JP5773404B2 - Method for producing chitin degradation products - Google Patents

Description

  The present invention relates to a method for producing a chitin degradation product from a chitin-containing raw material.

  Chitin is contained in crustaceans such as crabs and shrimps, insects, and fungi, and is synthesized almost 100 billion tons every year on the earth. Chitin is an insoluble polysaccharide bound with the monosaccharide N-acetyl-D-glucosamine, and chitin polysaccharides and oligosaccharides and monosaccharides derived from them are superior in antibacterial properties, moisture retention, biocompatibility, safety and Has chelating properties. For this reason, the oligosaccharide or monosaccharide is used in a wide variety of fields such as medical materials, medicines, cosmetics, fibers, agriculture, water treatment, foods, and the like.

  A method has been reported in which crystalline chitin polysaccharide is treated with supercritical water (400 ° C.) to produce oligosaccharides and monosaccharides by amorphization and hydrolysis (see Non-Patent Document 1).

  In addition, a method has been reported in which crystalline chitin polysaccharide is made amorphous in advance by a strong alkali treatment, and the amorphous chitin polysaccharide is treated with subcritical water (220 ° C or lower) to produce oligosaccharides and monosaccharides. (Refer nonpatent literature 2).

Takashi Yoshida, Shiro Saka et al., Development and application of chitin and chitosan, Chapter 4 Section 1, CMC Publishing (2004) Kimihiko Sato, Development and application of chitin chitosan, Chapter 4 Section 2, CMMC Publishing (2004)

  However, according to the conventional method, the sugar chain length of the generated oligosaccharide cannot be completely controlled. In addition, the chemical structure of N-acetyl-D-glucosamine cannot be stably maintained because it is treated at high temperature for a long time. For this reason, the sugar chain length of the generated oligosaccharide is not constant and the selectivity is low. In addition, a side reaction such as a condensation / polymerization reaction or a by-product due to excessive decomposition is also generated.

  Then, this invention makes it a solution subject to provide the method of producing | generating the said chitin degradation product from a raw material, improving the selectivity of a chitin degradation product from a chitin containing raw material.

The method for producing a chitin degradation product according to the present invention for solving the above-mentioned problems is a range defined by approximate curves L1 and L2 shown in FIG. 4 on the temperature-time plane of a chitin-containing composition or a raw material comprising chitin . A first step of treating with subcritical water or supercritical water at a temperature and time included in a specified range, and a second step of hydrolyzing the raw material hydrothermally treated in the first step or chitin extracted from the raw material And further comprising a pulverization step of pulverizing the raw material hydrothermally treated in the first step before the second step .

  The present invention is based on the inventor's knowledge that the temperature and time when the raw material is treated with subcritical water or supercritical water greatly affects the level of selectivity of the chitin degradation product obtained through subsequent hydrolysis. ing. Therefore, according to the present invention, the combination of temperature and time when the first step is performed is set or adjusted so as to be within the specified range on the temperature-time plane, thereby improving the selectivity of the chitin degradation product. The chitin degradation product can be produced while being planned.

  The “designated range” is based on the point that the yield of the desired chitin degradation product shows a maximum value on the temperature-time plane, and the decrease rate from the maximum value is a predetermined value (for example, 0.10 to 0.20). A range defined by a pair of approximate curves L1 and L2 representing a point group that falls within the range is set as the designated range.

Further, according to the knowledge of the inventor, when the raw material is crushed after the first step and before the second step, the chitin decomposition is compared with the case where the raw material is crushed before the first step. The yield of the product is high .

  Prior to the second step, chitin is obtained by subjecting the raw material comprising the chitin-containing composition hydrothermally treated in the first step to at least one of protein removal treatment, inorganic salt removal treatment, and pigment removal treatment. You may further provide the 1st intermediate process to extract. The second step may be a step including a treatment of causing an enzyme that hydrolyzes a β-1,4 glucoside bond to act on chitin. The second step may include a step of causing at least one enzyme among chitinase, lysozyme and chitobiase to act on chitin. The second step may be a step including a treatment of causing hydrochloric acid to act on chitin.

  In the first step, the raw material may be hydrothermally treated using supercritical or subcritical water as a solvent.

  You may further provide the 3rd process of deacetylating the acetyl group of one or both acetylglucosamine residues among the chitin derived from the said raw material, and its low molecular weight product.

  As the chitin-containing composition, one or both of shellfish shells or crab shells and shrimp shells as shellfish shells may be used.

Explanatory drawing regarding the production | generation method of the chitin decomposition product as one Embodiment of this invention. Schematic explanatory drawing regarding hydrothermal treatment and hydrolysis treatment. Explanatory drawing regarding the production | generation result of a chitin decomposition product. Explanatory drawing about the specified range. Explanatory drawing regarding the production | generation result of the chitin decomposition product according to a grinding process timing. Explanatory drawing regarding the production | generation result of the chitin decomposition product according to a grinding | pulverization processing time. Explanatory drawing regarding the production | generation result of the chitin decomposition product according to enzyme usage-amount.

(Method for producing chitin degradation product as one embodiment of the present invention)
The method for extracting a chitin degradation product according to an embodiment of the present invention includes a first step (STEP01) and a second step (STEP02) subsequent thereto as shown in FIG.

  In the “first step”, the raw material is treated with subcritical water or supercritical water at a temperature and time within a specified range in the temperature-time plane. Supercritical or subcritical water may be used as the solvent. As a result, the chitin before hydrothermal treatment (high crystallinity) schematically shown in FIG. 2 (a) is lowered in crystallinity as schematically shown in FIG. 2 (b). As a raw material, a shellfish shell (such as crab shell or shrimp shell) as a chitin-containing composition is employed.

  In the “second step”, the raw material hydrothermally treated in the first step or chitin extracted from the raw material is hydrolyzed. As a result, as schematically shown in FIG. 2 (c), the enzyme reacts with the chitin (having low crystallinity) after hydrothermal treatment to produce the selected chitin compound.

  The second step may be a step including a treatment of causing an enzyme that hydrolyzes a β-1,4 glucoside bond to act on chitin. The second step may be a step including a treatment of causing at least one enzyme of chitinase, lysozyme and chitobiase to act on chitin. The second step may be a step including a treatment for causing hydrochloric acid to act on chitin.

  The “designated range” is based on the point that the yield of the desired chitin degradation product shows a maximum value on the temperature-time plane, and the decrease rate from the maximum value is a predetermined value (for example, 0.10 to 0.20). The range that extends to a point that is within the range was determined by the following experiment.

(Experiment method to set the specified range)
First, 0.20 [g] of chitin and 3.0 [g] of water are stored in a batch reactor (internal volume 6 [ml]) that supports high temperature and high pressure, and each temperature falls within the range of 200 to 400 [° C]. The reaction was started by placing it in a molten salt furnace set to. Moreover, after making it react over a certain time which falls in the range of 0-5 [min], the reactor was taken out from the molten salt furnace, the reaction was stopped by putting it into a cold water bath and cooling the reactor. The chitin sample collected from the reactor was dried for 24 [h] under an environment of a temperature of 90 [° C.].

  Furthermore, 20 [mg] of chitin after hydrothermal treatment was added to 1.8 [ml] of 10 [mM] M sodium phosphate buffer (pH 6.0). Chitinase enzyme preparation 0.2 [ml] (10 [mg / ml]) dissolved in the same buffer was added to make the total amount of the reaction solution 2 [ml]. The reaction solution was sampled at a constant time while shaking at 40 [° C.] and 1500 [rpm], and analyzed by high performance liquid chromatography (HPLC) to measure the yield of chitobiose as the selected chitin degradation product. .

  In FIG. 3, chitin hydrothermally treated with 300 [° C.] subcritical water, 350 [° C.] subcritical water and 400 [° C.] supercritical water, respectively, is subjected to enzymatic saccharification (48 [h]). The result of the measurement of the yield of chitobiose is shown.

  When the hydrothermal treatment temperature of chitin is 300 [℃], when the hydrothermal treatment time is tmax (T = 300 [℃]) = 10 [min] (or 10 ± 0.20 [min]), It was found that the yield showed a maximum value and a maximum value. That is, for example, when the hydrothermal treatment time is about 1 to 3 [min] shorter than the time tmax (T = 300 [° C.]), the chitobiose yield is as low as half or less of the maximum value.

  When the hydrothermal treatment temperature of chitin is 350 [℃], when the hydrothermal treatment time is tmax (T = 350 [℃]) = 3 [min] (or 3 ± 0.15 [min]), It was found that the yield showed a maximum value and a maximum value. That is, for example, when the hydrothermal treatment time is about 1 to 1.5 [min] shorter than the time tmax (T = 350 [° C.]), the chitobiose yield is as low as half or less of the maximum value. When the hydrothermal treatment time exceeds the time tmax (T = 350 [° C.]) and is about 5 [min], the chitobiose yield is as low as half or less of the maximum value.

  When the hydrothermal treatment temperature of chitin is 400 [℃], when the hydrothermal treatment time is tmax (T = 400 [℃]) = 1 [min] (or 1 ± 0.10 [min]), It was found that the yield showed a maximum value and a maximum value. That is, for example, when the hydrothermal treatment time is 0 [min] shorter than the time tmax (T = 400 [° C.]) or 2 [min] exceeding the time tmax (T = 400 [° C.]), the chitobiose yield is increased by performing the hydrothermal treatment. Without being reduced to the same level as when the hydrolysis treatment was performed by the enzyme over 48 [h].

  FIG. 4 shows the experimental results shown in FIG. 3 three-dimensionally. The point at which the rate of decrease from the maximum value falls within a predetermined value (for example, 0.10 to 0.20) on the temperature-time plane, based on the point where the yield of chitobiose shows the maximum value (see ●) A range defined by a pair of approximate curves L1 and L2 representing a group is set as the designated range.

(Operational effect of the method for producing a chitin degradation product of the present invention)
The present invention is based on the inventor's knowledge that the temperature and time when the raw material is treated with subcritical water or supercritical water greatly affects the level of selectivity of the chitin degradation product obtained through subsequent hydrolysis. ing. Therefore, according to the present invention, the combination of temperature and time when the first step is performed is set or adjusted so as to be within the specified range on the temperature-time plane, thereby improving the selectivity of the chitin degradation product. It is possible to produce chitobiose as the chitin degradation product. Chitobiose can produce higher chitin oligosaccharides by transglycosylation and is expected to be used for medical materials.

(Other embodiments of the present invention)
According to the inventor's knowledge, when the raw material is crushed after the first step and before the second step, compared to the case where the raw material is crushed before the first step, The yield is high. Therefore, before the second step, a pulverization step of pulverizing the raw material hydrothermally treated in the first step with a high energy mill or the like may be further provided.

  FIG. 5 shows the treatment conditions before execution of the second step and the concentration of chitobiose when the second step (enzyme concentration 0.1 [%]) was carried out for 3 hours, 24 hours and 48 hours. The relationship with rate is shown. The yield of chitobiose when the second step is performed without performing the first step and the pulverization step is shown for reference (see x).

  When the first step and the second step are sequentially executed after the pulverization step (see ▲), when the second step is executed after the first step (see △) and the pulverization step over the unit time T The yield of chitobiose is lower than in the case where the second step is performed later (see ○).

  Here, the first step was performed at 400 [° C.] for 1 [min]. The unit time T is a pulverization time required to reach an average particle diameter of the raw material of 50 [μm] or less or a crystallinity of 70 [%] or less. The predetermined value is constant regardless of the difference in the crushing function of the machine or instrument used for crushing, and can be set to a different value such as 5 minutes, 10 minutes or 12 minutes depending on the difference in the machine or instrument.

  The average particle diameter (D50) was measured using a particle size distribution measuring apparatus “X-100” (manufactured by Microtrack, USA) based on the principle of laser diffraction / scattering method using methanol as a dispersion medium. The crystallinity was measured using an X-ray diffractometer “JDX-3530” (manufactured by JEOL Ltd.), using Cu—Kα rays for X-rays, setting conditions of tube current 30 mA and tube voltage 30 kV. did. The crystallinity is based on the diffraction angle-scattering intensity plot diagram, and based on the scattering intensity (height) A of the first peak of chitin and the scattering intensity B of 2θ = 16 °, Therefore, it is a calculated value.

   (A−B) / A × 100 [%] .. (1).

  On the other hand, when the second step is performed after the first step is performed and the pulverization step is performed over the unit time T (see ◎), the yield of chitobiose has reached 90%, When the first step and the second step are sequentially executed after the pulverization step (see ▲), when the second step is executed after the first step (see △), and after the pulverization step, the second step The yield is higher than any of the cases where (see ○).

  The inventor predicted that the yield of chitobiose could be further improved by lengthening the execution time of the grinding step. However, contrary to this prediction, if the second step is executed after the first step is executed and the pulverization step is executed for three times the unit time T (see ●), the yield of chitobiose is reduced. The rate remained at around 70%. This is to improve the yield of chitobiose by adjusting the execution time of the crushing process when the second process is executed after the crushing process is executed after the first process is executed. Means you can.

  FIG. 6 shows the relationship between the processing conditions before execution of the second step and the yield of chitobiose when the second step is executed for 3 hours, 24 hours, and 48 hours. The yield of chitobiose when the second step is performed without performing the first step and the pulverization step is shown for reference (see x).

  When the first step and the second step are sequentially executed after the pulverization step over the unit time T (see ▲), the second step is executed after the pulverization step over the unit time T without executing the first step. The yield of chitobiose is lower than that of the case (see ○). On the other hand, when the first step and the second step are sequentially performed after the grinding step over three times the unit time T (see ■), the second step is performed after the grinding step over the unit time T. The yield of chitobiose is higher than that of the case (see ○). This is to improve the chitobiose yield by adjusting the execution time of the crushing step when the second step is executed after the first step is executed after the crushing step is executed. Means you can.

  In addition, when the second step is sequentially executed after the pulverization step over three times the unit time T (see □), the first step and the first step after the pulverization step over three times the unit time T are performed. The yield of chitobiose is higher than when the two steps are executed in sequence (see ■) and when the second step is executed after the pulverization step over the unit time T (see ○). This is because, when the first step is omitted and the second step is executed after the pulverization step is executed, the execution time of the pulverization step is adjusted, thereby improving the chitobiose yield. Means.

  FIG. 7 shows the relationship between the enzyme concentration and the yield of chitobiose when the second step was carried out for 3 hours, 24 hours and 48 hours, respectively. The yield of chitobiose is shown for reference when the second step is performed at an enzyme concentration of 0.1% without performing the first step and the grinding step (see x).

  In addition, when the second step is performed at the enzyme concentration of 0.1% through the pulverization step after the first step (see ◎), the second step is performed at the enzyme concentration of 0 after the pulverization step after the first step. The yield of chitobiose is shown for each run at .01% (see circles).

  From this experimental result, it can be seen that a sufficient amount of chitobiose can be produced even if the enzyme concentration is lowered to some extent. That is, it can be seen that the extraction efficiency of chitobiose can be maintained sufficiently high while reducing the cost required to use the enzyme (not negligible).

  Extracting chitin by at least one of protein removal treatment, inorganic salt removal treatment and pigment removal treatment for the raw material comprising the chitin-containing composition hydrothermally treated in the first step before the second step. One intermediate step may be further provided.

  You may further provide the 2nd intermediate | middle process of deacetylating the acetyl group of one or both acetylglucosamine residues among the chitin derived from a raw material, and its low molecular weight product.

S: Designated range.

Claims (8)

A chitin-containing composition or a raw material comprising chitin is treated with subcritical water or supercritical water at a temperature and time included in a specified range which is a range defined by approximate curves L1 and L2 shown in FIG. A first step of
A second step of hydrolyzing the raw material hydrothermally treated in the first step or chitin extracted from the raw material ,
The method for producing a chitin decomposition product, further comprising a pulverization step of pulverizing the raw material hydrothermally treated in the first step before the second step . The method of claim 1 , wherein
Prior to the second step, chitin is obtained by subjecting the raw material comprising the chitin-containing composition hydrothermally treated in the first step to at least one of protein removal treatment, inorganic salt removal treatment, and pigment removal treatment. A method for producing a chitin degradation product, further comprising a first intermediate step of extraction. The method according to claim 1 or 2 , wherein
The said 2nd process is a process including the process which makes the enzyme which hydrolyzes a β-1,4 glucoside bond act on chitin, The production method of the chitin degradation product characterized by the above-mentioned. The method according to any one of claims 1 to 3 ,
The method for producing a chitin degradation product, wherein the second step includes a treatment of causing at least one enzyme of chitinase, lysozyme and chitobiase to act on chitin. In the method according to any one of claims 1 to 4 ,
The method for producing a chitin degradation product, wherein the second step includes a treatment of causing hydrochloric acid to act on chitin. The method according to any one of claims 1-5 ,
In the first step, the raw material is hydrothermally treated using supercritical or subcritical water as a solvent. The method according to any one of claims 1-6 ,
A method for producing a chitin degradation product, further comprising a second intermediate step of deacetylating one or both acetylglucosamine residues of chitin derived from the raw material and its low molecular weight product. The method according to any one of claims 1 to 7 ,
A method for producing a chitin degradation product, wherein one or both of a crustacean shell or a crab shell and a shrimp shell as a shell of the shellfish are used as the chitin-containing composition.

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