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

Abstract

To provide a method enabling production of poly(3-hydroxybutyric acid) (PHB) having enhanced sweetness.SOLUTION: A method for producing poly(3-hydroxybutyric acid), characterized by separating a PHB component from a wet microbial mass containing microbial cells and PHB accumulated in the microbial cells, recovering the PHB component, optionally drying the recovered PHB component, and thereafter performing heat treatment at 100°C to 185°C for 10 to 80 minutes.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Poly(3-hydroxybutyrate) (also known as "poly-3-hydroxybutyrate" or "PHB") is a "biodegradable plastic" that is eventually completely decomposed into water and carbon dioxide by the action of microorganisms. It is known as a "marine biodegradable plastic" that is biodegradable in the ocean.

[0003] PHB is a polyester made from 3-hydroxybutyric acid (a ketone body) and is known to be produced by bacteria living in the sea and lakes, and to accumulate as granules in the cytoplasm of the bacteria as an energy substrate. PHB can be easily extracted from bacteria. It has attracted attention because it can be produced relatively easily and in large quantities by a variety of bacteria, including root nodule bacteria such as Sinorhizobium, as well as species of Alcaligenes, Athiorhodium, Azotobacter, Bacillus, Nocardia, Pseudomonas, Rhizobium, and Spirillum. PHB is also known to be insoluble in water and resistant to hydrolysis. While most currently known biodegradable plastics are water-soluble, PHB has distinct characteristics compared to conventional biodegradable plastics, which are susceptible to moisture. Furthermore, PHB is highly resistant to ultraviolet light, but is susceptible to acids and alkalis, and is soluble in chlorinated hydrocarbons such as chloroform. It is also biocompatible and non-toxic, making it suitable for medical applications. Its melting point is 175°C and its glass transition temperature is 15°C. Its tensile strength is 40 MPa, similar to that of polypropylene. However, unlike polypropylene, it sinks in water and is susceptible to anaerobic decomposition in sediments.

[0004] As mentioned above, PHB is known to accumulate as granules in the cytoplasm of bacteria. Although cells containing PHB can be used as is, for many applications, it is desirable to separate and purify PHB from the cells.

[0005] A known method for separating PHB from cells is to treat an aqueous suspension of PHB-containing microbial cells (also called "wet microbial cells") with a cell disruption device to disrupt the microbial cells and release the accumulated PHB outside the cells, and then separate the PHB components from the microbial cells by centrifugation, filtration, or the like 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 cause poly-3-hydroxybutyric acid granules to leak out of the cells, and then bacterial components other than poly-3-hydroxybutyric acid are separated from the high-pressure homogenizer-treated liquid to obtain a poly-3-hydroxybutyric acid fraction, and then the poly-3-hydroxybutyric acid fraction is treated with an oxygen-based bleach.

[0007] Patent Document 2 discloses a method for purifying polyhydroxybutyric acid, which comprises passing an aqueous solution containing the microorganisms that have produced polyhydroxybutyric acid through a cell disruption device to disrupt the microorganisms, 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, 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] Japanese Patent Application Publication No. 7-177894 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-193940 Summary of the Invention [Problem to be solved by the invention]

[0009] As mentioned above, PHB is a biodegradable and biocompatible polymer, and its properties can be adjusted, so it is expected to be used, for example, as animal feed and pet food. It is known that some animals are more sensitive to sweetness than humans, and therefore, when considering applications for pets and feed, a PHB with a strong sweetness is preferable.

[0010] Therefore, an object of the present invention is to provide a method for producing highly sweet poly(3-hydroxybutyric acid) (PHB) and a method for enhancing the sweetness of poly(3-hydroxybutyric acid). [Means for solving the problem]

[0011] In order to solve the above problems, the present invention proposes the following aspects.

[0012] [1] A first aspect of the present invention is a method for producing PHB, comprising separating and recovering PHB components from bacterial cells and wet bacterial cells containing PHB accumulated by the bacterial cells, optionally drying the PHB components, and then subjecting the PHB components to a heat treatment at 100 to 185°C for 10 to 80 minutes. [2] A second aspect of the present invention is a method for producing PHB according to the first aspect, wherein the heat treatment is performed 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.

[0013] [3] A third aspect of the present invention is a method for enhancing the sweetness of PHB, comprising recovering PHB components by separating them from the bacterial cells and wet bacterial cells containing PHB accumulated by the bacterial cells, optionally drying the recovered PHB components, and then subjecting the recovered PHB components to a heat treatment at 100 to 185°C for 10 to 80 minutes. [4] A fourth aspect of the present invention is a method for enhancing the sweetness of PHB according to the third aspect, 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. [Effects 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, it is possible to produce PHB with a strong sweetness, which is suitable as a raw material for feed, pet food, and the like. DETAILED DESCRIPTION OF THE INVENTION

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

[0016] <Method of producing PHB of the present invention> A method for producing PHB as an example of an embodiment of the present invention (also referred to as the "method for producing PHB of the present invention") is a method for producing PHB, characterized in that PHB components are separated and recovered from bacterial cells and wet bacterial cells containing PHB accumulated by the bacterial cells, the recovered PHB components are dried as necessary, and then heat-treated 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 bacteria) The wet bacterial cells containing the bacterial cells and PHB accumulated by the bacterial cells can be obtained by introducing a known PHB synthesis gene into a known bacterial cell capable of producing PHB, such as Escherichia coli, to produce a genetically modified strain (PHB-producing strain), inoculating this into a known medium, providing a known carbon source such as glucose, and culturing it by a known method to produce and accumulate PHB, and then adding an alkaline aqueous solution such as a sodium hydroxide aqueous solution to kill the bacterial cells. However, the method for obtaining wet fungal cells is not limited to this method, and any known method can be appropriately adopted. In this case, even if wet fungal cells are obtained by other methods, the PHB produced will be 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 to the wet cells, if necessary, and the mixture may be heated and stirred. For the purpose of improving the efficiency of cell disruption, the solution can be diluted with water. The stirring may be carried out by a known method, for example, stirring at a temperature of 70° C. or less.

[0020] (Method for separating and recovering PHB components from wet bacteria) The means for separating and recovering the PHB component from the wet bacterial cells is not particularly limited, and any known separation means can be used, such as a means for releasing PHB granules outside the bacterial cells using a cell disruption device and then separating and recovering the PHB, or a means for treating the cells with an enzyme, disrupting the cells to obtain a cell lysate, and then separating and recovering the PHB component from the cell lysate.

[0021] [Method using a cell disruption device] By performing a bacterial cell disruption treatment using a cell disrupter, the bacterial cells can be disrupted, and the PHB granules accumulated in the bacterial cells can be released outside the bacterial cells. Examples of cell disruption devices include high-pressure homogenizers, ultrasonic homogenizers, blender devices such as a Waring blender, and bead-type cell disruption devices that disrupt microorganisms using beads. Treatment with a cell disruption device may be repeated.

[0022] When performing bacterial cell disruption treatment using a cell disrupter, heating or cooling may or may not be performed. However, since the temperature of the treatment solution increases when treated using a cell disrupter, it is preferable to cool the solution as needed. For example, bacterial cell disruption treatment may be performed while cooling the solution to maintain the solution temperature at 60°C or less, preferably 35°C or less. In addition, it is generally known that soluble proteins become insoluble in pH ranges that are extremely acidic or alkaline, so the pH of the wet bacteria (bacterial cell suspension) may be adjusted to 5 to 8 in advance, if necessary.

[0023] After passing the solution through the cell disruption device as described above, the aqueous solution containing the insoluble residue including PHB granules and water-soluble impurities is subjected to solid-liquid separation means such as filtration or centrifugation to separate the insoluble residue including PHB granules from the aqueous solution containing water-soluble impurities, and the PHB granules are recovered. In this case, examples of the solid-liquid separation method for separating the PHB fraction from the bacterial cells include centrifugation, ultrafiltration, and membrane filtration.

[0024] After recovery, the PHB granules are preferably washed with alcohol or water as needed. In this case, examples of the alcohol that can be used include ethanol, methanol, and 2-propanol. The water used in this case may be distilled water, ultrapure water, tap water, or the like. It is preferable to repeat the above separation and recovery method.

[0025] [Method using enzyme treatment] When treating wet fungal cells with enzymes, it is preferable to use a cell wall-degrading enzyme, and it is more preferable to use a protease in addition to the cell wall-degrading enzyme. The cell wall-degrading enzyme can destroy the cells and turn the PHB components into spherical particles. The combined use of the cell wall-degrading enzyme and the protease can make the PHB particles even more spherical.

[0026] The cell wall-degrading enzyme is not particularly limited as long as it is an enzyme that degrades bacterial cell walls, and examples thereof include lysozyme, amylase, cellulase, maltase, saccharase, α- and β-glycosinase. The proteolytic enzyme is not particularly limited, but examples thereof include alcalase, pepsin, trypsin, papain, chymotrypsin, aminopeptidase, and carboxypeptidase. Furthermore, an enzyme stabilizer, a surfactant, an anti-redeposition agent, etc. may be added.

[0027] After the enzyme treatment, the cells are disrupted by a known method to obtain a cell lysate, and the PHB component is recovered from the cell lysate. Known methods can be used for disrupting cells, such as disrupting cells using enzymes, applying mechanical shearing force, or using surfactants or alkalis. The PHB component can be recovered from the cell lysate by the above-mentioned solid-liquid separation method. Furthermore, it is preferable to wash the recovered PHB component in the same manner as above, and it is preferable to repeat the above separation and recovery method.

[0028] (Dry) The PHB component recovered as described above, for example, PHB granules, is wet and has low purity, so it is preferable to dry it. The drying method can be any known drying method, such as spray drying, drying under reduced pressure, drying under heat, or drying under reduced pressure and heat.

[0029] (heat treatment) Surprisingly, it was confirmed that the sweetness of the PHB components increased when they were heated at 100 to 185°C for 10 to 80 minutes. From this viewpoint, the heat treatment temperature is preferably 100°C or higher, more preferably 120°C or higher, and even more preferably 150°C or higher, while the upper limit is preferably 185°C or lower, more preferably 178°C or lower, and even more preferably 175°C or lower in consideration of the fact that the melting point of PHB is around 175°C and that PHB may decompose. The above temperature during the heat treatment is the temperature of the object to be heated, i.e., the product temperature, and includes the set temperature inside the furnace and the set temperature of the hot plate, which can be considered to be the same temperature as the product temperature.

[0030] The heating time at the above heating temperature is preferably 10 minutes or more, more preferably 15 minutes or more, and even more preferably 17 minutes or more. On the other hand, in consideration of the decomposition of PHB, the heating time is preferably 80 minutes or less, more preferably 60 minutes or less, and even more preferably 50 minutes or less.

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

[0032] The heating method may be any known method, such as a method of heating in a thermostatic bath, an electric furnace, or a hot plate. The heat treatment may be carried out in conjunction with a step having another purpose, such as drying, or may be carried out consecutively to the drying step.

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

[0034] <Explanation of terms, etc.> In the present invention, when it is written "α to β" (α and β are arbitrary numbers), unless otherwise specified, it means "not less than α and not more than β", and also means "preferably greater than α" or "preferably smaller than β". Furthermore, when it is stated that the value is "α or more" (α is any number), it also means that the value is "preferably greater than α" unless otherwise specified, and when it is stated that the value is "β or less" (β is any number), it also means that the value is "preferably smaller than β" unless otherwise specified. [Example]

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

[0036] <Culture and wet bacteria> E. coli with the PHB synthesis gene system inserted was inoculated into LB medium containing glucose and antibiotics in an Erlenmeyer flask and cultured at 30°C to obtain a seed culture. This seed culture was then cultured in MR medium containing glucose and antibiotics in an Erlenmeyer flask. This MR medium contains a small amount of trace metal solution. Aqueous sodium hydroxide solution was added to the culture solution (a suspension of bacterial cells that had accumulated PHB) and stirred, and then sulfuric acid was added to neutralize the solution, and the death of the bacterial cells was confirmed. The culture medium was centrifuged to separate it into a bacterial cell component and a culture medium component, and the wet bacterial cells as the bacterial cell component were collected, while the culture medium component was discarded.

[0037] Examples 1 to 7 The wet bacterial cells obtained as described above were added with a purification agent, and the mixture was heated to 70°C or below while stirring. The bacterial cells were then disrupted using a high-pressure homogenizer, followed by centrifugation using a high-speed refrigerated centrifuge to recover the PHB fraction. Purified water was then added to the PHB fraction, which was then suspended and washed, and centrifuged again to recover the PHB fraction. The recovered PHB fraction was dried, and the resulting PHB was heat-treated (annealed) in an electric furnace (FO100, manufactured by Yamato Scientific Co., Ltd.) under the conditions shown in Tables 1 and 2 to obtain purified PHB (evaluation sample). The electric furnace was heated with the minimum amount of PHB necessary, so the temperature inside the furnace can be considered the product temperature of PHB.

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

[0039] (Comparative Example 2) The recovered PHB fraction was dried, and purified PHB (evaluation sample) was obtained in the same manner as in the Examples, 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 heat treatment (annealing) conditions were set to 155° C. for 5 minutes.

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

[0042] [Table 1]

[0043] [Table 2]

[0044] (Consideration) From the above examples and comparative examples and the results of tests conducted by the inventors, it has been found that poly(3-hydroxybutyrate) (PHB) with a strong sweet taste can be produced by separating and recovering PHB components from bacterial cells and wet bacterial cells containing PHB accumulated by the bacterial cells, and then subjecting the PHB components to a heat treatment at 100 to 185°C for 10 to 80 minutes.

Claims

1. A method for producing poly(3-hydroxybutyric acid) (also referred to as "PHB") is provided, which comprises separating and recovering the PHB component from wet bacterial cells containing bacterial cells and accumulated poly(3-hydroxybutyric acid) (also referred to as "PHB"), optionally drying the PHB component, and then subjecting the PHB component to a heat treatment at 100 to 185°C for 10 to 80 minutes.

2. The method for producing poly(3-hydroxybutyric acid) according to claim 1, 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.

3. The method for enhancing the sweetness of poly(3-hydroxybutyric acid) comprises separating and recovering the PHB component from the bacterial cells and wet bacterial cells containing the PHB accumulated in the bacterial cells, optionally drying the PHB component, and then subjecting the PHB component to a heat treatment at 100 to 185°C for 10 to 80 minutes.

4. The method for enhancing the sweetness of poly(3-hydroxybutyric acid) according to claim 3, 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.

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