KR101322282B1 - Composition for bone growth promotion comprising Gelatin hydrolysates - Google Patents

Abstract

The present invention relates to a composition for promoting bone growth containing a gelatin-derived enzymatic degradation product, and more particularly, it can be usefully used as a food source for growing children by significantly proliferating osteoblasts and effectively growing bone length. In addition, the present invention relates to a composition for promoting bone growth, including a gelatin-derived enzyme (low molecular peptide) that can be easily used for promoting bone strength and promoting growth, as an active ingredient.

Gelatin, Enzymes, Bone Growth

Description

Composition for bone growth promotion containing gelatin-derived enzymatic digestion {Composition for bone growth promotion comprising Gelatin hydrolysates}

The present invention relates to a composition for promoting bone growth containing a gelatin-derived enzymatic degradation product.

In recent years, as bone age and busy modern life increase the imbalance in food intake among the elderly, various bone diseases such as an increase in osteoporosis have become social problems. It is estimated that about 10 million osteoporosis patients in Korea and 16 million in 2010.

42.6% of Korean women show osteoporosis or osteopenia, and the rate of osteoporosis is more than doubled as they progress to the 50s, 60s, and 70s.

Osteoporosis was diagnosed by korea women's society and women's women in their 30s and 70s, and osteoporosis (bone density -1.0 ~ -2.5%) was 233 patients (35.3%), and osteoporosis (bone density -2.5 or more) was 48 patients (7.3%). In addition, 5.1% of those in their 50's were osteoporosis, 12.4% in their 60's and 26% in their 70's.

Osteoporosis is occurring in young people due to aging osteoporosis due to aging and postmenopausal osteoporosis due to female hormone deficiency, as well as poor eating habits such as lack of calcium intake. In addition, the fracture rate of elementary and middle school students has more than doubled in recent 20 years, and it is important to increase and manage the maximum bone mass through food during the growing season to prevent osteoporosis.

Nutrition plays a very important role in maintaining bone's structural resistance to external physical loads [Bonjour JP, J. Am Coll Nutr. 2005, 24 (6), 526S-536S. In addition to the proper intake of vitamin D, along with calcium, protein is a very important nutrient for bone health and plays a role in the prevention of osteoporosis. In particular, studies have shown that animal protein promotes chronic metabolic acid, ie calcium depletion in urine (calciuric), deteriorates bone health and destroys minerals.However, decreased protein intake has a negative effect on bone health. Turned out. Thus, the selective deficiency of protein can be attributed to the rapid decrease in bone weight, microstructure and bone strength and osteoporosis. In elderly patients, hip bone fractures were frequently observed with decreased protein intake, and feeding these patients with kesin protein reduced bone loss after fracture, increased muscle strength, reduced complications, and reduced hospitalization dates. Taken together, whether it is a laboratory or clinical trial, the relatively high protein intake, ie, animal or vegetable protein, increases bone mineral content, reduces the rate of fractures in osteoporosis-induced osteoporosis, and protein intake enhances IGF-1. It acts as a positive role in influencing skeletal development and bone formation. In conclusion, protein along with calcium and vitamin D are essential for the prevention of osteoporosis.

Various calcium-reinforcing protein or peptide materials have been developed to enhance bone strength, promote growth and prevent osteoporosis. To date, casein phosphopeptide (CPP) derived from milk is representative and is known to help calcium absorption by enhancing the solubility of calcium when ingested.

CPP is obtained by the degradation of casein in the digestive tract and is known to aid in the calcification of bone in rats. This CPP chelates calcium and prevents the precipitation of calcium phosphates, increasing dissolved calcium availability.

There is also a study showing that phosphite, a protein present in egg yolk, enhances calcium solubility and promotes calcium absorption, thereby promoting bone metabolism.

The binding ability of oligophosphate peptides and calcium extracted from fish bones was increased by proteolytic enzymes and suggested as a calcium absorption promoter [Jung et al., British Journal of Nutrition, Vol 95, p 124-128, 2006].

Research has been reported to improve bone mineral density (BMD) and physical bone strength in soymilk [Omi et al., Journal of Nutritional Science and Vitaminology. Vol 40, p 201-211, 1994.] This is thought to be due to the peptide contained in soymilk, and divided into four groups to experimental animals that induced osteoporosis by separating into a group of high-molecular and low-molecular weight peptides. Mineral density (BMD) and bone strength were increased in both soy milk, high molecular weight soy milk peptide fed groups and low molecular weight peptide fed groups. In addition, the calcium absorption in the small intestine increased significantly. Based on these results, peptides in soymilk were found to increase calcium absorption rate, which is thought to enhance BMD and bone strength.

Accordingly, the present inventors have completed the present invention by confirming that gelatin enzymatic products significantly increase osteoblasts and effectively increase bone length as a result of research efforts to solve the above problems.

Accordingly, an object of the present invention is to provide a composition containing gelatin enzymatic degradation products which can be used as a growth promoting food source, medicine, or animal feed material by significantly proliferating osteoblasts and effectively increasing bone length.

The present invention is characterized by a composition for promoting bone growth containing a gelatin-derived enzymatic degradation product.

In addition, the present invention also includes a functional food or animal feed for promoting bone growth containing a gelatin-derived enzymatic degradation product.

The composition for promoting bone growth using the gelatin enzymatic degradation product according to the present invention as an active ingredient, which can be orally administered, can be used as a feed for functional foods, medicines, and animals, and in particular, can prevent osteoporosis and improve symptoms. It is useful as a material.

Hereinafter, the present invention will be described in detail.

The present invention not only can be usefully used as a food source for growing children by significantly proliferating osteoblasts and effectively growing bone length, and in particular, gelatin-derived enzymes that can be easily used for bone strength enhancement and growth promotion. It relates to a composition for promoting bone growth comprising (small molecule peptide) as an active ingredient.

An object of the present invention is to provide a material that can be used in a wide range of foods, beverages, pharmaceuticals, animal feeds, etc. as a composition for promoting bone growth and easy to ingest easily and inexpensive in daily life.

Gelatin used as a raw material of the enzymatic digestion of the present invention can be used in the epidermis, bones, intestines, etc. of animals, and in particular can be obtained from the shells of various livestock such as cowhide, pork skin, chicken shell, chicken wings, chicken feet. The bone strengthening composition used in the present invention was purchased from Geltec Co., Ltd. as a substance obtained by gelatinizing collagen derived from donpi.

The main ingredients of Ponpipes are as follows: 61.48% of moisture, 34.44% of crude protein, 2.71% of crude fat, 1.42% of crude protein and more than 70% of crude protein consist of type I collagen.

Collagen is the main protein of the connective tissue and accounts for 30% of the proteins in the human body. It is distributed in skin and bones, and it accounts for 70% of skin protein and 50% of cartilage. It is present in the human body in the bones and skin, in the surrounding membranes of the gut, in articular cartilage, in the cornea of the eye, and in particular as an adhesive for the calcium that constitutes the bones.

Bones are complex tissues whose main function is to resist physical forces and damage. The strength of bones depends not only on the amount of bone tissue but also on the quality, which is characterized by the geometry and morphology of the bones, the microstructure of the cancellous bone, minerals and collagen. Other determinants of bone quality are closely related to each other, especially minerals and collagen. Thus, in front of physical force, the strength of bone depends on the correlation between Type I collagen and minerals.

In humans, there are osteoblasts that form bones and osteoclasts that destroy bones. Old bones, which are the processes of bone resorption (osteoblasts), or new bones form at the rate of destroying unnecessary bones (osteoblasts, external cells of bone). Osteoporosis can occur if it is faster than the rate at which the matrix is formed and connected, and it also regulates continuous mineralization. In addition, the growth cartilage layer, which is a collection of chondrocytes, at both ends of the bones of the growing children and adolescents, which grows vertically because the osteoblasts are formed and turn into bone.

With proteolytic enzymes used for the enzymatic degradation of the gelatin can be used at least one selected from alkali race ^(alcalase?), Other pro-Mex ^(protamex?) And plastisol Bor atom ^{(flavorzyme?).}

Protease and gelatin are preferably used in a weight ratio of 1: 50 to 500. The reaction time, temperature, and pH of the enzyme may be different depending on the enzymes used, but it is preferable to carry out under conditions of 2 to 24 hours, 30 to 60 ° C, and 6.0 to 9.0, respectively. After desalting the obtained enzymatically decomposed product appropriately, it is preferable to separate and purify by ultrafiltration, gel filtration and various chromatography, membrane filter, and method using isoelectric point.

The gelatin hydrolyzate separated by the above method has a molecular weight distribution of 200 to 50,000 Da, preferably 200 to 3,000 Da, preferably by gel filtration and membrane filter.

In the bone-enhancing composition, the gelatin protein hydrolyzate is preferably at least 20.00% higher than the control group in which the osteoblast proliferation promoting activity is not added to the gelatin protein hydrolyzate treatment group. In addition, it is desirable to increase the length of the tibia of the experimental animal rat 20 um / day or more. The osteoblast proliferation promoting activity was measured by a method using MC3T3-T1 cells, which are mouse-derived osteoblasts, or U2OS cells, which are osteoblasts derived from humans, and bone length was measured using Sprague-Dawley rats.

The composition containing the gelatine enzymatic degradation product thus obtained as an active ingredient can be used as a substance for preventing and treating bone diseases as well as growth promoters for growing children. As the bone disease, osteoporosis, fracture, bone mineral density reduction and the like are preferable.

The present invention contains 0.1 to 50% by weight of the gelatine enzymatic degradation product of the present invention as an active ingredient based on the total weight of the composition, and may include a pharmaceutically acceptable carrier, excipient or diluent.

The compositions of the present invention may be in various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention may be administered orally or parenterally, and when parenteral administration, it is preferable to select an external skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection method, most preferably. Preferably for oral administration.

The dosage of the composition of the present invention varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate and severity of the disease of the patient, the daily dosage is the amount of gelatin degrading product 0.1 to 500 mg / kg, preferably 30 to 80 mg / kg, more preferably 50 to 60 mg / kg, and may be administered 1 to 6 times per day.

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions.

In addition, the present invention includes a functional food for promoting bone growth and preventing and improving bone disease containing a gelatin enzymatic degradation product as an active ingredient.

Functional foods according to the present invention, for example, chewing gum, caramel products, candy, ice cream, confectionery, such as various food products, soft drinks, mineral water, alcoholic beverages, beverage products such as vitamins and minerals Can be.

The gelatin enzymatic degradation product of the present invention may be added as it is or used in combination with other food or food ingredients, and may be appropriately used according to conventional methods. The blending amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or hygiene). Generally, in the preparation of food or beverages, the gelatin enzymatic products of the present invention are added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the raw materials. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

The functional food of the present invention may contain various flavors, natural carbohydrates, and the like as additional ingredients. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is preferably 0.01 to 0.04 part by weight, more preferably 0.02 to 0.03 part by weight per 100 parts by weight of the health food of the present invention.

In addition to the above, the functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, , A carbonating agent used in carbonated drinks, and the like. In addition, the functional food of the present invention may contain fruit flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components can be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

In addition, the present invention may include an animal feed additive for promoting bone growth and preventing and improving bone disease, which contains a gelatin enzymatic degradation product as an active ingredient.

Gelatin enzymatic degradation products of the present invention are organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymeric phosphate), polyphenols, catechin, It may further include any one or more of natural antioxidants such as alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid and the like.

Animal feed containing gelatin degrading products of the present invention is amino

Grains, such as wheat, oats, barley, corn and rice, in which various adjuvants such as acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes, live microbial agents and the like are crushed or crushed; Vegetable protein feedstuffs, for example, based on rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; Sugars and dairy products, for example, a dry ingredient consisting of various powdered milk and whey powder, and a drying additive, all mixed together with a liquid component, and a component which becomes a liquid after heating, that is, a lipid, for example, an animal liquefied by heating. In addition to the main components such as fats and vegetable fats can be used with substances such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like.

In addition, the animal feed comprising gelatin enzymatic products may be any protein-containing organic cereal meal commonly used to meet the dietary needs of animals. These protein-containing flours usually consist mainly of corn, soy flour or corn / soy flour mix.

The present invention is applicable to a number of animal diets including mammals, poultry, and fish. In the animal feed blending method including the gelatin enzymatic degradation product according to the present invention, the gelatin enzymatic degradation product is incorporated into the animal feed in an amount of about 1 g to 100 g per kg of feed on a dry weight basis.

Hereinafter, the present invention will be described in detail based on the following examples, but the present invention is not limited thereto.

Example  One

(1) hydrolysis of gelatin screen fraction

As shown in FIG. 1, 5 times of water was added to donpi gelatin (Note, Geltec) and left for 10 minutes, followed by hydration by stirring at 80 ° C. at a speed of 200 rpm for 1 hour. After that, the white foam was rolled out to obtain a yellow gelatin solution.

(2) Preparation of Gelatin Enzyme Degradation Products
Alcalase (A), Protamax (P) and Flavorzyme (F) used in the examples are trade names of proteolytic enzymes sold by Novozyme. The alcalase is an enzyme obtained by culturing bacillus licheniformis strain. Protamax is an enzyme obtained by culturing bacillus licheniformis and bacillus amyloliquefa strains. The flavorzyme is an enzyme obtained by culturing aspergillus oryzae strains.

The pH of the gelatine solution obtained above was adjusted to 7.0, and 50 g of gelatin solution was added to 1 g of AP (Alcalase + Protamex), PF (Protamex + Flavorzyme), FA (Flavorzyme + Alcalse) and APF (Alcalase + Protamex + Flavorzyme [Novozyme Co.]). After 4, 12, and 24 hours of digestion, the salts were removed, and the gelatine enzymatic product was separated into decomposition products having a size of 50 kDa or less, between 50 kDa and 3 kDa and 3 kDa or less using a membrane filter. 36 digests were prepared by dividing the enzyme digestion by digestion time, treatment enzyme and molecular weight size, and gelatin was prepared before enzymatic digestion as a positive control. Descriptions of these samples are shown in Table 1 below.

[Table 1]

(3) Measurement of osteoblast proliferation promoting activity of gelatin enzymatic degradation products

The proliferative activity of MG-63 osteoblasts, mouse osteoblasts of the gelatin hydrolyzate obtained in (2), was measured using BrdU.

That is, it measured using the Roche (Germany) Cell Proliferation ELISA, BrdU (colorimetric) Kit. Subcultured MG-63 osteoblasts were stabilized by dispensing into 96 well plates around 1 × 10 mm 3. Thereafter, 37 samples were appropriately treated and BrdU was treated in each well and incubated for 12-16 hours. Each cell was then fixed with a fixed solution, followed by finding an BrdU using an anti BrdU solution and reacting with the substrate solution. After sufficient color development, 25 μl of 1M H ₂ SO ₄ solution was treated to stop color development and measured at absorbance at 450 nm. Diluted gelatin hydrolyzate separated by various enzymes, decomposition temperature, and decomposition time was measured 10 times. , A significantly higher osteoblast proliferation was observed at # 30, and the osteoblast proliferation effect was generally higher than the sample digested with enzyme 2 (Protamex + Flavorzyme) compared with the sample digested with other enzymes (Fig. 3). By molecular size, the osteoblast proliferation was relatively high in the treatment group below 3 kDa, and the decomposition time was high in the 4 hours or 12 hours enzyme treatment group. Not observed. Finally, combining the properties of the most economical and high osteoblast proliferating effect, the molecular size is less than 3 kDa and the sample treated with PF enzyme is the most stable and excellent, only after 4 or 12 hours of treatment. Although there was an effect, there was no significant difference, so the treatment time was shorter and the economical 4-hour treatment sample was selected as the fraction with the highest osteoblast proliferation ability. In the case of further growth experiments using animals, it was determined that this sample # 26 (44.12% growth effect) was the most preferable and was selected.

(4) bone growth promoting effect

Bone growth promoting activity was evaluated using Sprague-Dawley rats. Normal group (control) orally administered distilled water to the gelatin hydrolyzate (# 26), which showed a high osteoblast proliferation effect of 44.12%, and sample A administration group 2 concentrations (5 and 1 ml / kg, BW), respectively. The sample B administration group was administered once daily for 5 days from the start of the experiment at two concentrations (200 and 40 mg / kg, BW), respectively. Thereafter, the promoting effect was evaluated in comparison with the bone height when the base sample (control) was administered. During the test period, rats were free to drink and feed, and MF (trade name, oriental yeast) was used as a basic feed. In order to observe the efficacy of Samples A and B on bone growth in rats, Hansson's first method was used to measure bone growth. This is a method for observing normal bone metabolism by using the phenomenon that the fluorescent factor is deposited on the bone through calcium and chelate binding. In the growth stage where bone formation is most active, it is most deposited on the bone angiogenesis at the bottom of the bone growth plate to form a line. After administration by one time, the length between the fluorescent line and the growth plate is observed by fluorescence microscope after a certain period of time. It was. All the experimental groups were administered calcein (calcein: 10 mg / kg, i.p .: Sigma, U.S.A.) 4 days after the start of the experiment.

One day after the administration of the fluorescent agent, the rat was dissected. The left and right tibia of the rats were removed and fixed in 0.1 M phosphate buffered formalin fixative for 2 hours, then demineralized and left for 2-3 days, then soaked in 30% sucrose for protection against freezing at 4 ° C. It was kept overnight at. After freezing the fixed bone tissue, a sagittal section of the proximal part of the tibia was collected every 60 μm using a sliding microtome (HM440E, Zeiss, Germany). The collected sections were placed on a slide glass and dried, and the length between the line formed by the deposition of the fluorescent material in the bone tissue using a fluorescence microscope and the growth plate was measured and used as a bone growth index. The long bone growth of the rats was found to be 404.0 ± 11.6 ㎛ in normal group, 468.4 ± 27.3 ㎛ in sample A administration group and 450.2 ± 29.1 ㎛ in length, respectively, and sample B (200 and 40 mg / kg) administration group showed a length growth of 406.1 ± 21.5 ㎛ and 385.0 ± 12.9 ㎛, respectively. The tibial length of rats fed with high and low concentrations of gelatin hydrolyzate (# 26) increased to 64.4 ㎛ and 46.2 ㎛, respectively, compared to the control group. The tibial length of rats showed a difference of 2.1 μm and -19 μm, respectively, compared to the control group, and it was confirmed that ingestion of gelatin hydrolyzate (less than 3 kDa, 4 hours degradation, protamex + flavorzyme enzyme treatment) increased bone growth promoting activity. (FIG. 4).

(5) Growth plate chondrocyte metabolism promoting effect

As described above, the sections collected every 60 μm were dried on a slide glass, dried, dyed with cresyl violet, and the height of the growth plate was measured. As a result of measuring the height of the growth plate, it showed 561.4 ± 5.7 ㎛ growth in the normal group, 586.9 ± 15.1 ㎛ and 626.3 ± 10.7 ㎛ in the sample A (5 and 1 ml / kg, po) administration group, respectively In the sample B (200 and 40 mg / kg) administration group, the growth of 561.5 ± 5.0 ㎛ and 596.9 ± 4.5 ㎛, respectively, to increase the bone length through promoting the growth plate cartilage metabolism (Fig. 5). Growth plate is a special tissue that produces bone tissue explosively and is essential for growth only in infants and adolescents. Therefore, in order to promote growth, the function of the growth plate should be promoted, and along with this, the structure of the growth plate is also proliferated to increase the size of the growth plate. The upper arrow of the photograph A shows the growth plate chondrocytes of the resting phase waiting for the growth plate chondrocytes to proliferate, and the growth plate chondrocytes of the proliferation portion of the proliferation part where several flat nuclei are clustered below. Under the proliferation, growth plate chondrocytes can be seen 2 to 5 times larger, which is hypertrophy. The down arrow is the osteophyte, which bones into the bone (skeleton) in the chondrocytes. Osteoporosis is flattened and the chondrocytes are removed through the natural death process, leaving only the bones to complete bone growth. In addition, later remodeling involves osteoblasts and osteoclasts to refine their shape.

Example  2: toxicity test

Toxicity experiments were performed on the gelatine enzymatic degradation products of the present invention as follows. Gelatin enzyme digest was dissolved in dimethylsulfoxide (DMSO), diluted with water, and then administered to each mouse (10 mice per group) at 10 g / kg and observed for 7 days, but no rats died.

Formulation example  1: Preparation of pharmaceutical preparations

<1-1> Sanje  Produce

2 g of gelatin enzyme digestion of the present invention

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of tablets

100 mg of gelatin enzyme digestion of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

<1-3> Preparation of the ring

1 g of gelatin enzymatic degradation product of the present invention

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

Formulation example  2: Preparation of Functional Foods

Foods containing the gelatin enzymatic degradation products of the present invention were prepared as follows.

<2-1> Production of flour food

0.5 to 5.0 parts by weight of the gelatin enzymatic degradation product of the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

<2-2> Dairy products ( dairy products Manufacturing

5 to 10 parts by weight of the gelatin enzyme digest of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-3> Preparation of Drink

1000 mg of gelatin degradate of the present invention

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Purified water was added to a total of 900 ml

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare beverage compositions of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Formulation example  3: Preparation of Animal Feed

Gelatin Enzyme Degradation ............... 240 g

Excipients (powder) ......... 760 g

It prepared according to the Korean Pharmacopoeia powder preparation method with the above components.

Although the composition ratio of the mixture is a relatively suitable composition for the feed composition in a preferred embodiment, the composition ratio may be arbitrarily modified, and after mixing the above components according to a conventional feed composition manufacturing method, It can be used for manufacture according to the powder preparation method.

Figure 1 is a schematic diagram showing the separation process of gelatin enzyme digestion for promoting bone growth.

Figure 2 is a photograph of the gelatin decomposed product of 3 kDa or less, 4 hours decomposition, PF enzyme treatment.

Figure 3 shows the osteoblast proliferation effect of gelatin enzyme digestion.

Figure 4 shows the effect (um / day) of the gelatin enzyme digestion (# 26, 3 kDa or less, 4 hours degradation, PF treatment) by concentration concentration treatment of animals tibia.

Figure 5 shows the effect on the growth plate of gelatin enzyme digest (# 26, 3 kDa or less, 4 hours degradation, PF treatment) (A: growth plate of normal group, B: growth plate of # 26 sample administration group).

Claims (15)

It contains gelatin-derived enzyme digest as an active ingredient, The enzyme digest has a molecular weight of 200 ~ 3,000 Da, The enzymatic digest is a protein consisting of flaborzyme obtained by culturing protamex and aspergillus oryzae strains obtained by culturing bacillus licheniformis and bacillus amyloliquefa strains. Food composition for promoting bone growth, characterized in that the decomposition product using a hydrolase simultaneously. delete delete The method of claim 1, wherein the enzymatic digest is 1) Step of Hydrolyzing Gelatin to Obtain Gelatin Solution 2) enzymatically degrading the gelatine solution at 30-60 ° C. for 4-12 hours after addition of protamex and flaborzyme proteolytic enzymes under pH 6-9; and 3) The food composition, characterized in that prepared by the method for producing an enzymatic degradation product comprising the step of desalting the degradation product to produce a degradation product for each molecular weight. delete The food composition according to claim 4, wherein the proteolytic enzyme and gelatin are used in a weight ratio of 1:50 to 500. A functional food for promoting bone growth, comprising the food composition of claim 1. delete It contains gelatin-derived enzyme digest as an active ingredient, The enzyme digest has a molecular weight of 200 ~ 3,000 Da, The enzymatic digest is a protein consisting of flaborzyme obtained by culturing protamex and aspergillus oryzae strains obtained by culturing bacillus licheniformis and bacillus amyloliquefa strains. Pharmaceutical composition for promoting bone growth, characterized in that the decomposition product using a hydrolase simultaneously. The method of claim 9, wherein the enzymatic digest is 1) Step of Hydrolyzing Gelatin to Obtain Gelatin Solution 2) enzymatically digesting the gelatin solution at 30-60 ° C. for 4-12 hours after adding protamex and flaborzyme proteolytic enzymes under pH 6-9; and 3) The pharmaceutical composition for promoting bone growth, characterized in that it was prepared by a method for producing an enzymatic degradation product comprising the step of desalting the degradation product to produce a degradation product for each molecular weight. The pharmaceutical composition for promoting bone growth according to claim 9, wherein the proteolytic enzyme and gelatin are used in a weight ratio of 1:50 to 500. It contains gelatin-derived enzyme digest as an active ingredient, The enzyme digest has a molecular weight of 200 ~ 3,000 Da, The enzymatic digest is a protein consisting of flaborzyme obtained by culturing protamex and aspergillus oryzae strains obtained by culturing bacillus licheniformis and bacillus amyloliquefa strains. Bone growth promoting feed composition, characterized in that the decomposition product using a hydrolase simultaneously. The method of claim 12, wherein the enzymatic digest is 1) Step of Hydrolyzing Gelatin to Obtain Gelatin Solution 2) enzymatically degrading the gelatine solution at 30 to 60 ° C. for 4 to 12 hours after the addition of protamex and flaborzyme proteolytic enzymes under pH 6-9; and 3) The bone growth promoting feed composition, characterized in that it was prepared by a method for producing an enzymatic degradation product comprising the step of desalting the degradation product to produce a decomposition product for each molecular weight. The composition for promoting bone growth according to claim 13, wherein the proteolytic enzyme and gelatin are used in a weight ratio of 1:50 to 500. Animal feed for promoting bone growth, comprising the feed composition of claim 12.

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