JPH0873396A - Lipid having high natural menaquinone-7 content - Google Patents

Abstract

PURPOSE: To obtain the subject lipid having high safety as an (auxiliary)food, being colorless and odorless, free from problem on taste and useful for preventing and treating hemorrhage and osteoporosis by taking out from a food raw material fermented with Bacillus subtilis represented by Bacillus natto. CONSTITUTION: This lipid is taken out from a food raw material fermented, e.g. at >=42 deg.C for >=48hr with Bacillus subtilis such as Bacillus natto under conditions not causing decomposition of natural menaquinone-7 using a purification method such as solvent extraction with solvent, fractionation with solvent, absorption fractionation, molecular distillation, steam distillation, chromatography or separation with membrane. This lipid is obtained by adding isopropyl alcohol to fermented soybean, powdering the fermented soybean, adding n-hexane thereto, re-powdering the fermented soybean, filtering the filtrate, collecting n-hexane layer, distilling the solvent away to afford the residue and subjecting the residue to steam treatment so as to remove odor.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a lipid containing a high content of natural menaquinone-7 obtained from a food material fermented with Bacillus subtilis typified by Bacillus natto, a method for producing the lipid, and a main component of the lipid. The present invention relates to a composition for preventing or / and treating osteoporosis.

[0002]

2. Description of the Related Art Vitamin K has long been known as a vitamin involved in blood coagulation. Synthetic vitamins K ₁ and K ₂ are used as medicines for the treatment of vitamin K deficiency hemorrhage, and natural vitamin K ₁ is used as food for prevention. A concentrate is used. Osteoporosis is a pathological condition in which bones become brittle due to aging, diseases, and the like, and is becoming a major social problem in terms of medical care for the elderly, with fractures and severe pain. Calcium and vitamin D are mainly used as medicines or foods for the treatment and prevention of osteoporosis. Recently, vitamin K alone or in combination with a known active substance has been used for the treatment and prevention of osteoporosis. It was found to be effective. Vitamin K required for the treatment and prevention of hemorrhage is extremely small (on the order of μg per day), but for the treatment and prevention of osteoporosis, it is several to several tens per day.
You need mg vitamin K.

Of the vitamin Ks, only the vitamins K ₁ and K ₂ are naturally present. K ₁ is mainly synthesized by plants. Menaquinone (MK) -1 to 14 are known in the K ₂ group due to the difference in side chain length, MK-4 is mainly synthesized by intestinal bacteria in the animal body, and MK-7 is mainly Synthesized by Bacillus natto. Among foods, K ₁ is particularly abundant in green vegetables, vegetable oils, seaweed, etc., and MK-7, which is a typical substance of K ₂ , is abundant in natto. Vitamin K content of the main food or in food materials, vitamin K ₁ is a few tens of ppm, such as in seaweed, laver, brown leaf, vitamin K ₁ is a few ppm to, such as soybean oil, spinach, broccoli, MK-7 in natto It is several to ten and several ppm. Soybean oil or soybean, which is a raw material of natto, contains only ₁ ppm or less of vitamin K ₁ .

[0004]

When ingesting an effective amount of vitamin K for osteoporosis from a commercially available food, it is assumed that 1 day a day from a food or food material containing 1 ppm of vitamin K.
If you try to take 0 mg of vitamin K, 10 kg per day
It is impossible because it is necessary to eat a large amount. Even natto containing a few dozen ppm must eat several hundred g per day, and it is difficult to eat such an amount every day because of taste. Since a large amount of natural vitamin K ₁ concentrate that is added to powdered milk formula for preventing hemorrhage on the market is expensive, a large amount of vitamin K is required.
It is difficult to take ₁ in terms of price. On the other hand, synthetic vitamin K, which is a drug, cannot be used in foods.

[0005] Natural MK-7 is extremely difficult to isolate because it exists in the natural world in a very small amount, and thus far no examples of preparing lipids with a high MK-7 content have been known. Although there is an example in which MK-7 existing in the form of a complex with a protein is separated by extraction with water, there is a problem in extraction efficiency because only a part of MK-7 is present in the complex.
There is also an example in which a composition containing sterol as a main component is extracted from natto, but a saponification reaction is indispensable to increase the sterol content, and MK-
7 decomposes, so MK-7 in the obtained composition
Is hardly contained.

[0006]

The object of the present invention is produced by natural vitamin K which cannot be ingested in a sufficient amount from ordinary foods or is difficult to ingest, particularly edible Bacillus subtilis represented by Bacillus natto. To provide a concentrated lipid having a high content of natural MK-7, which allows the natural MK-7 to be easily ingested daily as a food or a supplement, and a food or supplement containing the same as a main component. .

The inventors of the present invention have conducted extensive studies to achieve the above object, and as a result, extracted natural lipids from the food material fermented with Bacillus subtilis or extracted or extracted and purified the natural MK-7 The present invention has been completed by finding that a lipid content can be obtained. In the present invention, the raw material for the lipid can be used as long as it can ferment and ferment Bacillus subtilis typified by Bacillus natto, but since the lipid taken out from the fermented product is edible, it is a food material. Is preferred. As the food material, by-products such as lees and broth produced during the processing thereof may be used in addition to the materials for general foods. As the general food, grains such as soybean are particularly preferable. Other than the above, it is not particularly limited as long as it can be eaten and can reproduce Bacillus subtilis.

As the Bacillus subtilis to be inoculated, edible Bacillus subtilis is preferable for the same reason as the above material. Bacillus subtilis is the most common edible Bacillus subtilis. Commercially available Bacillus natto can be used, and among them, those having a high natural MK-7 producing ability are preferable. In addition, it is also possible to use those artificially enhanced in natural MK-7 production ability. As the fermentation method, in addition to the generally known fermentation method for natto, a known fermentation method such as liquid culture may be used. Usually, the fermentation of natto is carried out at 36 to 39 ° C for 15 to 20 hours, but the fermentation of the fermented soybeans at a temperature of 42 ° C or more for a long time of 48 hours or more enhances the content of natural MK-7 in the lipid. be able to.

As a method of taking out, a method of separating by utilizing differences in physical properties such as extraction, squeezing and centrifugation can be selected, and the extraction method is particularly effective. The extraction method may be selected the most effective method for the fermented product and is not particularly limited, but if the solid content of the fermented product is high, the solid-liquid extraction method is used, and if the solid content is low, the liquid-liquid extraction is used. The law is effective. When the solid-liquid extraction method is selected, the fermented product contains a large amount of water even if it is a solid. Therefore, the water content is measured in advance, and the hydrophilic organic solvent has an equal to 10 times the water content. Water is removed by mixing or heating at a temperature of 100 ° C. or lower under reduced pressure, and then a lipophilic organic solvent is added in an equal to 10 times amount, and pulverization / extraction improves extraction efficiency. .

When dehydration is carried out by mixing with a hydrophilic organic solvent, lipid may be extracted after separating / removing the hydrophilic solvent-water mixture, or may be extracted without being removed. Hydrophilic solvent-
When the extraction is performed after removing the water mixture, the separated hydrophilic solvent-water mixture may be further extracted with a lipophilic organic solvent to extract the lipid component. The dehydrated fermented product is crushed, extracted, filtered, and the lipophilic organic solvent layer of the filtrate is collected, and then the solvent is distilled off under reduced pressure to obtain an extracted lipid.

In the liquid-liquid extraction method, extraction can be carried out without removing water, but it is necessary to select a solvent or a mixed solvent having a low affinity for water to separate the two layers at the time of extraction. is there. After mixing a solvent or a mixed solvent with a low affinity for water that is 1/10 to 10 times the amount of the fermentation liquor with the fermentation liquor, the mixture is allowed to stand, or acceleration is applied to forcibly separate the two layers and lipophilic organic After separating the solvent layer, the solvent is distilled off under reduced pressure to obtain an extracted lipid. It is more effective to repeat the same operation for the remaining water layer.

As the organic solvent, a hydrocarbon having 1 to 10 carbon atoms or a mixture of two or more selected from the group consisting of alcohols, ethers, esters and ketones can be used. The extraction method may be a batch type or a continuous type for both solid-liquid and liquid-liquid. The extraction temperature can be used as long as it is not higher than the boiling point of the solvent, but a temperature of about room temperature to 100 ° C. is desirable in order to suppress chemical decomposition of natural MK-7.

Only by the above extraction operation, the content of natural MK-7 in the extracted lipid obtained is increased to about 200 ppm to 1%. At this time, MK- contained in the lipid
Substances other than 7 include fats and oils, sterols and their derivatives,
Examples include tocopherols, phospholipids, hydrocarbons and the like.

In order to obtain a higher content of lipid, it is necessary to carry out further purification treatment. As a purification method, a solvent fractionation, an adsorption fractionation, a distillation, a chromatography, a membrane separation operation alone or a combination of two or more thereof is effective, but a general alkali purification as a lipid purification method in fats and oils. The method is not preferable because MK-7 is decomposed by the strong alkali used. There are the following two types of solvent fractionation methods. Two solvents that are immiscible with each other are selected in the above-mentioned extraction solvent range or a mixture thereof, and the extracted lipid is dissolved in either one or both of them, and mixed or separated in batch or continuously between the two liquid phases. By repeating the process, the natural MK-7 is concentrated in the solvent layer having a high distribution rate of the natural MK-7. It is separated and the solvent is distilled off under reduced pressure to obtain a natural MK-7-rich lipid. The mixing ratio of the solvent is preferably 1/5 to 5 times the amount of the other solvent with respect to 1 time of the one solvent. Alternatively, the sterols mainly produced by heating and dissolving the extracted lipid in a solvent in the range of the above-mentioned extraction solvent or a mixture thereof in an amount of 1/2 to 10 times the amount of the extracted lipid are mainly used. The insoluble matter as a component is removed by a method such as filtration or centrifugation to obtain a lipid having a high content of natural MK-7.

The adsorption fractionation method is a method of adsorbing and eluting by utilizing the property that MK-7 is selectively adsorbed by a carbon-based adsorbent. Activated carbon was added to the organic solvent solution of the extracted lipid.
After mixing or passing a solution of the extracted lipid in an organic solvent through a column packed with activated carbon to adsorb MK-7 on the activated carbon, wash the activated carbon with the organic solvent to wash away impurities, and then wash with an organic solvent. Elution of MK-7 gives a lipid with a high content of natural MK-7. The organic solvent used here is a solvent in the above extraction solvent range or a mixture thereof, but a solvent having a weak elution action for adsorption / washing, mainly alcohols,
By using a solvent having a strong elution action, mainly hydrocarbons, elution can be performed more efficiently.

As the distillation method, since natural MK-7 is a substance that has a high boiling point and decomposes at high temperatures, the molecular distillation method or steam distillation method, which is high vacuum distillation, is effective. In both methods, it is possible to reduce the vapor pressure of the object to be vaporized so that even a substance having a high boiling point can be vaporized at a low temperature. These distillation methods may be batch type or continuous type. The lipid with a high content of natural MK-7 obtained by applying this method has no solvent remaining and is odorless, and thus can be directly used for food. In the molecular distillation method, 20 at a pressure of 5 Pa or less
The extracted lipids are subjected to a molecular distillation apparatus in a temperature range of 0 ° C to 300 ° C, and the evaporation fraction is collected at a stepwise temperature to obtain a lipid having a high content of natural MK-7. In the steam distillation method, distillation is carried out at a pressure of 1 kPa or less at a temperature range of 200 to 350 ° C. while introducing water or steam, and an evaporation fraction is collected at a stepwise temperature to obtain a natural MK-7.
A high content of lipids is obtained.

As the chromatographic method, a column chromatographic method is effective. The stationary phase packed in the column may be a commercially available packing material for chromatography. Practically, a silica gel-based adsorbent or a synthetic adsorbent having an organic polymer in the skeleton is desirable. The mobile phase is selected from solvents in the above extraction solvent range or mixtures thereof. A natural MK-7-rich lipid can be obtained by performing the above purification methods alone or in combination. At this time, the MK-7 content is 1% or more. Almost 100% can be prepared by repeating purification. The natural MK-7-rich lipid obtained by distillation may contain a small amount of solvent as a substance other than the lipid and also an odor derived from the fermentation product.
To get rid of both of these completely
Steam distillation may be performed at a temperature of up to 250 ° C. Steam distillation is difficult to apply unless it has fluidity. Therefore, when it becomes a paste or a solid when the solvent is distilled off, it is dissolved in 1/2 to 5 times the amount of vegetable oil before steam distillation treatment.

[0018]

The natural MK-7-rich lipid obtained according to the present invention is a natural material and is edible, so that it is effective for preventing not only hemorrhage but also osteoporosis. It is highly safe as a food or supplement and can be easily taken on a daily basis. Further, since this lipid is tasteless and odorless, it can be applied to various forms of food without any problem in taste.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will now be described in detail by way of examples, which should not be construed as limiting the invention thereto. In the following examples, the MK-7 content is (goods)
The values measured according to the high performance liquid chromatograph method of the Japan Food Analysis Center, and the other analysis values are the values measured according to the official method.

Example 1 Commercially available ground natto (MK-7 content 17.2 pp at room temperature)
2.3 L of isopropyl alcohol was added to 3 kg of m; water content 57.7%) and pulverized, and then 4.6 L of n-hexane was added and pulverized again. After filtering the whole amount, the n-hexane layer (upper layer) separated by allowing the filtrate to stand was separated. The upper layer was 5.2L. The upper layer solvent was distilled off at 2 kPa and 60 ° C. to obtain 153 g of residue. The residue is 3 hPa, 170
The mixture was deodorized for 60 minutes at 1.5 ° C steam / hour at 1.5 ° C to obtain 144 g of a yellow, tasteless, odorless and oily lipid (). This lipid contains MK-7 351 ppm, tocopherol
When it was observed by TLC (thin layer chromatography), most of it was triglyceride and trace amounts of sterols and their derivatives, phospholipids and hydrocarbons were found. This lipid had an acid value of 1.2 and a peroxide value of 0.6. Recovery of MK-7

Example 2 A soybean juice by-produced during the production of natto was inoculated with the natto bacterium and cultured under the same conditions as in the production of natto (MK-7 content 1
After 10 L of diethyl ether was added to 10 kg of 0.8 ppm) and shaken, the diethyl ether layer (upper layer) separated by standing was separated. The upper layer solvent was distilled off at 2 kPa and 60 ° C. to obtain 7.9 g of residue. This residue is 5hPa, 180
Deodorize at 0.08g steam / hour for 60 minutes,
7.2 g of oily lipid () which was yellow, tasteless, odorless and oily was obtained.
This lipid contains MK-7 1.4%, tocopherol
When it was observed by TLC, triglyceride, sterols and their derivatives, phospholipids and hydrocarbons were found in addition to the above. The lipid had an acid value of 0.8 and a peroxide value of 0.2. The recovery rate of MK-7 was 93%.

Example 3 10 kg of commercially available natto (MK-7 content 8.5 ppm) was added at 80 ° C.
After vacuum drying for 24 hours, 4.75 kg of a dried product was obtained. While the temperature of the dried product did not decrease, it was attached to a twin-screw extruder manufactured by Suehiro Iron Works to obtain 315 g of crude oil. 15 mL of warm water was added to the crude oil at 80 ° C., the mixture was stirred, and after centrifugation, the upper oil layer was separated. Further, 6 ml of 20% aqueous sodium hydroxide solution was added and stirred, and after centrifugation, the upper oil layer was separated. After vacuum drying the oil layer, add 15 g of activated clay at 100 ° C,
It was stirred and filtered. The filtrate was deodorized at 3 hPa, 180 ° C., 3 g steam / hour for 60 minutes to obtain 296 g of a yellow, tasteless, odorless and oily lipid. This lipid contains MK-7 1
It contained 72 ppm and 752 ppm of tocopherol, and as a result of observation by TLC, most of it was triglyceride, and trace amounts of sterols and their derivatives, phospholipids, and hydrocarbons were found. This lipid had an acid value of 0.2 and a peroxide value of 0.1. The recovery rate of MK-7 was 60%.

Example 4 50 g of the lipid () obtained in Example 1 above was placed in a falling film type molecular distillation apparatus. 200 ° C to 280 at a pressure of 3Pa
The temperature was raised to 10 ° C in steps of 10 ° C. The evaporate distilled off at 230 ° C to 280 ° C was collected to obtain 10.3 g of a yellowish brown, tasteless, odorless and pasty lipid. This lipid contains MK
-7 1560 ppm and tocopherol 163 ppm were contained, and when observed by TLC, triglyceride, sterol and its derivative, and hydrocarbons were found in addition to the above. This lipid has an acid value of 0 and a peroxide value of 0.
It was 1. The recovery rate of MK-7 was 92%.

Example 5 The lipid () obtained in Example 1 above was placed in a column packed with 50 g of commercially available granular activated carbon in isopropyl alcohol.
A solution prepared by dissolving 50 g in 150 ml of isopropyl alcohol was passed at 40 ° C. and a space velocity (SV) of 1.0 to give MK.
-7 was adsorbed. After washing 1 L of isopropyl alcohol at the same temperature and flow rate to wash the column,
5 of the liquid flowing out of the column after passing 500 ml of toluene
Fractions of 0-500 ml were collected. This fraction is 2kPa, 6
The solvent was distilled off at 0 ° C. to obtain 4.5 g of residue. To this residue was added 9 ml of acetone and dissolved at 50 ° C, then at -20 ° C for 1 hour.
After cooling for an hour, the formed precipitate was filtered off. 2kP of filtrate
The solvent was distilled off at a temperature of 60 ° C. to obtain a residue of 3.9 g. To this residue, add 3 g of soybean oil, add 3 hPa, 180 ℃, 0.0
Deodorization was performed for 60 minutes with 7 g steam / hour to obtain 6.8 g of a yellow, tasteless, odorless and oily lipid. In this lipid,
It contained 2200 ppm of MK-7 and 12 ppm of tocopherol, and as a result of TLC observation, most of the triglyceride was found to contain trace amounts of sterols and their derivatives and hydrocarbons. This lipid had an acid value of 1.5 and a peroxide value of 1.8. The recovery rate of MK-7 was 85%.

Example 6 5 g of the lipid () obtained in Example 2 above was added to 20 ml of n-
After dissolving in hexane and adding 20 ml of 5% hydrous ethanol and shaking, separate the hexane layer (upper layer) and the hydrous ethanol layer (lower layer), respectively, and 20 ml of 5% hydrous ethanol for the upper layer and the lower layer for 20 ml of n-hexane was added and the mixture was shaken again. Separate each upper layer 2kPa
The solvent was distilled off at 60 ° C. to obtain 4.5 g of residue. 10
A column filled with 0 g of silica gel with n-hexane,
A solution prepared by dissolving the above residue with 5 ml of n-hexane was added to SV.
After passing through 1.0, 500 ml of n-hexane: diethyl ether (20: 1) was passed through, and 320 to 480 ml fractions of the liquid flowing out from the column were collected. 2 for this fraction
The solvent was distilled off at kPa and 60 ° C. to obtain 0.31 g of lipid.
This lipid contained 15.4% of MK-7, and when observed by TLC, triglyceride, sterol and its derivative, and hydrocarbons were found in addition to the above. The recovery rate of MK-7 was 68%.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location A61K 31/12 ADT 9455-4C C07C 46/10 C11B 1/10 3/10 3/12

Claims (13)

[Claims] 1. A natural menaquinone-7 high-content lipid extracted from a food material fermented with Bacillus subtilis. 2. The natural menaquinone-7 high-content lipid according to claim 1, wherein the extracting method is extraction. 3. The natural menaquinone-7-rich lipid according to claim 1, wherein the extracting method is extraction and purification under a condition that natural menaquinone-7 is not decomposed. 4. The natural menaquinone-7 high-content lipid according to any one of claims 1 to 3, wherein the food material is an edible plant or a by-product such as meal or broth produced in the process of processing them. 5. The high content of natural menaquinone-7 according to any one of claims 1 to 4, wherein the fermentation condition with Bacillus subtilis is a temperature of 42 ° C. or higher and a time of 48 hours or longer. Lipids. 6. The natural menaquinone-7-rich lipid according to any one of claims 1 to 5, wherein the Bacillus subtilis is Bacillus natto. 7. The content of natural menaquinone-7 is 200 ppm.
The natural menaquinone-7 high content lipid according to any one of claims 1 to 6, which is the above. 8. A method of extraction and purification under the condition that natural menaquinone-7 is not decomposed includes solvent extraction, solvent fractionation,
The natural menaquinone-7 high-content lipid according to any one of claims 3 to 7, which is a method of adsorption fractionation, distillation, chromatography or membrane separation alone or a combination of two or more thereof. 9. The natural menaquinone-7 high-content lipid according to any one of claims 2 to 8, wherein the solvent used for extraction or purification is an organic solvent or a water-containing organic solvent. 10. The adsorption fractionation method comprises adsorption on activated carbon,
The natural menaquinone-7-rich lipid according to claim 8, which is formed by elution. 11. The natural menaquinone-7 high content lipid according to claim 8, wherein the distillation method is high vacuum distillation such as molecular distillation or steam distillation. 12. The natural menaquinone-7 high content lipid according to claim 9, wherein the organic solvent is a hydrocarbon having 1 to 10 carbon atoms or alcohol, ether, ester or ketone. 13. A composition for preventing or / and treating osteoporosis, which comprises a natural menaquinone-7-rich lipid according to any one of claims 1 to 12 as a main component.