JP2008266259A - Fat accumulation inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fat accumulation inhibitor to prevent or improve obesity, and drugs and foods and drinks containing the same. <P>SOLUTION: This fat accumulation inhibitor contains a micellar calcium phosphate/phosphopeptide complex as an effective component. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a fat accumulation inhibitor containing a micellar calcium phosphate-phosphopeptide complex.

  Metabolic syndrome is a civilized disease caused by typical lifestyles in developed countries, such as overnutrition and lack of exercise. It is thought that the risk of arteriosclerotic diseases such as myocardial infarction and cerebrovascular disease is increased by 2 to 3 times. ing. In Japan, the proportion of those who are strongly suspected of having metabolic syndrome (built-in fat syndrome) and those considered to be reserves is 23.0% for men over 20 years old and 7.8% for women. . The diagnostic criteria for metabolic syndrome in Japan was established in April 2005, with abdominal obesity and visceral fat accumulation as essential items, hypertension, abnormal lipid metabolism (high TG or low HDL cholesterol), hyperglycemia If two or more items are present, it is diagnosed as metabolic syndrome.

  Visceral fat type obesity has been reported as a risk factor for metabolic syndrome. In visceral fat-type obesity, free fatty acids and glycerides, which are triglyceride degradation products, are excessively released from the visceral fat during fasting, and the visceral fat is linked to the portal vein. Influx causes hyperlipidemia, hyperglycemia, and catabolism of insulin, leading to insulin resistance.

  Moreover, there are living environment factors and genetic factors as factors of metabolic syndrome. Living environment factors include meals, and recently, meals that are difficult to become metabolic syndrome have been attracting attention. According to an American study by Zemel et al. (Non-Patent Documents 1 and 2), increased dairy intake for obese individuals reduced total body fat and visceral fat. Moreover, calcium in dairy products was clearly more effective than other calcium sources. However, in the above study, dairy intake was effective in reducing fat mass, but what actually is effective in suppressing fat accumulation is what is a milk component in dairy products, or calcium derived from milk. It is not clear whether it is due to this.

  On the other hand, various studies and patent applications have been made on calcium derived from milk. For example, a calcium-phosphopeptide complex that is excellent in absorbability and bioavailability and is soluble in a neutral aqueous solution (Patent Document 1 and Patent Document 2), a method for producing a calcium complex (Patent Document 3), and the like.

JP-A-10-290682 JP-A-11-310599 JP 2004-215521 A Sun X, Zemel MB, Calcium and dairy products inhibit weight and fat regain during ad libitum consumption following energy restriction in Ap2-agouti transgenic mice, J Nutr. 2004 Nov; 134 (11): 3054-60. Zemel MB, Miller SL, Dietary calcium and dairy modulation of adiposity and obesity risk, Nutr Rev. 2004 Apr; 62 (4): 125-31. Review.

  The objective of this invention is providing the fat accumulation inhibitor which prevents or improves obesity, the chemical | medical agent containing the same, and food-drinks.

  The inventors of the present invention provide a micellar calcium phosphate-phosphopeptide complex, which is a complex of micellar calcium phosphate in which calcium in milk is incorporated into casein micelles in the form of calcium apatite and a phosphopeptide derived from casein. The present invention was completed by finding that it has an effect of suppressing accumulation.

The features of the present invention are summarized as follows.
(1) A fat accumulation inhibitor comprising a micellar calcium phosphate-phosphopeptide complex as an active ingredient.
(2) A drug containing the fat accumulation inhibitor according to (1).
(3) Food / beverage products containing the fat accumulation inhibitor as described in (1).

  In the present specification, the term “drug” or “food or drink” is not limited to those intended for humans, but animals other than humans, for example, mammals such as domestic animals, pets, non-human primates, and rodents. , Medicines and foods and drinks (for example, pet food, feed) for birds, fish and the like.

  The fat accumulation-inhibiting agent of the present invention can suppress fat accumulation in the body when a human or non-human animal ingests food or drink or feed, thereby preventing or improving visceral fat-type obesity. As a result, metabolic syndrome can be prevented. The drug or food or drink containing the fat accumulation inhibitor of the present invention can be safely and easily ingested by animal bodies including the human body, and the commercial value of the drug or food or drink can be increased.

  The fat accumulation inhibitor of the first aspect of the present invention is characterized by containing a micellar calcium phosphate-phosphopeptide complex as an active ingredient.

  The micellar calcium phosphate-phosphopeptide complex used in the present invention is a complex of micellar calcium phosphate in which calcium in milk is incorporated into casein micelles in the form of calcium apatite and a phosphopeptide derived from casein. For example, it can be produced from milk of mammals such as milk, goat milk, buffalo milk and sheep milk, preferably from milk.

  Calcium in milk is divided into soluble calcium and calcium present in casein micelles. The calcium present in casein micelles is further divided into casein-binding calcium and micellar calcium phosphate. The micellar calcium phosphate is also called colloidal calcium phosphate.

  Casein-binding calcium is calcium bound to casein micelles, for example in the form of a calcium phosphate-casein complex or a calcium phosphate-citrate-casein complex. As described above, micellar calcium phosphate has calcium incorporated into casein micelles in the form of calcium apatite.

  Regarding the absorption of calcium by form, soluble calcium also has good absorption, but it has been reported that casein-binding calcium and micellar calcium phosphate are more useful (Nutr. Rep. Int., 21,6738 (1980)). And about 60-70% of these calcium contained in milk is in the form of casein-binding calcium and micellar calcium phosphate.

  Casein micelles generally refer to those in which casein associates to form a stable micelle structure in solution, but in this specification, casein micelles containing casein-binding calcium and / or micellar calcium phosphate are included. Say a fraction.

  The micellar calcium phosphate-phosphopeptide complex used in the present invention is preferably a casein micelle (preferably milk casein micelle or rennet casein micelle) hydrolyzed with protease, and then an acid is added to pH 5.4 or less. Subsequently, the solution obtained by removing insoluble peptide and undegraded casein can be formed in the solution by adjusting the pH to 6.6 or higher. As used herein, “micellar calcium phosphate-phosphopeptide complex” is meant to include a complex of a phosphopeptide and a micellar calcium phosphate and optionally casein-binding calcium. Specifically, the micellar calcium phosphate-phosphopeptide complex can be produced as follows.

  First, casein micelles are prepared from milk of mammals such as milk, goat milk, buffalo milk and sheep milk. For example, when preparing rennet casein as casein micelles, an appropriate amount of rennet is added to skimmed milk having a pH of 5.5 or more in order to coagulate the milk according to a conventional method, and then finely cut with a card knife. Next, the cut card is heated and allowed to stand, and then whey is eliminated. And after adding water to a card | curd and stirring for several minutes and wash | cleaning a card | curd, it leaves still and excludes water 2-3 times, and rennet casein is obtained. For example, when preparing casein from which lactose and soluble minerals are removed as casein micelles, whole milk and skim milk having a pH of 5.5 or higher are subjected to ultrafiltration membranes (fractionated molecular weight of 50,000 Da or higher) Casein from which lactose and soluble minerals have been removed is obtained by treatment with a membrane such as a filtration membrane or reverse osmosis membrane, or by electrodialysis. The prepared casein micelle contains casein-binding calcium and micellar calcium phosphate.

  Next, the casein micelles thus prepared are hydrolyzed with a proteolytic enzyme. As the proteolytic enzyme, trypsin, pancreatin, chymotrypsin, pepsin, papain, pronase, thermolysin and the like can be used. Add 0.001 to 2% of proteolytic enzyme to the weight of casein and incubate at 15-60 ° C for about 5 minutes to 100 hours. After the reaction, the enzyme reaction is stopped by heating at 80 to 120 ° C. for about 2 seconds to 10 hours.

  Next, the pH of the reaction product is adjusted to 5.4 or lower, and then the insoluble peptide and undegraded casein are removed to separate and recover casein-binding calcium and micellar calcium phosphate from casein micelles.

  Examples of the method of adjusting the pH to 5.4 or lower include a method of adding an acid and a method of using lactic acid fermentation by adding a lactic acid bacterium. Specifically, there is a method in which an inorganic acid such as hydrochloric acid or an organic acid such as acetic acid, lactic acid or citric acid is added to casein micelles so that the pH is 5.4 or lower. At this time, it is preferable to use a homomixer, a colloid mill, or the like to prevent the formation of nodules and to sufficiently stir, knead, grind, etc. so that the acid is uniformly dispersed.

  After pH adjustment by acid treatment or the like in this manner, for example, centrifugation is performed at 1000 × g for 15 minutes to remove insoluble peptide and undegraded casein (casein that has not been treated with proteolytic enzyme). be able to. Such an insoluble peptide and undegraded casein may be removed using a conventional method known to those skilled in the art.

  The casein micelle solution thus obtained may be further concentrated by ultrafiltration or the like before the pH is adjusted to 6.6 or higher as described later. Thereby, casein-binding calcium and micellar calcium phosphate can be separated and recovered from casein micelles. For example, after adjusting the pH of casein micelles to 5.4 or less, the solution is stirred and homogenized, and the solution is ultrafiltered using an ultrafiltration membrane (fractionated molecular weight of 50,000 Da or less). Casein-binding calcium and micellar calcium phosphate are collected on the permeate side, and the permeate is subjected to the next pH adjustment. In addition, since casein-binding calcium and micellar calcium phosphate obtained by ultrafiltration contain a large amount of anions such as chloride ions, it is better to remove the anions by treatment such as electrodialysis to increase the calcium content. .

  The casein micelle solution prepared by removing the insoluble peptide and undegraded casein as described above contains micellar calcium phosphate, optionally casein-binding calcium, and phosphopeptide. By adjusting the pH of this solution to be 6.6 or more, a complex of micellar calcium phosphate and phosphopeptide can be formed. Examples of a method for adjusting the pH to 6.6 or higher include a method of adding an alkali to the above solution. As the alkali to be used, sodium hydroxide is preferable. When the pH of the solution from which insoluble peptide and undegraded casein are removed is less than 6.6, a complex of micellar calcium phosphate and phosphopeptide is hardly formed.

  The micellar calcium phosphate-phosphopeptide complex thus obtained is usually liquid. In the present invention, this liquid micellar calcium phosphate-phosphopeptide complex can be used directly as a fat accumulation inhibitor. The liquid micellar calcium phosphate-phosphopeptide complex may be further concentrated by an evaporator, ultrafiltration, or the like, and the resulting micellar calcium phosphate-phosphopeptide complex-containing concentrate may be used as a fat suppression and accumulation agent. Further, the liquid micellar calcium phosphate-phosphopeptide complex can be dried (preferably after being concentrated) and used in a solid or powder form. Examples of the drying treatment method include arbitrary methods such as an air drying method, a freeze drying method, a spray drying method, a reduced pressure drying method, and a heat drying method. In addition, by treating with a membrane such as an ultrafiltration membrane or a microfiltration membrane, monovalent ions are removed, and free casein-binding calcium, micellar calcium phosphate, and phosphopeptide that do not form a complex are removed. Since it can be removed, a higher purity complex can be obtained.

  The micellar calcium phosphate-phosphopeptide complex obtained by the above method exhibits high solubility at both neutral pH and powder. Since the micellar calcium phosphate-phosphopeptide complex obtained by this method can be obtained from casein micelles in a single step, it can be produced not only at a very low cost but also directly from milk, for example. It is easier for consumers seeking safety and security.

  The amount of the fat accumulation inhibitor containing the micellar calcium phosphate-phosphopeptide complex in the present invention as an active ingredient can be adjusted depending on the type and purpose of the composition, but the total amount of the composition On the other hand, for example, 100% by weight, 99% by weight, 98% by weight, 95% by weight, 90% by weight, 80% by weight, 70% by weight, 60% by weight, and 50% by weight are not limited thereto.

  The medicine which is the 2nd mode of the present invention contains the above-mentioned fat accumulation inhibitor. For example, additives such as excipients, binders, disintegrants, lubricants, colorants, and corrigents are added to the fat accumulation inhibitor, and oral preparations are preferred by conventional methods, specifically capsules, granules It can be produced in a dosage form such as an agent, a pill, and a tablet. What is necessary is just to use what is generally used in the said field as such an additive, for example, as an excipient | filler, lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, As succinic acid, binders are water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, calcium phosphate, polyvinylpyrrolidone, etc. Is dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose, etc. As lubricants, purified talc, stearate, borax, polyester Glycol or the like, caramel color as the colorant, paprika dye, cochineal dyes, indigo carmine etc., sucrose as a flavoring agent, orange peel, citric acid, can be exemplified tartaric acid. Further, it may be produced in a liquid preparation form, and a liquid preparation, syrup preparation and the like may be produced by a conventional method by adding a flavoring agent, a buffering agent, a stabilizer and the like to the fat accumulation inhibitor. In this case, the flavoring agents may be those listed above, examples of the buffer include sodium citrate, and examples of the stabilizer include tragacanth, gum arabic, and gelatin.

  The agent of the present invention may be contained so as to be administered, for example, in the range of 40 mg to 400 mg / Kg body weight, more preferably 120 mg to 400 mg / Kg body weight per day as a micellar calcium phosphate-phosphopeptide complex. It can be appropriately adjusted according to the degree of obesity, sex, age, etc., and in the case of animals other than human beings, in addition to the species of animals. The drug of the present invention is administered in advance to a person who is prone to obesity due to factors such as lifestyle habits, constitution, genetics, etc., to suppress fat accumulation in the body, prevent obesity, or administer to obese patients. Thus, for example, the state of visceral fat obesity can be improved. Moreover, when using as a preventive agent, it is good also as a small quantity-the same amount compared with the case where it uses as an obesity improving agent.

The food / beverage products which are the 3rd aspect of this invention are characterized by including the said fat accumulation inhibitor.
The fat accumulation inhibitor can be contained in a food or drink by any appropriate method available to those skilled in the art. For example, the fat accumulation inhibitor may be processed into a liquid, solid or granular form and then contained in food. Or you may mix or melt | dissolve directly in food / beverage products, and may be embedded in food / beverage products. Moreover, you may apply | coat, coat | cover, infiltrate, or spray a fat accumulation inhibitor on foodstuffs. The fat accumulation inhibitor may be uniformly distributed in the food or drink, or may be unevenly distributed. Alternatively, the fat accumulation inhibitor may be unevenly distributed at a specific site in the food. When the preparation for fat accumulation is dissolved in a beverage in a freeze-dried state, it is preferable to dissolve the preparation in water, for example, mix uniformly by stirring, and then add to the beverage, water, and the like. Moreover, when mixing a fat accumulation inhibitor with a solid food, for example, it is preferable to add to a food material, and after mixing uniformly by stirring, it is processed. Moreover, the food-drinks containing the fat accumulation inhibitor can be further processed. Such processed products are also encompassed within the scope of the present invention. Alternatively, the fat accumulation inhibitor itself may be solid-molded or formulated into a capsule, sugar-coated tablet or the like in the food or drink of the present invention.

  For example, in the case of a food or drink intended for humans, the food or drink of the present invention contains 2.4 to 24 g, more preferably 10 to 24 g as the daily intake of the micellar calcium phosphate-phosphopeptide complex. As described above, it is preferable to add a fat accumulation inhibitor, but it can be appropriately adjusted according to the person to be ingested, etc., and in the case of animals other than humans, in addition to the animal species. In the case of animals other than humans, for example, in the case of pet food for cats, a fat accumulation inhibitor is added so that the daily intake of micellar calcium phosphate complex is in the range of 1 to 2 g. However, it can be appropriately adjusted depending on age and sex. By ingesting the food and drink of the present invention, it is possible to suppress fat accumulation in the body, prevent visceral fat type obesity, or improve the state of obesity.

  In the production of the food and drink of the present invention, various additives conventionally used in food and drink may be used. Additives include, but are not limited to, color formers (sodium nitrite, etc.), coloring agents (garden pigment, red 102, etc.), perfumes (orange flavors, etc.), sweeteners (stevia, aster palm, etc.), storage (Sodium acetate, sorbic acid, etc.), emulsifier (sodium chondroitin sulfate, propylene glycol fatty acid ester, etc.), antioxidant (disodium EDTA, vitamin C, etc.), pH adjuster (citric acid, etc.), chemical seasoning (inosine) Sodium phosphate, etc.), thickeners (xanthan gum, etc.), swelling agents (calcium carbonate, etc.), antifoaming agents (calcium phosphate, etc.), binders (sodium polyphosphate, etc.), nutrient enhancers (calcium enhancer, vitamin A, etc.) ) And the like. Furthermore, you may add functional raw materials, such as a ginseng extract, an Ezocogi extract, a eucalyptus extract, and a Tochu tea extract.

  The kind of beverage of the present invention is not particularly limited. Examples of the beverage of the present invention include milk beverages (coffee milk, fruit milk, functional milk, etc.), lactic acid bacteria beverages (drink yogurt, etc.), tea-based beverages (non-fermented tea, fermented tea, semi-fermented tea, Tochu tea, etc.) Beverage), fruit and vegetable beverages (fruit juices such as oranges, apples, grapes, peaches, beverages containing vegetable juices such as tomatoes, carrots, cabbage, celery), alcoholic beverages (beer, sparkling wine, whiskey, Beverages including wine, liqueurs, etc.), carbonated beverages, soft drinks, low calorie beverages and the like can be specifically exemplified. About the manufacturing method etc. of various drinks, the existing reference books, for example, "Latest soft drinks" (2003) (Kotsu Co., Ltd.) etc. can be referred.

  The kind of food of the present invention is not particularly limited. The food of the present invention may be a fresh food or a processed food. Preferably, dairy products such as skim milk powder, yogurt, cheese, butter, baked confectionery such as cookies, bread, cakes, rice crackers, tablet confectionery such as ramune confectionery, Japanese confectionery such as mutton, pudding, jelly, ice cream, etc. Sweets such as frozen confectionery, chewing gum, candy, snacks such as crackers and chips, noodles such as udon and buckwheat, fish paste products such as kamaboko, ham and fish sausage, seasonings such as miso, soy sauce, dressing, mayonnaise and sweeteners And various kinds of prepared dishes such as tofu, konjac, other boiled rice cakes, dumplings, croquettes, salads, soups, stews.

  The food or drink containing the fat accumulation inhibitor may be a functional food. The “functional food” of the present invention means a food having a certain function, such as a food for specified health use, a health function food including a conditional tokuho (food for specified health use) and a nutritional function food, a food for special use ( In addition to food for the sick, breast milk for pregnant and lactating women, infant formula, food for the elderly, etc., so-called health foods such as nutritional supplements, health supplements, supplements and beauty foods (eg diet foods) Is included. The functional food of the present invention also includes a health food to which a health claim based on the food standards of Codex (FAO / WHO Joint Food Standards Committee) is applied.

  The functional food of the present invention may be in the form of solid preparations such as tablets, granules, powders, pills and capsules, liquid preparations such as liquids, suspensions and syrups, or preparations such as gels. Or the shape (for example, drink, confectionery, etc.) of normal food-drinks may be sufficient.

  About the compounding quantity of a fat accumulation inhibitor to a functional food, a compounding method, and the additive which can be mix | blended with a functional food, it is the same as said food / beverage products.

  Although the functional food of the present invention is not limited, it is particularly preferable to prevent fat accumulation. The description or indication that it is a food or drink suitable for fat accumulation prevention is, for example, a functional indication (nutritional component function indication or It may be according to the indication of health use. Examples of such indications include "suppress body fat accumulation (increase)", "not susceptible to visceral fat obesity", "suitable for those who care about obesity", etc. Is not to be done.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, these Examples do not limit this invention.

  In addition, all the measurement results in the examples are shown as mean ± standard deviation. Tukey-Kramer's multiple comparison method was used to test the difference between the mean values. The significance level was 5%. Statistical analysis was performed using stat view ver5.0 (SAS Institute).

  In Example 1, paying attention to the difference in quality of calcium (mainly absorption rate), glucones containing calcium carbonate, gluconic acid and lactic acid, which are used to enhance calcium in dairy products, are reported to have a high absorption rate. Comparison of the effects of intake of various calcium materials on body fat accumulation in KK mice using calcium lactate and micellar calcium phosphate-phosphopeptide complex (hereinafter referred to as MCP-PP complex), which is one of the calcium forms in milk did. About 80% of KK mice are urinary sugar positive at 17 to 18 weeks of age even with general feed. Since an increase in blood sugar level occurs when the body weight exceeds 35-40 g, it is considered that body fat accumulation (obesity) plays a primary role in the development of hyperglycemia. These symptoms are thought to develop due to the involvement of multiple genes.

1. Calcium material As the calcium material, calcium carbonate (CaCO ₃ ), calcium lactate gluconate, and MCP-PP complex were used.
For calcium carbonate (CaCO ₃ ), a reagent from Wako Pure Chemical Industries, Ltd. was used. The calcium content was 80.0%.
As the calcium lactate gluconate, PURACAL XPerform (Pureac) was used. The calcium content was 10.4%.

  The MCP-PP complex used was prepared by the following method. After 5 kg of rennet casein (New Zealand NZMP) was suspended in 80 liters of water in a milk sterilizer, 60 g of trypsin (Novo Nordisk Bioindustrial P / L, type PTN 6.0S) was added to 1 liter of water. What was dissolved in was added. This was held at 50 ° C. for 4 hours with stirring so as not to precipitate rennet casein to decompose casein, and then heated at 90 ° C. for 10 minutes to stop the enzyme reaction. After cooling to 25 ° C., the pH is adjusted to 4.6 with 1N-HCl to release calcium micellar phosphate (MCP), and the resulting insoluble peptide and undegraded casein are removed by filtration through a nylon mesh, followed by Advantec No. 6 Was filtered through a filter paper to obtain a transparent filtrate. After adjusting the pH to 9 by adding 1N-NaOH to this filtrate, it is concentrated to about 8 liters with a GR60PP membrane (cut-off value: 25 kDa) using a Sanyo Co., Ltd. DDSS RO / UF device and lyophilized. did. The obtained MCP-PP complex was 1.24 kg. The prepared MCP-PP complex contained 61.69% protein and 8.66% calcium.

2. Animal Experiment The control feed was based on the AIN-93G composition and was a low calcium feed with a calcium content of 250 mg / 100 g. The lard was replaced with corn starch and added so that the fat energy ratio was 25%. The test group was formulated with calcium carbonate, calcium gluconate lactate, and MCP-PP complex so that the calcium content of the control feed was 500 mg / 100 g. Table 1 shows the feed composition.

  Using 4-week-old male KK mice (CLEA Japan, Inc.), after pre-breeding for 1 week, they were divided into 4 groups of 8 per group. The experimental feed and water shown in Table 1 were freely taken for 58 days, and the body weight and feed intake were measured. The breeding environment was a room temperature of 22 ± 1 ° C., a humidity of 50 ± 5 ° C., and a 12-hour light / dark cycle (12-24 hours). On the day of dissection, after measuring body weight and feed intake, fast for 4 hours, open under ether anesthesia, collect blood from the heart, and extract liver, retroabdominal wall fat, fat around the epididymis, and mesenteric adipose tissue The weight was measured. Thereafter, the liver was freeze-dried and pulverized, and the retroabdominal wall fat, fat around the epididymis, and mesenteric adipose tissue were fixed with a 10% formalin solution, and used as samples for analysis.

  The growth results of the mice are shown in Table 2. There was no significant difference in the initial and final weights in either group. The weight gain was slightly higher when the calcium lactate gluconate group was compared with the other three groups. There was no significant difference in feed intake and feed efficiency in either group.

  Table 3 shows mouse organ weights. There was no significant difference in liver weight. The abdominal wall adipose tissue weight was significantly lower in the MCP-PP complex group than in the calcium and calcium lactate gluconate groups. The adipose tissue weight around the accessory testicle was significantly lower in the MCP-PP complex group than in the calcium lactate gluconate group. There was no significant difference in mesenteric fat. Therefore, it was confirmed that the MCP-PP complex has an inhibitory effect on intraperitoneal fat accumulation in KK mice.

3. Serum biochemical analysis Serum was separated from the collected blood, and serum cholesterol, serum free fatty acid, and serum triglyceride concentrations were analyzed by an enzymatic method. "Cholesterol E-Test Wako" for serum total cholesterol, "Triglyceride E-Test Wako" for serum triglycerides, "NEFAC-Test Wako" for serum free fatty acids, "Glucose CII-Test Wako" for blood sugar, serum Calcium was measured using “Calcium C-Test Wako” (both Wako Pure Chemical Industries, Ltd.) according to the instructions for each kit.

  Serum leptin concentration is determined using mouse leptin immunoassay kit (R & D Systems), and serum parathyroid hormone (PTH) concentration is determined using mouse intact PTH ELISA kit (Immutopics) according to the instructions for each kit. Analysis was performed by ELISA.

Serum biochemical values and hormone concentrations are shown in Table 4.
There was no significant difference in serum lipid levels. Serum triglyceride concentration was slightly lower in the calcium carbonate group than in the low calcium group. The serum free fatty acid concentration was slightly higher in the calcium gluconate lactate group than in the low calcium and MCP-PP group. There was no difference in serum cholesterol concentration between the groups.

  There was no significant difference in blood glucose level between the groups. The blood glucose level of rats reaches a peak at about 30 minutes after eating, then declines, and returns to the original level after 3 hours. In the present example, the blood sugar level was the blood sugar level after 4 hours because it was dissected after fasting for 4 hours. The blood glucose level of normal rats was 50 to 135 mg / dl, whereas in this example, all groups showed higher values than normal values.

  There was no significant difference in serum calcium concentration between groups. The low calcium group showed a slightly higher value than the MCP-PP complex group. Since the normal value of serum calcium concentration was 7.2 to 13.9 mg / dl, all groups were in the normal range.

  The leptin concentration was not significantly different between the groups. The low calcium group showed a higher value than the other groups, and the calcium gluconate lactate group showed the lowest value.

  PTH was not significantly different between groups. The low calcium group and MCP-PP complex group showed slightly higher values than the calcium carbonate group and calcium lactate gluconate group. However, since the normal range of PTH was 10 to 65 pg / ml, all groups were within the normal range.

4). Analysis of liver lipid The liver was freeze-dried and then pulverized, and the liver lipid was extracted by the Folch method using a chloroform: methanol (2: 1) solution. After washing with water by the Folch method, it was dissolved in isopropanol containing 10% Triton X-100, and total cholesterol and triglycerides were analyzed by an enzymatic method. “Cholesterol E-Test Wako” was used for quantification of total cholesterol, and “Triglyceride E-Test Wako” (both Wako Pure Chemical Industries, Ltd.) was used for triglycerides.

  Liver lipids are shown in Table 5. The amount of triglycerides and cholesterol per liver in the MCP-PP complex group was significantly lower than that in the low calcium group, and the MCP-PP complex group was considered to have an effect of suppressing triglyceride accumulation in the liver. .

5. Analysis of Fecal Lipids and Calcium in Feces Feces were freeze-dried and then crushed, and the fecal lipids were extracted and washed with water by the Folch method using a 4% acetic acid chloroform: methanol (2: 1) solution. Thereafter, the total lipid amount was measured by a gravimetric method. Apparent digestion and absorption rate was determined from the amount of lipid intake and total lipid excretion.
Table 6 shows lipid intake, lipid excretion, and apparent digestion and absorption rate. Lipid excretion was significantly higher in the calcium lactate gluconate group than in the MCP-PP complex group and the low calcium group. Apparent digestive absorption rate was significantly lower in the gluconate lactic acid Ca group than in the MCP-PP complex group.

  Table 7 shows the intake calcium amount, excretion calcium amount, apparent calcium digestion absorption amount and absorption rate. Intake calcium and fecal excretion calcium were significantly lower in the low calcium group than in the other three groups. The apparent amount of digestion and absorption of calcium gluconate was significantly higher than that of the low calcium and calcium carbonate groups. In addition, the MCP-PP complex group showed a significantly higher value than the low calcium group. Apparent calcium absorption was high in the gluconate lactate Ca group and the MCP-PP complex group, but no significant difference was observed.

6). Analysis of fat cell size and calculation of fat cell count After immersing and fixing posterior abdominal wall fat, epididymal fat, and mesenteric adipose tissue in 10% formalin solution, dehydration, defatting, paraffin penetration, paraffin embedding, thin After cutting, extending and drying, hematoxylin and eosin were stained and encapsulated. Each adipose tissue was observed with an optical microscope, and the specimen was photographed with a digital camera. Measure the diameter of about 100 cells per mouse using Scion Image (alpha4.0.3.2) from the scales of the scale-prepared preparations. Asked.

The number of adipocytes was calculated by the method of MORIN, CL et al. (Endocrinology, 139, 4998, 1998).
g / adipocyte = average cell size (pl) × 0.95 (ng fat / pl) × g / 10 ⁹ ng
That the value obtained by dividing the adipose tissue weight in g / adipocytes were fat cell number (× 10 ⁶ cells in the display).

  The size of fat cells and the number of fat cells are shown in Table 8. The size of adipocytes in the abdominal wall adipose tissue was significantly larger in the calcium gluconate lactate group than in the calcium carbonate group and in the MCP-PP complex group as compared with the low calcium and calcium carbonate groups. There were no differences between the groups in the adipocyte size of the accessory testicles and mesenteric adipose tissue.

  The number of adipocytes in the abdominal wall adipose tissue was significantly lower in the MCP-PP complex group than in the low calcium and calcium carbonate groups. There was no difference between the groups in the adipose tissue around the accessory testicles. In mesenteric adipose tissue, calcium lactate gluconate showed a slightly higher value than other groups.

7). MRNA analysis of lipid metabolism-related enzymes in posterior abdominal wall adipose tissue and liver RNA was extracted from posterior abdominal wall adipose tissue and liver using Sepasol RNA I Super (Nacalai Tesque), chloroform, and isopropanol. Reverse transcription was performed using ReverTra Ace (Toyobo) and random primers (Invitrogen) to prepare cDNA. For the abdominal wall fat, the expression levels of hormone sensitive lipase (HSL), diacylglycerol acyltransferase 2 (DGAT2), fatty acid synthase (FAS), and lipoprotein lipase (LPL) are expressed. For the liver, DGAT1, DGAT2, and FAS are used. The mRNA expression level of glucose 6-phosphate dehydrogenase (G6PD) was quantified by real-time PCR (7300 Real Time PCR System, Applied Biosystems). Quantitative values were relatively compared with a C57BL / 6J mouse that did not exhibit obesity fed with AIN-93G diet as 1.

  Figure 1 shows the mRNA expression levels of hormone-sensitive lipase (HSL), diacylglycerol acyltransferase 2 (DGAT2), fatty acid synthase (FAS), and lipoprotein lipase (LPL) in the abdominal wall adipose tissue, and DGAT1, DGAT2, and FAS in the liver. FIG. 2 shows the mRNA expression level of glucose 6-phosphate dehydrogenase (G6PD). In posterior abdominal wall adipose tissue, KK mice had significantly higher levels of mRNA for fat degradation (HSL) and triglyceride synthesis (DGAT2) than normal mice (C57BL / 6J mice). No specific phenomenon was observed. Even in the liver, the mRNA expression of lipid synthase was increased by a factor of 10 compared to normal mice, but no phenomenon specific to the MCP-PP complex was observed.

  In Example 2, for the purpose of investigating whether components other than MCP-PP complex exist that affect body fat accumulation, the comparison was made under the same conditions when the calcium source was skim milk and MCP-PP complex. I went there.

1. Experimental Sample As the calcium source, calcium carbonate (CaCO ₃ ), skim milk powder, and MCP-PP complex were used. The same calcium carbonate and MCP-PP complex as in Experiment 1 was used. Non-fat dry milk used by Snow Brand Milk Products Co., Ltd. was used. The protein content in skim milk powder was 36.7%, and the calcium content was 1.2%.

2. Animal Experiment The control feed was based on the AIN-93G composition and was a low calcium feed with a calcium content of 100 mg / 100 g. The lard was replaced with corn starch and added so that the fat energy ratio was 25%. In the test group, calcium carbonate, nonfat dry milk, and MCP-PP complex were blended so that the calcium content of the control feed was 500 mg / 100 g. Table 9 shows the feed composition.

  Using 4-week-old male KK mice (CLEA Japan, Inc.), after pre-breeding for 1 week, they were divided into 4 groups of 8 per group. The experimental feed and water shown in Table 9 were freely taken for 56 days, and the body weight and feed intake were measured. The breeding environment was a room temperature of 22 ± 1 ° C., a humidity of 50 ± 5 ° C., and a 12-hour light / dark cycle (12-24 hours). On the day of dissection, after measuring body weight and feed intake, fast for 4 hours, open under ether anesthesia, collect blood from the heart, remove liver, retroabdominal wall fat, peripheral testicular fat, mesenteric adipose tissue, The weight was measured. Thereafter, the liver was freeze-dried and then pulverized, and the abdominal wall adipose tissue was fixed with a 10% formalin solution and used as a sample for each analysis.

  The growth results of the mice are shown in Table 10. There were no significant differences between the groups in initial weight, final weight, weight gain, feed intake, and feed efficiency.

  Table 11 shows mouse organ weights. Although the liver weight was not significantly different, the MCP-PP complex group showed a lower value than the other groups.

  The abdominal wall adipose tissue weight was significantly lower in the MCP-PP complex group than in the low calcium group. The non-fat dry milk group showed the same tendency, but no significant difference was observed. There were no significant differences between the groups in the weight of adipose tissue around the epididymis and mesenteric adipose tissue. Therefore, it was confirmed that the MCP-PP complex has an inhibitory effect on intraperitoneal fat accumulation in KK mice. However, the non-fat dry milk group did not show the same effect as the MCP-PP complex, so the contribution of other components in the non-fat dry milk is considered to be small in this experimental system.

3. Serum Biochemical Analysis Serum was separated from the collected blood, and serum cholesterol, serum free fatty acid, serum triglyceride, calcium concentration and leptin concentration were measured in the same manner as in Experiment 1. Serum biochemical values and leptin concentrations are shown in Table 12.

  There was no significant difference in serum lipid levels. Serum cholesterol and triglyceride concentrations tended to be lower in the skim milk powder group and the MCP-PP complex group. Serum free fatty acid concentrations tended to be lower in the low calcium group and the MCP-PP complex group. Although there was no significant difference in blood glucose level between the groups, the low calcium group showed high values. There was no significant difference in serum calcium concentration between groups. Serum leptin concentrations were not significantly different between the groups, but the MCP-PP complex group showed a low value.

4). Analysis of liver lipids Liver cholesterol and triglycerides were analyzed in the same manner as in Experiment 1.
Liver lipids are shown in Table 13. Liver cholesterol levels were significantly higher in the low calcium group than in the other three groups. The amount of triglyceride per liver was significantly lower in the skim milk powder group and the MCP-PP complex group than in the low calcium group. In particular, in the skim milk powder group, the amount of accumulation decreased significantly. The low calcium group seems to promote the accumulation of liver fat, as the accumulation of both liver cholesterol and triglycerides increased. In particular, the amount of liver triglyceride was significantly lower in skim milk powder and MCP-PP complex group, and thus it was revealed that calcium and the like in milk components suppress the accumulation of liver triglyceride.

5. Analysis of size and number of adipocytes The size and number of fat cells in the abdominal wall adipose tissue were analyzed in the same manner as in Experiment 1.
Table 14 shows the size and number of fat cells in the abdominal wall adipose tissue. There was no difference in the size of adipocytes between the groups. The number of adipocytes in the abdominal wall adipose tissue was significantly lower in the MCP-PP complex group than in the low calcium and calcium carbonate groups. Example 1-6. From the results of this Example, it was confirmed that the inhibitory action of MCP-PP complex on the accumulation of intraperitoneal fat was not due to at least suppression of adipocyte hypertrophy, and MCP-PP complex It was thought not to suppress fat cell hypertrophy but to reduce lipid weight through a decrease in the number of fat cells.

  Conventionally, as a mechanism of reducing body fat by milk components, reports have been made that body fat decreases when calcium intake increases due to the hormonal action of vitamin D, and energy increases when calcium intake increases in mice with hereditary obesity. There is a report that fat is hard to be accumulated by dissipating as heat. However, in the above examples using KK mice, both the serum calcium concentration and PTH were within the normal range, and this result could not be explained by this mechanism. Further, in the above examples, the MCP-PP complex group had a large amount of lipid intake, but rather the amount of excretion was rather small. Therefore, the suppression of the accumulation of liver lipids and rear abdominal wall fat was caused by the excretion of dietary fat. Instead of decreasing, it was thought to be decreasing by burning fat in the body and using it as energy.

  Since the decrease in the number of fat cells was estimated from the results of the size and number of fat cells in Examples 1 and 2, the fat accumulation suppression effect of the MCP-PP complex is mainly due to the suppression of fat cell differentiation. It was estimated that the form of micellar calcium phosphate might have affected the bioavailability of the body, and thus the metabolism of the liver and adipose tissue.

  The fat accumulation inhibitor of the present invention can be widely applied to drugs, foods and drinks, feeds and the like.

The mRNA expression level of a lipid metabolism-related enzyme in retroabdominal wall fat is shown. Shows the mRNA expression level of liver lipid metabolism-related enzymes.

Claims (3)

  A fat accumulation inhibitor comprising a micellar calcium phosphate-phosphopeptide complex as an active ingredient.   A drug comprising the fat accumulation-suppressing agent according to claim 1.   Food-drinks containing the fat accumulation inhibitor of Claim 1.

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