in various ways. In particular, studies on compound feeds and feed additives for the production of high-quality meat have been continuously carried out. For example, diverse feed-related products such as loess feed, bamboo feed, green tea, and brown rice have been studied And is commercially available.

In addition, with the development of animal breeding business and feed industry, the use of antibiotics for the prevention of diseases and promotion of growth, as well as the treatment of diseases, has greatly improved livestock productivity and could be converted to a large-scale specification system. However, Livestock is increasingly susceptible to stress, and the importance of antibiotic substitute development has emerged, regulating the use of antibiotics in the course of time. Therefore, it is becoming more and more important to increase the productivity through improvement of immunity and improvement of blood characteristics of livestock using natural materials.

Oxidative stress is not only a general process of aging but also a primary cause of many diseases. Reactive oxygen species (ROS) and inflammatory cytokines have been implicated in oxidative stress and play an important role as mediators in the pathology of various diseases, including endotoxin stimulation. When oxidative stress is applied to cells by ROS, it causes peroxidation of cell membrane lipids, changes in cell membrane permeability, or DNA damage. To eliminate these ROS, antioxidant enzymes and nonenzymatic antioxidants in cells protect cells from oxidative damage by reactive oxygen species. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR) , And non-enzymatic antioxidant compounds such as vitamin C and E, phenolic compounds such as caffeic acid, chlorogenic acid and ferulic acid, tannins such as catechine, quercetin quercetin, campferol, etc., and chalotinoids maintain a balance between the production and elimination of reactive oxygen species and maintain cell function. In other words, in vivo, oxidative attack and defense of antioxidants harmonize and maintain homeostasis. Therefore, studies for discovering new antioxidants capable of protecting biological membranes from oxidative stress have been actively conducted.

On the other hand, licorice (Licorice, Glycyrrhiza uralensis Fisch) is a perennial herbaceous plant belonging to the rosemary bean family of dicotyledonous plants. It is about 30 ~ 70㎝ in height and 1m in length. It has been written. Root is used as a medicinal substance. Its characteristic is that it has no toxic and warm aura with taste, it is known to act on cardiopulmonary and gastric ganglion, and it is known to be effective on eugen, It is used as an expectorant and an emollient. It is also known that the pain relieving effect caused by the sudden tension of muscles and tissues, weight gain, leukocyte increase, diuretic action, and anti-inflammatory action.

Korean Patent No. 10-0725926

In the present invention, functional and natural nutrient feed additives and feeds having excellent safety and efficacy were developed using Ricocalcone A and Ricocalcone B, which are natural plant extracts isolated from licorice roots, and furthermore, And the present invention has been completed on the basis thereof.

Accordingly, the present invention provides a pharmaceutical composition containing, as an active ingredient, any one of ricocalcolone A, ricocalcone B and mixtures thereof isolated from licorice root to inhibit oxidative stress and increase the growth ability of livestock, A feed additive for improving meat quality of a livestock and a feed composition using the feed additive.

In order to achieve the above object, the present invention provides a feed additive for improving meat quality of a livestock comprising one of components of Licochalcone A, Licochalcone B and mixtures thereof as an active ingredient .

The present invention also provides a feed composition for improving meat quality of a livestock containing the feed additive.

The present invention also provides a method for improving meat quality of a livestock comprising feeding the feed composition to livestock.

According to the present invention, it is possible to produce and supply high quality meat by suppressing oxidative stress and improving meat quality, including natural plant extracts, ricocalcone A and ricocalcone B, isolated from licorice roots.

FIG. 1 is a graph showing the results of 1-electrophoresis and silver staining according to the present invention, in which expression patterns of proteins expressing in a grade-specific porcine donkey are measured.
FIG. 2 shows the results of the analysis of the proteins expressed in the grade-I pork loin using LC-MS / MS and then using Upiprot KB and NCBI data base to determine the protein quality of HQLD (low quality of longissimus dorsi) dorsi), HQLD & LQLD.
FIG. 3 is a photograph showing cultivation of C 2 Cl 2 in a proliferation-related culture medium (DM) and a differentiation-related culture medium (DM) in order to establish the differentiation condition of muscle cells according to an embodiment of the present invention.
FIG. 4 is a graph showing the cell survival rate of myofilaments after 24 and 48 hours after H ₂ O ₂ concentration treatment of root cells to establish oxidative stress conditions according to an embodiment of the present invention. FIG.
FIG. 5 is a graph showing the cell shape and condition of 7 days-old cells treated with H ₂ O ₂ at a concentration of ₂₀₀ μM in order to establish the undifferentiated condition of myoblasts by oxidative stress according to an embodiment of the present invention. Fig.
FIG. 6 is a graph showing the results of confirmation of root canal cells and undifferentiated stem cells by immunostaining after treatment with H ₂ O ₂ at a concentration of ₂₀₀ μM in order to establish conditions for undifferentiation of myoblasts by oxidative stress according to an embodiment of the present invention Fig.
FIG. 7 shows mRNA expression (A) and protein expression (B) patterns in MyoD and Myog in the root cell group and the root cell group subjected to oxidative stress (H ₂ O ₂ treatment) according to an embodiment of the present invention .
FIG. 8 is a graph showing the results of RT-PCR of the mRNA level of LDH (Lactate dehydrogenase A) in the source cells through the differentiation inducing medium according to an embodiment of the present invention.
FIG. 9 is a graph showing the results of experiments showing that in order to establish conditions for maintenance of source cell undifferentiation by oxidative stress, ricocalcone A, ricocalcone B, and ricocalcone B in vitro in accordance with an embodiment of the present invention, + B (3: 1), Rico chalcone a + B (1: 1) , Rico chalcone a + B (1: 3) single treatment with Rico chalcone a + H ₂ O _2, Rico chalcone B + H ₂ O ₂ of , Rico chalcone A + B (3: 1) + H 2 O 2, Rico chalcone A + B (1: 1) + H 2 O 2, Rico chalcone A + B (1: 3) + H 2 O 2 mixed treatment Lt; RTI ID = 0.0 > cell / cell < / RTI >
10 is a graph showing the activity of LDH in pig muscle tissue using an LDH activity measurement kit according to an embodiment of the present invention.
FIG. 11 is a graph showing the results of a proliferation induction culture, a differentiation inducing culture solution, H ₂ O ₂ , Ricocalcone A, Ricocalcone B, Ricorcone A + B (3: 1) B (1: 1), Rico chalcone A + B (1: 3) , Rico chalcone A + H ₂ O _2, Rico chalcone B + H ₂ O _2, Rico chalcone A + B (3: 1) + H 2 O _2, Rico chalcone a + B (1: 1) + H 2 O 2, Rico chalcone a + B (1: 3) + H 2 O after each treatment the _two MyoD, the Myog immune staining, RT-PCR and wester blot. < / RTI >
FIG. 12 is a graph showing the results of grouping the differential expression proteins identified by LC-MS / MS among the proteins expressed in the grade-specific porcine donkey by biological and molecular functions.
FIG. 13 is a graph showing the effect of Ricocalcone A or Ricolacon B on oxidative stress on stress-related proteins (TPM3, PRDX6, HSP70) through Western blot according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a feed additive for improving meat quality of a livestock containing, as an active ingredient, any one of Licochalcone A, Licochalcone B and a mixture thereof.

The term "livestock" as used in the present invention means domestic animals, such as mammals, poultry, and the like. In the present invention, the mammal is exemplified by cattle, pigs, goats, sheep, horses and the like, and examples of the poultry include, but are not limited to, chickens, ducks and turkeys.

The term " Ricoh chalcone A "or" Ricoh chalcone B "used in the present invention can be used in the form of a pharmaceutically acceptable salt other than a polyphenolic compound having chalcone as a basic skeleton, And the acid addition salt formed by a pharmaceutically acceptable free acid is useful as a salt. As the free acid, inorganic acid and organic acid can be used. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid and the like can be used. As the organic acid, citric acid, acetic acid, maleic acid, fumaric acid, , Acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid and arpartic acid. Preferably, hydrochloric acid is used as the inorganic acid, and methanesulfonic acid is used as the organic acid. The above-mentioned ricocalcone A includes not only pharmaceutically acceptable salts but also all salts, hydrates and solvates which can be prepared by conventional methods.

Commercially available products may be purchased and used for the above-mentioned Ricocalcone A and Ricocalcone B, but it is particularly preferable to use those extracted or isolated from licorice.

When the licorice is heated, the non-polar substance including the active ingredient ricocalcone A or ricocalcone B increases, and the ricocalcone A or the ricocalcone B is contained in a large amount in the root of the licorice root.

Thus, Ricocalcone A and Ricocalcone B can be isolated from the root of licorice root using extraction or fractionation methods known in the art.

As a suitable solvent for extracting or separating Ricocalcone A and Ricocalcone B, any pharmaceutically acceptable solvent may be used, and water or an organic solvent may be used, but the present invention is not limited thereto. For example, the solvent may be selected from the group consisting of purified water, alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropanol, and butanol, Can be used. Preferably, methanol is used as the solvent.

As the extraction method, methods such as room temperature extraction, hot water extraction, cold extraction, reflux extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression may be used, and more preferably hot water extraction or reflux extraction Do.

The licorice extract may also be obtained by fractionating the organic solvent from nonpolar to polar.

Suitable solvents for fractionation of the licorice fraction are methylene chloride, ethyl acetate, butanol, water or mixtures thereof, and it is particularly preferred to separate Ricocalcone A and Ricocalcone B from the methylene chloride fraction.

The extracts or fractions thus extracted or fractioned can be purified by medium pressure liquid chromatography (MPLC), silica gel column chromatography, thin layer chromatography, high performance liquid chromatography performance liquid chromatography and the like to further isolate Ricocalcone A and Ricocalcone B from each other.

For example, Ricocalcone A and Ricocalcone B of the present invention can be separated as follows. 600 g of licorice is added with 800-1,000 mL of methanol, extracted twice, and methanol is removed. Dissolve in 1,000 mL of water and remove the fat with 300 mL of hexane. The remaining aqueous layer is subjected to solvent extraction twice with 300 mL of methylene chloride, ethyl acetate and butanol sequentially twice. The methylene chloride layer was then subjected to medium pressure liquid chromatography (MPLC) using a solvent system of hexane: ethyl acetate: methanol = 2: 1: 0.1 to 1: 1: 0.1 to 0: (M1 to 3), and the first fraction (M1) was fractionated into three small fractions (M1-1 to M1-3) using a solvent system of hexane: ethyl acetate: methanol = 2: Separate the two small fractions (M2) to obtain three small fractions (M2-1 to M2-3) using a solvent system of hexane: ethyl acetate: methanol = 1: 1: 0.1. Each of the small fractions thus obtained was compared with a high performance liquid chromatography (HPLC) profile and the standards of Ricocalcone A and B to obtain ricocalcone A as a small fraction of M1-3 and ricocalcone B , Thereby separating Ricoh chalcone A and Ricoh colone B from each other.

Ricocalcone A or Ricocalcone B used in the feed additive of the present invention reduces oxidative stress by decreasing lactate dehydrogenase (LDH) level. The feed additive containing the feed additive or the feed, It is possible to prevent the problem that the enemy stress is suppressed and the meat quality is lowered due to the stress.

Ricocalcone A, Ricocalcone B, or a mixture thereof may be contained in the feed additive in an amount of 0.01 to 95% by weight, preferably 1 to 80% by weight. When the content is within the above range, the oxidative stress can be remarkably suppressed by reducing the lactate dehydrogenase level, and the effect of improving the meat quality can be excellent.

It is further preferable to use ricocalcone A or ricocarcone B together with ricocalcone A or ricocarcon B alone in combination with feed additive rather than with ricocalcone A or ricocarcone B in order to improve the meat quality by alleviating the stress of the livestock. At this time, the ricocalcone A and the ricocarcon B are mixed in a weight ratio of 1 to 50:50 to 1, and they are preferably included in the feed additive, more preferably mixed in a weight ratio of 1 to 3: 3 to 1. As described above, when the mixing ratios of Ricorcocone A and Ricorcone B are as described above, it is better to alleviate the oxidative stress of the livestock and improve the ability of the livestock to grow.

The livestock may be cattle such as cattle, pigs, goats, sheep, horses, chickens, ducks, turkeys and the like, preferably pigs. In the following examples of the present invention, finishing pigs were used as livestock.

The present invention also provides a feed composition for improving meat quality of a livestock containing the feed additive.

The feed additive may be included in the feed composition in an amount of 0.1 to 50% by weight, preferably 1 to 30% by weight. When the content is included in the above range, the effect of improving the meat quality and improving the growth ability of the livestock can be exhibited more efficiently.

On the other hand, the feed composition may include general basic feeds fed according to the type of livestock. These basic feeds are known in the art and are commercially available depending on the type of livestock, so that any person skilled in the art will be able to manufacture or purchase them very easily.

Specifically, the basic feed may include some or all of a starch-containing substance, a protein-containing substance, a fat-containing substance, a vitamin-containing substance, an inorganic substance-containing substance, an antioxidant substance, an antibiotic,

The starch-containing material may be included in the feedstuff of the rabbit as long as it can sustain the growth of the animal. Such starch-containing material is generally obtained from corn, soybean, wheat, sorghum, barley, oats and the like. Examples of suitable starch-containing substances include corn flour or powder, oat flour or powder, bean flour or powder, wheat flour or powder. Suitable suitable starch containing materials are oat flour, corn flour, wheat flour, soy flour, and the like. The concentration of such a starch-containing substance is not particularly limited as long as it can effectively maintain the growth of the animal. Generally, the starch-containing material may be included in the basic feed in a range of about 30 to 80% by weight.

Protein-containing materials can also be included in animal feed as long as they can sustain the growth of the animal. For economic reasons, protein-containing substances such as fish meal and soybean powder have been used. Others include soy protein concentrate, blood meal, plasma protein, skim milk dry product, milk protein concentrate, corn gluten powder, wheat gluten Powder, yeast, sunflower seed powder and the like. Preferred protein-containing substances are fish meal, blood meal, plasma protein, soybean powder and the like. As long as the protein-containing material can effectively maintain the growth of the animal, its concentration in the animal feed is not important. Generally, the protein-containing material can be included in the basic feed in a range of about 10 to 50% by weight.

The fat-containing material includes, but is not limited to, lard, tallow, soybean oil, lecithin, coconut oil and the like. The concentration of the fat-containing substance is not limited as long as it can maintain the growth of the animal, and it can be included in the basic feed in a range of about 2 to 20% by weight.

The base feed can also contain water-soluble, fat-soluble vitamins and trace minerals. Examples of the vitamins include vitamin A, vitamin D, vitamin E, vitamin K, riboflavin, pantothenic acid, niacin, vitamin B12, folic acid, biotin and vitamin C. Copper, zinc, iodine, selenium, manganese, iron, cobalt and the like can be used as a trace amount of an inorganic substance. Here, "trace" means that the amount of these ingredients used in the livestock base feed is substantially less than the amount of the other ingredients. Generally, when a small amount of vitamin or mineral is included in the basic feed, it may be contained in an amount of about 0.0001 to 5% by weight based on the total weight of the composition.

In addition, the base feed may include antioxidants such as BHT (Butylated Hydroxytoluene), BHA (Butylated Hydroxyanisole), vitamin E and ascorbic acid, and these antioxidants may be contained in a very small amount of about 0.0001 to 1% by weight.

The invention also provides a method for improving meat quality of a livestock comprising feeding the feed composition to the livestock.

As described above, the feed composition comprising the feed additive for improving the meat quality of the livestock according to the present invention has an advantage that the growth rate of livestock can be increased and the meat quality can be improved at the same time by feeding to the livestock.

Hereinafter, the present invention will be described in more detail with reference to examples. These embodiments are for purposes of illustration only and are not intended to limit the scope of protection of the present invention.

Example 1 Isolation of Ricocalcone A and Ricocalcone B

To 600 g of licorice, 800-1,000 mL of methanol was added, and the mixture was extracted twice. After removal of methanol, the mixture was dissolved in 1,000 mL of water and the fat was removed with 300 mL of hexane. The remaining aqueous layer was subjected to solvent extraction twice with 300 mL of methylene chloride, ethyl acetate and butanol sequentially twice each. The methylene chloride layer was subjected to medium pressure liquid chromatography (MPLC) using a solvent system of hexane: ethyl acetate: methanol = 2: 1: 0.1 to 1: 1: 0.1 to 0: Fractions (M1 to 3) were separated. Subsequently, three small fractions (M1-1 to M1-3) were separated by medium pressure liquid chromatography using a 1 part fraction (M1) using a solvent system of hexane: ethyl acetate: methanol = 2: 1: 0.1 . The second fraction (M2) was further eluted with hexane: ethyl acetate: methanol = 1: 1: 0.1 through medium pressure liquid chromatography to obtain three small fractions (M2-1 to M2-3). High performance liquid chromatography (HPLC) profiles of each fraction were compared with those of Ricoh colcons A and B to confirm that ricochalcone A was contained in M1-3 small fraction and ricochocolon B was contained in M2-2 small fraction And separated by preparative liquid chromatography, respectively, and used in the experiment.

Example 2. Confirmation of Expression Pattern of Protein Expressed from Swine Muscle Tissue (Ribs) by Grade

In order to compare the expression patterns of the proteins expressed in the pig muscle tissues (sirloin) of each grade, six pigs were selected from the pig meat slaughtered from the good meat grade to the low grade pig meat sirloin, respectively, and 1Dimension-electrophoresis And silver staining. The results were shown in Fig. 1. The results are shown in Fig.

As shown in Fig. 1, protein expression patterns in the pig muscle tissues of the grade (sirloin) were evaluated by using HQLD (high quality of longissimus dorsi (1 to 6)) having good meat quality grade and LQLD (a *) and 34 kDa (b #), respectively, when the cells were classified as low quality of longissimus dorsi (7-12)).

Example 3: Elucidation of new marker factors by LC-MS / MS analysis of proteins expressed from pig muscle tissue (sirloin)

The proteins (130 kDa, 34 kDa) corresponding to the respective sizes expressed in the pork loin in the above-mentioned Example 1 were identified using LC-MS / MS, and the function of each protein was analyzed using Upiprot KB and NCBI data base And grouped into HQLD, LQLD, HQLD & LQLD, and the results are shown in FIG. 2 and Table 1 below.

As shown in Fig. 2 and Table 1, the HQLD (a *) and MYBPC2 (Myosin binding protein C) predicted as candidates of new marker factors among the proteins overexpressed in each HQLD and LQLD, LDH (Lactate dehydrogenase A) were selected. For development of the epigenetic markers, LDH was selected as a cause of degradation due to oxidative stress.

Example 4. Establishment of Differentiation Conditions by Source Cell Culture and Differentiation in Root Canal Cells and Oxidative Stress on New Marker Factors Related to Meat Quality

C2C12, known as a representative myoblast, is a mouse-derived myoblast that is differentiated into myotubes by house serum stimulation. Therefore, in this embodiment, the present inventors tried to establish the conditions of differentiation of C2Cl2 using growth media (GM) and differentiation media (DM) which induce continuous proliferation by using these characteristics.

GM was generally replaced with DMEM medium (10% fetal bovine serum (FBS), 1% antibiotics supplement) used for culturing C2C12 cells at the same time for 7 days. In the case of DM, house serum was added DMEM medium (2% house serum, 1% antibiotics) was replaced at the same time for 7 days. The above-mentioned GM and DM were used to cultivate C 2 Cl 2 cells, and the results are shown in FIG.

As shown in FIG. 3, GM showed a proliferation in the form of a general cell, and in DM, C2CC2 cells were differentiated into canaliculus cells. From these results, it was confirmed that the culture medium supplemented with house serum was differentiated into root canal cells from the myoblast cells by replacing the culture medium for 7 days under differentiation conditions using GM and DM.

Example 5: Evaluation of environmental factors of LDH expressed by oxidative stress in vitro Establish conditions for performing functional studies on

C2C12 was cultured with H ₂ O ₂ at concentrations of 12.5, 25, 50, 100, 400, 600, 800 and 100 μM in order to establish conditions that do not differentiate into canaliculus cells due to oxidative stress Cell viability was measured at 24 and 48 hours after treatment on one 96-well plate. For cell viability measurement, MTS analysis was performed, and a 30: 1 mixture of MTS and PMS in a ratio of 20: 1 was added to each well. The cells were incubated at 37 ° C for 3 hours in a dark state, and cell viability was measured at a wavelength of 490 nm using an ELISA reader. The results are shown in FIG.

As shown in FIG. 4, it was confirmed that the suitable concentration of H ₂ O ₂ for oxidative stress induction, which can maintain the normal condition of the source cells and perform in vitro studies, is about 200 μM.

Then, the source cells treated with H ₂ O ₂ at a concentration of ₂₀₀ μM and the normal group were continuously observed for 7 days in terms of the cell shape and condition so that the source cells were oxidatively stressed when the root cells were differentiated into canaliculus cells The results are shown in Fig. Fig. 6 shows the canaliculus cells and undifferentiated myoepithelial cells through immunostaining.

As a result, as shown in FIG. 5, it was confirmed that the root cells that underwent oxidative stress (H ₂ O ₂ treatment) did not differentiate into root canal cells as compared with the normal culture condition group on the fifth day. These results indicate that oxidative stress is involved in differentiation from root cells into root canal cells, and as shown in Fig. 6 showing the result of immunohistochemical staining, single nucleus does not form polynuclear nucleus, Could know.

Example 6. Expression Patterns and Changes of Related Proteins Related to Root Cell Maintenance and Root Cell Differentiation by Oxidative Stress

RT-PCR and western blot were performed for MyoD and Myog, respectively, which are involved in proliferation and differentiation at each condition caused by oxidative stress. For the RT-PCR analysis, primers for MyoD and Myog were constructed (MyoD; F: GAT GGC ATG ATG GAT TAC AGC; R: GAC TAT GTC CTT TCT TTG GGG; Myog; F: GCT CAG CTC CCT CAA CCA G, R : ATG TGA ATG GGG AGT GGG GA) and the expression of MyoD (528 bp) and Myog (424 bp) was confirmed for each sample. For western blot analysis, each sample was lysed and quantitated at the same concentration, and protein expression of MyoD and Myog was confirmed by 10% SDS-PAGE containing 30% Bis-acrylamide. The results are shown in Fig.

As shown in Fig. 7, the myofibroblast group subjected to the oxidative stress (H ₂ O ₂ treatment) showed mRNA expression (A) and protein expression (MyoD and Myog) in proliferation and differentiation, B) There was no significant difference in the aspect.

RT-PCR was performed to confirm the mRNA level of LDH in each condition of the source cells through the differentiation inducing medium, and the results are shown in FIG.

As a result, as shown in FIG. 8, it was confirmed that LDH mRNA levels were not significantly different under normal conditions and in vitro conditions.

Example 7. Confirmation of Oxidative Stress Inhibitory Effect of Ricorcone A, Ricorcone B and Ricorcone A + B

7-1. in vitro  Under conditions, Ricoh chalcone A, Ricoh chalcone B, and Ricoh chalcone A + B and oxidative stress were used to establish conditions for maintaining undifferentiated source cells.

In order to investigate LDH function, which is known to be caused by oxidative stress, which is the cause of degradation of meat quality, Ricorcone A, Ricorcone B, Ricorcone A + B (3: 1), Ricorcone A + B (3: 1) + H ₂ (1: 1), Ricoh chalcone A + B (1: 3) and Ricoh chalcone A + H ₂ O ₂ , Ricorcone B + H ₂ O ₂ , was treated with H ₂ O ₂ + conditions of blending O _2, Rico chalcone a + B (1:: 1 ) + H 2 O 2, Rico chalcone a + B (3 1). At this time, ricocalcone A, B and A + B were treated at concentrations of 2.5, 5, 10 and 30. 60 μM, and H ₂ O ₂ was treated at a concentration of ₂₀₀ μM. The cell viability was measured 24 hours and 48 hours after the treatment, and the results are shown in FIG.

Experimental results for establishing undifferentiated maintenance conditions in which oxidative stress does not damage root cells with ricocalcone A and ricocarcone B as described above. As shown in Fig. 9, the single treatment of ricocalcone A, B and A + B And Ricoh chalcone A, B, and A + B and H ₂ O ₂ , respectively, when ricocalcone A, B and A + B were used at a concentration of about 10 μM, It would not give.

7-2. in vivo LDH activity from graded pork loin

In order to confirm the activity of LDH in the pig muscle tissue of each grade, an enzyme-based LDH activity measurement kit was used. LDH is an enzyme present in the cytoplasm and usually does not pass through the cell membrane, but when the cell membrane is damaged, it is released outside the cell, that is, into the culture medium. Using the LDH activity measurement kit, the released LDH catalyzes the dehydrogenation of lactic acid and produces pyruvate and NADH. This NADH reduces the tetrazolium salt (INT) by a diapholase catalyst and forms a red formazan dye with an absorption of 490 nm, and thus the LDH activity can be measured by increasing the absorbance at 490 nm. C2C12 cells were cultured in 96-well plates for 24 hours. The culture medium was changed to 50 μl / well and LDH standard solution (0.05 U / ml) was added at 50 μl / well. Next, Solution C was added at 100 μl / well, and the absorbance was measured using an ELISA reader to confirm LDH activity, which is a factor of meat quality degradation. The results are shown in FIG.

As a result, as shown in FIG. 10A, it was confirmed that the LDH level, which is a factor of meat quality deterioration in low quality meat, is higher than in vitro . In addition, LDH activity in the in vivo level through the source cells was found to be decreased in the differentiation inducing medium (DM) and in the ricocalcone A or B treatment group, which is an oxidative stress reducing substance, as shown in Fig. 10B.

7-3. Identification of oxidative stress inhibitory effect

Multiply the source cell induced cultures, differentiation-inducing culture solution, H ₂ O _2, Rico chalcone A, Rico chalcone B, Rico chalcone A + B (3: 1) , Rico chalcone A + B (1: 1) , Rico chalcone A + B (1: 3), Ricorcone A + H ₂ O ₂ , Ricorcone B + H ₂ O ₂ , Ricorcone A + B (3: 1) + H ₂ O ₂ , Ricorcone A + B ) + H ₂ O ₂ , and ricocalcone A + B (1: 3) + H ₂ O ₂ , respectively. The proliferation and differentiation of the genes involved in oxidative stress at each condition were examined by immunostaining , RT-PCR and wester blot. The results are shown in Fig.

Immunostaining was performed as follows. After washing with 100% ethanol, a cover slip sterilized with an alcohol lamp was placed in a 6-well plate, and C2C12 cells were cultured on the 6-well plate. After washing with phosphate-buffered saline (PBS), the cells were fixed with 1 ml of cytofix (cytofix / cytoperm) (1 h, 4 ° C) and washed with PBS. After blocking with 0.5% Bovine serum albumin (BSA), the cells were incubated with the primary antibody at 4 ° C, washed with PBS, and then incubated with the secondary antibody at 4 ° C. Finally, the cells were stained with DAPI solution and observed with a microscope. RT-PCR and western blot were performed as shown in FIG.

As shown in Fig. 11, the proliferation inducing culture medium, differentiation inducing culture broth, H ₂ O ₂ , ricocalcone A, ricocalcone B, ricocalcone A + B (3: 1), ricocalcone A + B (1: 1), Rico chalcone A + B (1: 3) , Rico chalcone A + H ₂ O _2, Rico chalcone B + H ₂ O _2, Rico chalcone A + B (3: 1) + H 2 O 2, Rico chalcone Myoc and MyoD, which are involved in the differentiation and proliferation of A + B (1: 1) + H ₂ O ₂ , Ricorcone A + B (1: 3) + H ₂ O ₂ , The increase and decrease were clearly confirmed. It was confirmed that the Myog level gradually recovered with the differentiation date in the source cells which received oxidative stress (H ₂ O ₂ ) during treatment with Ricorcone A and Ricorcone B. Specifically, the ratio of 3: 1 ratio of ricocalcone A and B was found to be higher in myocytes treated with stress (H ₂ O ₂ ) than in the other groups.

From these results, it was predicted that the ingestion of the feed additive containing ricocalcone A or ricocalcone B of the present invention would improve the degradation of meat quality caused by oxidative stress.

Example 8. Analysis of Molecular Mechanism of Ricocalcone A and Ricocalcone B on Functional Changes of Selected Proteins Related to Lowering of Meat Quality and Improvement of Meat Quality According to Stress Induction

8-1. Selection of expression proteins related to degradation of meat

(1-dimensional electrophoresis), and the proteins identified by LC MS / MS from Gene Ontology (Gene Ontology), which differ more than 1.5 times in expression level, (http://www.geneontology.org) and UniProt (http://www.expasy.uniprot.org) to group related biological processes and molecular functions to analyze related mechanisms And the results are shown in Fig.

Based on the grouped information, as shown in Fig. 12, the related pathways between the differentially expressed proteases are deduced, and proteins deduced to be involved in muscle development and meat quality formation among proteins differentially expressed in the proteome are shown in Table 2 below .

8-2. Analysis of Molecular Mechanism of Ricocalcone A and Ricocalcone B on Functional Changes of Selected Protein Related to Lowering of Meat Quality and Improvement of Meat Quality by Stress Induced

In order to analyze the different functional changes in stress induced by the LC-MS / MS, the control group differentiated into root cell control group and root canal cell group, A group treated with an optimal H ₂ O ₂ concentration (200 μM) which does not cause damage, a group treated with a single treatment group of Ricorcone A, a group treated with Ricorcone B alone, a group treated with Ricorcone A + B (3: 1) (TPM3, PRDX6, HSP70) involved in the differentiation and stress of canaliculus cells were treated with a mixture of 1: 1 mixture of B (1: 1) and ricocalcone A + B The results are shown in Fig.

As shown in FIG. 13, the values of proteins (TPM3, PRDX6, HSP70) involved in differentiation and stress of canaliculi, which were reduced by oxidative stress by treatment with ricocalcone A or B, And it was confirmed that it recovered to the level similar to the numerical change. In particular, the Ricoh chalcone A + B mixture muscle differentiation efficiency confirmed through western blot of results Rico chalcone A and B a 3 via a TPM3 showing the most pronounced significance in the differentiation efficiency: stress when mixed in a ratio of 1 treatment (H ₂ O ₂ ), the amount of TPM3 expression recovered higher than that of the other groups.

These results indicate that the ingestion of the feed additives containing Ricocalcone A or Ricocalcone B of the present invention can improve the degradation of meat quality caused by oxidative stress.

Example 9. Preparation of feed 1

Feeds were prepared by adding 3.5% by weight of Ricocalcone A isolated from licorice root in Example 1 to the base feed having the composition ratios shown in Table 3 below.

Example 10. Production of feed 2

In Example 9, 3.5% by weight of Ricocalcone B isolated from licorice root was added to produce a feed.

Example 11 Production of feed 3

In Example 9, 2.0% by weight of Ricocalcone A and 2.0% by weight of Ricocalcone B isolated from Licorice root were added to produce a feed.

Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.

Claims (8)

A feed additive for improving meat quality of a livestock containing one of the components of Licochalcone A, Licochalcone B and mixtures thereof as an active ingredient. The method according to claim 1,
The feed additive for improving meat quality of livestock according to any one of claims 1 to 3, wherein the feed additive comprises 0.01 to 95 wt% of any one of ricocalcolon A, ricocalcone B and mixtures thereof. The method according to claim 1,
Characterized in that any one of the components Ricocalcone A, Ricocalcone B and mixtures thereof is obtained from licorice. The method according to claim 1,
The feed additive for improving meat quality of livestock according to any one of the above-mentioned components, wherein the component is one or more of lactic acid dehydrogenase (LDH). The method according to claim 1,
Wherein the livestock is at least one selected from the group consisting of cattle, pigs, goats, sheep, horses, chickens, ducks, and turkeys. A feed composition for improving meat quality of livestock comprising the feed additive of any one of claims 1 to 5. The method according to claim 6,
Wherein the feed additive is included in the feed composition in an amount of 0.1 to 50 wt%. A method for improving meat quality of a domestic animal, comprising feeding the feed composition of claim 6 to the domestic animal.

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