JPH0517796A - Recovery of triglyceride - Google Patents

Abstract

PURPOSE:To remarkably efficiently and safely recover triglyceride in triglyceride-containing microorganismic cells by crushing the microorganismic cells being dispersed in water, dehydrating the resultant crushed cells and subsequently extracting them by using a solvent. CONSTITUTION:Triglyceride-containing microorganismic cells are dispersed in water, crushed in that state and dehydrated by using a spray drier, etc. The resultant dehydrated crushed cells are subsequently extracted by using a solvent such as n-hexane or ethanol to recover triglyceride contained in the microorganismic cells with a simple process. In addition, the above-mentioned extraction in contact with the solvent is preferably carried out in a column.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for recovering triglycerides, and more particularly to a method for extremely efficiently and safely extracting and recovering fats (triglycerides) in microbial cells.

[0002]

PRIOR ART AND PROBLEMS TO BE SOLVED BY THE INVENTION
Since microorganisms such as yeast have the ability to produce fats and oils, a method for extracting fats and oils from microbial cells having such ability to produce fats and oils has been performed. That is, triglyceride, which is the main component of fats and oils, accumulates in the microbial cells, so that the cells may be destroyed directly from the cells or mechanically or enzymatically,
A method of extracting is known.

As such a technique, for example, microbial cells capable of producing fats and oils are suspended in ethanol and crushed,
A method has been proposed in which ethanol is removed by filtration and centrifugation, then an extraction solvent is added to the suspension, and the suspension is crushed and extracted (JP-A-61-170397, JP-A-61-170397).
227790, 62-44170, and 6
No. 2-179598). However, these methods have a drawback in that, in addition to using two kinds of solvents, an operation for separating bacterial cells and an organic solvent is required, which makes the operation complicated. Moreover, in these methods, since they are crushed in an organic solvent, there is a risk of fire, and an excessive amount of equipment is required.

[0004]

Means for Solving the Problems As a result of intensive studies to solve such conventional problems, the present inventors crushed and dehydrated triglyceride-containing microbial cells in a state of being dispersed in water. After that, it was found that triglycerides can be obtained very efficiently by preferably carrying out the solvent extraction after filling the cells with the column, and based on this finding, the present invention has been completed.

That is, according to the present invention, in extracting triglyceride from microbial cells containing triglyceride, the microbial cells are crushed in a state of being dispersed in water and dehydrated,
The present invention provides a method for recovering triglyceride, which comprises extracting with a solvent.

The triglyceride-containing microbial cell used in the method of the present invention is obtained by culturing a microorganism capable of producing triglyceride by a conventional method. Examples of the microorganism having a triglyceride-producing ability include various types such as filamentous fungi and yeast.

For example, as a microorganism having the ability to produce γ-linolenic acid-containing fats and oils, (1) JP-A-60-16839 is used.
Mortierel described in Japanese Patent No. 1 etc.
la) microorganisms belonging to genus (2) JP-A-63-28358
Examples thereof include microorganisms belonging to the genus Mucor described in JP-B No. 9 and the like, and (3) microorganisms belonging to the genus Rhizopus described in JP-A-63-133994.

Examples of microorganisms having the ability to produce dihomo-γ-linolenic acid-containing fats and oils and arachidonic acid-containing fats and oils include, for example, (4) Japanese Patent Laid-Open Nos. 63-14696, 63-12290 and 63-63. Microorganisms belonging to the genus Mortierella described in JP-A-44891 and the like, (5) JP-A-64-47384, JP-A-64-47385, and JP-A-63-102688. Examples thereof include microorganisms belonging to the genus Conidiobolus.

More specifically, Mortierella (Mortier
Examples of microorganisms belonging to the genus ella include Mortierella isabellina IFO
7824 and Mortierella
ramaniana var.angrispora) IFO 8187 and the like.

[0010] Examples of the microorganisms belonging to the genus Mucor include, for example, Mucor silcineroides (Mucor).
・ Circinelloides) HUT1121
MP-9359) and Mucor Javanics (Mucor
javanicus) HUT1162 (Microtech Lab.
9360) and the like.

Further, examples of the microorganisms belonging to the genus Conidiobolus include Conidiobolus heterosporus ATCC.
12941, Conidiobo
lus ・ nanodes) CBS 183/62, Conidiobolus lamprauges A
TCC 12585 etc. are mentioned. All of these have the ability to produce fats and oils (triglycerides), and accumulate a considerable amount of fats and oils (triglycerides) in the cells.

Culturing of such microorganisms may be carried out by a conventional method. That is, the culture medium for culturing the above-mentioned microorganism may be any one as long as the microorganism can grow well and produce the target oil and fat (triglyceride).
Examples thereof include glucose and starch as carbon sources, and ammonium sulfate and urea as nitrogen sources, as well as organic nitrogen sources such as defatted soybean powder and defatted rice sugar. In addition, culture may be performed in a medium to which metal salts such as phosphates, magnesium salts, manganese salts, calcium salts are added, if necessary, while controlling pH and temperature.

Thus, microbial cells containing triglyceride are obtained. Since triglyceride is usually accumulated in the microbial cells, the microbial cells are recovered from the culture solution by filtration or centrifugation after the culture of the microorganism is completed.

In the method of the present invention, the recovered bacterial cells are crushed in a state of being dispersed in water. In particular,
The collected bacterial cells are dispersed / suspended in water again, or after dispersed / suspended in water, the bacterial cells are crushed. At the time of dispersion / suspension in this water, it is better that the concentration of the bacterial cells is high, but in consideration of the fluidity at the time of transfer by a pump, the concentration of the bacterial cells is adjusted to 5-18%, preferably 8-15%. To do. Further, in order to prevent clogging in the crushing process using a crusher or the like, it is desirable to perform rough crushing beforehand using a disperser such as a disper mill. If the bacterial concentration in the culture solution is sufficiently high and there are no by-products in the culture solution that may cause problems such as contamination in the post-process or the product, the bacterial cells can be directly disrupted without separating the cells. Can also be supplied to.

Crushing of bacterial cells is usually carried out mechanically,
For example, it can be carried out by using a French press, an ultrasonic crusher, or the like, or by homogenizing in the presence of glass beads, or by using a ball mill.

In order to continuously disperse / suspend / crush the cells, a high-pressure homogenizer type generally used for emulsification and homogenization of fine powder suspension is effective. Also, a dyno mill, a bar mill, etc. can be used.

It should be noted that the temperature of the suspension is 50 to 50 instead of water.
The lipase possessed by the microorganism may be inactivated by suspending it in warm water so that the temperature becomes 80 ° C. By this, after crushing,
It is also possible to prevent the decomposition of triglyceride by lipase, which occurs before drying, and to prevent the increase in the acid value of fats and oils.

Next, the cells thus crushed are dehydrated. Here, dehydration may be carried out so that the water content of the bacterial cells is 10% or less, preferably 5% or less, and it is not always necessary to perform it until the water content of the bacterial cells becomes 0%, that is, until completely dried. Absent. This dehydration may be performed using a freeze dryer, a vacuum dryer, an airflow dryer, or a spray dryer, a paddle dryer, a drum dryer, or the like.

After dehydration in this way, triglyceride is extracted and recovered using a solvent. Examples of the solvent used for extraction include n-hexane, ethanol,
Acetone, methyl ethyl ketone, cyclohexane, diethyl ether, ethyl acetate, etc. may be mentioned.
From the viewpoint of application to food, n-hexane is preferable.

The extraction method using a solvent is not particularly limited, but it is particularly preferable to carry out the extraction using a packed column. For extraction and recovery of triglyceride using a packed column, for example, dehydrated crushed bacterial cells are packed in a cylindrical packed column, the extraction solvent is allowed to flow down from the top, and the solvent flowing out from the bottom of the packed column is collected and concentrated. Can be done by. By doing so, it is possible to reduce the amount of solvent used. The bottom of the packed column is preferably covered with a wire mesh of an appropriate size or filled with a filter aid such as diatomaceous earth in order to prevent the bacterial cells from leaking out.

The amount of the solvent used is 2 to 12 per 1 kg of bacterial cells.
The ratio is preferably 1 liter, and more preferably 3 to 5 liters, especially in consideration of the extraction rate and economy.

The crude oil (crude triglyceride) can be obtained by distilling off the solvent flowing out from the bottom of the packed column by a conventional method such as vacuum concentration. If necessary, it can be further purified by a conventional method.

[0023]

EXAMPLES The present invention will now be described in more detail with reference to examples.

Reference Example In the following Examples and Comparative Examples, the total amount of fats and oils in the cells as a reference for calculating the extraction ratio of the fats and oils was measured by the following method. Mucor circinel
loides) HUT1121 (Microtechnology Research Institute Bacteria-based FERM P-
The cells of 9359) were cultured in a large amount in a 30-liter culture tank under the conditions shown in Table 1, and the obtained γ-linolenic acid-containing cells were collected by filtration. 1 of these collected cells
It was dispersed again in water so that the concentration became 2%, and a part of this was dehydrated by a double drum dryer to obtain a dry microbial cell having a water content of 4%. To 5 g of the dried cells, 100 ml of glass beads having a diameter of 0.6 mm and 100 ml of n-hexane were added, and the number of revolutions was 10,000 by a homogenizer (Nippon Seiki Co., Ltd., Excel Auto Homogenizer DX-3).
After homogenizing at rpm for 3 minutes, glass beads and cell fragments were removed by filtration. Then, 100 ml of n-hexane was added to the cells again, and the same homogenization as above was repeated twice. The obtained filtrates were collected and concentrated under reduced pressure to obtain 1.9 g of a yellow oil and fat. As a result,
It was found that the cells contained oil and fat in a proportion of 38%.

Example 1 Mucor circinelloides
HUT1121 (Microtechnology Research Institute, FERM P-935
The cells of 9) were cultured in a large amount in a 30-liter culture tank under the conditions shown in Table 1, and the resulting γ-linolenic acid-containing cells were collected by filtration. 10% of the collected cells
After uniformly dispersing and suspending in water so that the concentration becomes
It was crushed at a pressure of 700 kg / cm ² using a crusher (Microfluidizer M110Y type, manufactured by Microfluidize Co.). Then, freeze-dry the crushed cells to obtain the water content.
3.4% dry cells were obtained. 10g of this dried bacterial cell
As shown in the figure, the glass column having an inner diameter of 20 mm was packed and 100 ml of n-hexane was flown from the top. The n-hexane / fat / oil mixture (miscella) flowing out from the bottom of the column was recovered, and n-hexane was distilled off by concentration under reduced pressure. As a result, 3.4 g of oil / fat was obtained. The extraction rate at this time is 90%
Met.

Example 2 Mucor circinelloides
HUT1121 (Microtechnology Research Institute, FERM P-935
The cells of 9) were cultured in a 300 liter culture solution under the conditions shown in Table 1, and the obtained cells were collected by filtration.
The collected bacterial cells are uniformly dispersed and suspended in water to
A 2% suspension was made. Then, a high-pressure homogenizer (HV-OH-0.7- manufactured by Izumi Food Machinery Co., Ltd.)
3.7S) was crushed at a pressure of 700 kg / cm ² and a flow rate of 60 liters / hr. The crushed bacterial cells were dehydrated using a double drum dryer to obtain a dry bacterial cell powder having a water content of 3.8%. 700 g of this dried bacterial cell powder was used in a column similar to that shown in FIG. 1 (however, inner diameter 55 mm, total length 140
It is 0 mm, and the difference is that no cock is provided. ), The 4-fold amount (2.8 liters) of n-hexane was made to flow down with respect to the dried bacterial cell powder, and 2.3 liters of effluent from the hexane flowing out from the bottom until the yellow color of the oil and fat disappeared. Recovered. This was concentrated under reduced pressure to obtain 215 g of oil and fat. The extraction rate at this time was 94%.

Examples 3 to 8 In the same manner as in Example 1 except that various oil-and-fat-producing microorganisms cultured under the conditions shown in Table 1 were used, culturing, crushing, drying,
Extraction was performed. The extraction rate is shown in Table 2.

Comparative Example 1 Mucor circinelloides
HUT1121 (Microtechnology Research Institute, FERM P-935
The bacterium of 9) was cultured in a large amount in a 30-liter culture tank, and the obtained γ-linolenic acid-containing bacterium was collected by filtration. The collected bacterial cells were dispersed again in water so as to have a concentration of 12%, and a part thereof was dried by a double drum dryer to obtain dried bacterial cells having a water content of 4%. 10g of this dry cell
Was packed in a column and extracted in the same manner as in Example 1 to obtain 1.2 g of a yellow oil and fat. Therefore, the extraction rate was 31%.

Comparative Example 2 Wet cells (water content 65%) 10 obtained in Comparative Example 1
300 ml of ethanol was added to 0 g, and the mixture was crushed for 5 hours in a ball mill having a volume of 1 liter. After crushing, ethanol was removed by filtration under reduced pressure, the remaining bacterial cells were collected, 300 ml of n-hexane was added, and crushed and extracted again with a ball mill for 5 hours. The bacterial residue and n-hexane were separated by vacuum filtration, and the resulting hexane fraction was concentrated under reduced pressure to recover 9.2 g of oil and fat. The extraction rate at this time was 69%.

[0030]

[Table 1]

[0031]

[Table 2]

[0032]

INDUSTRIAL APPLICABILITY The method of the present invention has high extraction efficiency and can efficiently extract fats and oils (triglycerides) in microbial cells. In addition, the method of the present invention has the advantages of using less solvent, being safe in operation, and being a simple process.

[0033]

[Brief description of drawings]

FIG. 1 is an explanatory view showing a column used in Example 1.

[Explanation of symbols]

a bacterial body b absorbent cotton c cock valve

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location (C12P 7/64 C12R 1: 645)

Claims (3)

[Claims] 1. When extracting triglyceride from a triglyceride-containing microbial cell, the microbial cell is crushed in a state of being dispersed in water, dehydrated, and then extracted with a solvent. Recovery method. 2. The method according to claim 1, wherein the extraction with the solvent comprises contacting the solvent in a column. 3. The method according to claim 1, wherein the solvent is n-hexane.