JP4660650B2 - Ceramide extraction method - Google Patents

Description

  The present invention relates to a method for extracting ceramide from fungi, and more particularly, to a method for efficiently extracting ceramide using a dried product of fungi that has been newly developed this time. is there.

  When a physiologically active substance is extracted from fungi, if the extraction solvent is aqueous, it is not necessary to specifically examine the conditions for drying the fungi. However, when extracting lipids such as ceramide, an organic solvent is often used as an extraction solvent. However, moisture contained in the extractables (fungi) adversely affects extraction efficiency. Therefore, it is required that the extractables are dried to have low moisture.

  One method for drying and pulverizing fungi is to freeze-dry the cells and then pulverize them to obtain a powder. Freeze-drying is a method that freezes the cells once and further sublimates the moisture to obtain a dried product.The material to be dried follows a low-temperature process. This is a very good method for subsequent extraction. However, the energy required for freezing and vacuum sublimation is large, and the production cost is increased, which is not suitable for industrial production of ceramide in large quantities.

  On the other hand, in the method of drying by heating and physically pulverizing the dried product, the product temperature rises during drying and pulverization, and it is exposed to conditions contrary to freeze-drying, so a substance with low thermal stability is obtained. It is not an appropriate method. Spray dryers that dry by spraying the aerosol of the object to be dried in hot air will obtain fine dry particles with fine particles of the dried product, but processing per hour because the drying room occupying the drying facility will be large To increase the amount, the equipment volume also increases.

Several methods have been proposed for extracting ceramides from plants (see Patent Documents 1, 2, and 3). On the other hand, although it is known that cerebroside which is a kind of ceramide (a substance in which sugar is bound to ceramide) is contained in certain yeasts (see Non-Patent Document 1), cerebroside is industrially stable. A method for mass production from yeast has not yet been established. Of course, a method for extracting cerebroside very efficiently using a specific dried product of yeast that was newly developed this time has not been known so far, and the specific dried product itself is not known. is there.
Japanese Patent Laid-Open No. 11-92781 JP-A-11-193238 JP2000-80394 FEMS Yeast Research vol.2 (2002) 533−538

  As described above, when a physiologically active substance is extracted from fungi such as yeast, it is not necessary to examine a drying method if the extraction solvent is aqueous. For example, in the case of hot water extraction, the target product can be obtained in the filtrate fraction by heating the yeast suspension as it is, extracting and filtering. However, when the extraction solvent is a hydrophobic or polar solvent, the moisture contained in the extraction object has an adverse effect on the extraction efficiency. Therefore, the to-be-extracted object needs to be a fine powder form with low moisture and a large contact area. We found that the dry product obtained by lyophilization is porous, which has improved the lipid extraction efficiency. However, although lyophilization is a suitable method for lipid extraction, it has a high drying cost and a large amount of yeast. It is not an excellent method for drying the body.

  The object of the present invention is to newly develop a system for efficiently extracting ceramide, and since ceramide is a lipid, organic solvent extraction is suitable for this purpose. ) Is preferably dried, and conventionally a freeze-drying method has been used. However, as described above, the freeze-drying method is costly and complicated in operation, so it is difficult to say that it is an industrial method.

  An object of the present invention is to newly develop a new system for efficiently extracting ceramide from fungi at an industrial scale and at low cost. As a result of examination from each side, we focused on fungi and developed a completely different drying method from the freeze-drying method, in other words, to find new drying conditions equivalent to the freeze-drying method. It was aimed.

  That is, an object of the present invention is to efficiently perform ceramide extraction from fungi on an industrial scale. Specifically, in order to obtain a large amount of ceramide concentrate, a method for producing a dried product using dried fungi and having a large surface area, low dry moisture, and little oxidation was studied. The problem was how to extract ceramide efficiently.

  In order to solve the above-mentioned problems, it is necessary to produce a dry product that satisfies all the above-mentioned conditions, but it is very difficult to achieve one of the conditions. Naturally, if the surface area is increased, it becomes easy to be oxidized, but on the other hand, the condition that oxidation is low is required, and these conditions are inherently contradictory and are not compatible or incompatible. Nevertheless, it is increasingly difficult to find the optimum value because these conditions must be met. And it is no exaggeration to say that it is a difficult task, such as finding the optimal value that can satisfy all the conditions in a well-balanced manner.

The present inventors have tried to solve a very severe technical problem that is difficult to solve or impossible to solve, and satisfy all the above-mentioned various conditions for extracting ceramide from a dried fungus. As a result of reconfirming that it is important and diligently studying from various directions, the dried product preferably has a moisture content of 2 to 15 wt% and a specific surface area of 7,000 to 11,000 cm ² / g. A dried product having an average particle size of 400 to 620 μm, more preferably, a water content of 2 to 10 wt%, a specific surface area of 8,000 to 10,000 cm ² / g, and an average particle size of 400 to 550 μm Was found for the first time to be suitable for extracting ceramide from fungi. This dried product needs to be in a fine powder form in order to increase the contact area with the extraction solvent. The reason for this is that low moisture facilitates elution of lipids into hydrophobic or polar extraction solvents, and fine powder can reduce the amount of solvent required by increasing the contact area with the extraction solvent. Can be mentioned.

The present invention has not only found a specific dried product of such a fungus for the first time, but has also succeeded in identifying a dryer, a drying apparatus, drying conditions, etc. for producing this specific dried product, and thus It was also confirmed that ceramide could be efficiently extracted from the specific dried product of the produced fungi, and was finally completed.
Hereinafter, the present invention will be described in detail.

In the case of a fungus dryer, the dried product preferably has a moisture content of 2 to 15 wt%, a specific surface area of 7,000 to 11,000 cm ² / g, and an average particle size of 400 to 620 μm. Any dry form may be used. For example, stirring fluidized bed drying method (eg Anhydro spin flash dryer: Matsubo Co., Ltd.), airflow drying method (eg trade name Drymeister: Hosokawa Micron), vacuum stirring dryer, microwave dryer, paddle dryer, etc. It is done. The drying method is not particularly limited as long as it has a performance equivalent to these.

  As the fungi introduced into the dryer, a solution, a slurry, a cake, a solid, and the like can be used according to the type of the dryer. In order to obtain a cake-like product, the fungal culture solution is separated using a drum type dehydrator, a filter press or the like to obtain a fungal cake. The stirring fluidized bed drying method is suitable for drying cake-like fungi, and the airflow drying method is suitable for drying slurry.

  An ultrasonic homogenizer or the like is used for efficiently contacting the dried cells with a solvent and extracting lipids. As long as the similar contact extraction efficiency is good, it is not limited to the ultrasonic homogenizer. After lipid extraction, solid-liquid separation is performed by centrifugation to obtain a supernatant liquid containing lipid. However, when a fine solid remains in the supernatant, a cartridge filter, a membrane filter or the like is used.

  Therefore, drying conditions were determined by drying and pulverizing fungi such as live yeast to yield lipid extraction efficiency equivalent to that of freeze-dried products. Since lipids may be oxidized by heating during drying, the drying conditions were examined and the conditions for obtaining a dried product that yielded lipid extraction efficiency equivalent to the freeze-drying method were determined. In examining lipid extraction efficiency, the extraction efficiency of ceramide, which has recently been found to have many bioactive effects, was used as an index, but cerebroside in which hexose such as glucose is bound to ceramide is also extracted by the same extraction method. Therefore, it is possible to extract efficiently from the dried product according to the present invention.

  In this way, not only the various conditions of dried fungi suitable for ceramide extraction were determined, but also their industrial implementation was finally successful.

  The ceramide referred to in the present invention refers to lipids excluding fat, glycerolipid and sterol lipid, and includes those represented by the structural formula of the following chemical formula 1.

In the formula, R _{1 to 3} represent the following.
R ₁ : alkyl group R ₂ : hydrogen, or sugar, or phosphoric acid and phosphonic acid derivatives R ₃ : hydrogen or fatty acid residue

Specifically, sphingosine bases (such as sphingosine), sphingolipids (sphingoglycolipids, sphingophospholipids), ceramide (ceramide in the narrow sense; R ₂ = H), cerebroside (for example, sugar Glucosylceramide (glucocerebroside) bound with glucose, ganglioside having a sugar chain larger than a monosaccharide bond, lactosylceramide, cloboshiceramide, etc.), and various other ceramide derivatives (ceramides) are widely exemplified. .

  Examples of the fungi used as the raw material for extraction include yeast, filamentous fungi (molds), and basidiomycetes (mushrooms), and the site is not particularly limited.

  Examples of yeast include the genus Saccharomyces, specifically Saccharomyces kluyveri IFO 1685, and the like, such as Kluyveromyces, specifically Kluyveromyces lactis IFO 1090, Tolerance (Kluyveromyces thermotolerans IFO 10067), Kluyveromyces waltyi IFO 1666, Kluyveromyces marxianu musik Imi 1 ii IFO 1675), such as Zygosaccharomyces genus, specifically Zygosaccharomyces Shidori (Zygosaccharomyces cidri IFO 1990), Zygosaccharomyces Fell Mentha tee (Zygosaccharomyces fermentati IFO 1996), and the like.

  Examples of molds include Aspergillus genus, specifically Aspergillus oryzae IFO 30104, Aspergillus niger, and other genus Rhizopus, specifically Rhizopus ory Examples include the genus Penicillium, specifically Penicillium roqueforti IFO 8889, and the genus Monascus, specifically Monascus anka IFO 6540.

  The mushroom is not particularly limited as long as it is an edible mushroom, and either a fruiting body or a mycelium can be used. Examples of mushroom Maitake (Grifola frondosa), shiitake mushroom (Lentinus edodes IFO 31866), oyster mushroom (Pleurotus ostreatus IFO 30776), Pleurotus eryngii (Pleurotus eryngii), Pleurotus cornucopiae (Pluerotus cornucopiae IFO 30528), Flammulina velutipes (Flammulina velutipes IFO 31862), nameko ( Basidiomycetes belonging to the genus Griphora, Lentinus, Pleurotus, Framlina, Foliota, and Agaricus are widely used, such as Pholiota nameko IFO 30373) and Tucritake (Agaricus bisporus IFO 30874).

  If these fungi are used as the dried product specified in the present invention and this is extracted with a solvent, ceramide can be extracted efficiently. Solvent extraction may be performed according to a conventional method in lipid extraction. For example, solvent extraction can be performed as follows.

Organic solvent amount: 2 to 100 times, preferably 3 to 10 times.
Extraction temperature: not particularly limited, generally 20 to 100 ° C., more preferably 50 to 70 ° C., even at room temperature.
Time: 5 minutes to 24 hours, preferably 30 minutes to 4 hours.
Number of extractions: 1 to 10 times, preferably 1 to 3 times, but continuous extraction is also possible.
Kind of organic solvent: ethanol or water-containing ethanol (preferably 70 to 99% ethanol) is preferable, but methanol, water-containing methanol, hexane, acetone, chloroform, chloroform-methanol mixed solution, benzene, isopropanol and the like can also be used.
Extraction method: stirring extraction, reflux extraction, immersion extraction, shaking extraction, ultrasonic extraction.

The extract thus obtained can be concentrated by an appropriate concentration operation, for example, by a vacuum concentrator such as an evaporator, or by removing the solvent by heating, to obtain a concentrated solution. Further, it can be concentrated and dried to obtain a concentrated dried product. The concentrated dry product thus obtained is usually in the form of a yellow solid fat.
A ceramide fraction can be obtained from the extract thus obtained by an appropriate purification means, and if desired, further purification, fractionation means can be combined or repeated to separate each component, Can be acquired.

  According to the present invention, ceramide can be produced industrially and efficiently by providing a dried product of fungi with good extraction efficiency.

  Specific drying conditions are as follows. A yeast cake whose water content per unit weight is adjusted to 60 to 75%, preferably 63% to 73%, more preferably 66% by a method such as a drum type dehydrator or a filter press, is added in small portions under dry aeration. This aeration rate is a parameter that can be changed according to the volume and shape of the drying chamber, and is an important parameter in consideration of lipid extraction efficiency or alteration due to lipid oxidation. is not. Important parameters are the aeration temperature and the amount of heat applied to the material to be dried.

  The temperature when flowing into the drying chamber is 200 ° C. to 240 ° C., preferably 205 ° C. to 210 ° C. Excluding heat loss in the drying chamber, the amount of heat used to evaporate moisture from the yeast cake that is to be dried is from 40K. It is an important condition for obtaining the desired lipid extraction efficiency to be in the range of 160K, preferably 63K to 93K.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Example 1: Drying of Saccharomyces cerevisiae)
Dry cells were produced for lipid extraction from baker's yeast Saccharomyces cerevisiae. Baker's yeast (produced by Nippon Sugarcane Co., Ltd.) is cultured using sugarcane molasses, nitrogen sources such as ammonium sulfate and potassium phosphate, dehydrated with a dehydrator (Alfa Laval VUS-8M type), and a yeast cake with a moisture content of 66%. did. A part of the yeast cake was freeze-dried to prepare dry cells for comparison with the drying method according to the present method.

  A stirring fluidized bed drying method was employed as a drying method, and a spin flash dryer (manufactured by APV Nordic Anhydro) was used as an apparatus. According to this apparatus, the hot air heated by the heater enters the drying chamber from the tangential direction and becomes a high-speed swirl flow, while the raw material (yeast cake) crushed by the rotor rides the swirl flow, and only the dried product is above On the other hand, the undried product settles down by its own weight (this action is further promoted by the classification mechanism), returns to the fluidized bed, and is further dried.

  In order to measure the heat loss of the spin flash dryer (manufactured by APV Nordic Anhydro), the exhaust air temperature was measured while changing the inflow air temperature, and the heat loss in the apparatus itself was 50K (Table 1).

The dried yeast product was produced by changing the temperature of hot air. The amount of heat applied to the yeast was varied depending on the set temperature of the warm air and the supply amount of the yeast cake as the material to be dried (Table 2).
The moisture content of the dried product was 2.32 to 6.45%, the highest, which was 6.45% of Test No. 2, but this level of moisture content does not hinder the solvent extraction. About others, it was 2 to 6%, and it was a good dry state at about 2 to 3%.
The moisture evaporation rate is 9.60 kg / hour at the maximum, and the ability to dry 28 kg per hour for a 66% moisture yeast cake.

  On the other hand, for freeze-drying, sample preparation at a laboratory level is performed, and about 300 g of freeze-dried product is prepared using the same yeast cake. For spray drying, a yeast suspension with a dry matter concentration of 20% is used. Then, a dried product of about 1.1 kg was prepared at an atomizer rotational speed of 13,000 rpm, a supply liquid amount of 80 ml / min, and a ventilation temperature of 125 ° C.

(Example 2: Lipid extraction from dried sample)
About 0.5 g of dried cells were collected in a centrifuge tube with a stopper, and after weighing, 6 ml of methanol was added. A 10-minute ultrasonic homogenizer (SONIFIER 250 type manufactured by BRANSON) was performed while cooling with ice, and then 12 ml of chloroform was added, followed by a 10-minute ultrasonic homogenizer while cooling with ice. After centrifugation at 3000 rpm for 10 minutes, the supernatant was transferred to another test tube (A). To the residue, 6 ml of a 2: 1 mixture of chloroform / methanol was added, subjected to ultrasonic homogenizer treatment for 10 minutes while cooling with ice, centrifuged at 3000 rpm for 10 minutes, and the supernatant was transferred to the same test tube as A. To the residue, 6 ml of a 1: 2 mixture of chloroform / methanol was added, subjected to ultrasonic homogenizer treatment with ice cooling for 10 minutes, centrifuged at 3000 rpm for 10 minutes, and the supernatant was transferred to the same test tube as A.

  6 ml of chloroform and 9 ml of 1% potassium chloride were added to the supernatant of 3 times, and the test tube was well stirred. After centrifuging at 3000 rpm for 10 minutes, the chloroform layer was transferred to a 100 ml eggplant type flask and concentrated on a rotary evaporator. The concentrate was dissolved in a 2: 1 mixture of chloroform / methanol, transferred to a weighed test tube, concentrated in a nitrogen stream, dried in a desiccator for about 1 hour, and the lipid yield was measured. The lipid content in the sample was 3.64% of the lyophilized product, while the sample No. 7 was approximately equal to 3.50%. The spray-dried product was 3.19%, and the sample No. according to the present invention was used. It was lower than 7 (Table 3).

(Example 3: Measurement of phospholipid content)
A useful lipid-related substance that is expected to have biological functions such as skin preservation effects is that ceramide, a cell membrane component, is contained in the phospholipid fraction. By quantifying the phosphorus content in the extracted total lipid, The extraction efficiency of the ceramide fraction was estimated.

  About 1 mg of lipid extracted in Example 2 was precisely weighed and collected in a Pyrex (registered trademark) length test tube. 0.7 ml of a 1: 1 mixture of 60% perchloric acid / concentrated sulfuric acid was added and wet cracked. After the solution turned pale yellow, it was allowed to cool and 0.5 ml of demineralized water was added. 1% ammonium molybdate aqueous solution was added and 0.2 ml reducing reagent (prepared by dissolving 250 mg of 1-amino-2-naphthol-4-sulfonic acid and 1 g of sodium sulfite in 15% sodium metabisulfite aqueous solution and filtering) Were added and mixed, and reacted in boiling water for 10 minutes to develop color. The absorbance at 820 nm was measured, and the phosphorus content was calculated from a known phosphorus calibration curve. (Reference: Chemistry and Biological Experiment Line 23 Lipid Analysis Method (Yodogawa Shoten) p72).

  The total amount of lipid recovered was the spin flash dry sample no. Except for conditions 9 and 10, there was no difference due to the drying method. Spray drying was lower than other drying methods in the phosphorus content of total lipids measured to be an index of membrane lipid extraction where functional lipids are localized. That is, in order to improve the yield of lipid-related substances such as ceramide, which is expected to have functionality, it is necessary to attach importance to the drying conditions or the physical conditions of the sample in which the phosphorus content in the lipid is increased. In the drying method using a spin flash dryer, sample no. It is presumed that the phospholipid recovery efficiency equivalent to that of the freeze-drying method can be obtained under the drying condition of a heat amount of 76K with respect to 7.

(Example 4: Measurement of peroxide value POV)
In the lyophilization method, the drying process is performed under a low temperature condition and a low oxygen partial pressure, and therefore, it is estimated that the oxidative effect on lipids and lipid-related substances is very low. On the other hand, since the spin flash dryer performed under the heated aeration condition is dried under the condition where the oxidation action is very easy to work, the situation where the difference in the drying condition affects the oxidation of the lipid was investigated. The Saccharomyces cerevisiae that was tested was a bacterium with a lipid composition of a small amount of unsaturated fatty acids in the genus / species lipid composition, so it was thought that there would be no significant difference. ) Measurement was carried out by the method described in 40 (1) Volume 20-23.

  200 μg of the lipid extracted in Example 2 was collected in a 5 ml brown volumetric flask and dissolved in 2 ml of a mixture of chloroform / acetic acid / ethanol = 4: 4: 1. To this was added 10 μl of saturated potassium iodide solution, and the volume was increased with a mixture of chloroform / acetic acid / ethanol = 4: 4: 1. After standing in a cool dark place for 30 minutes, absorbance at 360 nm was measured using a mixed solution of chloroform / acetic acid / ethanol = 4: 4: 1 as a control (absorbance a). In the reagent control group, the lipid sample was not added (absorbance b1), and in the sample control group, the saturated potassium iodide solution was not added (absorbance b2). The absorbance obtained from iodine produced by the reaction of peroxide and potassium iodide in the sample was determined by a-b1-b2, and the amount of iodine was calculated from a calibration curve. The peroxide value (meq / kg) was obtained by 1000 × iodine milliequivalent (meq / 5 ml) / sample amount.

  The extracted samples have higher peroxide values for sample numbers 3, 4, and 5 (heated amounts 42K and 52K), and sample numbers 6, 7, and 8 (heated amounts 63K, 76K, and 93K) that are lower or equivalent to lyophilization. Met. Considering the lipid extraction efficiency depending on the temperature condition and the oxidation state of the sample, it is considered that the condition that the amount of heat in the range of 63K to 93K is applied to the yeast cake to be dried is appropriate as the drying condition. Since spray drying was performed in an atmosphere of 125K, the peroxide value was the highest.

(Example 5: Measurement of surface area of various dry yeast samples)
When extracting useful lipid-related substances from yeast cells, a large contact area with the extraction solvent is considered to be an important factor for increasing the extraction efficiency. By controlling the particle size at the time of drying, it was considered possible to produce a dried product exhibiting good extraction efficiency, and it was considered that the particle size could be used as a parameter at the time of production.

  The specific surface area was measured using a powder specific surface area measuring apparatus SS-100 manufactured by Shimadzu Corporation using a constant pressure air permeation method. The table shows the average values obtained by measuring five times for each sample (Table 5).

When the correlation between this data and the lipid extraction amount of Example 2 (vs. sample%) was examined, the specific surface area and the lipid extraction amount were positively correlated with a determination coefficient r ² = 0.6405 (FIG. 1), the average particle size A negative correlation (FIG. 2) with a coefficient of determination r ² = 0.7339 was observed between the diameter and the lipid extraction amount. However, the spray-drying data was excluded because the average particle size was largely different from other drying methods and could not be correlated.

  No correlation was found between the peroxide value, specific surface area, and average particle size in Example 4.

To obtain lipid-related substance extraction efficiency equivalent to lyophilization, the lipid extraction efficiency does not change even if the specific surface area is as small as spray drying, but the average particle size is as large as spray drying. Is considered to decrease the amount of phospholipid extracted. Therefore, it is necessary to adjust the sample under conditions where the specific surface area is larger than that of sample numbers 9 and 10 and the average particle size is small, that is, the specific surface area is 6,210 cm ² / g or more and the average particle size is 686 μm or less. . The production of dry powder by controlling the specific surface area and particle size is widely used for the purpose of obtaining a sufficient contact area with the extraction solvent in the drying method prepared this time and in the case of pulverization by pulverization of the dried product. It can be used.

The correlation between specific surface area and lipid extraction is shown. The correlation between the average particle size and the lipid extraction amount is shown.

Claims (8)

A specific surface area of 7,000 to 11, characterized in that a fungal cake dehydrated to a moisture content of 60 to 75% is dried under conditions of an aeration temperature of 200 to 240 ° C. and a heat amount of 40 to 160 K applied to the cake . 000cm ² / g, an average particle diameter of 400～620Myuemu, and method for producing moisture dried product of fungi 2 to 15 wt%. 2. The fungal dried product according to claim 1, wherein the fungal dried product is produced by at least one of a stirring fluidized bed drying method, an air flow drying method, a vacuum stirring drying method, a microwave drying method, and a paddle dryer drying method . Manufacturing method . The method for producing a dried product of fungi according to claim 1 or 2, wherein the fungus is at least one selected from yeast, filamentous fungi, and basidiomycetes. The method for producing a dried product of fungi according to claim 3, wherein the yeast belongs to at least one of the genera Saccharomyces, Kluyveromyces, and Tigosaccharomyces. The method for producing a dried product of fungi according to claim 3, wherein the filamentous fungus is a filamentous fungus belonging to at least one of the genus Aspergillus, Penicillium, Rhizopus, and Monascus. The basidiomycete is a basidiomycete belonging to at least one of the genus Griphora, Lentinus, Plurotus, Framlina, Foriota, and Agaricus, The production of a dried fungal product according to claim 3, Way . The dried fungi produced by the method according to any one of claims 1 to 6 are extracted with an organic solvent without being destroyed or destroyed, and a ceramide or a ceramide-containing fraction is fractionated and collected from the extract. A method for extracting fungal ceramides.   The ceramide extraction method according to claim 7, wherein the ceramide or the ceramide-containing fraction is a phospholipid fraction.

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