JP6179769B2 - Method for extracting betaine and / or glucosylglycerol from halophilic microorganisms - Google Patents

Description

  The present invention relates to a method for extracting betaine and / or glucosylglycerol from halophilic microorganisms such as halophilic bacteria and algae.

  Betaine (also known as glycine betaine or trimethylglycine) has been used as a food additive for the purpose of improving the storage stability, taste quality, umami penetration, etc. of foods. It is a substance that is also expected as a functional food such as prevention of food. As a method for obtaining betaine, a method of extracting and purifying from cultivated sugar beet is common, but sugar beet cultivation depends on the weather, and the number of harvests is limited to about twice a year.

  A certain halophilic microorganism is known as an organism containing betaine in addition to sugar beet (Patent Document 1). Betaine is present in the cells of halophilic microorganisms and functions as an osmotic pressure regulating substance. A halophilic microorganism can be cultured throughout the year by adjusting the culture conditions. Therefore, by culturing a halophilic microorganism capable of producing betaine and extracting betaine from this microorganism, it can be expected to obtain betaine more efficiently than extracting from sugar beet. In addition, glucosylglycerol (glyceryl glucoside) useful as a material for cosmetics, skin preparations and the like can be expected to be extracted from the above microorganisms as well as betaine.

JP 2001-48776 A

  However, since halophilic microorganisms are cultured using a culture solution containing a large amount of salt, a desalination treatment is not necessary when extracting and purifying betaine and / or glucosylglycerol from microorganisms collected (collected) from the culture solution. There is a problem that is needed. That is, in order to use the ion exchange method, which is a general method for purifying extracts from plants, it is first necessary to remove high-concentration salinity contained in the culture solution attached to microorganisms. However, the method of washing microorganisms with water for desalting cannot be employed because the cells of the microorganisms are ruptured by an osmotic pressure difference and target betaine and glucosylglycerol are diffused into the washing water.

  The present invention has been made in view of the above circumstances, and provides a method for efficiently extracting betaine and / or glucosylglycerol from a microorganism collected from a culture solution having a high salt concentration.

In order to achieve the above object, the present invention provides the following means. In the present invention, “betaine” means trimethylglycine.
In the present specification and claims, “betaine and / or glucosylglycerol” means “at least one of betaine and glucosylglycerol”.

(1) A washing step for washing halophilic microorganisms containing betaine and / or glucosylglycerol using an isotonic solution containing salts that are hardly soluble in alcohol, and collecting the washed halophilic microorganisms A collecting step, a dissolving step of dissolving betaine and / or glucosylglycerol in the collected halophilic microorganism in the alcohol, a removing step of removing a substance not dissolved in the alcohol, A method for extracting betaine and / or glucosylglycerol from a halophilic microorganism, comprising a separation step of recovering dissolved betaine and / or glucosylglycerol.

  According to the method of (1) above, in the washing step, the culture components adhering to the halophilic microorganism are washed so that the hardly soluble salts with respect to the alcohol are mainly attached to the halophilic microorganism. can do. Thereafter, in the dissolution step, betaine and / or glucosylglycerol is dissolved in the alcohol from the halophilic microorganism to which the salts are attached. On the other hand, since the salts are difficult to dissolve in the alcohol, the salts can be removed as an alcohol-insoluble substance together with the residue of halophilic microorganisms in the subsequent removal step. Thereafter, in the separation step, the target betaine and / or glucosylglycerol can be efficiently and simply obtained by separating betaine and / or glucosylglycerol from the alcohol.

(2) In the separation step, betaine and / or glucosylglycerol is recovered as an evaporation residue by evaporating the alcohol.
According to the method (2), betaine and / or glucosylglycerol extracted from a halophilic microorganism can be efficiently and simply obtained as an evaporation residue remaining after evaporation of alcohol.

(3) In the above (1) or (2), the betaine and / or glucosylglycerol is purified by an ion exchange method from an aqueous solution obtained by dissolving the recovered betaine and / or glucosylglycerol in water. The method described.
According to the method of (3) above, the target betaine can be purified from the component extracted from the halophilic microorganism into the alcohol to obtain high-purity betaine.

(4) The method according to any one of (1) to (3), wherein the alcohol is ethanol.
According to the method (4), betaine and / or glucosylglycerol can be easily extracted from a halophilic microorganism.

(5) The method according to any one of (1) to (4), wherein the salt is sodium chloride.
According to the above method (5), sodium chloride is efficiently removed as an alcohol-insoluble substance in the removal step, and the salt concentration contained in betaine and / or glucosylglycerol obtained in the separation step is further reduced. Can do.

  According to the method for extracting betaine and / or glucosylglycerol from a halophilic microorganism of the present invention, betaine and / or glucosylglycerol are efficiently extracted from a microorganism collected from a culture solution having a high salt concentration, and the extracted betaine is extracted. And / or glucosylglycerol and the salt in a culture solution can be isolate | separated. For this reason, the extracted betaine and / or glucosylglycerol can be easily purified by an ion exchange method or the like, and high-purity betaine and / or glucosylglycerol can be obtained at low cost.

It is a HPLC chart of a standard sample in a reference example. 2 is a HPLC chart of a filtrate obtained by extraction from microorganisms in Example 1. FIG. 3 is a HPLC chart of a filtrate obtained by extraction from microorganisms in Example 2.

Hereinafter, the present invention will be described based on preferred embodiments.
An embodiment of the method according to the present invention extracts betaine and / or glucosylglycerol from a halophilic microorganism, which includes at least a washing step, a collection step, a lysis step, a removal step, and a separation step. Is the method.
First, a series of flows of these steps will be described.

  Hereinafter, a case where betaine is extracted will be described. Extraction of glucosylglycerol can be performed in the same manner as in the case of betaine (“betaine” can be read as “glucosylglycerol”), and thus description of extracting glucosylglycerol is omitted. In addition, when the microorganisms to be used contain both betaine and glucosylglycerol, both can be extracted simultaneously. The extracted betaine and glucosylglycerol can be separated by a known method such as chromatography.

  In <washing step>, various isotopes such as culture components from the cells and subsequent purification inhibition by using an isotonic solution (washing solution) having an osmotic pressure equal to that of the halophilic microorganisms collected from the culture solution The salt can be washed.

  In the subsequent <bacterial collection step>, the cells can be collected by separating the isotonic solution used for washing and the cells by a treatment such as centrifugation or filtration. In the collected cells, salts that are hardly soluble in the alcohol contained in the isotonic solution are mainly attached.

  In the next <lysis step>, the washed bacterial cells are suspended in the alcohol and the cell membrane of the bacterial cells is dissolved by the alcohol, or betaine contained in the bacterial cells by treatment such as ultrasonic disruption or homogenization. And the like can be eluted out of the cells. As a result, betaine is dissolved in the alcohol.

  Thereafter, in <removal step>, the residue of the microbial cells that did not dissolve in the alcohol and a substance that is insoluble in the alcohol such as the salts contained in the isotonic solution are centrifuged or filtered, etc. Is removed from the alcohol.

  Next, in the <separation step>, the alcohol is evaporated (distilled) by a process such as evaporation, distillation, drying, vacuum drying or freeze-drying to recover betaine dissolved in the alcohol as an evaporation residue. be able to.

  Prior to the washing step, a culturing step for culturing halophilic microorganisms containing the target betaine may be provided in advance. Moreover, you may provide the collection | recovery process which collect | recovers the halophilic microorganisms containing the target betaine from a culture solution or salt water before a washing | cleaning process.

The halophilic microorganisms to be washed in the washing process may be microorganisms cultured in a high salt concentration culture solution, or naturally in a high salt concentration environment (salt water) such as the ocean, salt lake, salt field, hot spring, etc. Microorganisms that live in Here, the halophilic microorganisms include halophilic algae, actinomycetes, and the like that contain glycine and / or betaine in the body.
The method for collecting (collecting) the halophilic microorganism containing the target betaine from the salt water is not particularly limited, and examples thereof include means such as centrifugation, precipitation, and filtration. Usually, the collected halophilic microorganisms are collected as wet pellets. This pellet contains a small amount of a culture solution or salt water, and when the salts contained in this pellet coexist with betaine, it becomes difficult to purify the betaine by an ion exchange method.

  In the washing process, the culture solution attached to the halophilic microorganisms recovered from the culture solution, etc. is washed with an isotonic solution adjusted to a salt concentration that does not cause the cells of the halophilic microorganisms to burst due to osmotic pressure difference. To do. Here, the salts contained in the isotonic solution are hardly soluble salts having very low solubility in the alcohol used in the dissolving step.

  The solubility of the salt contained in the isotonic solution in the alcohol is preferably as small as possible. For example, 0.5 g / 100 mL or less is preferable, 0.1 g / 100 mL or less is more preferable, and 0.07 g / 100 mL is more preferable.

  Examples of the salts contained in the isotonic solution include sodium chloride and potassium chloride.

  The method for washing halophilic microorganisms with an isotonic solution is not particularly limited as long as it can wash excess culture components adhering to halophilic microorganisms without destroying halophilic microorganisms. . For example, a method of suspending a pellet obtained by collecting halophilic microorganisms in an isotonic solution can be mentioned. By suspending the pellet in an isotonic solution in an excessive amount with respect to the volume of the pellet, unnecessary components adhering to the halophilic microorganism can be diffused and diluted in the isotonic solution. As a specific example, for example, an unnecessary component adhering to a pellet obtained by recovering a halophilic microorganism after suspending the pellet in an isotonic solution 20 times the volume of the pellet is theoretically Decrease by a factor of 20 before washing.

  The method for preparing the isotonic solution is not particularly limited, and the salts may be dissolved in a predetermined amount in pure water (purified water). The salt concentration in the isotonic solution may be appropriately adjusted according to the type of the halophilic microorganism to be used so that the isotonic solution does not rupture the cells of the halophilic microorganism to be used. As a specific example, for example, when Actinopolyspora which is a kind of actinomycete as a halophilic microorganism or Dunaliella which is a kind of green algae is used, an isotonic solution in which sodium chloride is dissolved in pure water at 100 to 200 g / L can be mentioned. . The sodium chloride concentration is preferably adjusted so as to be an isotonic solution for the cells of the halophilic microorganism.

  As a method for collecting halophilic microorganisms in the collection process, for example, there is a method in which halophilic microorganisms suspended in an isotonic solution are collected as wet pellets by means of centrifugation, precipitation, filtration, or the like. Can be mentioned.

  In the lysis step, betaine in the collected halophilic microorganism is dissolved in the alcohol by suspending the collected halophilic microorganism pellet in the alcohol, and the cell membrane of the microorganism is naturally dissolved. And the method of waiting for betaine contained in a cell to elute in the said alcohol is mentioned. In addition, after suspending the pellet in the alcohol, the suspension may be vigorously stirred or the cell membrane may be actively destroyed by impact with ultrasonic waves. Alternatively, betaine may be positively eluted from the cells by suspending the pellet in the alcohol and then applying the suspension to a homogenizer to grind the cells.

  The alcohol used in the dissolution step is not particularly limited as long as it can dissolve betaine extracted from halophilic microorganisms and hardly dissolve salts contained in an isotonic solution used in the washing step. The solubility of betaine with respect to 100 g of alcohol used in the dissolving step is preferably 1 g / 100 g or more, more preferably 4 g / 100 g or more, and further preferably 7 g / 100 g or more. Since this solubility is preferably as high as possible, the upper limit is not particularly limited, but it is considered that the upper limit is usually about 100 g / 100 g. Specific examples of the alcohol used in the dissolution step include primary alcohols having about 1 to 5 carbon atoms such as ethanol, methanol, and isopropanol. The solubility of betaine in 100 g of ethanol is about 8.7 g (20 ° C.).

  The mass ratio of the solubility of betaine to the alcohol used in the dissolution step and the solubility of the salt contained in the isotonic solution is preferably as large as possible from the viewpoint of increasing the yield of betaine and increasing the salt removal rate. The mass ratio of the solubility (solubility of betaine / solubility of salts contained in the isotonic solution) is, for example, preferably 10 or more, more preferably 50 or more, and further preferably 100 or more. The upper limit value of the mass ratio is not particularly limited, and it is considered that the standard value is usually about 10,000.

For example, since the solubility of betaine in 100 g of ethanol is about 8.7 g and the solubility of sodium chloride in 100 g of ethanol is about 0.065 g, the mass ratio of the solubility is about 133.8 (8.7 ÷ 0.06). It is.
Although the solubility of glucosylglycerol in ethanol is different from that of betaine, it is much better soluble than sodium chloride.

  Betaine is dissolved in the alcohol solution obtained after the dissolution step. On the other hand, the alcohol-insoluble substances such as the salts constituting the isotonic solution contained in a small amount in the bacterial cell residue and the bacterial cell pellet are floating or precipitated in the alcohol solution.

  In the removal step, the method for removing these alcohol-insoluble substances is not particularly limited, and examples thereof include a method of removing residues and the like as pellets or aggregates by means of centrifugation, precipitation, filtration, and the like. The alcohol solution after removing the alcohol-insoluble substance is subjected to a separation step.

  In the separation step, the method for separating betaine from the alcohol solution is not particularly limited. Suitable methods include, for example, a method of recovering the target betaine as an evaporation residue by evaporating an alcohol, which is a solvent for dissolving betaine, from the solution.

Examples of the method for evaporating alcohol from an alcohol solution containing betaine include a method of subjecting the alcohol solution to a treatment such as heating, distillation, drying, vacuum drying, and freeze drying.
It is preferable to set the heating temperature and the distillation temperature at the time of evaporation as high as possible within the range where betaine is not decomposed by heat. By setting in this way, evaporation of alcohol can be promoted.

  The evaporation residue containing betaine contains components that can be dissolved in the alcohol used in the dissolution step among the cell components of halophilic microorganisms. This component may include glucosylglycerol. By putting the evaporation residue into water or an aqueous solvent, a water-soluble substance such as betaine is dissolved, and a water-insoluble substance is not dissolved but floats or precipitates in water. This water-insoluble substance can be removed by means such as centrifugation, precipitation, and filtration. High-purity betaine and glucosylglycerol can be separated and obtained from an aqueous solution in which betaine is dissolved by a known purification method such as an ion exchange method.

  The above-described method for extracting betaine from halophilic microorganisms can be applied as a method for extracting glucosylglycerol from halophilic microorganisms instead of betaine.

[Reference example]
A sample prepared by adding sodium chloride (NaCl) at 0 wt%, 0.01 wt%, 0.02 wt%, 0.06 wt% or 0.08 wt% to a standard aqueous solution containing glucosylglycerol (components I & II) and betaine at a predetermined concentration. Prepared each. The components contained in each sample were separated by known chromatography (HPLC). The result is shown in FIG. If 1% or more of NaCl is present, the peak becomes very large, and the peaks of glucosylglycerol (components I & II) and betaine are covered and cannot be distinguished.
From the chart in FIG. 1, it was confirmed that a trace amount of NaCl contained in the sample could be detected separately from the betaine and glucosylglycerol peaks.

[Example 1]
One genus of halophilic Geitlerinema (a kind of cyanobacteria) containing betaine and glucosylglycerol was isolated from a tide pool along the sea where the tide is full, and cultured in an osmotic pressure corresponding to 10 wt% NaCl. The microorganisms were washed with a 20 wt% NaCl aqueous solution, and the microorganisms collected by a conventional method were dispersed in ethanol to dissolve the cell membrane, and the components contained in the cells were extracted into ethanol by ultrasonic disruption. The ethanol extract was centrifuged to remove insoluble components, and the filtrate obtained by filtration using filter paper was subjected to HPLC in the same manner as in Reference Example. The result is shown in FIG.

From the chart of FIG. 2, NaCl contained in the filtrate is very small (0.1 wt% or less), and the NaCl concentration is sufficiently reduced as compared with the 10 wt% NaCl aqueous solution used as the microorganism cleaning solution. It could be confirmed. It was also confirmed that betaine was recovered in the filtrate. By fractionating the fraction corresponding to each peak of HPLC, betaine and NaCl could be separated and recovered. The target betaine could be obtained as an evaporation residue by evaporating ethanol as a solvent of the obtained fraction with an evaporator.
From these results, it was confirmed that the extraction method according to the present invention can easily remove the salts used for culturing halophilic microorganisms and recover the target betaine with high purity.

[Example 2]
A halophilic Cyanobacterium genus containing betaine and glucosylglycerol was isolated from a tidal pool along the sea where the tides were intense, and cultured in an osmotic pressure corresponding to 5 wt% NaCl. This microorganism was washed with a 5 wt% NaCl aqueous solution, and the subsequent treatment was carried out in the same manner as in Example 1. The result is shown in FIG.

  From the chart of FIG. 3, the NaCl contained in the filtrate is very small (0.1 wt% or less), and the NaCl concentration is sufficiently reduced compared to the 5 wt% NaCl aqueous solution used as the microorganism cleaning solution. It could be confirmed. It was also confirmed that glucosylglycerol was recovered in the filtrate.

  The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention.

Claims (5)

A washing step of washing a halophilic microorganism containing betaine and / or glucosylglycerol using an isotonic solution containing a salt that is sparingly soluble in alcohol;
A collection process for collecting the washed halophilic microorganisms;
A dissolution step of dissolving betaine and / or glucosylglycerol in the collected halophilic microorganism in the alcohol;
A removal step of removing the substance that did not dissolve in the alcohol;
A separation step of recovering betaine and / or glucosylglycerol dissolved in the alcohol;
A method for extracting betaine and / or glucosylglycerol from a halophilic microorganism.   The method according to claim 1, wherein in the separation step, betaine and / or glucosylglycerol is recovered as an evaporation residue by evaporating the alcohol.   The method according to claim 1 or 2, wherein the betaine and / or glucosylglycerol is purified by an ion exchange method from an aqueous solution obtained by dissolving the recovered betaine and / or glucosylglycerol in water.   The method according to claim 1, wherein the alcohol is ethanol.   The method according to claim 1, wherein the salt is sodium chloride.

Images