Method for producing poly(3-hydroxybutyric acid) and method for enhancing sweetness of poly(3-hydroxybutyric acid)

Abstract

Provided is a production method capable of producing poly(3-hydroxybutyric acid) (PHB) with a strong sweetness. 【Solution means】A method for producing poly(3-hydroxybutyric acid), characterized in that the PHB component is separated from wet cells containing cells and PHB accumulated by the cells to recover the PHB component, and after drying if necessary, heat treatment is performed at 100 to 185 ° C for 10 to 80 minutes.

Description

Technical Field

[0001] The present invention relates to a method for producing poly(3-hydroxybutyric acid) that can be suitably used as a raw material for, for example, feeds, pet foods, etc., and a method for enhancing the sweetness of poly(3-hydroxybutyric acid).

Background Art

[0002] Poly(3-hydroxybutyric acid) (also referred to as "poly-3-hydroxybutyric acid" or "PHB") is known as a "marine biodegradable plastic" that exhibits biodegradability even in the ocean among "biodegradable plastics" that are ultimately completely decomposed into water and carbon dioxide by the action of microorganisms.

[0003] PHB is a polyester composed of 3-hydroxybutyric acid (a ketone body), is produced from bacteria inhabiting the sea or lakes, and is known to be accumulated as granules in the cytoplasm as an energy substrate in the bacteria. PHB can be easily extracted from bacteria. For example, in addition to rhizobia such as the genus Sinorhizobium, it has attracted attention because it can be produced relatively easily and in large quantities by various bacteria such as the genus Alcaligenes, the genus Athiorhodium, the genus Azotobacter, the genus Bacillus, the genus Nocardia, the genus Pseudomonas, the genus Rhizobium, and the genus Spirillum. In addition, PHB is known to be insoluble in water and have hydrolysis resistance. Many of the currently known biodegradable plastics are water-soluble, and PHB has characteristics different from those of conventional biodegradable plastics that are vulnerable to moisture. Furthermore, PHB has high ultraviolet resistance, but is weak against acids and alkalis and is soluble in chlorinated hydrocarbons such as chloroform. Also, since it has biocompatibility and no toxicity, it is also suitable for medical applications. Its melting point is 175°C and its glass transition temperature is 15°C. Its tensile strength is 40 MPa, which is close to that of polypropylene. However, unlike polypropylene, it sinks in water and is thus easily anaerobically decomposed in sediments.

[0004] As described above, PHB is known to accumulate as granules in the cytoplasm within bacteria. Although it is possible to directly use cells containing PHB, in many applications, it is desirable to separate and purify PHB from the cells.

[0005] As a method for separating PHB from cells, an aqueous suspension of PHB-containing microbial cells (also referred to as "wet bacterial cells") is treated with a cell disruption device to disrupt the cells of the microbial cells and release the accumulated PHB extracellularly, and then the PHB component is separated from the bacterial cells by centrifugation, filtration, etc. to recover PHB.

[0006] For example, Patent Document 1 discloses a method for separating and purifying poly-3-hydroxybutyric acid, in which a suspension of microbial cells containing poly-3-hydroxybutyric acid is treated with a high-pressure homogenizer to disrupt the microbial cells and leak poly-3-hydroxybutyric acid granules outside the cells, then the cell components other than poly-3-hydroxybutyric acid are separated from this high-pressure homogenizer treatment solution to obtain a poly-3-hydroxybutyric acid fraction, and then the poly-3-hydroxybutyric acid fraction is treated with an oxygen-based bleaching agent.

[0007] Patent Document 2 discloses a method for purifying polyhydroxybutyric acid, which comprises subjecting an aqueous solution containing the microorganism that has produced polyhydroxybutyric acid to a cell disruption device to disrupt the microorganism, obtaining an insoluble residue containing the polyhydroxybutyric acid and water-soluble impurities, suspending the insoluble residue in a neutral aqueous solution to obtain a suspension containing the polyhydroxybutyric acid, adding an enzyme and a surfactant to the suspension and mixing them, precipitating the polyhydroxybutyric acid contained in the mixed suspension, and washing the precipitated polyhydroxybutyric acid with a washing liquid.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0009] As described above, PHB is a biodegradable and biocompatible polymer, and since its properties can be adjusted, it is expected to be used, for example, as feed, pet food, etc. Some animals are known to be more sensitive to sweetness than humans, and considering applications for pets and feed, it is preferable that PHB has a strong sweetness.

[0010] Therefore, an object of the present invention is to provide a production method capable of producing poly(3-hydroxybutyric acid) (PHB) with a strong sweetness, and a method for enhancing the sweetness of poly(3-hydroxybutyric acid).

Means for Solving the Problems

[0011] To solve such problems, the present invention proposes the following aspects.

[0012] [1] The first aspect of the present invention is a method for producing PHB, which comprises separating the PHB component from wet bacterial cells containing bacterial cells and PHB accumulated by the bacterial cells to recover the PHB component, drying it if necessary, and then performing a heat treatment at 100 to 185 °C for 10 to 80 minutes. [2] In the second aspect of the present invention, in the first aspect, the heat treatment is performed such that the value (°C × minute) obtained by multiplying the heating temperature (°C) by the heating time (minute) is 1,200 or more and 14,000 or less. This is a method for producing PHB.

[0013] [3] The third aspect of the present invention is a method for enhancing the sweetness of PHB, which comprises separating the PHB component from wet bacterial cells containing bacterial cells and PHB accumulated by the bacterial cells to recover the PHB component, drying it if necessary, and then performing a heat treatment at 100 to 185 °C for 10 to 80 minutes. [4] In the fourth aspect of the present invention, in the third aspect, the heat treatment is performed such that the value (°C × minute) obtained by multiplying the heating temperature (°C) by the heating time (minute) is 1,200 or more and 14,000 or less. This is a method for enhancing the sweetness of PHB.

Advantages of the Invention

[0014] According to the method for producing PHB or the method for enhancing the sweetness of PHB proposed by the present invention, PHB with a strong sweetness can be produced, so it is suitable as a raw material for feeds, pet foods, etc.

Modes for Carrying Out the Invention

[0015] Hereinafter, an example of an embodiment of the present invention will be described. However, the present invention is not limited to the embodiments described below.

[0016] <Method for Producing PHB of the Present Invention> A method for producing PHB (also referred to as "the method for producing PHB of the present invention") as an example of an embodiment of the present invention is a method for producing PHB, which comprises separating and recovering the PHB component from wet bacterial cells containing bacterial cells and PHB accumulated by the bacterial cells, drying the recovered PHB component if necessary, and then performing a heat treatment under predetermined conditions.

[0017] The method for producing PHB of the present invention can be used as a method for enhancing the sweetness of PHB.

[0018] (Wet cells) Wet cells containing cells and PHB accumulated by the cells can be obtained by introducing a known PHB synthesis gene into a known cell capable of producing PHB, such as Escherichia coli, to obtain a recombinant (PHB-producing strain), inoculating this into a known medium, providing a known carbon source such as glucose, culturing by a known method to produce and accumulate PHB, and then adding an alkaline aqueous solution such as an aqueous sodium hydroxide solution to kill the cells. However, the method for obtaining wet cells is not limited to such a method, and a known method can be appropriately adopted. At this time, even when wet cells are obtained by other methods, the produced PHB is the same.

[0019] (Heating and stirring) Before separating and recovering the PHB component from the wet cells, water, a known purifying agent, etc. may be added and heated and stirred as necessary. Water can be added and diluted for the purpose of improving cell disruption efficiency, etc. Stirring at this time may be carried out by a known method. For example, a treatment of stirring at a treatment temperature of 70 °C or lower can be exemplified.

[0020] (Means for separating and recovering the PHB component from wet cells) The means for separating and recovering the PHB component from wet cells is not particularly limited, and a known separation means can be adopted. For example, means for releasing PHB granules outside the cells of the cells by using a cell disruption device and separating and recovering the PHB, or means for treating with an enzyme and then disrupting the cells to obtain a cell disruption solution and separating and recovering the PHB component from this cell disruption solution, etc. can be mentioned.

[0021] [Means using a cell disruption device] By performing a cell disruption treatment on the cells with a cell disruption device, the cells of the cells can be disrupted, and the PHB granules accumulated in the cells can be released outside the cells of the cells. Examples of the cell disruption device include a high-pressure homogenizer, an ultrasonic homogenizer, a blender device such as a Waring blender, and a bead-type cell disruption device that disrupts microorganisms with beads. The treatment with the cell disruption device may be repeated.

[0022] When performing the cell disruption treatment on the cells, heating or cooling may or may not be performed. However, since the temperature of the treatment liquid increases by treating with the cell disruption device, it is preferable to cool as necessary. For example, the cell disruption treatment may be performed while cooling to maintain the liquid temperature at 60°C or lower, especially 35°C or lower. In general, it is known that soluble proteins are insolubilized in a pH range that is extremely acidic or alkaline. Therefore, if necessary, the pH of the wet cells (cell suspension) may be adjusted to 5 to 8 in advance.

[0023] The aqueous solution containing the insoluble residue containing PHB granules and the water-soluble impurities after being subjected to the cell disruption device as described above is separated from the aqueous solution containing the water-soluble impurities by solid-liquid separation means such as filtration and centrifugation to recover the insoluble residue containing PHB granules. At this time, examples of the solid-liquid separation method for separating the PHB fraction from the cells include centrifugation, ultrafiltration, and membrane filtration.

[0024] The recovered PHB granules are preferably washed with alcohol or water as necessary. At this time, examples of the alcohol used include ethanol, methanol, and 2-propanol. Also, at this time, examples of the water used include distilled water, ultrapure water, and tap water. It should be noted that it is preferable to repeat the above separation and recovery method.

[0025] [Means by Enzyme Treatment] When treating wet bacterial cells with enzymes, it is preferable to use cell wall degrading enzymes as the enzymes to be used, and it is more preferable to use proteolytic enzymes in addition to cell wall degrading enzymes. By the cell wall degrading enzymes, the cells can be disrupted and the PHB component can be made into spherical particles. By using the cell wall degrading enzymes and proteolytic enzymes in combination, it becomes possible to further sphericalize the PHB particles.

[0026] The cell wall degrading enzyme is not particularly limited as long as it is an enzyme that degrades the cell wall of bacteria. For example, lysozyme, amylase, cellulase, maltase, saccharase, α- and β-glycosidase, etc. can be mentioned. The proteolytic enzyme is not particularly limited. For example, alcalase, pepsin, trypsin, papain, chymotrypsin, aminopeptidase, carboxypeptidase, etc. can be mentioned. Furthermore, an enzyme stabilizer, a surfactant, an anti-redeposition agent, etc. may be added.

[0027] After the enzyme treatment is carried out, the cells may be disrupted by a well-known method to obtain a cell disruption solution, and the PHB component may be recovered from the cell disruption solution. As the disruption means, known methods can be applied. For example, a method of disrupting cells using enzymes, a method of applying mechanical shearing force, a method of using a surfactant or an alkali, etc. can be mentioned. As the method for recovering the PHB component from the cell disruption solution, the solid-liquid separation method described above may be adopted. Also, the recovered PHB component is preferably washed in the same manner as above, and the separation and recovery method is preferably repeated.

[0028] (Drying) Since the PHB component recovered as described above, for example, PHB granules, is wet and of low purity, it is preferable to dry it. As the drying method, known drying methods can be adopted. For example, spray drying method, vacuum drying, heat drying, vacuum heat drying, etc. can be mentioned.

[0029] (Heat treatment) By subjecting the recovered PHB component to a heat treatment at 100 to 185 °C for 10 to 80 minutes, surprisingly, it was confirmed that the sweetness increased in the taste chart of PHB. From such a viewpoint, it is preferable that the heat treatment temperature be 100 °C or higher, more preferably 120 °C or higher, and still more preferably 150 °C or higher. On the other hand, the upper limit is preferably 185 °C or lower, more preferably 178 °C or lower, and still more preferably 175 °C or lower in consideration of the fact that PHB decomposes because the melting point of PHB is around 175 °C. Note that the above temperature during the heat treatment is the temperature of the object to be heated, that is, the product temperature, and includes the furnace internal setting temperature and the hot plate setting temperature that can be regarded as the same temperature as the product temperature.

[0030] Also, the heating time at the above heating temperature is preferably 10 minutes or more, more preferably 15 minutes or more, and still more preferably 17 minutes or more. On the other hand, considering the decomposition of PHB, it is preferably 80 minutes or less, more preferably 60 minutes or less, and still more preferably 50 minutes or less.

[0031] Moreover, the value (°C × min) obtained by multiplying the heating temperature (°C) and the heating time (min) is preferably 1,200 or more, more preferably 1,800 or more, and still more preferably 2,500 or more. The upper limit is preferably 14,000 or less, more preferably 10,000 or less, and still more preferably 8,700 or less. From the viewpoint of further enhancing the sweetness, the heat treatment in the method for producing the PHB of the present invention is preferably carried out by adjusting so that the value (°C × min) obtained by multiplying the heating temperature (°C) and the heating time (min) falls within the above range.

[0032] As the heating method, a known heating method may be employed. For example, methods such as heating with a constant temperature bath, an electric furnace, a hot plate, etc. can be mentioned. Also, the heat treatment may be carried out in combination with a process having another purpose such as drying, or the heat treatment may be carried out continuously with the drying process.

[0033] <Use> According to the method for producing PHB of the present invention, since PHB with a strong sweetness can be produced, it can be suitably used, for example, as a raw material for feeds, pet foods, pharmaceuticals, health foods, matrix materials for regenerative medicine, and the like.

[0034] <Explanation of terms, etc.> In the present invention, when described as "α to β" (α and β are arbitrary numbers), unless otherwise specified, it includes the meaning of "α or more and β or less" and also the meaning of "preferably larger than α" or "preferably smaller than β". Further, when described as "α or more" (α is an arbitrary number), unless otherwise specified, it includes the meaning of "preferably larger than α", and when described as "β or less" (β is an arbitrary number), unless otherwise specified, it also includes the meaning of "preferably smaller than β".

Example

[0035] Hereinafter, an example of an embodiment of the present invention will be described. However, the present invention is not limited to the embodiments described below.

[0036] <Culture and wet cells> Escherichia coli incorporated with the PHB synthesis gene system was inoculated into an LB medium containing glucose and an antibiotic in an Erlenmeyer flask and cultured at 30 °C to obtain a seed culture solution. This seed culture solution was cultured in an MR medium containing glucose and an antibiotic in an Erlenmeyer flask. This MR medium contains a small amount of trace metal solution. An aqueous sodium hydroxide solution was added to the culture solution (suspension of cells accumulating PHB) and stirred, and then sulfuric acid was added for neutralization to confirm the death of the cells. The culture solution was centrifuged to separate into a cell component and a culture solution component, the wet cells as the cell component were recovered, and the culture solution component was discarded.

[0037] (Examples 1 to 7) A purification agent was added to the wet bacterial cells obtained as described above, and the mixture was heated and stirred while maintaining the temperature below 70°C. Subsequently, after performing cell disruption treatment using a high-pressure homogenizer, centrifugation was carried out using a high-speed cooling centrifuge to recover the PHB fraction. Thereafter, purified water was added to the PHB fraction, and after suspension and washing, centrifugation was performed again to recover the PHB fraction. The recovered PHB fraction was dried, and the obtained PHB was heat-treated (annealed) under the conditions shown in Tables 1 and 2 using an electric furnace (FO100 manufactured by Yamato Scientific Co., Ltd.) to obtain purified PHB (evaluation sample). Note that the minimum necessary amount of PHB was placed in the electric furnace for heat treatment. Thus, the temperature inside the furnace can be regarded as the product temperature of PHB.

[0038] (Comparative Example 1) As an evaluation sample for Comparative Example 1, a commercially available non-woven fabric filter for food (polyethylene, polyester) was prepared.

[0039] (Comparative Example 2) The recovered PHB fraction was dried, and a purified PHB (evaluation sample) was obtained in the same manner as in the Example, except that the obtained PHB was not heat-treated (annealed).

[0040] (Comparative Example 3) A purified PHB (evaluation sample) was obtained in the same manner as in the Example, except that the conditions for heat treatment (annealing) were set to 155°C for 5 minutes.

[0041] (Analysis by Taste Sensor) The purified PHB (evaluation sample) obtained in the Example and Comparative Examples was dispersed in distilled water to a concentration of 1 wt%, heated and stirred at 55°C for 6 hours, stored at room temperature overnight (18 hours), and the filtrate obtained by centrifugation and filtration was used as a taste evaluation sample. The taste evaluation sample was analyzed using a taste sensor (TS-5000Z manufactured by Intelligent Sensor Technology Co., Ltd.) to obtain a taste chart, and the frequency of sweetness is shown in Tables 1 and 2. Note that the non-woven fabric filter for food in Comparative Example 1 was cut into a square with a length of 1 cm and a width of 1 cm using scissors, and then treated in the same manner as the evaluation sample in the Example to obtain a taste evaluation sample.

[0042]

Table 1

[0043]

Table 2

[0044] (Investigation) From the above Examples, Comparative Examples, and the test results obtained by the inventors so far, it was found that the PHB component can be separated from the wet cells containing the cells and PHB accumulated by the cells, and the PHB component can be recovered, and by performing heat treatment at 100 to 185 °C for 10 to 80 minutes, poly(3-hydroxybutyric acid) (PHB) with a strong sweetness can be produced.

Claims

1. The method for producing poly(3-hydroxybutyric acid) comprises separating and purifying poly(3-hydroxybutyric acid) (also referred to as "PHB") components from wet cells containing the fungus and accumulated PHB, recovering the PHB components, and subjecting the PHB components to a heat treatment at 100 to 175°C for 10 to 80 minutes.

2. However, the method for producing poly(3-hydroxybutyrate) according to claim 1, excluding a production method in which poly(3-hydroxybutyrate-co-3-hydroxyhexanoic acid) is separated and purified from wet cells containing the bacterial cells and poly(3-hydroxybutyrate-co-3-hydroxyhexanoic acid) accumulated by the bacterial cells to recover poly(3-hydroxybutyrate-co-3-hydroxyhexanoic acid), and then heat-treated at 100 to 175°C for 10 to 80 minutes.

3. The method for producing poly(3-hydroxybutyric acid) according to claim 1 or 2, wherein the heat treatment is carried out so that the value (°C x min) obtained by multiplying the heating temperature (°C) by the heating time (min) is 1,200 or more and 14,000 or less.

4. The method for producing poly(3-hydroxybutyric acid) according to claim 1 or 2, further comprising recovering the PHB component, drying the recovered PHB component, and then subjecting the dried PHB component to a heat treatment.

5. The method for enhancing the sweetness of poly(3-hydroxybutyric acid) comprises separating and purifying the PHB component from the bacterial cells and wet bacterial cells containing PHB accumulated by the bacterial cells, recovering the PHB component, and subjecting the PHB component to a heat treatment at 100 to 175°C for 10 to 80 minutes.

6. The method for enhancing the sweetness of poly(3-hydroxybutyric acid) according to claim 5, wherein the heat treatment is carried out so that the value (°C x min) obtained by multiplying the heating temperature (°C) by the heating time (min) is 1,200 or more and 14,000 or less.

7. 7. The method for enhancing the sweetness of poly(3-hydroxybutyric acid) according to claim 5 or 6, further comprising recovering the PHB component, drying the recovered PHB component, and then subjecting the dried PHB component to a heat treatment.

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