JP5399468B2 - Composition exhibiting lipolytic activity - Google Patents

Description

The present invention relates to a method for producing a composition exhibiting a lipolytic action.

Examples of fat accumulation include visceral fat and subcutaneous fat, both of which cause lifestyle-related diseases, and measures are urgently promoted from the viewpoint of health and beauty. Also in cosmetics, the accumulation of sebum lipids causes oxidation and skin health is also impaired.

Although the function of breaking down fat is also in the lipase itself, it is impossible for the intestine and skin to penetrate the polymer. There is a need for safer ingredients with lower molecular weight.

Although the enzyme activator that breaks down fat is also organically synthesized, there remains a problem with safety. For this reason, although various studies have been conducted, the function of lipolysis is limited, and the problem has not been solved.

As an invention relating to lipolysis, for example, there is a hypoglycemic and / or anti-obesity composition containing an acacia bark-derived material (for example, see Patent Document 1), but its application is limited.

  Japanese Patent Application No. 2008-49984

The lipolytic action of existing substances is mild, and there is a problem that their use in industry is limited. In addition, chemically synthesized substances have problems in safety and their use is limited.

Therefore, a production method for efficiently producing an organic acid derivative exhibiting an excellent lipolytic action with weak side effects is desired.

In order to achieve the above object, the invention according to claim 1 relates to a method for producing a composition having a lipolytic action.

Since this invention is comprised as mentioned above, there exist the following effects.

According to the manufacturing method of Claim 1, an organic acid derivative can be manufactured efficiently.

Hereinafter, embodiments embodying the present invention will be described in detail.

A method for producing a lipolytic composition comprising a step of fermenting Ashitaba leaves, soybean powder , fermented natto and gold powder , further fermenting with B. niger and further protease treatment To do.

When the obtained composition is used as a pharmaceutical material, it is preferable to separate and purify the target organic acid derivative exhibiting a lipolytic action from the viewpoint of increasing the purity of the target organic acid derivative and removing impurities.

Used as pharmaceuticals, parenteral preparations such as injections, oral preparations and coatings, and quasi-drugs used in tablets, capsules, drinks, soaps, coatings, gels, toothpastes, etc. Is done.

Examples of oral preparations include tablets, capsules, powders, syrups, and drinks. When mixed with the above-mentioned tablets and capsules, it can be used together with a binder, excipient, swelling agent, lubricant, sweetener, flavoring agent and the like. The tablets can also be coated with shellac or sugar.

Moreover, in the case of the said capsule, liquid carriers, such as fats and oils, can be further contained in said material. In the case of the above syrup and drink, sweeteners, preservatives, pigment flavoring agents and the like can be added.

Examples of parenteral preparations include injections in addition to external preparations such as ointments, creams, and liquids. Vaseline, paraffin, fats and oils, lanolin, macro gold, etc. are used as a base material for external preparations, and can be made into ointments, creams, and the like by ordinary methods.

Injections include liquids, and other lyophilization agents. This is used aseptically dissolved in distilled water for injection or physiological saline at the time of use.

It is used as a food preparation for health foods and beauty foods for the purpose of lipolysis and beauty. Moreover, as a health functional food, it is preferable to use it for a nutrition functional food or a food for specified health.

When the obtained food preparation is used for pets and livestock animals such as dogs and cats, it is used as feed or supplements for the purpose of maintaining fat decomposition and skin health.

As a cosmetic, it can be used together with a surfactant, a solvent, a thickener, an excipient and the like according to a conventional method. For example, it can be in the form of cream, gel for hair, facial cleanser, cosmetic liquid, lotion and the like.

The form of the cosmetic is arbitrary, and can be used as a solution, cream, paste, gel, gel, solid or powder.

The resulting cosmetic decomposes excess sebum and exerts a skin cleaning action.

In addition, since sebum becomes a nutrient for bacteria, degradation of the keratinous lipid and skin regeneration of atopic patients are promoted by degrading the lipid with this derivative.

This production method comprises a step of subjecting a fermented liquid obtained by mixing and fermenting Ashitaba leaf, soybean powder , natto bacteria and gold powder to a protease after being fermented with B. niger.

The raw materials are Ashitaba leaf, soybean powder, Bacillus natto, Beniculium fungus and protease.

Ashitaba here is the scientific name Angelica keiskei and is a plant native to Japan and native to the Pacific coast of the Kii and Izu Islands from the Boso Peninsula. Another name is Hachijo grass, which is also cultivated in Asia and America, and is also used for food.

Since Ashitaba leaves contain polyphenols and organic acids, they are preferred as raw materials for this derivative.

Ashitaba leaves may be from any country such as Japan, China, Taiwan, and the United States. In addition, those produced with low pesticides or reduced pesticides are preferred.

The leaves of Ashitaba are preferably dried and pulverized, and autoclaving before fermentation is preferable because fermentation is performed smoothly.

A powder having a particle size of 3 micrometers or less is preferable because it facilitates the fermentation process.

The soybean powder used as a raw material can be used in soybeans of any origin such as Japanese, Chinese, American, and Russian, but is preferably Japanese because of its reliable traceability and clear producers.

Of these, soybeans cultivated organically or without agricultural chemicals are more preferred because they do not contain harmful agricultural chemicals or metals.

When using soybeans, Nara Machinery Co., Ltd. free mill, super free mill, sample mill, goblin, super clean mill, micros, small vacuum dryer manufactured by Toyo Riko as vacuum dryer, small size manufactured by Matsui Co., Ltd. It is pulverized by a pulverizer such as a vacuum heat transfer dryer DPTH-40, clean dry VD-7, VD-20 manufactured by AKM Kyushu Technos Co., Ltd., DM-6 manufactured by Nakayama Technical Research Institute. Thereby, the process of fermentation tends to advance efficiently.

Further, it is preferable to sterilize the leaf and soybean of Ashitaba after pulverization by autoclaving or the like because propagation of germs can be prevented.

The Bacillus natto used is the scientific name Bacillus subtilis, which is widely used in the production of natto in Japan. Bacteria species from Japan such as Okinawa and Kagoshima, and from Southeast Asia such as China and Taiwan are used. Among these, natto bacteria manufactured by Natto Honpo are preferable because they exhibit high fermentability.

The Bacillus natto ferments organic acid and protein consisting of Ashitaba leaf and soybean at the same time to combine the organic acid and protein.

As for each addition amount regarding the said fermentation, 0.2 to 3 weights of soybean powder and 0.001 to 0.04 weight of natto bacteria are preferable with respect to 1 weight of dry powder of Ashitaba leaf. It is preferable to pre-cultivate natto bacteria before being fermented, because the initial time of fermentation is shortened and the fermentation time is shortened.

The fermentation is carried out in a clean culture tank, and it is preferable to mix the materials with sterilized tap water.

Moreover, this fermentation is heated at 33-40 degreeC, and fermentation is performed for 14 days from 2 days. It is preferable to carry out the process control of the fermentation by quantifying the target organic acid derivative by HPLC or TLC and confirming the growth of the cells.

The resulting Bacillus fermentation liquor is subsequently fermented by B. niger. The organic acid derivative is separated by the fermentation by the fungus, and absorption is also promoted.

The fungus used is a filamentous fungus of the scientific name Monasuc purpureus and has long been used in fermented foods such as red sake and tofu in Japan, China and Taiwan. In addition, bacterial species from Japan such as Okinawa and Kagoshima, and from Southeast Asia in China and Taiwan are used.

As for each addition amount regarding the said fermentation, 0.0001-0.005 weight is preferable with respect to 1 weight of said fermented products. It is preferable to pre-cultivate B. subtilis before fermentation because the initial time of fermentation is shortened and the fermentation time is shortened.

The fermentation is carried out in a clean culture tank, and it is preferable to mix the materials with sterilized tap water.

Moreover, this fermentation is heated at 35-47 degreeC, and fermentation is performed for 14 days from 1 day.

Although the protein binds to the organic acid by this fermentation process, the molecular weight is large, so that the protein is decomposed and reduced in molecular weight by protease for the purpose of promoting absorption and penetration from the skin.

Proteases are hydrolytic enzymes that break down proteins to produce peptides and amino acids, and are also used as food. Amano's protease N is preferred because of its high enzyme activity.

By adding a protease to the fermented product and heating it, the organic acid and protein are decomposed into a peptide-type organic acid derivative.

The amount of protease added is preferably 0.001 to 0.02 weight per 1 weight of the fermented product. The heating temperature is preferably 30 to 40 degrees. The heating time is preferably 1 to 6 hours.

It is preferable that the protease-treated decomposition product is extracted with water-containing ethanol because the product can be efficiently recovered, the protease can be deactivated, and the next step can be easily performed.

Moreover, since the product is easy to isolate | separate, it is preferable to ultrasonically treat the obtained fermented material. Moreover, it is preferable to concentrate by freeze drying or the like because the following steps can be performed in a short time.

Separating and purifying the target organic acid derivative from the reduction reaction product is preferable from the viewpoint that the intake can be reduced as a highly pure substance. As a purification method, it is preferable to use a purification operation such as a separation resin.

For example, the target organic acid derivative can be obtained by separation with a separation carrier or resin and separation. As the separation carrier or resin, porous polysaccharides, silicon oxide compounds, polyacrylamide, polystyrene, polypropylene, styrene-vinylbenzene copolymers, etc., whose surfaces are coated as described later, are used. Those having a particle size of 0.1 to 300 μm are preferred. The finer the particle size, the higher the accuracy of the separation, but the longer the separation time.

For example, a reverse phase carrier or resin whose surface is coated with a hydrophobic compound is used for separation of a highly hydrophobic substance. Those coated with a cationic substance are suitable for the separation of anionically charged substances. Also, those coated with an anionic substance are suitable for separating a cationically charged substance. When a specific antibody is coated, it is used as an affinity carrier or resin for separating only a specific substance.

The affinity carrier or resin is used for specific preparation of an antigen using an antigen-antibody reaction. A partitionable carrier or resin is used for isolation of a substance such as silica gel (manufactured by Merck) if there is a difference in partition coefficient between the substance and the solvent for separation.

Among these, an adsorbent carrier or resin, a dispersible carrier or resin, a molecular sieve carrier or resin, and an ion exchange carrier or resin are preferable from the viewpoint of reducing production costs. Furthermore, the reverse phase carrier or resin and the dispersible carrier or resin are more preferable because the difference in the distribution coefficient with respect to the separation solvent is large.

When an organic solvent is used as the separation solvent, a carrier or resin having resistance to the organic solvent is used. Moreover, the carrier or resin used for pharmaceutical manufacture or food manufacture is preferable.

From these points, Diaion (Mitsubishi Chemical Co., Ltd.) and XAD-2 or XAD-4 (Rohm and Haas) are used as the adsorptive carrier, and Sephadex LH-20 (Amersham Pharmacia) is used as the molecular sieve carrier. Silica gel as the distribution carrier, IRA-410 (Rohm and Haas) as the ion exchange carrier, and DM1020T (Fuji Silysia) as the reverse phase carrier are more preferable.

Of these, Diaion, Sephadex LH-20 and DM1020T are more preferred.

The obtained extract is dissolved in a solvent for swelling the carrier for separation or the resin before separation. The amount is preferably 1 to 35 times the weight of the extract from the viewpoint of separation efficiency, and more preferably 4 to 25 times the amount. The separation temperature is preferably 4 to 30 ° C., more preferably 10 to 25 ° C. from the viewpoint of the stability of the substance.

As the separation solvent, water or a lower alcohol containing water, a hydrophilic solvent, or a lipophilic solvent is used. As the lower alcohol, methanol, ethanol, propanol and butanol are used, and ethanol used for food is preferable.

When Sephadex LH-20 is used, a lower alcohol is preferable as the separation solvent. When silica gel is used, the separation solvent is preferably chloroform, methanol, acetic acid or a mixture thereof.

When Diaion and DM1020T are used, the separation solvent is preferably a lower alcohol such as methanol or ethanol or a mixed solution of lower alcohol and water.

It is preferable to collect a fraction containing an organic acid derivative and remove the solvent by drying or vacuum drying to obtain the desired organic acid derivative as a powder or a concentrated liquid because the influence of the solvent can be excluded.

Moreover, it is preferable to carry out the final extraction with fats and oils used for edible oils and cosmetics because the organic acid derivative obtained is stably maintained. For example, extraction with soybean oil, rice bran oil, grape seed oil, olive oil or jojoba oil is preferred.

Further, it is preferable to powder this organic acid derivative for the purpose of preserving.

Hereinafter, the embodiment will be specifically described with reference to examples and test examples. These are merely examples, and conditions can be changed within the range of common sense according to differences in materials, raw materials, and specimens.

Ashitaba leaf grown in Miyazaki Prefecture with reduced pesticides was used. After collecting the leaves, washed with tap water, dried in the sun, and pulverized with a pulverizer (Super Free Mill manufactured by Nara Machinery Co., Ltd.) to obtain 1.1 kg of dried powder pulverized product of Ashitaba leaf It was.

The soybeans from Hokkaido were used in a mixer (Cuisinate) to obtain 1.1 kg of soybean pulverized material. The above-mentioned Ashitaba leaf and soybean grind were subjected to autoclaving and sterilized at 121 ° C. for 20 minutes.

These were put into a clean fermentation tank (sterilized round 40-liter tank for fermentation), and 7 kg of sterilized tap water was added and stirred.

Separately, 10 g of powdered natto bacteria (manufactured by Natto Honpo) was supplied to a small fermentation tank, and a sterilized soybean powder and a precultured culture solution were prepared.

The pre-cultured Bacillus natto solution is added to the fermented tank containing the gold pulverized product, dried powder of Ashitaba and soybeans, stirred, and then heated in a temperature range of 40 to 43 ° C. for fermentation. I let you.

In the fermentation process, the fermentation liquor was sampled while bubbling and stirring by aeration.

To 1 kg of the fermented product of this Bacillus, 10 g of Beniculum was added and fermented at 37 degrees for 2 days.

10 g of Amano Protease N was added to 1 kg of the obtained fermented product and heated at 40 degrees for 2 hours.

This treated product was heated and ethanol was added to obtain 120 g of a composition containing the desired organic acid derivative.

Below, the test method regarding the structural analysis of an organic acid derivative and a result are demonstrated.
(Test Example 1)

The specimen 1 obtained as described above was dissolved in ethanol and analyzed by high performance liquid chromatography with a mass spectrometer (HPLC, Shimadzu Corporation).

Furthermore, it analyzed with the nuclear magnetic resonance apparatus (NMR, the Bruker make, AC-250). As a result of the structural analysis, sialic acid, polyphenol and a conjugate thereof were detected from specimen 1.

The confirmation test using human adipocytes is described below.
(Test Example 2)

Human adipocytes purchased from Kurabo Industries Co., Ltd. were used. As a culture solution, 1000 cells cultured using 5% fetal calf serum-containing MEM medium (manufactured by Sigma) were seeded in a 35 mm culture dish, and cultured at 37 ° C. under 5% carbon dioxide gas. To this, catechin was added at a final concentration of 0.1 mg / ml as Sample 1 obtained in Example 1 and as a control. This was cultured for 48 hours.

After detaching the cells, the number of cells was counted, the cell suspension was adjusted, and the amount of neutral fat in the cells was measured by ELISA (Wako Pure Chemical Industries). In addition, the petri dish calculated the average value using five sheets.

As a result, the addition of 0.1 mg / ml of Specimen 1 reduced the number of adipocytes to an average value of 34% compared to the control group. For catechin, the decrease was 88%, and Sample 1 was superior.

As for neutral fat, it decreased to 20% by Sample 1 compared to the control group. The catechin was 77%, and the decrease in Sample 1 was significant.

The composition obtained in the present invention degrades fat and skin lipids, reduces health problems due to excessive fat, and has fewer side effects. Therefore, it improves the national QOL and increases the healthy working population. And medical expenses can be reduced.

Since the composition obtained by the present invention has an action of improving skin lipids, it contributes to improvement of those suffering from excessive sebum, atopy and skin trouble as a cosmetic, and contributes to the development of the cosmetic industry.

Since the composition obtained by this invention can be utilized also as a foodstuff, it contributes to development of the food industry.

By using dried powder of Ashitaba, waste can be used effectively and agricultural resources can be used effectively, and agriculture and its secondary processing industry can be developed.

This production method uses advanced fermentation technology and contributes to the improvement of new fermentation technology and the advancement of the fermentation industry.

Claims (1)

A composition exhibiting a lipolytic action obtained by further fermenting a fermented liquid obtained by mixing and fermenting Ashitaba leaf, soybean powder , natto and gold powder, and further fermenting with B. niger.