JPS61170397A - Multi-stage extraction treatment of cell of mold belonging to mortierella genus - Google Patents

Abstract

PURPOSE:To extract and separate a lipid component in a highly refined state, in an improved extraction efficiency, by extracting the cells of mold belonging to Mortierella genus under specific condition, and carrying out the solid-liquid separation of the extract. CONSTITUTION:Cells of a mold belonging to Mortierella genus are disintegrated in an alcohol solvent in the presence of water by mechanical force. >=90% of the whole polar lipid contained in the cell and a part of neutral lipid are extracted and separated by this process. The extract is separated into the disintegrated cell component and the alcohol solvent component containing polar lipid by a solid-liquid separation step. The disintegrated cell component is again extracted with a hydrocarbon solvent to extract and separate the neutral lipid in high efficiency. The extraction product is separated into the microbial cells and the hydrocarbon solvent containing the neutral lipid by solid-liquid separation.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a multistage extraction treatment method for filamentous fungi of the genus Morteierella.

[Prior art]

By culturing filamentous fungi such as Isabelina, Vinacea, Lamaniana, Lamaniana anglispora, and Nana belonging to the genus Morteierella in a medium with a high concentration of carbohydrate as a carbon source, bacteria with a high content of lipids containing γ-1-Noronic acid can be produced. A method for producing cells at high density has already been proposed (Japanese Patent Application No. 59-22394).

By the way, in order to industrially advantageously separate valuable lipids such as γ-lylunic acid-containing lipids contained therein from the proliferating microorganisms obtained in this way, t=ti.

There is a need to develop a bacterial cell treatment method that is suitable for the bacterial cells. In the conventional method, a mixture of chloroform and methanol is used as a solvent, and homogenization is performed in the presence of glass beads to simultaneously crush the bacterial cells and extract the lipids. However, since such one-stage extraction method uses a mixed solvent of chloroform and methanol, it is very difficult to separate and recover neutral lipids and polar lipids from the resulting extract. This was difficult and was not yet satisfactory as an industrial method.

〔the purpose〕

An object of the present invention is to provide an industrially advantageous extraction method for extracting and separating lipid components contained in filamentous fungi of the genus Morteierella.

(Structure) That is, according to the present invention, in extracting the lipid components contained in a filamentous fungus of the genus Morteierella, (a) the fungus is crushed in an alcohol solvent in the presence of water; (b) A first solid-liquid separation step in which the extracted product obtained in the first extraction step is solid-liquid separated into bacterial cells and an alcohol solvent containing lipids.

(c) a second extraction step in which the bacterial cells obtained in the first solid-liquid separation step are extracted using a hydrocarbon solvent; A second solid-liquid separation step of separating the bacterial cells and a hydrocarbon solvent containing lipids.

Provided is a multi-stage extraction treatment method for Morteiherella genus microorganisms, which is characterized by comprising: 6. In the present invention, Morteiherella genus filamentous microorganisms are first extracted in a first extraction step using alcohol in the presence of water. Process. in this case.

As the bacterial cells used as the processing raw material, a water-containing bacterial cake with a water content of about 50 to 80% separated from the culture medium by centrifugation or filtration, or a dried product thereof can be used.
From the point of view of economy, it is advantageous to use a water-containing bacterial cell cake. In addition, in this first extraction process, it is necessary to crush the bacterial cells by applying one mechanical force in an alcohol solvent in the presence of water, and by crushing the bacterial cells, an efficient extraction process is achieved. Ru. As such an extraction device that involves crushing the bacterial cells, a conventionally known wet grinder such as a ball mill, a Masatsu disk mill, a Hensel mixer, etc. can be used. With such a crusher, the bacterial cells are subjected to mechanical forces such as compressive force and crushing, and some of them are damaged or crushed. In this case, it is not preferable to crush the bacterial cells too finely.

From the viewpoint of filterability, it is preferable to apply mechanical force to such an extent that the particle size of the bacterial cells does not substantially change.As the alcohol solvent, lower alcohols such as methanol, ethanol, and propatool are usually used. From the point of view of safety for the human body, it is preferable to use ethanol.The ratio of alcohol solvent used is 2 to 7 parts by weight, preferably 3 to 6 parts by weight, per 1 part by weight of bacterial cells (dry basis). be. In this first extraction process, it is necessary to perform the extraction process in the presence of water in order to elute polar lipids,
The amount of water present is 0.2 parts by weight of alcohol solvent.
-0.7 parts by weight, preferably 0.3-0.6 parts by weight. Addition of water to this first extraction treatment system.

It can be carried out using microbial cells containing water, or by adding it to an alcohol solvent.

Through such an extraction process, 90% or more of all polar lipids contained in the bacterial cells can be extracted and separated, and a portion of neutral lipids can also be extracted. In addition, in this first extraction step,
The lipid recovery rate is usually 5 to 30% by weight, preferably 8 to 25% by weight based on the total lipid recovery rate.

Next, the first extraction product obtained above is separated into a crushed bacterial cell component and an alcohol solvent component containing polar lipids in a first solid-liquid separation step. In this case, the solid-liquid separation method is
Conventional methods such as centrifugation and filtration separation methods are employed.

The bacterial cells obtained in the first solid-liquid separation step are extracted again in a second extraction step using a hydrocarbon solvent. In this case, the extraction method is the same as that described in the first extraction process.

It is preferable to perform the extraction process while further crushing the bacterial cells, but since the bacterial cells in this second extraction process have already been crushed and are easier to extract, the crushing process may be omitted. .

As the hydrocarbon solvent, n-hexane, cyclohexane, etc. are used, and the usage ratio is 2 to 8 parts by weight, preferably 3 to 6 parts by weight, per 1 part by weight of bacterial cells (dry basis). be. In this second extraction treatment step, the presence of water is not preferable, and 0
．． It is preferable to specify the amount in the range of 0.05 parts by weight or less, preferably in the range of 0.03 to 0 parts by weight.

Such adjustment of the water content in the second extraction step is similar to that in the first extraction step.
This can be carried out by adjusting the amount of liquid removed in the solid-liquid separation step, or by washing the bacterial cells obtained in the first solid-liquid separation step with a hydrocarbon solvent. By such a second extraction process, neutral lipids are efficiently extracted and separated into a hydrocarbon solvent.

The extracted product obtained in the second extraction step is separated into bacterial cells and a hydrocarbon solvent containing neutral lipids in a second solid-liquid separation step. In order to separate and recover neutral lipids from the hydrocarbon solvent containing neutral lipids obtained in this way, an adsorption treatment using an adsorbent such as activated carbon or activated clay is performed as necessary to remove the neutral lipids contained therein. After removing polar lipids, etc.
Perform distillation treatment.

〔effect〕

In the present invention, as described above, almost all of the polar lipids can be separated in the first extraction process, so the extract obtained in the second extraction process consists mostly of neutral lipids. It is what it is. Therefore, the extract obtained in the second step can be used directly as a bacterial cell oil product by subjecting it to a simple adsorption treatment and then removing the hydrocarbon solvent. On the other hand, the extract obtained in the first extraction treatment step consists of neutral lipids and polar lipids, which can be further separated by an appropriate separation method 1, for example, an extraction separation method using a solvent such as hexane and acetonitrile, or By using an adsorption separation method using an adsorbent such as silicic acid or alumina, polar lipids and neutral lipids can be separated.

In the present invention, the recovery amount of the extract consisting of a mixture of polar lipids and neutral lipids obtained in the first extraction treatment step is usually as low as 20% or less of the total lipid recovery amount. Therefore, in the case of the present invention, the handling amount of a mixture of polar lipids and neutral lipids that is difficult to separate is significantly reduced compared to the one-stage extraction method, so the apparatus for separating the mixture is small. It has the advantage of being standardized. Furthermore, in the case of the present invention, the alcohol and hydrocarbon used as extraction solvent are
Since they are used individually without being used as a mixture, the effects of each extraction solvent can be fully exhibited. Therefore, when crushing the bacterial cells, there is no need to specifically pulverize the bacterial cells, but the crushing process that involves squeezing the bacterial cells and crushing some of them using compressive force or massaging is sufficient to achieve high lipid recovery. The cost of crushing is reduced. Furthermore, when each extraction solvent is used alone, the separation of lipids from the extraction solvent becomes significantly easier than when a mixed solvent is used.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples. Note that all percentages in the examples are based on weight.

Example Morteierella filamentous fungus 3 (obtained by mass culture in an H1 culture tank and containing a high content of γ-lylunic acid-containing lipids was dehydrated and separated using a centrifugal dehydrator, and the water content was 50 to 7.
Obtain a 0% bacterial block (cake). This bacterial block (hereinafter referred to as wet bacterial cells) was placed in an autoclave for 120 min.
After sterilization at 2 atm at 0.degree. C. for 10 minutes, lipids were extracted as shown below.

1.0 to 1.7 kg of the wet microbial cells were placed in a stainless steel ball mill with an internal volume of 6Q, and 2 ml of ethanol was added as a solvent, and extraction was carried out for 4 hours while crushing the microbial cells with the ball mill. After filtering the extract, the obtained bacterial cells (water content 3.4%) were subjected to the same extraction treatment as above for 7 hours using hexane 2Q as a solvent again.

Table 1 summarizes the results of extracting lipids from the bacterial cells of the three bacterial strains using the two-step extraction method.

From the results in Table 1 above, it can be seen that the extraction amount in the first stage of ethanol extraction is 4.5 to 9.7% based on the dry weight of the bacterial cells, the lipid content of the bacterial strain or the bacterial cells, and the wet bacterial cell content. The recovery rate of lipids is 9.1, although it varies somewhat depending on the water content etc.
It is observed that ~21.3%, which is approximately 20% or less of the lipids present in the bacterial cells, is extracted in the first stage. In the second stage of hexane extraction, 29.0 to 42.5
%, a high bacterial cell extraction rate was obtained. A high lipid recovery rate of over 92% was obtained in the first and second stages.
It was revealed that this extraction method is extremely effective.

Next, the ethanol-extracted lipid (first stage) and hexane-extracted lipid (second stage) obtained in Experiment No. 2 in Table-1.
We investigated its lipid composition and fatty acid composition, and investigated the effectiveness of its two-step extraction method. First, each extracted lipid was subjected to column chromatography using silicic acid as a packing agent to separate it into a polar lipid category and a neutral lipid category, and the respective quantitative ratios were determined.
1)). Regarding the neutral lipid fraction, compositional analysis was carried out by a method combining thin layer chromatography and densitometer ["Oil Kagaku", 28.59 (1979)]. In addition, analysis of the fatty acid composition of each category was performed by gas chromatography [Oil Chemistry, Kan, 854 (1981)]. The results of these analyzes are summarized in Table 2 and Table 3.
Table 2 shows the quantitative ratio of the polar lipid category and the neutral lipid category, and also shows the neutral lipid composition. In addition, each code shown in Table 2 means the following.

TG...triglyceride DG...diglyceride MG...monoglyceride FFA...free fatty acid FS...free sterol SE...sterol ester Table 3 shows polar lipids (PL) and neutral lipids in each lipid category. (NL) Indicates fatty acid composition, and each code shown in the table means the following.

C-14:0...Myristic acid C-16:0...Palmitic acid C-16:1...Palmitoleic acid C-18:0.
... Stearic acid C-18:1 ... Oleic acid C-18:2 ... Linoleic acid C-111:3 ... γ-lylunic acid From the results shown in Table 2, in ethanol-extracted lipids, Polar lipid category is 14.1% and hexane extracted lipid is 0.8%
It was found that polar lipids were concentrated in the ethanol-extracted lipids.

In addition, in the neutral lipid composition of ethanol-extracted lipids, triglyceride (τG) is extremely low at 4.4%, diglyceride (66.6%) and free fatty acid (23.6%) are the main components, and free It can be seen that sterols are also concentrated in this category. That is, it can be seen that lipids having polar groups or functional groups that are highly compatible with water are extracted in the first stage using ethanol as a solvent. In terms of fatty acid composition, it is not limited to the polar lipid category or the neutral lipid category, but also the γ
-Lilunic acid (C-18:3) and linoleic acid (C-18:3)
: 2) The content is higher than that of hexane-extracted lipids;
Palmitic acid (C-16:0), stearic acid (C-1
8:0), and the content of oleic acid (C-18:1) was found to be small (see Table 3).

On the other hand, as shown above, the hexane-extracted lipids in the second stage contain almost no polar lipids at 0.8%, and only neutral lipids are obtained (Table 2). reference)
. Furthermore, as for the composition of the neutral lipid category, triglyceride is the main component, accounting for 89.3%, and only 9% of other diglycerides are included, and only a small amount of free fatty acids and free sterols are observed. , it can be seen that it can be used as it is as an oil and fat product (see Table 2).Also, as for the fatty acid composition of the neutral lipid category of the hexane-extracted lipid in the second stage, the γ-lylunic acid content is 6.2%, and evening primrose oil (See Table 3), and it is clear that this extraction method is effective as a method for extracting purified γ-lylunic acid-containing fats and oils from bacterial cells.

Furthermore, from Table 1-3, it is clear that ethanol-extracted lipids can be used as a raw material for purifying polar lipids (phospholipids, glycolipids, etc.) with a high content of γ-lylunic acid; , it is clear that it has uses as a diglyceride with a high content of γ-lylphinic acid or as a concentrated raw material for γ-lylinic acid.

As described above, it has been revealed that the multi-stage extraction method of the present invention has the advantage that the extraction efficiency is high and that the extracted lipids are extracted in a highly purified form in each extraction stage.

Claims (1)

[Claims] (1) In extracting the lipids contained in Morteierella filamentous fungi, (a) a first extraction process in which the fungi are crushed in an alcohol solvent in the presence of water; ) a first solid-liquid separation step in which the extracted product obtained in the first extraction treatment step is subjected to solid-liquid separation to separate the bacterial cells and an alcohol solvent containing lipid; (c) the first solid-liquid separation; a second extraction step in which the bacterial cells obtained in the step are extracted using a hydrocarbon solvent; (d) the extracted product obtained in the second extraction treatment step is subjected to solid-liquid separation to separate the bacterial cells and lipids; 1. A multi-stage extraction treatment method for a filamentous fungus of the genus Morteierella, comprising a second solid-liquid separation step in which the cells are separated into a hydrocarbon solvent and a hydrocarbon solvent.