JP7705633B2 - Visceral fat reducing agent - Google Patents

Description

The present invention relates to a visceral fat reducing agent.

Visceral fat is fatty tissue that is distributed between the stomach and liver and in the surrounding areas, and it is known that excessive accumulation of visceral fat is a risk factor for arteriosclerotic lesions. Currently, visceral fat accumulation is the first criterion for diagnosing metabolic syndrome. Managing the accumulation of visceral fat is extremely important in aiming to extend healthy life expectancy.

More than 1000 kinds and more than 100 trillion bacteria live in the human digestive tract, forming the intestinal flora. Recent research has revealed that the intestinal flora is involved in the pathogenesis of various diseases, including not only digestive diseases but also metabolic diseases, immune diseases, and mental disorders. In particular, it has been suggested that the composition of the intestinal flora may be closely related to obesity and metabolic diseases caused by obesity.
The two most predominant phyla of intestinal flora living in the digestive tract are Firmicutes and Bacteroidetes. When comparing the intestinal flora of obese mice and normal mice, it has been reported that the obese mice have more bacteria of the Firmicutes phylum and fewer bacteria of the Bacteroidetes phylum, while the normal mice have more bacteria of the Bacteroidetes phylum. It has also been reported that the ratio of Firmicutes/Bacteroidetes is high in obese humans. Furthermore, although there is no consistent correlation between the presence of bacteria in the phyla Firmicutes and Bacteroidetes and visceral fat, when bacterial species are analyzed by "genera," which are smaller than phyla, it has been reported that people with small visceral fat area, regardless of gender, have a higher proportion of Blautia bacteria in their intestines, and that among intestinal bacteria, Blautia bacteria are associated with visceral fat mass in both men and women (Non-Patent Document 1).

Blautia bacteria are the major bacteria that account for about 3 to 11% of the total intestinal bacteria in humans and animals. Blautia bacteria are known to produce short-chain fatty acids such as acetic acid, propionic acid, and butyric acid, which have the effect of eliminating obesity in the body, and have been reported to be reduced in patients with diabetes, liver cirrhosis, colon cancer, and rheumatoid arthritis. It has also been reported that strains of Blautia bacteria, such as Blautia hydrogenotrophica, are effective in increasing or maintaining microbiota diversity and in treating brain damage (e.g., Patent Documents 1 and 2).
However, there have been no reports on the effect of intake of Blautia bacteria on visceral fat accumulation.

Special table 2019-508363 publication Special Publication No. 2020-520916

npj Biofilms and Microbiomes 2019 5:28

The present invention relates to providing a visceral fat reducing agent that is useful for reducing visceral fat.

In light of the above problems, the present inventors conducted extensive research and found that oral intake of Blautia bacteria significantly reduces visceral fat.

That is, the present invention provides a visceral fat reducing agent that contains Blautia bacteria as an active ingredient.

The visceral fat-reducing agent of the present invention exhibits an excellent effect of reducing visceral fat. Therefore, it is expected to prevent, improve, or treat the onset or progression of health disorders or diseases closely related to the accumulation of visceral fat.

Graph showing the total visceral fat weight, epididymal weight, mesenteric weight, perirenal weight and retroperitoneal weight for each experimental group.

In the present invention, the bacteria of the genus Blautia are bacteria belonging to the genus Blautia, which is classified into the phylum Firmicutes.
Examples of bacteria belonging to the genus Blautia include Blautia hydrogenotrophica, Blautia stercoris, Blautia faecis, Blautia coccoides, Blautia glucea, Blautia hansenii, Blautia luti, Blautia producta, Blautia schinkii, Blautia wax, Blautia vaccinata ... Blautia wexlerae and the like. These may be used alone or in combination. Among these, Blautia hensennii is preferred from the viewpoint of improving the visceral fat reducing effect.
Blautia hensennii can be exemplified by JCM 14655 strain or a bacterium identical thereto. The term "same bacterium" refers to a bacterium of the same genus having a nucleotide sequence of 16S rRNA gene that has a homology of 98% or more, preferably 99% or more, with the nucleotide sequence of the 16S rRNA gene of the cell strain and having the same bacteriological properties.
Blautia hensennii JCM 14655 strain is publicly available from the Microbial Research Center, RIKEN BioResource Research Center (RIKEN BRC) based on the strain's JCM number listed in their online catalog (e.g., for JCM 14655 strain, see https://www.jcm.riken.jp/cgi-bin/jcm/jcm_number?JCM=14655).

Bacteria of the genus Blautia can be obtained by culturing them according to a method commonly used for culturing bacteria of the genus Blautia. For example, the bacteria are cultured in GAM medium or the like at a culture temperature of 30 to 45°C. The culture is preferably performed under anaerobic conditions. After the culture, the bacteria can be separated by a collection method such as centrifugation or membrane concentration.

The Blautia bacteria may be either live or killed, but from the viewpoints of quality stability and safety to living organisms, it is preferable to use killed bacteria.
The killed cells may be, for example, heat-sterilized cells obtained by heat-sterilizing cells after culture. The killed cells may be in the form of a suspension in water or the like, or in the form of a dried product. The drying method may be a conventional method such as spray drying or freeze drying.

As shown in the Examples below, when Blautia bacteria are ingested, the accumulation of visceral fat promoted by the intake of a high-fat diet is suppressed. That is, Blautia bacteria have the effect of suppressing the accumulation of visceral fat and reducing visceral fat. It is considered that the intake of Blautia bacteria affects the living conditions and bacterial count of Blautia bacteria existing in the intestine, thereby reducing visceral fat. By reducing visceral fat, it becomes possible to prevent, improve, or treat visceral fat-type obesity and metabolic syndrome (visceral fat syndrome) based on visceral fat accumulation.
Therefore, Blautia bacteria can be a visceral fat-reducing agent that reduces visceral fat, and can be used to produce a visceral fat-reducing agent. Furthermore, Blautia bacteria can be used to reduce visceral fat.
As used herein, "visceral fat" refers to fat accumulated in visceral tissues (e.g., stomach, liver, kidneys, etc.) in the abdominal cavity and around them. "Reducing visceral fat" refers to reducing the amount of visceral fat and inhibiting the accumulation of visceral fat.
"Use" may be administration to or ingestion by an animal, including a human, and may be therapeutic or non-therapeutic use. "Non-therapeutic" is a concept that does not include medical procedures, i.e., methods of surgery, therapy, or diagnosis of humans, and more specifically, methods of surgery, therapy, or diagnosis performed by a physician or a person under the supervision of a physician, on humans.

The visceral fat-reducing agent of the present invention can be a drug, quasi-drug, food, or feed for humans or animals that exerts a visceral fat-reducing effect when administered or ingested by animals, including humans, and can also be a material or preparation used by being mixed with the drug, quasi-drug, food, or feed.

The pharmaceutical (hereinafter, including quasi-drugs) contains Blautia bacteria as an active ingredient for reducing visceral fat. Furthermore, the pharmaceutical may contain a pharma- ceutical acceptable carrier, or other active ingredients, pharmacological ingredients, etc., as necessary, so long as the function of the active ingredient is not lost.
The pharmaceutical agent may be administered in any dosage form. Examples of dosage forms include oral solid preparations such as tablets (including chewable tablets), capsules, granules, powders, and lozenges, oral liquid preparations such as oral liquids and syrups, and parenteral preparations such as injections, suppositories, inhalants, transdermal agents, and external preparations. The preferred dosage form is oral administration. The size of the dosage form can be adjusted as desired depending on the purpose of use.
Such preparations in various dosage forms can be prepared according to standard methods by appropriately combining, as necessary, pharma- ceutically acceptable carriers such as excipients, binders, bulking agents, disintegrants, surfactants, lubricants, dispersants, buffers, osmotic pressure regulators, pH adjusters, emulsifiers, preservatives, stabilizers, thickeners, flow improvers, flavoring agents, foaming agents, fragrances, coating agents, diluents, and other medicinal ingredients.

The food contains Blautia bacteria as an active ingredient for reducing visceral fat.
Foods include foods that claim to reduce visceral fat and have been approved or notified to be labeled as such, as necessary (foods for specified health uses, foods with functional claims). An example of a label is "reduces visceral fat." Foods that have been approved or notified to be labeled as such can be distinguished from general foods.
The food may be in the form of a solid, semi-solid, or liquid (e.g., beverage). Examples include various food compositions (breads, cakes, noodles, confectioneries, frozen foods, ice creams, candies, toppings, soups, dairy products, shakes, beverages, seasonings, etc.), and nutritional supplement compositions in the same form as the oral preparations described above (solid preparations such as granules, powders, tablets, capsules, microcapsules, and lozenges).
Foods of various forms can be prepared according to standard methods by appropriately combining Blautia bacteria with any food material, or other active ingredients, or additives acceptable for foods (e.g., solvents, softeners, oils, emulsifiers, preservatives, acidulants, sweeteners, bittering agents, pH adjusters, stabilizers, colorants, UV absorbers, antioxidants, moisturizers, thickeners, adhesives, dispersants, flow improvers, humectants, aromatics, seasonings, flavor adjusters), etc.

The feed contains Blautia bacteria as an active ingredient for reducing visceral fat.
The feed is preferably in the form of pellets, flakes, mash or liquid, and examples thereof include livestock feed for cows, pigs, chickens, sheep, horses, etc., small animal feed for rabbits, rats, mice, etc., and pet food for dogs, cats, small birds, etc.
Feed can be prepared according to standard methods by appropriately combining Blautia bacteria with other feed ingredients such as meat, protein, grains, bran, meal, sugar, vegetables, vitamins, minerals, gelling agents, shape-retaining agents, pH adjusters, seasonings, preservatives, nutritional supplements, etc.

The content of Blautia bacteria in the visceral fat-reducing agent of the present invention may vary depending on the dosage form or shape, but can be set appropriately taking into consideration ease of administration or ingestion, etc.

The dosage or intake of the visceral fat reducing agent of the present invention may vary according to the species, body weight, sex, age, condition or other factors of the subject of administration or intake. The dosage, route, interval, and intake amount and interval can be appropriately determined by those skilled in the art, but in the case of oral administration or oral intake, from the viewpoint of improving the visceral fat reducing effect per day for an adult (body weight 60 kg), the number of bacteria of the genus Blautia is preferably 1 x 10 ⁴ or more, more preferably 1 x 10 ⁵ or more, even more preferably 1 x 10 ⁷ or more, and also preferably 1 x 10 ¹⁵ or less, more preferably 1 x 10 ¹² or less, and even more preferably 1 x 10 ⁹ or less. The dosage or intake amount of the visceral fat-reducing agent of the present invention is preferably within the range of 1 x ¹⁰ to 1 x ¹⁰ bacteria, more preferably 1 x ¹⁰ to 1 x ¹⁰ bacteria, and even more preferably 1 x ¹⁰ to 1 x ¹⁰ bacteria.
In the present invention, it is preferable to orally administer or ingest such an amount once, twice, or three or more times a day.
In the examples described later, it is explained that the Blautia bacteria administered to mice have a visceral fat reducing effect, but from the viewpoint of ethical and safety considerations, this is done by using mice, which have been established as a human-presuming model by official test methods or academic papers, rather than humans. Therefore, the visceral fat reducing effect in the present invention is a concept that indicates not only the visceral fat reducing effect in mice, but also activity on humans in general.

The visceral fat-reducing agent of the present invention can be administered or ingested according to any schedule.
The administration or ingestion period is not particularly limited, and may be a single administration or ingestion, or may be repeated or continuous administration or ingestion. In the case of repeated or continuous administration or ingestion, it is preferable to administer or ingestion continuously for 2 weeks or more, further 4 weeks or more, and further 8 weeks or more.

The visceral fat reducing agent of the present invention is preferably administered or ingested during, before, or after eating/feeding. When administered or ingested before or after eating/feeding, it is preferably administered or ingested within 10 minutes before or after eating/feeding. The contents of the food (feeding) are not particularly limited, and include meals or feed containing proteins, carbohydrates (including sugars), and lipids.
There are no particular limitations on the subjects of administration or intake, so long as they are humans or non-human animals who need or desire it. Preferred examples of subjects include humans with a high amount of visceral fat accumulation (e.g., humans with a visceral fat area of 100 ^cm2 or more), humans who tend to eat a high-fat diet, etc.

Test Example 1
1. Test diet and method Blautia hensennii JCM 14655 strain (RIKEN BRC) was killed by heat sterilization in an autoclave, then suspended in distilled water, washed repeatedly by centrifugation, and freeze-dried to obtain a powder.
Three-week-old normal male mice (C57BL/6J mice) were pre-bred for one week, and then experimentally bred for nine weeks with free access to food and water. The mice were fed a standard diet of AIN-93G, a basic diet prescribed by the National Institute of Nutrition, and a high-fat diet containing 20% lipid by mass, which was prepared with reference to the composition of the basic diet.
The experimental groups were divided into three groups, a normal diet control group (n=8), a high-fat diet control group (n=8), and a high-fat diet (with Blautia bacteria) group (n=8), so that the initial body weight was almost constant. The dose of Blautia bacteria was 1 x ¹⁰⁹ bacteria/mouse/day. During the experimental rearing period, body weight and water intake were measured, and feces were collected.
After the experimental feeding, the animals were euthanized by cervical dislocation and dissected to extract the visceral fat, including epididymal fat, mesenteric fat, perirenal fat, and retroperitoneal fat, and the weights of the various organs were measured. The sum of these weights was taken as the total visceral fat weight.

2. Method for identifying Blautia bacteria in foods containing Blautia bacteria It is possible to identify Blautia bacteria using real-time PCR. A specific example is shown below.
Step 1. DNA extraction and extraction method Pretreatment and crude extraction: Each sample is suspended in 4 M guanidine thiocyanate, 100 mM Tris-HCl (pH 9.0), and 40 mM EDTA, and the sample is pretreated using a cell disruption device (such as FastPrep FP100A (MP Biomedicals, USA)). Furthermore, DNA can be extracted from the bead-treated suspension using Magtration System 12GC and GC series MagDEADNA 200 (Precision System Science, Japan).

Step 2. PCR conditions Amplify a DNA fragment. A kit such as Blautia Hansenii Detection Kit (manufactured by Techno Suruga Lab Co., Ltd.) can be used.

Step 3. Real-time PCR The reagent used in the real-time PCR may be, for example, TB Green Premix Ex Taq 2 (Tli RNaseH Plus) (manufactured by Takara Bio Inc.). The device may be, for example, Rotor-Gene Q (manufactured by QIAGEN). The PCR conditions may be, for example, the method described in Dig Dis Sci, 2015, 60(9)2654-61.

A t-test was used to test for significant differences. The values obtained are shown as the mean ± standard error.

2. Results Figure 1 shows the total visceral fat weight, epididymal weight, mesenteric weight, perirenal and retroperitoneal weight for each group. Different letters (a, b, c) in Figure 1 indicate a significance level of P<0.05, and # indicates P=0.08 in comparison with the normal diet. As a result, the high-fat diet (with Blautia) group showed a decrease in visceral fat weight compared to the high-fat diet control group, confirming that Blautia has an excellent inhibitory effect on visceral fat accumulation promoted by the intake of a high-fat diet.

Claims (2)

A visceral fat reducing agent whose active ingredient is Blautia hensenii . 2. The visceral fat reducing agent according to claim 1 , which is administered or ingested in an amount of 1×10 ⁴ to 1×10 ¹⁵ cells of Blautia hensennii per day.