JP2018000073A - Production method of 3hb - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide 3HB production methods that can efficiently purify and recover 3HB with high purification from a culture solution which is derived from organism fermentation and contains 3HB.SOLUTION: A method of the present invention comprises: a preliminary concentration step of distilling off moisture from a fermented solution produced by fermenting 3HB from 3 hydroxybutyric-acid (3HB) production bacterium; an extraction step of extracting, with an organic solvent, 3HB-containing solution which contains 3HB; a concentration step of distilling the organic solvent from the 3HB-containing solution extracted by the extraction step to obtain a 3HB concentrated solution; and a crystallization step of adding a crystallization solvent containing as main component at least one selected from ethyl acetate, diethyl ether, and t-butyl methyl ether to the 3HB concentrated solution obtained in the concentration step, and of precipitating 3HB crystal from the crystallization solvent in which 3HB is dissolved.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing 3-hydroxybutyric acid (3HB).

  Since 3-hydroxybutyric acid and its salt (hereinafter referred to as 3HB, also referred to as 3HB, especially its monomer) is a substance that is originally present in the human body, it has high biocompatibility and is a sugar. Expected to be a groundbreaking energy source instead of quality. In addition, it has been found that 3HB not only serves as a mere energy source but also acts as a signal transmitter that affects the expression of various genes and the activity of proteins. 3HB is known to improve cognitive function and long-lasting memory by inhibiting histone deacetylase, for example, by regulating gene expression, and its effectiveness in preventing Alzheimer's has been confirmed. For example, 3HB produced by ingestion and metabolism in the body of medium chain fatty acids that are abundant in coconut oil has the effect of improving the symptoms of Alzheimer's disease and diabetic patients who cannot utilize carbohydrates in the brain and body. It has been known. In addition, 3HB is converted to energy more rapidly than carbohydrates in the body and has the effect of suppressing the absorption of fat and sugar into cells, so it can be applied to energy substances for athletes, diet and health foods. Can be expected.

  In addition to these uses, 3HB is known to be used as a raw material for biodegradable resins, and is a substance whose utility value is increasing in industrial uses.

  Such 3HB can be obtained by causing various microorganisms to produce poly-3-hydroxybutyric acid (hereinafter referred to as PHB) and then decomposing the obtained PHB with an enzyme or the like (Patent Document 1). In addition, it has been found that Halomonas bacteria as such microorganisms accumulate PHB under aerobic conditions and secrete and produce 3HB produced by decomposing PHB by shifting to anaerobic conditions in the culture solution. (Patent Document 2).

JP-T-2005-521718

Yutaka Tokiwa et al. J Biotechnol. (2007) 132, 264-72.

  It is difficult to recover 3HB or PHB derived from biological fermentation as a pure product from the culture solution, and it is actually recovered in a form containing various impurities. When the recovered 3HB is used for application, it needs to be further purified to increase the purity. However, in reality, 3HB with low purity (for example, 3HB derived from biological fermentation currently distributed is about 70% in purity) is used as it is, and 3HB with high purity has not yet been stably supplied.

  In order to stably supply such high-purity 3HB, an efficient method for producing 3HB using a culture solution derived from biological fermentation and containing 3HB is required.

  Then, in view of the said actual condition, this invention aims at providing the manufacturing method of 3HB which can refine | purify and collect highly purified 3HB efficiently from the culture solution derived from biological fermentation and containing 3HB.

In order to achieve the above object, the characteristic configuration of the 3HB manufacturing method of the present invention is as follows:
A fermentation process for obtaining a fermentation broth produced by fermentation of 3HB from a 3-hydroxybutyric acid (3HB) producing bacterium;
From the fermentation liquid obtained in the fermentation step, a preconcentration step for distilling off water,
An extraction step of extracting a 3HB-containing solution containing 3HB from the fermentation broth with an organic solvent;
A concentration step of distilling off the organic solvent from the 3HB-containing solution extracted by the extraction step to obtain a 3HB concentrate;
A crystallization solvent containing at least one selected from ethyl acetate, diethyl ether, and t-butyl methyl ether as a main component is added to the 3HB concentrate obtained in the concentration step, and 3HB crystals are obtained from the crystallization solvent in which 3HB is dissolved. A crystallization step for precipitation,
The point is to do.

  3HB obtained by the fermentation process is considered to have very high optical purity (close to 100%). When an extraction step of extracting this with an organic solvent is performed, a 3HB-containing solution with high optical purity can be obtained from the fermentation broth.

  Here, in order to separate and recover 3HB from the fermented liquor, an extraction step in which extraction with an organic solvent is performed through a pre-concentration step for distilling off water efficiently converts 3HB into a layer of organic solvent with a small amount of organic solvent. Can be migrated. When the organic solvent is distilled off and concentrated by a concentration step, a 3HB concentrate containing 3HB at a high concentration is obtained, and 3HB is crystallized very efficiently by performing a crystallization step using this 3HB concentrate. 3HB with high purity can be obtained.

Thus, when the crystallization process which precipitates 3HB is performed, 3HB can be recrystallized from the 3HB containing solution only extracted from the fermentation liquid, and 3HB with higher purity can be obtained. Specifically, by performing a crystallization step after the concentration step, 3HB recrystallization operation can be performed to obtain highly pure 3HB.
Here, as a result of diligent research, the inventors of the present invention have used the crystallization solvent as a main component of at least one selected from ethyl acetate, diethyl ether, and t-butyl methyl ether. It was found experimentally that 3HB can be crystallized very efficiently even if it is directly used as a starting material, and the present invention has been completed.

  That is, when any one of ethyl acetate, diethyl ether, and t-butyl methyl ether is used as the crystallization solvent, the solubility curve of 3HB (temperature dependence of solubility) is particularly large (the difference in solubility due to temperature). It is clear that 3HB can be easily crystallized by recrystallization and impurities can be removed. As a result, high purity 3HB can be efficiently produced.

  The 3HB-producing bacterium is preferably a 3HB-producing halomonas bacterium. In this case, the fermentation step comprises an aerobic fermentation step in which the halomonas bacterium is aerobically fermented, and an aerobic fermentation step. An anaerobic fermentation step of anaerobically fermenting the halomonas having accumulated (PHB).

  That is, when 3HB-producing halomonas are used as 3HB-producing bacteria, 3HB can be produced with high productivity. In addition, Halomonas bacteria perform the aerobic fermentation process for aerobic fermentation and the anaerobic fermentation process for anaerobic fermentation, so that polyhydroxybutyric acid (PHB) accumulated in the body by the aerobic fermentation process is in vitro (in the fermentation broth). Can be discharged. Therefore, by using Halomonas bacteria, 3HB can be transferred to an easily reactable form relatively easily, and 3HB can be produced more efficiently.

  Moreover, you may perform the said extraction process, after performing the filtration process which filters the fermentation liquor obtained at the said fermentation process.

  In addition to 3HB, the fermented liquid obtained in the fermentation process also includes cells that produced 3HB. Since such microbial cells do not migrate into the 3HB-containing solution when the extraction step is performed, it is considered that the presence of impurities as a cause of deterioration in purity. In addition, the amount of the extraction solvent is wasted and used. Therefore, if the ultrafiltration is performed in advance, recrystallization can be performed without containing large molecular weight impurities such as bacterial cells and proteins, so that the purity of recrystallization can be further improved. Conceivable.

  Moreover, after performing the pH adjustment process which adjusts the pH of the fermented liquor obtained at the said fermentation process to the acidity of 1-6, the said extraction process can also be performed.

  Since 3HB contained in the fermentation broth is acidic, a part thereof is dissolved as a salt in the fermentation broth close to neutrality. Such a form of 3HB is difficult to migrate into the 3HB-containing solution, and there is a concern that the extraction efficiency of 3HB may be reduced. Therefore, if a pH adjustment step for adjusting the pH of the fermentation broth to pH 1 to 6 is performed, 3HB can be present in the fermentation broth as a free acid, and 3HB easily migrates into the extracted 3HB-containing solution. It is thought that it contributes to the improvement of the recovery efficiency of 3HB.

  Moreover, the activated carbon treatment process which makes the said 3HB containing solution or the said 3HB concentrate liquid contact activated carbon can also be performed.

  If the filtration step is performed, a contaminant component having a relatively large molecular weight can be removed, but a colored component for 3HB having a relatively small molecular weight cannot be removed. Although such a colored component does not have a great influence on the actual purity of 3HB, 3HB with a high degree of purification is a white crystal, whereas crystals colored in brown to light brown are obtained. As a product of 3HB, it looks inferior. Therefore, if such coloring components are removed with activated carbon, 3HB obtained in the crystallization step can be obtained as white crystals and can be provided as high-quality 3HB.

  In addition, as said organic solvent, what has as a main component at least 1 type chosen from 1-butanol, 1-pentanol, ethyl acetate, isoamyl alcohol, and methyl ethyl ketone can be used.

  As the organic solvent, those having a high solubility in 3HB can be used regardless of the slope of the solubility curve. Examples of such an organic solvent include 1-butanol, 1-pentanol, ethyl acetate, isoamyl alcohol, or methyl ethyl ketone. Those having at least one selected from the above as a main component are preferred because of their high solubility in 3HB.

  In the crystallization step, 3HB seed crystals are added to a crystallization solvent in which 3HB is dissolved, and cooling can be performed while stirring.

  Crystallization by cooling while stirring can start crystallization from a uniform solution without temperature spots, so that crystals with a very narrow crystallization start temperature range can be taken out, contributing to increase the purity of 3HB. . In addition, when a seed crystal is used, crystallization can be started from an undercooled 3HB solution at an appropriate crystallization start temperature, which contributes to providing 3HB with more stable quality.

  Therefore, 3HB having high purity can be efficiently purified and recovered from a culture solution derived from biological fermentation and containing 3HB.

  Below, the manufacturing method of 3HB concerning embodiment of this invention is demonstrated. Preferred embodiments are described below, but these embodiments are described in order to more specifically illustrate the present invention, and various modifications can be made without departing from the spirit of the present invention. The present invention is not limited to the following description.

The manufacturing method of 3HB according to the embodiment of the present invention is as follows:
A fermentation process for obtaining a fermentation broth produced by fermentation of 3HB from a 3-hydroxybutyric acid (3HB) producing bacterium;
From the fermentation liquid obtained in the fermentation step, a preconcentration step for distilling off water,
An extraction step of extracting a 3HB-containing solution containing 3HB from the fermentation broth with an organic solvent;
A concentration step of distilling off the organic solvent from the 3HB-containing solution extracted by the extraction step to obtain a 3HB concentrate;
A crystallization solvent containing at least one selected from ethyl acetate, diethyl ether, and t-butyl methyl ether as a main component is added to the 3HB concentrate obtained in the concentration step, and 3HB crystals are obtained from the crystallization solvent in which 3HB is dissolved. A crystallization step for precipitation.
Here, 3HB-producing halomonas bacteria are used as 3HB-producing bacteria, and as a fermentation process, an aerobic fermentation process in which the halomonas bacteria are aerobically fermented, and polyhydroxybutyric acid (PHB) accumulated by the aerobic fermentation process An anaerobic fermentation process for anaerobically fermenting Halomonas bacteria.
Prior to the extraction step, a filtration step of ultrafiltration of the fermentation broth obtained in the fermentation step is performed, and a pH adjustment step of adjusting the pH of the fermentation broth to 1 to 6 acidity is performed.
Moreover, the activated carbon treatment process which makes 3HB concentrated liquid contact activated carbon is performed.
In addition, as an organic solvent, what has as a main component at least 1 type chosen from 1-butanol, 1-pentanol, ethyl acetate, isoamyl alcohol, and methyl ethyl ketone is used, and the crystallization which 3HB melt | dissolved in the crystallization process. Cooling can be performed while adding 3HB seed crystals to the solvent and stirring. The solvent used in the crystallization process may be the same as the solvent used in the extraction process.

[Fermentation process]
A fermentation process obtains the fermented liquor which produced and fermented 3HB from 3HB production microbe. Specifically, a 3HB-productive halomonas bacterium is added to a fermentation vessel containing a saccharide-containing culture solution, and an aerobic fermentation process is first performed while stirring and aeration. Thereby, carbohydrates can be assimilated by Halomonas bacteria to produce PHB, and PHB is accumulated in Halomonas bacteria. Next, when the saccharide is almost completely consumed, the anaerobic fermentation process is performed by stopping aeration into the fermentation vessel. As a result, Halomonas bacteria decompose and consume PHB accumulated in the body and release 3HB into the fermentation broth. As a result, a fermentation broth containing 3HB and bacterial cells is obtained.

[Filtration process]
In the filtration step, contaminants such as bacterial cell components and high molecular weight proteins in the obtained fermentation broth are removed by filtration. Specifically, the obtained fermentation broth is filtered through an ultrafiltration membrane (UF membrane). Then, contaminants of large molecular weight such as bacterial cells and proteins can be easily removed, and a fermentation broth mainly containing 3HB can be obtained. The fermentation broth obtained here contained a fermentation broth containing 4.1% by mass of 3HB. In this embodiment, the concentration of 3HB is determined by a chromatographic method (the same applies below).

[PH adjustment step]
In the pH adjustment step, the pH of the fermentation broth is adjusted to pH 1 to 6 so that 3HB in the fermentation broth can be easily transferred to the 3HB-containing solution as a free acid. Specifically, in the pH adjustment step, 1.4 kg of 96 mass% sulfuric acid was added to 64.3 kg of the fermentation broth obtained in the filtration step to adjust the pH to 3.0.
Here, various acids can be used in the pH adjustment step, but it is preferable to use an inorganic acid that hardly causes adverse effects in the subsequent purification step. Further, the pH may be adjusted to an acidic region of 1 to 6, but it is preferable to adjust the pH by adding an acid until pH becomes about pH 3 where 3HB is almost present as a free acid. From the viewpoint of

[Preconcentration step]
The pre-concentration step is a step of concentrating the fermentation broth by distilling off water as a solvent in the fermentation broth. Specifically, concentration was performed by distilling off water with an evaporator from 65.7 kg of the fermented liquid after pH adjustment. As a result, 12.4 kg of a 3HB-containing solution containing 20.8% by mass of 3-hydroxybutyric acid was obtained.

[Activated carbon treatment process]
In the activated carbon treatment step, powdered or granulated activated carbon is brought into contact with the 3HB-containing solution to remove impurities such as coloring substances. Here, the activated carbon treatment step is preferably performed in order to improve the quality by decolorizing 3HB to be obtained, and can be omitted in the sense of avoiding a decrease in yield and an increase in man-hours.

[Extraction process]
Next, using 2 kg of the obtained 3HB-containing solution, the extraction step of extracting with the same weight of various organic solvents 1-butanol, 1-pentanol, ethyl acetate, isoamyl alcohol, or methyl ethyl ketone was repeated three times to contain 3HB. A solution was obtained. When the extraction process was repeated three times with each organic solvent, the yield of recovered 3HB was as shown in Table 1. As the organic solvent, other general-purpose solvents can be used, but 1-butanol, 1-pentanol, ethyl acetate, isoamyl alcohol, or methyl ethyl ketone is preferably used because of its high solubility in 3HB.

[Concentration process]
In the concentration step, the organic solvent is distilled off from the obtained 3HB-containing liquid using an evaporator. In this concentration step, a 3HB concentrate containing 75% by mass of 3HB was obtained. The concentration step is preferably performed until the 3HB concentration of the 3HB concentrate is about 50% to 90%. More preferably, the lower limit of concentration is 60% or more, or 70% or more, and the upper limit of concentration is 85% or less, and it is particularly preferable that the concentration is concentrated to about 70% or more and 85% or less.

[Activated carbon treatment process]
Here, an activated carbon treatment step for removing impurities such as colored substances from the concentrated liquid using powdered or granular activated carbon may be performed. The activated carbon treatment step is preferably performed on either the 3HB-containing solution or the 3HB concentrated solution, but may be omitted or performed on both.

[Crystalling process]
Subsequently, 60 g of ethyl acetate as a crystallization solvent was added to 200 g of a concentrated solution containing 75% by mass of 3HB (extracted using 1-butanol as an organic solvent), and 3HB was homogeneous at 40 ° C. Solution was obtained. The 3HB solution was cooled to a saturated concentration of 3HB at 20 to 30 ° C. with stirring and mixing in a crystallizer. When the saturation concentration was reached, 3HB obtained as a crystal by purification in advance was added as a seed crystal. Further, cooling was carried out at a constant rate while stirring to precipitate 3HB. The precipitated 3HB was suction filtered and dried under reduced pressure to isolate 85 g of white crystals. The yield for the crystallization process was 57%.

  In addition, when ethyl acetate was changed to diethyl ether, t-butyl methyl ether, acetone, and methyl ethyl ketone, and a crystallization process was similarly performed on 200 g of a concentrated solution containing 3% by mass of 3HB, the results shown in Table 2 were obtained. It was.

  As the crystallization solvent, an ester-based or ether-based substance having a large temperature-dependent solubility gradient can be used, and the crystallization solvent selected from ethyl acetate, diethyl ether, and t-butyl methyl ether is methanol. As compared with various known easy-to-handle solvents such as ethanol, propanol, butanol, acetone, methyl ethyl ketone, etc., it was found that the solubility gradient was large and the crystallization solvent was extremely excellent.

  According to the 3HB production method of the present invention, 3HB with high efficiency and high purity can be produced.

Claims (8)

A fermentation process for obtaining a fermentation broth produced by fermentation of 3HB from a 3-hydroxybutyric acid (3HB) producing bacterium;
From the fermentation liquid obtained in the fermentation step, a preconcentration step for distilling off water,
An extraction step of extracting a 3HB-containing solution containing 3HB from the fermentation broth with an organic solvent;
A concentration step of distilling off the organic solvent from the 3HB-containing solution extracted by the extraction step to obtain a 3HB concentrate;
A crystallization solvent containing at least one selected from ethyl acetate, diethyl ether, and t-butyl methyl ether as a main component is added to the 3HB concentrate obtained in the concentration step, and 3HB crystals are obtained from the crystallization solvent in which 3HB is dissolved. 3HB manufacturing method which performs the crystallization process to make it precipitate.   The method for producing 3HB according to claim 1, wherein the 3HB-producing bacterium is a 3HB-producing halomonas bacterium.   The said fermentation process contains the aerobic fermentation process which carries out the aerobic fermentation of the said Halomonas bacteria, and the anaerobic fermentation process which carries out the anaerobic fermentation of the said Halomonas bacteria which accumulated polyhydroxybutyric acid (PHB) by the aerobic fermentation process. The manufacturing method of 3HB of description.   The manufacturing method of 3HB as described in any one of Claims 1-3 which perform the said extraction process after performing the filtration process which filters the fermented liquor obtained at the said fermentation process.   The manufacturing method of 3HB as described in any one of Claims 1-4 which perform the said extraction process after performing the pH adjustment process which adjusts the pH of the fermented liquor obtained at the said fermentation process to the acidity of 1-6. .   The method for producing 3HB according to any one of claims 1 to 5, wherein an activated carbon treatment step in which the 3HB-containing solution or the 3HB concentrate is brought into contact with activated carbon is performed.   The said organic solvent is what has as a main component at least 1 type chosen from 1-butanol, 1-pentanol, ethyl acetate, isoamyl alcohol, and methyl ethyl ketone, The manufacture of 3HB as described in any one of Claims 1-6. Method.   The method for producing 3HB according to any one of claims 1 to 7, wherein in the crystallization step, 3HB seed crystals are added to a crystallization solvent in which 3HB is dissolved, and cooling is performed while stirring.