KR20090093576A - A meat quality improvement method of livestock using subcutaneous implantation of soybean isoflavone genestein substance - Google Patents

Abstract

A method for improving meat quality of livestock is provided to subcutaneously implant a composition for improving the meat quality of the livestock in order to increase the amount of intramuscular fat accumulated in the muscular tissue. A method for improving meat quality of livestock comprises the following steps of: preparing a composition for improving the quality of meat containing soybean isoflavone genistein as an active ingredient; pelleting the composition with together glucose and additives; and subcutaneously implanting it to the livestock. A vegetable oil is preferred as the additive. The vegetable oil is selected from the group consisting of corn oil, rice oil and soybean oil. The composition for improving the quality of meat includes glucose.

Description

A meat quality improvement method of livestock using subcutaneous implantation of soybean isoflavone genestein substance}

The present invention relates to a drug delivery system for subcutaneous delivery of soy isoflavone genestane, and more particularly, to pelletized subcutaneous skin containing the genestine as an active ingredient and containing 2 uL to 6 uL vegetable oil per 50 mg of genestane as an adhesive. It relates to an edible drug delivery system. In addition, the present invention provides a method for improving meat quality of a livestock, comprising the step of subcutaneous transplantation on a livestock by applying the pelletized subcutaneous food delivery system.

1. Soybeans in Nutrition of Humans and Animals

1.1. Soybean Products and Soybean Food

Soybeans have long been consumed as a traditional food in Asia, while soybeans are still an important source of protein and dietary fiber, as well as a variety of plant chemicals such as isoflavones, phytic acid and saponins in Western countries. It is treated as unused food (Anderson and Wolf, 1995). Soy products, flour, soybean processed products, soybean tofu, and soybean fermented products are used as foods, and these foods are consumed by health-conscious people and are representative of the vegetarian diet. It is the main ingredient and is often used in infant formulas (Keinan et al., 2002).

Soy is a good source of protein with a balanced composition of amino acids and is used not only for human food but also for animal feed. Soy flour is commonly used as an efficient plant-derived protein in the animal feed industry. Soybean contains about 21% (dry weight) of oil, which is extracted and used for human food. High protein flour derived from soybean is a by-product of oil extraction, and economically, soy flour is typically heat treated to increase the value of the feed. Soy flour has a high crude protein content of more than 40% by content. Soy flour is rich in lysine and low in calcium, but contains a lot of phosphorus in most tests. Usually, more than half of the phosphorus in soy flour is in phytate form, which is produced by monogastrics. Incompletely used form. Soy flour usually contains vitamins at medium to moderate levels.

1.2. Soybean Plant Chemicals

Soy is high in protein and contains some minor ingredients such as trypsin inhibitors, phytic acid, saponins and isoflavones. These compounds are thought to have beneficial biological effects in food, such as lowering cholesterol in the blood and preventing cancer.

All soybeans are reported to contain 17-27 mg of trypsin inhibitor per gram based on 19 varieties and varieties (Hafez, 1983). Trypsin inhibitors are one of the antinutritional factors (ANFs) and trypsin inhibitors, proteins, are prone to degeneration and inactivation by heat.

Whole roasted soybeans have trypsin inhibitors at levels of only 8-9 mg per gram of flour or about 16 mg per gram of protein (Anderson and Wolf, 1995). Oriental soy foods have a low trypsin inhibitor content, and tofu is made by heating the soy milk to agglomerate the protein with salts such as calcium sulfate and then cool it, containing 9 mg of trypsin inhibitor per gram of protein.

Phytic acid is known to interfere with the absorption of minerals, especially zinc. However, much evidence suggests that phytic acid may have anticancer properties. Soy phytic acid content can vary widely. Field type cutivar, a common trade item, contains 1.0-2.3%, and the phytic acid content of oriental soybean foods such as soymilk and tofu is 0.5-1.2% okara tempeh. Except 1-3 (Anderson and Wolf, 1995).

Soy saponin has been reported to have hypocholesterolemia and anticancer effects (Messina and Barnes, 1991). Soy saponin is a complex compound consisting of a nonpolar triterpenoid alcohol aglycone linked to one or more polar oligosaccharides to form molecules with amphiphiles such as detergents. Soybeans typically contain 0.1-0.5% saponin, and saponin content in oriental foods such as soymilk, tofu skin, tofu and natto is 0.3-0.4%.

The most interesting soybean component is isoflavones. It is considered a phytoestrogen and is important for the health of humans and animals.

1.3. Antinutritional factors (ANFs)

Soy flour is commonly used as an efficient source of protein in the animal feed industry. However, various antinutritional factors (ANFs) such as trypsin inhibitors, lectins, and soybean globulins in soybeans limit the widespread use of soy flour as feed for animals, especially young animals (Li et al., 1990). Soy flour sensitization and subsequent feeding of powder-containing feeds can cause digestive problems such as impaired digestive movements and inflammatory reactions in the intestinal mucosa of pigs (Newby et al., 1984). In another study, pigs fed foods containing soy flour before weaning and foods containing soy flour after weaning showed excessive hypersensitivity to soy protein (Le et al., 1990). This response appears to be due to the presence of glycine in the soybean and antigenic proteins such as betacoglycine.

2. Isoflavones

2.1. Phytoestrogens and Isoflavones

Phytoestrogens are natural products with estrogen-like structures and activities derived from plants. Phytoestrogens are a group of biologically active plant substances with structures similar to estradiol, an endogenous estrogen. This structural similarity causes these compounds to bind to estrogen receptors in a variety of cells and exerts estrogenic or antiestrogenic effects in humans and animals. Phytoestrogens can be used as dietary estrogens and are chemically classified into four groups as shown in FIG. 1.

2.2. Classification of isoflavones

Soy isoflavones include aglycone genistein, daidzein, and glycidin, as shown in FIG. Aglycone is bioavailable as a non-sugar compound that remains when the glycosyl group is replaced in the glycosides by the hydrogen atom. The well-known isoflavones, Genestain and DydeZane, are found in plants like other isoflavones and are inactive glycosides (Tomasz and Leszek, 2005). Genestain is known to have a higher binding capacity than other derivatives and has a higher affinity than ERα in ERβ (Kuiper et al., 1997).

Formononetin and biochanin A are subclasses of isoflavones, such as genesteine, dydzein and glycidane. Equol, O-DMA (O-desmethylangolensin) is a metabolite of dydzein, 6'-Hydroxy-O-demethylangolensin, 6'-OH-Dma ) Is a metabolite of genenestein.

Of these isoflavones, the four isoflavones, Bioshanin A, Pomononetin, Genestain and Dyzedine, are considered important phytoestrogens. Their chemical structure is as shown in FIG. Most of the isoflavones that occur in the human body are genestains and dyedzeins. Methylated pomononetin and bioshanin A undergo demethylation in the intestine and are converted to dyed zein and genestain, respectively (Park, 2005).

2.3. Metabolites of Isoflavones

Glycosides, the inactive form of isoflavones in the intestine of animals, are metabolized into biologically active aglycones and absorbed in the intestinal tract. Hydrolysis of isoflavone glycosides is necessary for absorption from the intestines, and there is strong evidence that no glycosides were detected in the plasma. Although glycosidase enzymes are produced in the small intestine and symbiotic with the intestinal microflora, it is known (Day et al., 1998). The site of hydrolysis of glycosides is not yet clear (Day et al., 1998).

Aglycone performs further metabolic processes, and as shown in FIG. 4, genestain is composed of p-ethyl phenol (p-ethyl phenol) and 4-hydroxypebyl-2-phosphonic acid (4-hydroxyphebyl-2- porpionic acid), and Dyzedine is reduced to equol and O-DMA (O-desmethylangolensin). There is a marked variation between individuals in the production of certain metabolites, in particular equol and O-DMA. For example, Lampe et al., 1998 found that only about 35% of subjects secreted an effective amount of equol after soybean ingestion.

Processes for soy foods increase the net degradation of glycosides and increase the concentration of aglycone (Zhou and Erdman, 1997; Hutchins et al., 1995). Aglycones are absorbed faster in the gastrointestinal tract than glycosidic bonds. Aglycones derived from hydrolysis by intestinal bacteria from glycosides are absorbed in the large intestine, glucornidated and sulfated in the liver, perform the enteric cycle and are first excreted from the urine.

2.4. Dietary Isoflavones

Phytoestrogens are found in plants including legumes, seeds and grains consumed by various humans and animals. The most abundant source of isoflavin is soybean and soybean products. Soybeans have been treated as an important component of food in Asia for centuries, and soybean and soybean constituents, Genesstein and Dyzein, have been widely consumed without special side effects. Soy isoflavone content depends on the type of soybean, the geographic area of cultivation, and the harvest year (Wang et al., 1994-a; Axelson et al., 1984; Coward et al., 1993). The content of isoflavones also depends on the process method (Wang et al., 1994-b). In soy, isoflavones tightly bind to proteins. The protein content of soybean is 36% by weight (Haytowitz et al., 1986), and the nutritional value of soy protein is roughly equivalent to the high biological value of animal proteins (Young et al., 1991).

Processed soy protein and food provide genestained and dyedzein in varying amounts of bound glycoside form or aglycone form. Several studies have shown that commercially available soy products, such as matured and roasted soybeans, soy flour and textured protein, contain 0.10-0.5 mg isoflavones per gram of protein. Green soybean and temp are also sources of isoflavones, providing 0.3 mg isoflavones per gram of soy protein as shown in Table 1. Traditional soy foods provide 0.25-40 mg of isoflavones. Soy tofu isolated from soy protein and soy milk provide 0.1-2.0 mg of isoflavones per gram of soy protein. Other kidney beans, lentils, peas, and clover contain very little isoflavones. Red shamrock is a legume plant that is used for cattle, horses, and sheep, and its leaves and young shoots are used worldwide. Isoflavones isolated from red shamrock are not as effective as the entire red shamrock plant, but are used to treat menopause and also to treat hot flashes, cardiovascular health and bone loss (Howes et al., 2000).

Isoflavone Content in Soybean Products (ug / g) Soybean products Total isoflavone content Genestain Dyed Jane Glycinein Roasted soybean 2661 1426 941 294 Soy-protein isolate 987 640 191 156 Tempe 865 422 405 38 tofu 532 245 238 49 Protein concentration ¹ 73 19 0 54 soy milk 28 21 7 -

¹ alcohol extract.

3. Biological Activity of Genestain

Genestane is one of the important isoflavones, and has received great attention as a phytoestrogen with pharmacological effects of estrogen and antiestrogenic. Several studies of the uptake and distribution, metabolism and secretion of genestine have been studied in mice and humans (Piskula et al., 1999; Coldham and Sauer, 2000; Mallis et al., 2003).

Although Genestain is rapidly absorbed in the small intestine, it is poorly soluble in water and has low biocompatibility. During the circulatory process, genes are absorbed in the form of glucuronide and several molecules of sulfate conjugates, free genistein, and protein bonds. The most basic metabolites of Genestain are Genestain Gluconide and Genestain Sulfate. Metabolism occurs in epithelial cells of the liver and intestinal walls (Coldham and Sauer, 2000).

Genestain has been known to have a variety of beneficial effects on human and animal health. This function is via an estrogen receptor dependent pathway or an estrogen independent pathway (Szkudelska et al., 2002). There have been studies of several genes, but studies supporting such effects are still lacking, and most of the mechanisms are unknown.

3.1. Estrogen effect

Estrogens play an important role in bone maintenance (Turner et al., 1994; Riggs et al., 2002). In postmenopausal and postmenopausal women, a decrease in estrogen content is accompanied by various problems such as hot flashes, depression, osteoporosis, weight gain and slow motion. As a treatment method, hormone replacement therapy (HRT) has been used to treat estrogen deficiency symptoms, and as phytoestrogens, genestane may be a potential candidate for HRT.

Genestain is expected to act as an ERβ-selective competitor and binds to both ER isoforms with moderate affinity but preferentially binds to ERβ and is somewhat similar to estradiol (Pike et al., 1999). Evidence has recently been reported that genes play a role in bone protection, and this activity appears to be due to ERα-dependent mechanisms (Hertrampf et al., 2007). Furthermore, physical activity has a great impact on the bone protective effects of genestain (Hertrampf et al., 2007).

The loss of circulating estrogen after ovarian surgery results in an increase in fat weight. Genestain also inhibits fat gain after ovarian resection in young adult mice and rats (Mohamed and Abdel-Rahman, 2000; Uesugi et al., 2001; Nazz et al., 2003). Estrogen production and circulation levels in other species of men are lower than in women (Kelch et al., 1972). However, adipose tissue in men, like women, expresses estrogen receptors, and further, estrogen treatment reduces the size of adipocytes in male rats (Pederson et al., 1991).

The estrogen receptor signaling system has been studied to be important for the accumulation of white adipose tissue in male mice. Estrogens act on adipocytes, reducing the size of adipocytes and reducing the number of adipocytes (Cooke et al., 2001). These findings suggest that genesis acts on the white adipose tissue of male mice. The effects of genestain on animal tissues are related to the metabolism of lipids, as discussed below.

3.2. Anticardiovascular effect

Genestains lower cholesterol and protect the heart. It is not known how soy protein and other components of soybeans, including genestain, lower cholesterol and lipids. Because hormones regulate the metabolism of lipids, they have the potential to affect the endocrine system (Ali, 2004).

Soy protein, including isoflavones, has several beneficial effects on cardiovascular health. In a meta-analysis study, an average of 47 grams of soy protein containing Genestine daily reduced 9.3% total cholesterol, 10.5% triglycerides, and 12.9% LDL. (Anderson et al., 1995). Furthermore, two meta-analyses on the human lipid profile of isoflavones in soybeans, including genes, show that ingestion of soy protein results in total serum cholesterol, LDL cholesterol, and triglycerides, respectively. Lowering 9.3%, 5.3-12.9% and 7.3-10.5% and raising HDL cholesterol by 2.4-3.0%. This beneficial effect has been used as a diet for the treatment of hypercholesterolemia patients (Jenkins et al., 2003; Merritt, 2004). The total effect of soy protein on lipid metabolism was observed when soy protein contained isoflavones, indicating that both components are essential for reducing lipids in the blood (Carroll, 1982).

Diabetes mellitus is a complex metabolic disorder, which includes abnormal changes in the secretion and activity of insulin resulting in changes in the endocrine system. Endogenous glucose production leads to a change in glucose tolerance and causes hyperlipidemia problems. Type 1 diabetes (insulin-dependent) and type 2 diabetes (non-insulin-dependent) are problematic in lipid metabolism, which further increases the risk of premature cardiovascular disease. Insulin resistance is a common feature of obesity, so diabetes, obesity, and cardiovascular disease are correlated. There is increasing evidence that isoflavones, including genestains, have a beneficial effect against obesity and diabetes (Bhathena and Velasquez, 2002).

In studies of genetically obese mice, Aoyama et al. Have found that soy protein isolates (Genestain, hydrolysates of Genestain) are more efficient than Whey protein isolates in weight loss and plasma glucose concentrations. Reduction of body fat by soy protein isolate and its hydrolyzate was compared with casein in rats fed high-fat foods and genetically obese yellow KK mice (Aoyama et al., 2000). Plasma glucose decreased more than soy protein isolate and soy protein hydrolysate. These findings demonstrate that Genstein has an anti-obesity effect in obese animals.

Many human and animal studies suggest that soy isoflavones have a good effect on diabetes. Therefore, the effects of soy isoflavones, including most genes, on type 1 diabetes are being tested in experimental animals. In a study of type 1 diabetes in BB (BioBreeding) rats, Atkinson et al. Reported that for foods containing a mixture of animal and vegetable proteins and for foods containing soy protein containing genesis, The incidence frequency and whether delaying the onset of urination were compared. In a similar study, there was a study on whether soy protein with or without nicotinamide prevented the development of diabetes in diabetic mice that were not obese (Reddy et al., 1995).

Further studies of type 2 diabetes in Hersensen et al. Found that soy protein and fibrin with genestein lower LDL cholesterol and deficiency fat protein B- compared to those fed with casein with cellulose. 100 and triglycerides (Hermannsen et al., 2001). Another study (Vedavanam et al.) Predicted that soy isoflavones are beneficial for diabetes, preventing glucose uptake in the small intestine by preventing estrogen activity, peroxidation of their glucose-induced lipids, and reducing the number of sodium-dependent glucose carriers. prevent. It has been reported to reduce postprandial hyperglycemia (Vedavanam et al., 1999).

3.3. Anticarcinogenic effect

Some studies have reported that biologically active isoflavones such as genestain and dyedzein prevent the risk of breast, colon and prostate cancer (Martin MP et al., 1987; Lamartiniere et al., 1995-a, b; Wang and Kurzer, 1997).

Isoflavones inhibit aromatase activity and reduce the risk of estrogen dependent cancers (Adlercreutz et al., 1993; Wang et al., 1994; Campbell and Kurzer, 1993). Aromatase is an enzyme involved in the production of estrogens that catalyzes the conversion of testosterone (androgen) to estradiol (estrogen).

Although the physiological properties of isoflavones are recognized to make them potential as chemoprotectors in various types of cancers, usually early breast cancers (Messina et al., 1994), this is considered the antiestrogenic effect of genenestein ( Sarkar and Li, 2003). This potential role for breast cancer is attributed to epidemiological and erosive research in the early stages of sensory life, which shows the historically low incidence of breast cancer and the association between soy intake and reduced risk of breast cancer in Asian regions where soy foods are high. It may be one.

3.4. Other effects

Although some research has been conducted on the beneficial effects of soy isoflavones on various age-related diseases, little is known about the function of genestain. Injury of various types of nerves, including the consequences of aging, leads to neurorodegenerative disease with decreased cognition. Several studies have found that isoflavones from soybeans containing genes improve cognitive function in humans and mice, but the mechanism is unknown. However, because genes have an estrogenic effect, it is expected that genes enhance cognitive function by mimicking the effects of estrogens in the brain (Anderson et al., 1995; Zhan and Ho, 2005), (Jenkins et al., 2003).

Genestane is a type of phytoestrogen, so there has been a study on reproduction stability. Like most hormones, the effects of genesteine are cell type specific, agonistic or antagonistic, and dependent on the tissue and dosage investigated. In the uterine weight test and the estrogen receptor binding test, genes are known to be estrogen (Lamarticiere et al., 1998). The offspring of rats injected with Genestain at 16-20 days of gestation had a low birth weight, short urogenital length, and delayed vaginal opening (Levy et al., 1995). These effects were different compared to the neonatal treatment group, suggesting that the differences between high (25 mg / day) and low (5 mg / day) doses play the role of agonists and antagonists (Levy et al. , 1995).

Recently, in vitro and in vivo Genenstein, including in vitro experiments in embryo culture to assess developmental stability, have been used to study embryonic and fetal development of mice. Indices (McClain et al, 2007). There was no potential to cause malformations upon oral administration of large amounts of Genestain at 1000 mg / kg / day. In in vitro culture tests, Genestain had high teratogenic potential, but this was not expected in in vivo studies (McClain et al, 2007).

Some researchers have focused their research on the prevention of kidney disease (Setchell, 1998; Messina 1999). Geneticin's protein tyrosine kinase inhibitor activity and the activity of immune modulators were investigated through studies (Sorenson et al., 1994; Zhang et al., 1999). It is anticipated that the activity of this genegenstein is performed without binding to the estrogen receptor. It has been claimed that ingestion of genegenstein has a beneficial effect on kidney function. Other research suggests that soy protein has a role in reducing kidney damage and inflammation through other mechanisms, independent of the estrogen receptor-associated pathways involving soy isoflavones (Kotsopoulos et al., 2000; Anderson et al., 2001). Predicted (Tovar et al., 2002).

The major isoflavones, known as genostein and dyedzein, are predicted to reduce glomerular damage by preventing the oxidation of LDL molecules during nephropathy (Fori et al., 2005). Because isoflavones contain phenolic rings, they react competitively with hypochlorous acid and dinitric acid, reducing chlorination and nitrification of proteins and preventing glomerular damage (Tovar et al., 2002; Pedraza-Chaverri et. al., 2004). Furthermore, isoflavones from soybeans containing genes have a beneficial effect on the function of the kidneys and, in nephropathy in patients with or without diabetes, reduce the glomerular filtration rate and increase the action of removing creatine ( Bilo et al., 1989; Azadbkht et al., 2003).

Experimental evidence predicts that protein tyrosine kinases play a role in regulating insulin secretion in pancreatic β cells (Srenson et al., 1994). Several in vitro studies using genostein of isoflavones as protein tyrosine kinase inhibitors have shown that the compound performs a variety of functions on the secretion of insulin from islet cells of the pancreas (Sorenson et al., 1994; Hisatomi et al., 1997; Verspohl et al., 1995; Jones et al., 1994). For example, in cultured Langerhans islet cells, genegenin (100 umol / L) plays a role in increasing the secretion of basic insulin, but such doses of genegenin play a role in reducing the proliferation of islet cells (Szkudelska et. al., 2000). In a further study, genesine did not affect glucose metabolism and inhibited the activation of islet tyrosine kinases and the secretion of glucose and sulfonylurea-stimulated insulin (Szkudelska et. al., 2000).

Genesteine acts as a regulator of immunity in animals by inhibiting the activity of tyrosine kinases. In vivo, genestain elicits potential immunomodulatory effects, both positive and negative. Low concentrations of ex vivo genomestein induce large natural killer cell activity in cell activity (Zhang et al., 1999) and induce replication and adhesion of antiviral agents (Yura et al., 1993). . High concentrations of exogenous genes in vitro inhibit the activity of tyrosine kinases and reduce the rate of macrophages, natural killer cells and phagocytosis (Steele and Brahmi, 1988). Inhibition of topoisomerase II lowers T lymphocytes and B lymphocytes production (Chang et al., 1995).

Payne's work with isoflavones supplemented with pigs focused on the hormone-like functions of isoflavones that affect growth and meat composition in pigs. Soy isoflavones reduce fat and increase greasy lean meat in stop-growing castration (Payne et al., 2001). However, in Cook's study, supplementation of isoflavones (1,585 mg / kg diet) increases carcass muscle but does not affect carcass fat until 6-32 kg of body weight. (Cook, 1998).

In a study by Greener, dietary intake of soybean genes at concentrations of 200 to 400 ppm, as an oral active immune modulator, enhances the elimination of systemic serum viruses and increases body growth (Greiner). et al., 2001).

4. Lipid Metabolism

Adipose tissue is a loose connective tissue composed of cells filled with lipids and is connected and maintained by blood vessels, collagen fibers, and fibroblasts. Brown adipose tissue can be found in mammals such as newborn human babies and rodents. It is important for metabolism of heat in the animal body by high mitochondrial concentrations. White adipose tissue, on the other hand, is the basic type of adipose tissue in humans and animals and allows for unlimited accumulation of triglycerides.

Although lipids have various forms and roles in metabolism in humans and animals, triglycerides play an important role in energy metabolism. After eating, the ingested fat is mixed with bile salts in the small intestine and spread in the form of micelles with bile salts and triglycerides. As shown in Fig. 5, these triglycerides are hydrolyzed by lipases to form free fatty acids (FFAs) and 2-monoglycerides (2-monoglycerides). These molecules are absorbed in the intestinal wall, triglycerides are then regenerated and wrapped in the form of chylomicrons for transport in the blood. Digestion of triglycerides wrapped by lipoprotein lipase (LPL) produces FFA and glycerol. Glycerol is returned to the liver for use in gluconeogenesis. FFAs have a more diverse fate and, depending on the body's energy balance, can be stored in adipose tissue, heart or musculoskeletal or oxidized by the liver, heart and musculoskeletal (Kenneth, 2005).

4.1. Biosynthesis of Fat

The synthesis of fat consists of the synthesis of fatty acids followed by the synthesis of triglycerides and occurs in the liver and adipose tissue. When eating foods rich in carbohydrates and low in fat, excess carbohydrates must be metabolized. Some amounts are immediately used as substrates, but most are stored as glycogen, fueling the biosynthesis of many molecules or converted to triglycerides. The conversion of carbohydrates to fat is known as de novo lipogenesis and de novo lipid biosynthesis is not the first step in the intake of residual carbohydrates, and an important response to increased carbohydrate intake is the increased storage of glycogen. Increase and oxidation of carbohydrates throughout the body. The main daily cycle, which consists of daytime carbohydrate storage and nighttime carbohydrate release, shows that daytime de novo lipid biosynthesis ( de no. novo lipogenesis). De novo lipid biosynthesis plays an important role, for example in immature children, at high carbohydrate intake in certain situations, such as during fetal development.

The predominant fate of excess carbohydrates is the storage of glycogen or increased oxidation. However, excessive glucose is also used to form triglycerides. Excess glucose is ingested in liver or adipose tissue and converted to pyruvate by glycolysis, as shown in FIG. 6, and acetyl-CoA (acetyl-CoA) by pyruvate dehydrogenase complex (PDC), a pyruvate dehydrogenase complex. Becomes And it is combined with oxalo acetate (oxaloaccetate) to form a citrate (citrate). Citrate is passed out from the mitochondria and is cleaved by ATP-citrate lyase to form oxaloaccetate and acetyl-CoA (acetyl-CoA).

Acetyl-CoA is the substrate for the synthesis of fatty acids in the cytoplasm. Acetyl-CoA carboxylase (ACC) converts acetyl-CoA to malonyl-CoA (irreversible reaction). This is the first step in fatty acid synthesis and the only reaction that takes place outside of the multi-enzyme fatty acid synthase complex (FAS). The acetyl and malonyl groups are converted to acyl carrier proteins (ACPs) by acetyl transferases and malonyl trensferases, respectively. Acetyl-ACP and malonyl-ACP are known to beta-ketoacyl synthase, also known as acyl-malonyl ACP condensing enzyme. It forms a complex and becomes butyryl-ACP through three steps such as reduction, dehydrogenation and reduction. The following cycle brings in more malonyl-ACP units until the last 16-carbonpalmitoyl-ACP is made. And palmitate is released. Palmitate undergoes desaturation and undergoes elongation reactions to form other fatty acids. In the liver, newly formed fatty acids are esterified to glycerol-3-phosphate, incorporated into low density lipoprotein (VLDL) molecules, and transported to adipose tissue for storage. Adipose tissue can synthesize fatty acids and triglycerides directly from glucose via this pathway (Kenneth, 2005).

4.2. Breakdown of fat

Adipose tissue is the main reservoir for triglycerides in humans and animals. When fatty acids are needed for fuel, hormone-sensitive lipase (HSL) in adipocytes hydrolyses triglycerides to obtain FFAs and one glycerol molecule. Once released into the bloodstream, FFAs bind albumin to be transported to other tissues and spread to cells in other tissues.

Fatty acids are important fuels for the body. In fasting or starvation, and in the predominant substrate of the heart muscle, it supports aerobic exercise by the musculoskeletal system. Glycerol produced by hydrolysis of triglycerides is absorbed by the liver, and glycerol is used for glucose neosynthesis.

Fatty acids secreted from stored triglycerides of adipose tissue are regulated by reversible phosphorylation by HSL enzymes. When starving or exercising, HSL is activated through cAMP-dependent protein kinase (PKA) -mediated phosphorylation. As shown in FIG. 7, depending on the physiological state, the first hormone can be glucagon, epinephrine or beta corticosteroids, which stimulate cAMP production and PKA activation. High levels of insulin in foods dephosphorylate, deactivate, and prevent fat movement (Kenneth, 2005).

4.3. Genes associated with lipid metabolism

4.3.1. Genes Associated with Adipogenesis

Growth and development of muscle and fat are regulated by incompletely understood mechanisms for communicating information between complexes and cells (Hausman et al., 2001; Sorisky et al., 1999). In adipocytes, fibroblast-like cells, adipocytes are converted into lipid-rich adipocytes through development and differentiation (Hausman et al., 2001). During the differentiation of adipocytes, other markers are expressed when they reach the last morphological stage of the cell (Sorisky et al., 1999; Boone et al., 2000). Important regulators in the process of differentiation are insulin, glucocorticoids, and many other hormones (Sorisky et al., 1999; Boone et al., 2000). Muscle tissues and cells perform controlled growth, perform differentiation processes, as well as use of substrates and energy partitioning (Brooks et al., 1998; Hocquette et al., 1998).

The first step in adipogenesis is to commit progenitor cells to adipocyte lineage, after which they cannot return to less differentiated, stem cell-like cells (Boone et al., 2000; Thompson et al. al., 1998). After the first step, the adipoblasts perform replication exponentially and terminate the cell cycle at G1. Markers of early differentiation, such as lipoprotein lipase (LPL), are then expressed and the cells known as adipocytes undergo more cell proliferation (Boone et al., 2000). After adipocytes stop proliferating, the last signs of differentiation, such as glycerol-3-phosphate dehydrogenase (GPDH) and fatty acid synthase (FAS), are detected. And cells begin to accumulate lipids in the cytoplasm. The cells at that time are called adipocytes (Boone et al., 2000).

Furthermore, Ap2 is expressed during differentiation as an intracellular fatty acid binding protein. Fatty acid binding protein (FABP), a fatty acid binding protein, and fatty acid transferase (FAT), a fatty acid transferase, are also expressed during differentiation. And these molecules deliver fatty acids to adipocytes and subsequently accumulate lipids (Boone et al., 2000; Zimmerman et al., 2002).

4.3.2. Genes involved in lipid biosynthesis and degradation of lipids

In humans and animals, triglycerides play an important role in energy metabolism. Through metabolism of triglycerides, the wrapped triglycerides produce FFAs and glycerol by LPL (lipoprotein lipase). Glycerol goes back to the liver and is used for glucose biosynthesis. FFAs are stored in adipose tissue or in the heart or musculoskeletal system or are oxidized in the liver, heart and musculoskeletal system. Therefore, LPL plays an important role in metabolism of triglycerides.

The conversion of carbohydrates to fats is known as de novo lipid biosynthesis. It consists of the synthesis of fatty acids followed by the synthesis of triglycerides. Malonyl-CoA, the final stage of fat production, uses fatty acid synthase (FAS) to form fatty acids and triglycerides. In animal tissues, FAS is considered a direct marker for the synthesis of fatty acids. Under certain conditions, such as fasting, humans and animals need energy from stored fuel. In fat cells, fatty acids are needed for fuel, and in fat cells, hormone-sensitive lipases (HSLs) hydrolyze triglycerides into three free fatty acids (FFAs) and one glycerol molecule. Thus, the activity of hormone-sensitive lipase HSL (hormone-sensitive lipase) is an important factor in the breakdown of fat.

5. Metabolism of Genestain and Lipids

Many researchers have found that isoflavones, especially genegenes, are linked to glucose homeostasis and lipid metabolism. Human and laboratory animals have been investigated for their effects on the metabolism of glucose and lipids and on the regulation of hormones by the ingestion of soy-containing foods. Genestain has a direct effect on liver and adipose tissue. For example, in isolated liver specimens, the binding of [ ¹⁴ C] glucose to lipids is reduced by Genestain and the action of releasing fatty acids into the medium (Nogowski et al., 1998). This change is accompanied by a decrease in the content of triglycerides in the liver. Similarly, in cultured adipocytes of isolated mice with increasing doses of genestein (0.01, 0.3, 0.6 and 1 mmol / L), the conversion of [14C] acetate to lipids in both basic and insulin stimulating conditions Significantly reduced.

Genestain (0.1, 1 mmol / L) increases basal lipolysis and further increases lipolysis at low concentrations (0.01 mmol / L), which is stimulated by epinephrine of adipocytes. Thus, Genestain affects the metabolism of lipids in straight liver and adipose tissue by directly affecting the synthesis and degradation of lipids. This activity demonstrates that Genestain has a useful biological activity in the metabolism of glucose and lipids, which has a good effect on obesity and diabetes.

A recent study (Naaz et al., 2003) found that feeding gentane to ovarian depleted mice reduced the fat layer weight by 37-57% depending on the dose and also decreased LPL mRNA (lipoprotein lipase mRNA). There is a bar. In another study, in vitro and in vivo conditions, genestain induces apoptosis of adipocytes, and accounting for a small part of weight loss is the removal of fat cells, which allows them to sustain weight loss continuously (Kim et. al., 2006). Phytoestrogens, including isoflavones, affect lipid metabolism in the liver and musculoskeletal system, and alter lipid parameters in the plasma (Nogowski, 1999).

In the cells of the musculoskeletal system, genegenin inhibits glucose uptake stimulated by uncoupling protein 3 (Huppertz et al., 2001). The lipogenic and antilipogenic effects of genenestein are not clear and still controversial. Some studies have predicted that the estrogenic effects of genenesine depend on the concentration of genenesine (Wilson et al., 2004), endogenous estrogen levels (Ratna, 2002), and sex (Faughnan et al., 2004). ).

In in vitro studies, low concentrations of genesine bind efficiently to estrogen receptors, although they bind to ERβ with high affinity (Kuiper et al., 1998). At high concentrations it has been reported that genestain acts as a tyrosine kinase inhibitor (Huang et al., 1992; Hong et al., 2005), antioxidants (Hwang et al., 2003), and steroid-metabolase modulators. (Atkinson et al., 2003). In addition, high concentrations of genenesine inhibit the activity of estrogen receptors by activation of nuclear receptors such as the peroxisome proliferator activated receptors (PPARs) (Dang et al., 2004).

6. Improving Productivity of Economic Animals

Various efforts have been made to increase the productivity of livestock, and in particular, various studies have been conducted to improve productivity and high added value of economic animals. In the case of pork and beef among livestock products, carcass grades are determined according to meat quality, so researches on improving meat quality are ongoing. The biggest factor affecting meat quality is the accumulation of intramuscular fat, which can improve the meat quality by increasing the accumulation of muscle fat, which is a edible fat, and reducing the accumulation of celiac fat, which is inedible fat.

In the case of pork and beef among livestock products, the carcass grade is determined according to the meat quality. Therefore, in order to improve the meat quality, it is necessary to increase the accumulation of intramuscular fat and reduce the accumulation of celiac fat, which is an indigestible fat.

Conventionally used methods to increase livestock stocks in livestock include feed methods to increase energy supply and non-feeding methods to add synthetic hormones.

Various types of steroid hormones have been used to regulate animal growth and meat components, including glucocorticoids, leptin and anabolic steroids. Anabolic steroids used to control growth include testosterone, estradiol and progesterone. There is this. Androgen steroids such as testosterone promote the production of lean muscle, and estrogen steroids such as estradiol can improve meat quality by increasing the accumulation of muscle fat. However, the administration of synthetic hormone has a problem of causing residual problems of hormones, stability, and avoidance of consumers. For this reason, a method of replacing steroid hormones has been required to improve meat quality.

In addition, the feed method is safer than the hormonal input method, but there is a problem that can not control the metabolism of the livestock, resulting in over-accumulation of abdominal fat, which is an indelible fat, and thus waste of feed.

In the case of pigs and cattle raised for the purpose of fattening, males use males because they have better growth rate and muscle production than females. In this case, boars have a problem of dullness, which deteriorates the quality of livestock, and hydrogen has a rough nature. There is a problem of injuries of breeders and a decrease in the accumulation of internal fat. Therefore, in order to solve this problem, after raising the castration and breeding. However, castration has been a problem that causes a decrease in growth and a decrease in feed efficiency.

As salpin, genesine binds to estrogen receptors in the body and produces an estrogen-like effect. Estrogen, a steroidal female hormone, not only induces the synthesis of body fat, but also promotes the synthesis of bone tissue. Therefore, feeding genestane not only increases the growth rate of castrated livestock, but also increases the accumulation of intramuscular fat. Can induce an effect. However, there is a problem that it is difficult to maintain a constant concentration in the blood due to the limiting factors such as loss in digestion process, the limit of absorption rate when the gene is fed in the form of feed. In addition, when these expensive natural extracts are fed in the form of feed additives, economic problems do not fit.

The present inventors have led to the present invention as a result of intensive studies to overcome the problems of the feed and non-feed method that has been used to improve the meat quality of the conventional livestock as described above.

Accordingly, an object of the present invention is to improve the problems of stability and consumer choice according to the use of synthetic hormones for the meat quality improvement of livestock products, and to overcome the problems of economic efficiency and efficiency when feeding in the form of feed additives Genesstein It is to provide a composition for improving animal meat quality as an active ingredient.

It is another object of the present invention to provide a method for improving livestock meat by subcutaneous transplantation using the composition.

The object of the present invention is to measure the concentration of genes in the serum, observe that the concentration of genes in serum increases in proportion to the concentration of subcutaneous genes produced by the subcutaneous genes in the pellets Achieved by confirming proper secretion.

More specifically, the objectives of the present invention are to measure the effects of weight gain and feed intake of the dietary treatment group and subcutaneous transplantation group of genestain, and to confirm the high weight gain in the 100 mg genusstein subcutaneous treatment group, which is in the back muscles. It was confirmed that the result was similar to the crude fat content, and gene expression studies showed that the gene synthesis level of fatty acid synthase decreased in adipose tissue and increased in muscle tissue, and hormone-sensitive lipids in adipose tissue of 100 mg subcutaneous treatment group. The increase in the degrading enzyme gene was confirmed to be related to the mechanism of genesstein through the estrogen receptor, and to provide a method for improving the meat quality by confirming the increase in the expression level of the estrogen receptor in the muscle and the accumulation of fat.

The present invention is characterized by providing a composition for improving livestock meat as an active ingredient. The composition is preferably prepared by adding glucose and pressure-sensitive adhesive to pellets. In addition, when using the pressure-sensitive adhesive in the composition, the pressure-sensitive adhesive is preferably vegetable oil. In the embodiment of the present invention, although the jade oil is used as the pressure-sensitive adhesive, the same effect can be obtained by using rice bran oil, soybean oil, etc. which are commonly available from the viewpoint of those skilled in the art.

The present invention is also characterized by providing a method for improving livestock meat, characterized in that the subcutaneous transplantation by pelletizing with an active ingredient of Genestain. Preferably, the livestock meat improvement method may be carried out by subcutaneous transplantation to avoid further comprising glucose in gene stain.

The present invention provides a pelletized subcutaneous subcutaneous drug delivery system containing a natural soybean isoflavone genestane as an active ingredient, and has an effect of improving the quality of meat by increasing the accumulation of intramuscular fat, which is a edible fat in muscle tissue. It solves the problems of stability of the method of administering hormones to improve meat quality and avoidance of consumers, and solves problems such as loss in digestion and limitation of absorption rate when feeding expensive natural extracts in the form of feed. Since it is effective, it is an invention very favorable to the livestock and meat processing industry.

1 is a diagram illustrating the classification of phytoestrogens.

2 is a diagram showing the structure of isoflavones.

3 shows the chemical structures of the four important isoflavones.

Figure 4 shows the metabolic process of isoflavones in animals.

5 is a diagram illustrating the metabolic process of triglycerides.

Figure 6 illustrates the de novo lipid biosynthesis process.

7 is a diagram illustrating a process of lipolysis of fat.

8 is a diagram illustrating a mobile phase condition in HPLC analysis.

9 is a diagram showing the concentration of genegenstein in serum.

10 is a) a diagram showing the concentration of triglycerides in serum. b) Cholesterol concentration in serum.

Figure 11 a) shows the concentration of high density lipoprotein cholesterol (HDL cholesterol) in the serum. b) shows the concentration of LDL cholesterol in serum.

Fig. 12 shows a) expression of the Ap2 gene in the posterior peritoneal tissue, and b) shows the expression of the Ap2 gene in the back muscle tissue.

Figure 13 shows a) expression of the FAS gene in the posterior peritoneal tissue, and b) shows the expression of the FAS gene in the back muscle tissue.

Fig. 14 shows a) expression of LPL genes in posterior peritoneal tissues, and b) shows expression of LPL genes in back muscle tissues.

Fig. 15 shows a) expression of the HSL gene in the posterior peritoneal tissue, and b) shows the expression of the HSL gene in the back muscle tissue.

Fig. 16 shows a) expression of the ERα gene in the posterior peritoneal tissue, and b) shows the expression of the ERα gene in the back muscle tissue.

Fig. 17 shows a) expression of the ERβ gene in the posterior peritoneal tissue, and b) shows the expression of the ERβ gene in the back muscle tissue.

Figure 18 shows a) expression of the PR gene in peritoneal posterior tissue, and b) shows the expression of the PR gene in back muscle tissue.

Hereinafter, preferred embodiments of the present invention have been described in detail through examples, but the scope of the present invention is not limited to these examples.

In the following, ADFI means daily feed intake, ADG is weight gain per day, BW is body weight, F1 is Genenstein 500ppm diet, F2 is Genenstein 750ppm diet, F3 is Genenstein 1000ppm diet, and I1 is 100mg subcutaneous. Implants, I2 refers to the Genstein 200mg subcutaneous treatment, I3 refers to the Genstein 300mg subcutaneous treatment.

Example 1 : In genenestain  by Weight gain  And effects of feed intake

A 6-week-old white paper was cast and seven groups of rats were assigned to each group. After surgery, they were recovered and adapted to experimental conditions. They are kept in stainless steel wire cages with temperature (21 ± 1 ° C) and humidity (45 ± 5%) controlled and maintained to switch between dark cycles (9AM to 9PM) and light cycles (9PM to 9AM) for 12 hours. Reared. They allowed free access to water and the AIN-93G diet (see Table 2).

AIN-93G Dietary Component (g / kg) casein 200.000 Cornstarch 397.486 Dyetrose 132.000 Sucrose 100.000 Cellulose 50.000 Soybean oil 70.000 t-Butylhydroquinone 0.014 Salt mix 35.000 Vitamin mix 10.000 L-Cystine 3.000 Choline Bitartrate 2.500

Genetics were added to final concentrations of 0, 500, 750 and 1000 ppm for the dietary isoflavone free AIN-93G diet. Corn oil (corn oil) was added to the dried Genestain powder (50uL per 50mg Genestain), it was molded into 100mg Genestain pellets using a self-made extruder (30kg / cm2) shown in FIG. In addition, it is possible to produce a mixture of Genestain pellets powder and glucose in a ratio of 9: 1, and then add corn oil (corn oil) 4uL per 50 mg of Genestain.

One (100 mg), two (200 mg), or three (300 mg) of Genestain pellets were implanted subcutaneously in the nape of the rat on the first day of the experiment. The control group received a 1 cm surgical incision stress on the nape of the neck. The diet and transplant experiments lasted six weeks, respectively. Body weight was measured weekly during the experiment week, feed intake was measured once every two days. The mean starting weight was 240.4 ± 2.06 g, and the animal experiments were conducted according to the guidelines for management and use of experimental animals of Hanyang University.

In this example, gain gain decreased as feed dosage increased and was not significant (P <0.05). Surgical stress affected the growth of the animals, delaying growth for a week. It was not significantly influenced by Genestain during both the entire experimental week and 1-6 weeks, respectively. Daily weight gain (ADG) tended to be similar to that of the experimental group. There was no significant difference in the average daily feed intake, weight gain, and salary rate between the treatments.

As shown in Table 3 below, the effect of the weight gain by genestain did not appear significantly in all treatments. However, 100 mg of Genestain subcutaneous treatment group showed higher weight gain than the control group, which showed significantly higher weight gain than all treatments during the 1-6 weeks. In addition, there was no significant effect of the dosage according to the method of dietary or transplantation of genestain.

Effect of Genestain on the Growth of Castration White Papers Items C F1 F2 F3 I1 I2 I3 C-OS SEM ¹ Weight gain (g) 0-6 week 152.8 152.5 142.7 138.3 161.7 145.2 149.9 151.0 1.99 1-6 week 110.0 ^b 115.7 ^b 104.2 ^b 101.7 ^b 130.3 ^a 111.2 ^b 113.4 ^b 116.3 ^b 1.91 Daily gain (g / d) 0-6 week 3.638 3.631 3.397 3.294 3.849 3.456 3.568 3.595 0.05 1-6 week 3.143 3.305 2.976 2.905 3.724 3.176 3.241 3.321 0.06 Daily feed intake (g / d) 0-6 week 21.97 21.46 21.27 20.08 22.42 21.39 21.45 20.59 0.32 1-6 week 21.40 21.08 20.81 19.46 22.34 21.01 21.09 20.09 0.36 Salary 0-6 week 0.166 0.169 0.160 0.164 0.172 0.162 0.166 0.175 0.003 1-6 week 0.148 0.157 0.144 0.152 0.171 0.153 0.154 0.166 0.003

Example 2 : In serum Genenestain  density

Animals were anesthetized under carbon dioxide conditions and then punctured the heart to collect blood. Serum samples were obtained by centrifuging blood samples at 2500 rpm for 15 minutes and stored at -80 ° C until entering the assay.

Serum Genesstein analysis to determine the concentration of Genestain was extracted serum isoflavones by the Yellayi, 2002 method at -80 ℃. That is, 200 uL of serum sample was mixed with the same volume of acetonitrile, sonicated for 10 minutes, and centrifuged at 15,000 rpm for 10 minutes. The supernatant was mixed with sodium citrate buffer (25 mM, pH 5.0) containing 4 mL of 500 IU glucuronidase and sulfatase to release the binding of genenesine. After 4 hours of incubation at 37 ° C, the samples were loaded on an activated C-18 SPE column (Merck, USA) and washed with 10 mL of distilled water. The washed column was separated by dissolving in 1.4 mL of methanol and evaporated by vacuum centrifugation. And resuspended with 100 uL methanol for HPLC analysis.

Prepared samples were separated using a gradient solvent system by HPLC (Agilent 1200 HPLC, Agilent Technologies) equipped with a Nova Pack C-18 column (3.9 × 150 mm column, Waters). 0.5% acetic acid is mobile phase A and methanol is mobile phase B. The flow rate was 0.6 mL / min at 30 ° C., and the detection of Genistein was measured by absorbance of UV at 254 nm using a UV detector.

As a result, the concentration of genes in serum showed a dose-dependent tendency, as shown in FIG. Serum concentrations of genusstein in the 300 mg Genstein subcutaneous diet were median between the 500 ppm and 750 ppm Genstein diets, and the control group was treated with AIN-93G feed without phytoestrogens. The concentration of genes in soybean oil was below detectable limits, and feed could not be the cause of plasma genes in controls. Samples were analyzed by HPLC-MS.

The concentration of genes in serum was 1.22-3.63 uM measured by high performance liquid chromatography in the genes of subcutaneous subcutaneous treatment, and it was increased in proportion to the concentration of subcutaneous genes produced in the animal. It seems to be secreted properly. According to the experimental results, the Genstein subcutaneous transplantation treatment group can be used as an efficient and economical transportation system of phytoestrogens.

Example 3 : Serum Lipid Profile

Blood lipids were measured using Selectra E (Vital Scientific, Netherland), and total cholesterol, triglycerides, HDL, and LDL in serum were determined by blood chemical assay reagents according to the manual (Diasys, Germany). ). After digestion with lipoprotein lipase (LPL), triglycerides were quenched by quinoneimine and quantified by spectrophotometer. Similarly, total cholesterol was determined after hydrolysis and oxidation by cholesterol esterases, cholesterol oxidase, and peroxidase enzymes. Antigen-antibody responses were used to analyze HDL cholesterol by using HDL specific antibodies. A two-step method was applied to the LDL cholesterol assay, in which the LDL was selectively protected from enzymatic reactions. And LDL was destroyed and determined in the second stage.

As a result, as shown in Figure 10a, the concentration of serum triglycerides of F1 was significantly lower than F2, F3, control group. However, the concentrations of serum triglycerides in F2 and F3 were significantly higher than the control stress group (P <0.05).

Therefore, 500 ppm of Genestain diet treatment (F1) is effective in lowering triglycerides in the blood, and this result is a result of a higher interaction with ERs than F2 and F3. The concentration of triglycerides was not significantly different in the Genstein subcutaneous treatment group (I1, I2, I3). As shown in FIG. 10B, the change in serum cholesterol concentration was not significant.

As shown in FIG. 11A, the concentrations of HDL cholesterol in the 750 ppm Genstein treatment group (F2) and the Genstein subcutaneous treatment group (I1, I2, I3) were significantly higher than the control group (P <0.05). According to Figure 11b, the concentration of LDL cholesterol was significantly lower in the experimental group. This result is in agreement with other findings (Zhan and Ho, 2005). Genestain contained in complete soy protein acts as a naturally selective estrogen receptor modulator and promotes the efficiency of lipid metabolic pathways through the expression of estrogen receptor dependent genes by biological similarity with estrogens (Potter, 1995).

In other words, the genegen concentration used in the present invention significantly reduced LDL and increased HDL, but did not affect total cholesterol. In serum triglycerides, there was a significant decrease in 500 ppm dietary genes added, but there was no significant difference in subcutaneous groups.

Example 4 : Weight of adipose tissue

White adipose tissue (WAT) in the posterior peritoneum, white adipose tissue (WAT) in the epididymis, and back muscles were collected and stored at -80 ° C until frozen and analyzed by liquid nitrogen. White adipose tissue (WAT) in the posterior peritoneum and white adipose tissue (WAT) in the epididymis were weighed before freezing.

As a result, as shown in Table 4, the fat layer was not significantly affected by the treatment of Genestain, and the fat layer weight of the posterior peritoneum and epididymis was 500-1000 ppm of the Genestain diet treatment and 100-300 mg of the Genestain subcutaneous treatment treatment. There was no significant change according to the treatment of Genestain. However, the weight of genes treated with Genstein stain in the fat layer in the posterior peritoneum was lower than the control group (P> 0.05).

Effect of Genestain on Adipose Tissue Treatment ^a Posterior Peritoneal Fat (%) ¹ SEM ² Epididymal Fat (%) ¹ SEM ² C 2.902 0.076 2.266 0.070 F1 2.885 0.096 2.156 0.080 F2 2.619 0.095 2.363 0.164 F3 2.773 0.177 2.055 0.111 I1 2.896 0.144 2.268 0.108 I2 2.585 0.089 2.043 0.118 I3 3.046 0.208 2.037 0.060 C-OS 2.538 0.181 1.947 0.163

Example 5 : On the back  there is Crude  content

Crude fat content in the back muscles was measured by official methods by AOAC analysis (association of official analytical chemist). Muscle samples were ground in liquid form on liquid nitrogen, 500 mg of powdered muscle samples in filter paper weighed (W1) and dried at 100 ° C. for 6 hours. After cooling, the weight (W2) of the dried sample was reweighed and extracted with ether for 11 hours. After extraction the samples were dried at 100 ° C. for 3 hours and then weighed after cooling (W3). Crude fat content was calculated by the following equation.

Fat (%) = (W2-W3) × 100 / W1.

As shown in Table 5, there was no significant difference in the crude fat of the back muscle of the castrated rats of the castrated rats compared to the genes of the genusstein diet, but the crude fat content of the back muscle of the I1 group was the highest. This is in line with the highest gain in the I1 group. Crude fat content in the back muscles of the experimental group was significantly higher than that of the control group. However, the 1000 ppm Genstein diet treatment (F3) is excluded.

Comparison of Crude Fat Contents of Back Muscles in Gentian Treatment in Castration Rats Treatment ^a CF (%) ¹ SEM ² C 2.87 ^ab 0.16 F1 2.46 ^bc 0.12 F2 2.54 ^abc 0.14 F3 2.01 ^cd 0.15 I1 3.28 ^a 0.15 I2 2.32 ^bc 0.10 I3 2.60 ^abc 0.10 C-OS 1.35 ^d 0.12

Example 6 : Comparison of Expression of Lipid Biosynthesis-associated Genes and Expression of Hormone Receptor-associated Genes

For RNA extraction, tissue samples were homogenized on liquid nitrogen using a tissue riser (Tissue Lyser, Qiagen, USA), and total RNA was extracted according to the manual using TRIzol reagent (Invitrogen, USA). The extracted RNA was dissolved in DEPC-treated water and quantified by spectrophotometer. Pure total RNA was indicated by agarose gel electrophoresis, and cDNA was synthesized from a total of 500 ng of RNA using SuperScript II Reverse Transcriptase (Invitrogen, USA). Oligo (dT) primers were used for reverse transcription (RT) reactions and cDNA was stored at −20 ° C. until use.

Gene expression in rat tissues was examined by quantitative real time PCR. Primer sequences used for PCR are shown in Table 6 below.

gene primer AmpliconSize (bp) Ta ⁸ (℃) GenBank No. GAPDH F 5’-ggtctacatgttccagtatgactct-3 ’ 80 62 NM017008 R 5'-gttgatgaccagcttcccattct-3 ' Ap2 ¹ F 5＇-gacgacaggaaggtgaagagc-3 ＇ 138 60 NM_024406 R 5＇-gcctttcataacacattccacc-3 ＇ FAS ² F 5＇-ggaacaactcatccgttctctgtgt-3 ＇ 127 60 M76767 R 5＇-ggaccgagtaatgccgttca-3 ＇ LPL ³ F 5＇-agatggagagcaaagccctgc -3 ＇ 377 61 NM_012598 R 5＇-ggcagacactggataatgttgc -3 ＇ HSL ⁴ F 5＇-ctcctcatggctcaactccttcc-3 ＇ 467 61 X51415 R 5＇-aggggttcttgactatgggtg-3 ＇ ERα ⁵ F 5＇-gggcttccccaacaccat-3 ＇ 65 59 Y00102 R 5＇-cgtttcagggattcgcagaa-3 ＇ ERβ ⁶ F 5＇-tgctggatggaggtgctaatg-3 ＇ 82 59 U57439 R 5＇-cgaggtcgggagcgaaa-3 ＇ PR ⁷ F 5＇-gggcactggctgtggaatt-3 ＇ 77 59 NM_022847 R 5＇-catgcccgccaggatct-3 ＇

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and real-time PCR was performed on light cycler fast-start DNA master sheaves using the LightCycler 1.5 instrument system (Roche, Germany). Le Green I (LightCycler FastStart DNA Master SYBR Green I, Roche, Germany). The reaction included ₁ × SyBR Green reaction mix (1 × SYBR Green reaction mix, 4 mM MgCl ₂ ), 0.5uM forward and reverse primers, respectively, and the cDNA product. The PCR reaction is followed by denaturing cycles for 10 minutes at 95 ° C., followed by 45 cycles of annealing and extension. Fluorescence was quantified during the annealing process, and product formation was confirmed by melting curve analysis (65 ° C.-95 ° C.). The size of the PCR product was confirmed by 1% agarose gel stained with ethidium bromide, and the amount of target gene involved was measured using the following equation after normalization to GAPDH (Kenneth and Thomas). , 2001).

2 ^{-ΔΔ CT}

In order to investigate the effects on the peritoneal posterior fat and the back muscle tissue by the genestain treatment, the expression of lipid metabolism associated genes and the expression of hormone receptor associated genes were investigated using real-time PCR. Fold induction of each experimental group was made in comparison with the control group.

Fat cell Binding protein  ( Ap2 , Adipocyte binding protein  2) expression of genes

Ap2 gene expression of posterior peritoneal fat did not have a significant effect in the treatment with genestein as shown in FIG. 12A. However, mRNA levels of Ap2 in F2, F3, I1 and I2 groups showed similar or slightly higher tendency than the control group.

As shown in FIG. 12B, the expression of Ap2 gene in the back muscle tissue was significantly higher than the control group as well as the other groups. Ap2 is a protein that binds intracellular fatty acids and is expressed upon cell division. Thus, the increase in the expression of Ap2 gene means the division of adipocytes, active in the F3 group.

Fatty acid synthase FAS ( Fatty acid synthase Expression of genes

As shown in FIG. 13A, the expression of FAS mRNA was decreased in the treatment of genestain regardless of the treatment method of genestain. Although the expression of FAS mRNA in adipose tissue was significantly higher than that of the other groups in the 500 ppm Genestain diet (F1), it was still lower than the control group. Genestain reduces the expression of FAS in the peritoneal adipose tissue and has an antilipogenic effect. The expression of FAS in the genusstein treatment of back muscle tissue was significant and highest in the F3 group (P> 0.05). The data showed similar trends as Ap2 mRNA data in the back muscle of castration rats.

Lipoprotein  Degrading enzyme LPL  ( lipoprotein lipase Expression of genes

As shown in FIG. 14A, the expression of the LPL gene in the genus subcutaneous subcutaneous treatment group was the same or lower than that of the control group. In the I2 group, the expression of LPL mRNA was lower than that of the Genstein diet (F1, F2, F3). These results are consistent with the Naaz (2003) report. In the Naz study, genes treated with degenerated ovary reduced LPL mRNA by 60-70% compared to controls. The 80 mg / kg BW · d used in this study was higher than the amount used in this study. Recent studies have shown that estradiol inhibits LPL mRNA, and the antilipidic effects mediated by changes in LPL are associated with adipose tissue. It is an important mechanism. Genesteine reduces LPL mRNA in adipose tissue, and therefore one of the important roles in genome's adipose tissue involves inhibiting LPL, such as estrogen.

As shown in FIG. 14B, LPL mRNA levels of the F3 group were higher in the back muscle tissue than in the other groups. In terms of fat formation in muscle tissues, the Ap2, FAS and LPL genes associated with adipose formation and lipid biosynthesis were increased in F3 compared to other groups. However, the F3 group is not only affected by fat formation and lipid biosynthesis, but also by lipolysis in the back muscle, and is examined in the following examples.

Hormone sensitivity Lipase HSL ( Hormone sensitive lipase Gene expression

As shown in FIG. 15A, the expression of the HSL gene was higher in the I1 and F3 groups than in the other controls. In the I1 group, HSL mRNA was increased by three times compared to the control group, and the low amount of Img, a 100 mg genesine transplant, affected the lipolysis in adipose tissue.

As shown in FIG. 15B, the HSL mRNA levels of the F3 group in the back muscles were significantly higher than in the other groups. HSL is a lipolytic enzyme and therefore lipolysis was active in the back muscles in the F3 group. However, in F3, as shown in the above example, Ap2, FAS, and LPL genes related to adipose formation and lipid biosynthesis were increased in the F3 group compared to other control groups. Therefore, the F3 group not only affects fat formation and lipid biosynthesis, but also affects lipolysis of the back muscles. These results need to be compared with the crude fat content in the back muscles.

As shown in Table 5, the crude fat content in the F3 group was significantly lower than the control group. Therefore, the experimental group exposed to 1000 ppm of genesine affects lipid metabolism, and in particular, it affects the lipolysis and lower crude fat content of the back muscle of castration rats.

There is a lack of research on genestein in muscle tissue, and researchers have found that dietary intake of genesteine at a concentration of 50-200000 ug / kg / day in young and healthy male or female rats has not altered the musculoskeletal system. (Penza et al., 2006).

In the present study, the F3 group affected lipid metabolism-associated mRNA levels and reduced crude fat content in muscle tissue in castrated rats. However, other genes did not affect the expression of lipid metabolism related genes in the back muscles.

ER α ( Estrogen receptor alpha Gene expression

The ERα mRNA of the peritoneal posterior fat of the castration rats was not significantly changed during the genestein treatment as shown in Fig. 16A. However, the I1 group showed higher ERαmRNA levels than the other groups.

The ERα mRNA levels of the F3, I1 and I2 groups in the back muscle tissue were similar or slightly higher than those of the control group as shown in FIG. 16B, and the expression of these groups of genes was much higher than that of the control stress group. Therefore, ERα binding affinity of group I1 is thought to be similar or higher in muscle and adipose tissue than in control group.

ER β ( Estrogen receptor beta Expression of genes

The ERβ mRNA levels of posterior peritoneal fat in castration rats were similar or higher than those in the control group, as shown in FIG. 17A. Genestain has a higher binding affinity for ERβ than for ERα in adipose tissue (Kuiper et al., 1997). In this study, ERα mRNA levels in adipose tissue were higher in the I1 group than in the control group. However, ERβ mRNA levels were higher in most groups. In addition, the expression of the ERβ gene was higher in I1 than in the other groups.

In the back muscle, the expression of the ERβ gene shows a similar tendency to the expression of the ERα gene, as shown in FIG. 17B. That is, in the F3, I1, and I2 groups, the level of ERβ mRNA was higher than that of the other groups, and only the I1 group had higher ERβ mRNA levels than the control group.

PR ( Progesterone receptor Expression of genes

The expression of PR mRNA of I1 in adipose tissue was higher in the peritoneal adipose tissue than in the other groups as shown in FIG. 18A. These results are consistent with the expression of ERα and ERβ genes was significantly higher in I1 than in the other groups. The expression of this hormone receptor gene is associated with the expression of the HSL gene because the I1 group showed high expression in the mRNAs and HSL mRNAs of the three hormone receptors. Thus, in the I1 group, genestein binds to ER, ERβ, which upregulates HSL mRNA. This phenomenon appears to affect lipolysis in the posterior peritoneum, although the weight of the fat layer in the I1 group did not significantly decrease compared to the control group.

PR mRNA levels in the back muscles are not significantly affected by the generation of genes. However, as shown in FIG. 18B, the expression of the PR gene was relatively higher in the F3, I1, and I2 groups than in the other groups. This tendency is very similar to the expression of ERα and ERβ genes in muscle tissue. Unlike adipose tissue, the expression of HSL genes in muscle tissue is not necessarily associated with hormone receptors. As shown in FIG. 15B, the HSL mRNA levels in the back muscles of the F3 group were significantly higher than those of the other groups. The ERα, ERβ, and PR mRNA levels of the F3 group were not significantly higher than those of the other groups. Seemed. Thus, lipolysis in muscle tissues appears to be less affected by genesine through hormone receptors than lipolysis in adipose tissue.

As salping in the above examples, genesis did not significantly affect the peritoneal, epididymal fat weight of castrated rats. However, the fatty acid synthase (FAS) gene levels of the genes treated group were significantly lower than those of the control group. In addition, the 100 mg subcutaneous treatment group increased the level of hormone sensitive lipolytic enzyme (HSL) genes, which seems to be related to hormone receptors including estrogen receptor and progesterone receptor.

That is, the crude fat content in the back muscle of group I1 was higher than that of the control, which is similar to the result of weight gain. Genetic level of fatty acid synthase in the back muscle of the genestine treatment was significantly higher than that of the control. In addition, the gene levels of hormone-sensitive lipolytic enzymes in the Genstein added diets in 1000 ppm diets were significantly higher than those in the control treatments, which seemed to affect the reduction of crude fat content in the back muscles of these treatments. However, lipolysis in muscle tissues seems to be less affected by genesine through hormone receptors than lipolysis in adipose tissue.

Claims (5)

Livestock meat quality improving composition comprising the gene as a active ingredient. The composition for improving animal meat according to claim 1, wherein the composition is pelletized by including glucose and an adhesive in gene stain. The composition for improving livestock meat according to claim 1 or 2, wherein the pressure-sensitive adhesive is plant oil. A method for improving livestock meat, characterized in that subcutaneous transplantation is carried out by using Genistein as an active ingredient and pelletizing with an adhesive. 5. The method of claim 4, wherein the gene further comprises glucose.