JP6872841B2 - Fat reduction composition - Google Patents

Description

The present invention relates to a composition for suppressing body fat accumulation, a composition for suppressing visceral fat accumulation, and a method for suppressing fat accumulation, which comprises an antibody against human pathogenic bacteria and a bacterial toxin as an active ingredient.

In recent years, the number of obese people has been increasing due to changes such as an increase in fat intake in the diet and a decrease in the amount of exercise in daily life. Obesity is a condition in which body fat is accumulated. The fat in the abdomen is called visceral fat, which causes lifestyle-related diseases such as diabetes, arteriosclerosis, hypertension, cerebral infarction, and dyslipidemia.

Metabolic syndrome (visceral fat syndrome) is a condition in which any two or more of hyperglycemia, hypertension, and dyslipidemia are combined with visceral fat obesity. Metabolic syndrome is the result of excess energy accumulated as fat due to the positive energy balance between energy intake and lack of exercise, but there is an irregular lifestyle as the cause, and it is prevented and improved by improving the lifestyle. It is believed that it can be done.

On the other hand, recent studies have reported that the intestinal flora is a factor involved in the accumulation of visceral fat, first in the 2004 Gordon JI et al. Study using sterile mice, and further in human studies from 2006 onwards. It came to.

Studies on gut microbiota, animals and humans can be summarized in the following two categories, A) and B).
A) In the human intestinal flora, there are resident multiple bacterial species such as dietary fiber that decompose food components that cannot be used as an energy source by humans and use them as an energy source that can be used by humans. These bacterial species and abundance are affected by genetic factors and dietary components, and affect human energy balance.

B) The gut microbiota is classified into bifidobacteria, lactic acid bacteria and other bacilli that are symbiotic with humans, and bacilli that cause harmful effects such as Clostridium perfringens and bacilli, which are pathogenic bacteria, and opportunistic bacteria. To. Opportunistic bacteria do not have harmful effects when the intestinal flora is mainly good bacteria, but they have harmful effects as bad bacteria in situations where bad bacteria are the main constituents. Non-pathogenic Escherichia coli, Eubacterium, streptococcus (Peptococcus) and the like. The ratio of good bacteria, opportunistic bacteria, and bad bacteria progresses to bad bacteria (Dysbiosis) due to the decrease of bifidobacteria and the increase of bad bacteria such as Clostridium perfringens and Escherichia coli after the middle age. With the progress of bad bacteriolysis, endotoxin, which is an index of bad bacteriolysis, progresses into the body after the 50s, and the state becomes chronically inflamed.

All intestinal bacteria are divided into Gram-positive bacteria and Gram-negative bacteria according to the constituents of the bacterial cells. Endotoxin is a cell wall component of Gram-negative bacteria, is chemically a lipoprotein polysaccharide, lipopolysaccharide, and is sometimes described by the abbreviation of LPS. Looking at the relationship between gram-negative bacteria and their pathogenicity to humans, they are widely distributed from pathogenic bacteria to opportunistic bacteria and non-pathogenic bacteria. Bacterial Gram-negative Bacterial growth increases endotoxin levels in the gastrointestinal tract.

The gastrointestinal tract is a barrier that avoids the transfer of foreign substances such as food and bacteria into the body, and the transfer of endotoxin in the digestive tract into the body is blocked by the barrier function. The barrier function is impaired by the conversion of the intestinal flora into bad bacteria, and the transfer of endotoxin into the body increases. Endotoxin is the most active inflammatory substance resident in the gastrointestinal tract, and in the bad bacteria state of the intestinal flora, the chronic transfer of endotoxin into the body leads to a state of chronic inflammation.

Chronic inflammation enhances lipase activity derived from vascular endothelial cells, promotes lipid synthesis in adipocytes, and causes an increase in obese people after middle and old age.

As a means of eliminating bad bacteria in the intestinal flora and suppressing obesity and fat accumulation by turning them into good bacteria, probiotics (Non-Patent Document 1), which are already living lactic acid bacteria, and nutrition of lactic acid bacteria Therefore, an attempt to use prebiotics (Non-Patent Document 2), which is an indigestible polysaccharide that acts on the growth of lactic acid bacteria, is known.

Antibodies are also known to be used as another means of improving the gut microbiota. For example, in Japanese Patent Application Laid-Open No. 2007-330193, a fermented microorganism is added after adding a milky protein to the fermented milk raw material mother liquor and sterilizing or sterilizing by a method in which 70% by mass or more of the anti-Welsh bacterium antibody in the mother liquor remains. It is disclosed that a fermented dairy product infused with a starter and fermented has an effect of improving the intestinal bacterial flora (Patent Document 1).

JP-A-2007-330193

Kadoka et al. , (2010): Regulation of abdominal adipose tissue by probiotics (Lactobacillus gasseri, SBT2055) in adults with obesity ten-dens. 64,636-43. Cani et al. , (2007): Selective increases of bifidobacteria in gut microflora irnprove high-fat-diet-induced diabetic diabetes in microbiota

The present invention is made by ingesting a composition containing an antibody as a main component, particularly an antibody against human pathogenic bacteria and bacterial toxins (endotoxin (endotoxin) and exotoxin (exotoxin)). An object of the present invention is to provide a composition for reducing fat and visceral fat.

In order to solve the above problems, the present inventors eliminate human pathogenic bacteria and endotoxin-producing bacteria by ingesting antibodies against human pathogenic bacteria and endotoxin-producing bacteria and antibodies against the bacterial toxins endotoxin and exotoxin. The study was conducted based on the hypothesis that body fat and visceral fat can be reduced by lowering the level of gastrointestinal endotoxin and reducing the transfer of endotoxin into the body. Then, they came to the finding that body fat was reduced by ingestion of the antibody, and completed the present invention.

The "bad bacteria", "human pathogenic bacteria", and "endotoxin-producing bacteria" in the present invention are defined below.

An "endotoxin-producing bacterium" is a bacterium having endotoxin on the cell wall as a structural component of a bacterial cell. All bacteria classified as Gram-negative bacteria are included in the classification of bacteria by Gram-pigment staining. For example, this includes gram-negative pathogenic bacteria and opportunistic bacteria such as toxin-producing pathogenic Escherichia coli O-157, non-pathogenic bacteria Escherichia coli, and Pseudomonas aeruginosa.

"Human pathogenic bacteria" are highly pathogenic pathogenic bacteria that do not normally inhabit healthy people, include opportunistic bacteria, and are bacteria other than good bacteria that have a symbiotic relationship with humans. Examples include gram-negative and gram-positive bacteria such as Vibrio cholerae, Shigella dysentis, Streptococcus pneumoniae, and Staphylococcus.

"Bad bacteria" are bacteria other than good bacteria that are resident in humans, and include pathogenic bacteria and opportunistic bacteria. It should be noted that pathogenic bacteria that are not normally resident in healthy subjects, such as Shigella dysentis and Vibrio cholerae, are not included.

The present invention provides a fat-reducing composition that contains an antibody as an active ingredient and is used to reduce fat accumulation.

According to the present invention, the antibody regulates the intestinal bacterial flora and suppresses the transfer of endotoxin into the body, thereby suppressing the accumulation of body fat and visceral fat.

The fat reduction composition according to the present invention will be described. The fat reduction composition according to the present embodiment contains an antibody as an active ingredient and is used to reduce fat accumulation.

The antibody used in the present embodiment is a product of an immune system that binds to pathogens and their constituents that are foreign substances to the living body, eliminates the foreign substances from the body, and detoxifies them, and is chemically immunoglobulin. Is.

For antibody selection, plasma of domestic animals such as cows, buffaloes, horses, pigs, goats, rabbits and rabbits, and eggs such as first milk, raw milk and chickens are the starting materials for preparing antibody compositions. Various human pathogenic bacteria can also be inoculated as vaccines to increase the concentration of antibodies. The starting materials are further freeze-dried and spray-dried after defibrinant treatment in the case of plasma, and after removing milk fat, lactose and casein in the case of first milk and raw milk, water removal and concentration by ultrafiltration and sterile filtration. Further, it is pulverized by freeze-drying and spray-drying. These technologies have already been established and implemented.

Since the activity of an antibody is rapidly lost by heating in a liquid state, it is necessary to manage the sterilization by heating to satisfy both the sterilization and the prevention of heat denaturation of the antibody. Practical forms of the composition are aqueous solutions or dry powders.

Quality assessment is done by quantification of antibodies. For the measurement of the total antibody amount, for example, a dual antibody enzyme immunoassay method, an immunodiffusion method, a protein A or protein G column method, etc. are recommended according to the text. In addition, the enzyme immunoassay is used to measure antibodies against specific bacteria and bacterial toxins.

It is important to check the antigen specificity of the antibody in order to ensure the function of the composition having the function of lowering endotoxin in blood and feces. That is, it is an essential requirement to include an antibody against endotoxin-producing bacteria, a human-derived pathogenic bacterium, an antibody against endotoxin, and an antibody against exotoxin.

Antibodies to endotoxin-producing bacteria promote removal by excreting endotoxin-producing bacteria from the intestinal tract, and antibodies to endotoxin bind to endotoxin and act to prevent endotoxin translocation into the body.

On the other hand, Gram-positive human pathogens that do not produce endotoxin (Clostridium perfringens, Staphylococcus, etc.) produce exotoxin, destroy the barrier of the digestive tract, and enhance the translocation of endotoxin into the body. It binds to toxins and blocks the translocation of endotoxins into the body.

The endotoxin-producing bacterium is a bacterium corresponding to the following genera. For example, Fusobacterium, Veillonella, Megasphaera, Neisseria, Moraxella, Branhamella, Branhamella, Acinetobacter (Acinetobacter) (Escherichia), Hafnia, Klebsiella, Morganella, Proteus, Providencia, Salmonella, Salmonella, Serratia, Serratia (Vibrio), Aeromonas, Plesiomonas, Haemofilus, Pasteurella, Pseudomonas, Legionella and the like can be mentioned.

Formalin-treated or heat-killed bacteria of endotoxin-producing bacteria are selected as an antigen for antibody detection and quantification, if necessary. Formalin treatment is required for the detection and quantification of antibodies against finbrier and flagella. As the endotoxin, each of the above-mentioned bacterial cells can be used as it is, or endotoxin (LPS) can be extracted from these bacterial cells by the trichloroacetic acid method or the phenol method according to the textbook and used.

The activity of anti-endotoxin antibody can be evaluated by measuring the amount of antibody by the ELISA method using endotoxin extracted from each bacterial cell or heated or formalin-treated bacterial cells as an antigen, and the endotoxin activity can be evaluated by the Limulus method on a commercially available kit. Can be used and measured.

The activity of the anti-endotoxin antibody is such that the anti-endotoxin antibody neutralizes the endotoxin and causes the activity of the endotoxin to be lost. By measuring with, the total endotoxin neutralizing (= detoxifying) activity of various antibody mixtures having different antigen specificities can be quantitatively measured.

In the present embodiment, it is preferable to use whey protein (WPC) containing an innate immune antibody antibody derived from raw milk as a material for the antibody. "Innate immune antibody" refers to an antibody obtained as a result of natural exposure of an animal's immune system to a foreign antigen. WPC is a collection of proteins contained in whey, which is a by-product of cheese production, and innate immune antibodies are concentrated in this WPC by using a production method with a small heat history. As the WPC containing the innate immune antibody, a commercially available WPC can be used. As a commercially available WPC, for example, "Asama Whey Protein" (manufactured by Asama Kasei Co., Ltd.) can be used.

"Asama milk clear protein" is, among other natural immune antibodies, a) endotoxin derived from 33 pathogenic bacteria selected from human pathogenic bacteria, b) pathogenic Escherichia coli O-26 strain, O-55 strain, O An antibody selected using endotoxin derived from the −111 strain, four endotoxins derived from Salmonella Minesota, Lipid A, c) enterotoxin B derived from Staphylococcus aureus, and enterotoxin derived from Clostridium perfringens as indicators. (Yoshiko Kijima et al., Journal of Japan Society for Food Chemistry, 2009, 56: 475-482). Therefore, "Asama whey protein" contains a large amount of innate immune antibodies against human pathogenic bacteria and bacterial toxins. This was obtained as a result of the newly developed ELISA method, which is a highly sensitive and accurate measurement method superior to the ELISA method, which made it possible to select whey protein products containing antibodies against human pathogenic bacteria. It is a thing.

The 33 types of spontaneous immune antibodies against human pathogenic bacteria in "Asama milk clear protein" are Escherichia coli O-111, intestinal hemorrhagic Escherichia coli O-157, and Streptococcus salmonella. Streptococcus, Shiga, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus Minnesota R595), Streptococcus epidermidis, Streptococcus aureus, Streptococcus cerevinum, Streptococcus profringens, Campylobacter, Campylobacter Streptococcus sanguis, Streptococcus salivarius, Streptococcus mutans, Aerogenes, Enterobacter aerogenes, Alkagenes aeruginosa), Proteus, Helicobacter streptococcus, Group A Streptococcus type 1 (Group A Streptococci type-1), Group A Streptococcus type 12 (Group Acpic12) Streptococcus pyogenic type 22 (Group A Streptococcus type-22), Streptococcus viridans, Streptococcus pneumoniae, Pneumoniae pneumoniae It is an antibody against Haemophilus influenzae, and the innate immune antibody may include other anti-human pathogenic bacterial antibodies.

The natural immune antibodies against bacterial toxins in "Asama milk clear protein" are endotoxins derived from pathogenic Escherichia coli O-26, O-55 and O-111 strains, Lipid A derived from Salmonella Minnesota, and Staphylococcus aureus. It is an antibody against enterotoxin B derived from S. aureus and enterotoxin derived from Clostridium perfringens, and the natural immune antibody may include other antibacterial toxin antibodies.

The selection of innate immune antibodies is based on the fact that the antibody titer is 8 μg or more in 1 g of whey protein for a) above, and the antibody titer is 1 μg or more in 1 g of whey protein for b) and c) above. did.

In the fat reduction composition according to the present embodiment, the antibody content required for reducing body fat is preferably 0.001% or more, more preferably 0.01% or more. The intake of the antibody is preferably 10 mg or more, more preferably 100 mg or more per day for an adult.

In order to further make the removal of adverse effects of intestinal pathogens and bacterial toxins more effective using the fat-reducing composition according to the present embodiment, an active ingredient other than an antibody can be blended. Examples of active substances other than antibodies include gliadin-treated SOD, prebiotics, and probiotics of antioxidant food materials. Prebiotics are indigestible food ingredients that show beneficial effects on human health by specifically growing limited bacteria such as bifidobacteria that live in the intestinal tract, such as lactulose and raffinose. Oligosaccharides can be exemplified. Probiotics are live or dead bacteria additives that benefit the host by improving the balance of intestinal microorganisms, such as Lactobacillus, Enterococcus, and Streptococcus. , Bifidobacterium and other lactic acid bacteria, including bacteria that produce lactic acid as a metabolite), butyric acid bacteria and the like can be exemplified.

The form of ingestion of the antibody is not particularly limited, and examples thereof include powders, tablets, capsules, granules, cookies, ice cream, and beverages. However, it is premised that the active ingredient antibody is ingested under the condition that it is not inactivated.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

1. 1. Effect of antibody intake on fecal endotoxin, intestinal flora and suppression of fat increase To investigate the effect of oral administration of antibody on reduction of fecal endotoxin, improvement of intestinal flora, and suppression of increase of abdominal visceral fat and subcutaneous fat. The test was conducted as follows.

(1) Feed As feed to be fed to mice, a high-fat feed with a fat content of 60% calorie ratio (HFD-60 manufactured by Oriental Yeast Co., Ltd.) and a natural immune antibody (manufactured by Asama Kasei Co., Ltd.) are added to this high-fat feed. A mixed diet (high-fat diet + natural immune antibody) was prepared by mixing 5% by weight of Asama whey protein. In addition, as a placebo, innate immune antibody contained in Asama Whey Protein (manufactured by Asama Kasei Co., Ltd.) is added to a high-fat diet (HFD-60 manufactured by Oriental Yeast Co., Ltd.) with a fat content of 60% calorie. A mixed diet (high-fat diet + placebo) was prepared by mixing 5% of the 100% inactivated product.

(2) Experimental method As the mouse, C57BL / 6J mouse ♀ 12 weeks old purchased from Sankyo Lab Co., Ltd. was used. The mice were divided into 3 groups so that there were 7 to 9 mice in each group. Among them, the first group was a group that was fed a high-fat diet so as to exhibit obesity (high-fat diet group), and the second group was a group that was fed a mixed diet in which 5% by weight of Asama whey protein was mixed with the high-fat diet. (High-fat diet + natural immune antibody group), and the third group was a group (high-fat diet + placebo group) in which a mixed diet diet in which 5% by weight of inactivated antibody was mixed with the high-fat diet was ingested.

These mice were fed a diet for 1 to 4 weeks so as to exhibit obesity, and the amount of fecal endotoxin (LPS), the number of intestinal bacteria, the abdominal visceral fat and the subcutaneous fat were measured, respectively.

(3) Amount of fecal endotoxin (LPS) The amount of fecal endotoxin (LPS) was quantified by the Limuras method using the feces collected at 8 am 4 weeks after ingestion of each feed as a sample. Table 1 shows the amount of endotoxin (LPS) in feces before feeding each feed and during the period up to 4 weeks after the start of feeding. There was no difference in the amount of LPS between the groups for 2 weeks after the start of feeding, but a significant decrease in LPS level was observed in the innate immune antibody-administered group until 3 and 4 weeks.

(4) Number of intestinal bacteria In order to examine the effect of antibody administration on the intestinal flora, the total number of fecal bacteria, lactic acid bacilli, Escherichia coli, bifidobacteria, and Clostridium perfringens were measured by the FISH method. That is, the cells were stained with a fluorescently labeled DNA probe for 16s ribosomal RNA of the bacterium and measured with a microscope. As a result, the bifidobacteria were 5.52 ± 0.28 cfu / g in the high-fat diet group and 5.87 ± 0.35 cfu / g in the placebo group, whereas they were 6.44 ± 0. A significant increase of 28 cfu / g was observed. The results are shown in Table 2. Since no significant difference was observed in the other measured bacterial species, it was judged that the intestinal bacterial balance was improved.

(5) Visceral fat and subcutaneous fat The abdominal visceral fat and subcutaneous fat were measured by CT scan before and 5 weeks after each feed was ingested. Mice were fasted from 16 hours after CT scan, and anesthetized by intraperitoneal administration (10 μg / g BW) of somnopentyl (6.48 μg / ml to 6.5 μg / ml) diluted with physiological saline. An abdominal CT scan was performed using an Explore Locus CT system (manufactured by GE Healthcare). From the X-ray CT image, each cross-sectional slice image from the lowermost part of the diaphragm to the uppermost part of the inguinal region was obtained. In each cross-sectional slice image, a fat region (lower: 10489, upper: 13989) was selected from the CT value histogram in the abdominal circumference, and the obtained area was taken as the total fat area.

Similarly, in each cross-sectional slice image, a fat region (lower: 10489, upper: 13989) was selected from the CT value histogram in the abdominal cavity, and the obtained area was used as the visceral fat area. The difference between the area of total fat and the area of visceral fat was defined as the subcutaneous fat area. Each area was calculated by accumulating from the lowermost part of the diaphragm to the uppermost part of the inguinal region.

Table 3 shows changes in visceral fat mass and subcutaneous fat mass before and after feeding each feed. Compared to before feeding each feed, in the "high-fat diet group" in which only the high-fat diet was ingested, the visceral fat increased significantly by 130.2 ± 14.9%, whereas the high-fat diet inactivated the antibody. In the "high-fat diet + placebo group" to which the added placebo was added, 121.3 ± 28.9%, and further, the "high-fat diet + natural immune antibody group" to which the natural immune antibody was added to the high-fat diet. , The increase of visceral fat was suppressed in the order of 105.7 ± 14.0%.

Subcutaneous fat did not change at 99.9 ± 32.5% in the “high fat group” compared to before feeding each feed, but 132.8 ± in the “high fat diet + placebo group”. It increased to 20.4%. On the other hand, in the "high fat diet + innate immune antibody group", it decreased significantly to 80.6 ± 11.4%.

From this, it can be expected that ingestion of innate immune antibody suppresses the increase of visceral fat and subcutaneous fat through the decrease of endotoxin in feces.

2. Effect of oral administration of antibody to reduce periovarian fat mass Animals were purchased from Sankyo Lab Co., Ltd., and C57BL / 6J mice ♀ 12 weeks old were used. In these mice, the amount of periovarian fat with and without antibody was compared, and the effect on visceral fat accumulation was evaluated.

(1) Feed AIN-93M manufactured by Oriental Yeast Co., Ltd. was used as a general feed (control). In addition, asama whey protein was used as an innate immune antibody.

(2) Experimental method The mice were divided into two groups so that there were 6 to 7 mice in one group, one group was the "control group" fed with general feed, and the other group was fed with general feed and 5 weights of Asama whey protein. The group was defined as the "innate immune antibody group" in which the mixed feed was ingested.

Before feeding each feed and 6 weeks after feeding, autopsy was performed after fasting for one day. Somnopentyl (sodium pentobarbital 64.8 mg / mL) was intraperitoneally administered for anesthesia. After laparotomy, the lower aorta was perfused with 0.9% physiological saline sterilized using an indwelling needle, and the periovarian fat was removed.

(3) Results Table 4 shows the amount of periovarian fat after 6 weeks of feeding each feed. In the control group that ingested the general diet, the periovarian fat mass was 0.304 ± 0.114 g. On the other hand, in the innate immune antibody group in which the innate immune antibody was added to the general feed, the amount decreased significantly to 0.169 ± 0.046 g.

As described above, the administration of innate immune antibody showed an improvement in fecal bacterial balance by an increase in bifidobacteria and a decrease in LPS levels. The decrease in LPS affected the fat metabolism of mice, and there was a decrease in body fat, especially a decrease in visceral fat due to a decrease in ovarian fat accumulation. Furthermore, in the dynamic observation of the mice in each group, the disorder of the coat was remarkable in the high-fat group mouse, but the normal coat in the innate immune antibody-administered group also symbolized the effect of the antibody on reducing body fat. Met.

Claims (7)

A fat-reducing composition used to reduce fat accumulation by containing anti-human pathogenic bacterial antibodies and anti-endotoxin antibodies as active ingredients, lowering gastrointestinal endotoxin levels and reducing endotoxin translocation into the body. There,
The anti-human pathogenic bacterial antibody includes all the antibodies against each human pathogenic bacterium described in a) below.
A fat-reducing composition, wherein the anti-endotoxin antibody contains all the antibodies against each endotoxin described in b) below.
a) Escherichia coli O-111, intestinal hemorrhagic Escherichia coli O-157, Salmonella, Shigella dysensis, Seleus, Ersina, Seratia, Salmonella minesota R595, Salmonella, Bacteroides, Aerogenes, Alkalinegenes, Enterobacter B) Pathogenic Escherichia coli O-26 strain, O-55 strain and O-111 strain-derived endotoxin, Salmonella Minesota-derived lipid A. The fat-reducing composition according to claim 1, wherein the antibody is an innate immune antibody. The fat-reducing composition according to claim 1 or 2, wherein the fat is visceral fat. The antibody is derived from an antibody produced by at least one selected from the group consisting of cows, goats, sheep, horses and chickens, and consists of milk, first milk, blood and chicken eggs of these animals. The fat-reducing composition according to any one of claims 1 to 3, which is produced from at least one selected from the group as a raw material. The fat-reducing composition according to any one of claims 1 to 4, wherein the content of the antibody is 0.001% or more. The fat-reducing composition according to any one of claims 1 to 5, wherein the antibody is selected based on the antibody titer against endotoxin. The fat-reducing composition according to any one of claims 1 to 6, further comprising at least one selected from the group consisting of prebiotics, probiotics and gliadin-coated superoxide dismutase (SOD).