WO2021090982A1 - Procédé de préparation de chitosane dérivé de mouche soldat noire - Google Patents

Procédé de préparation de chitosane dérivé de mouche soldat noire Download PDF

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Publication number
WO2021090982A1
WO2021090982A1 PCT/KR2019/015019 KR2019015019W WO2021090982A1 WO 2021090982 A1 WO2021090982 A1 WO 2021090982A1 KR 2019015019 W KR2019015019 W KR 2019015019W WO 2021090982 A1 WO2021090982 A1 WO 2021090982A1
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WIPO (PCT)
Prior art keywords
dongae
chitin
larvae
chitosan
genus
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PCT/KR2019/015019
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English (en)
Korean (ko)
Inventor
김태훈
권민정
정병옥
이유석
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농업회사법인푸디웜주식회사
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Priority to PCT/KR2019/015019 priority Critical patent/WO2021090982A1/fr
Publication of WO2021090982A1 publication Critical patent/WO2021090982A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

  • the present invention relates to a method for producing chitosan derived from Dongae et al., and more specifically, after undergoing pretreatment processes such as slaughter, pulverization and compression from larvae on Dongae et al., and then extracting chitin by a biological process, and deacetylating the chitosan. It relates to a method of manufacturing.
  • Chitin is a structure in which N-acetyl-D-glucosamine units are connected by beta bonds (1 ⁇ 4), and is the second most abundant polysaccharide on earth after cellulose.
  • Such chitin is a component that mainly constitutes the exoskeleton of crustaceans or insects, or part of the cell wall of fungi or bacteria.
  • chemical extraction methods for obtaining chitin by treating with hydrochloric acid solution and sodium hydroxide solution from crustaceans such as crabs and shrimps are mainstream. Achieved.
  • the chemical substances used in the above chemical extraction method are harmful to the environment and human body.
  • the protein removal process treated with sodium hydroxide solution requires a high temperature and consumes a lot of energy, and is recycled as a large amount of contaminated wastewater is discharged in the washing process. This has the disadvantage of being difficult and expensive.
  • the chemical extraction method of chitin there is a possibility that chitin may be denatured during the process, and thus the purity of chitin is deteriorated.
  • the present inventors while studying the process of biologically extracting chitin derived from insects, pretreatment processes such as special slaughter, pulverization and compression from larvae of Dongae et al., before performing biological processes such as enzyme hydrolysis or microbial fermentation. It was found that the purity of chitin as well as the degree of deacetylation in the process of conversion to chitosan can be increased, and the present invention has been completed.
  • the present invention was conceived in consideration of the above problems, and an object of the present invention is to extract chitin by a biological process after undergoing pretreatment such as slaughtering, crushing and pressing from larvae in Dongae, etc., and deacetylating it. It is intended to provide a method for producing chitosan with high purity and deacetylation.
  • the heating and drying process in the microwave is characterized in that it is carried out for 0.5 to 1 hour at 70 to 80 °C.
  • the pulverization and compression process is characterized in that the larva particles are adjusted to a size of 150 to 200 ⁇ m in Dongae, etc. using a pulverizer, and then compressed with a double screw compactor.
  • the enzyme used in the enzymatic hydrolysis process is characterized in that it is a single protease or a mixture of proteases.
  • the protease is characterized by at least one selected from the group consisting of aminopeptidase, metallocarboxypeptidase, serine endopeptidase, cysteine endopeptidase, aspartic acid endopeptidase, and metalloendopeptidase. do.
  • the enzymatic hydrolysis process is characterized in that it is performed for 5 to 10 hours at 35 to 40 °C in an acidic medium of pH 1.5 to 2.5.
  • the microorganism used in the microbial fermentation process is characterized in that it is a mixture of lactic acid-producing microorganisms and protease-producing microorganisms.
  • the lactic acid-producing microorganism is characterized in that it is a microorganism of the genus Lactobacillus or Saccharomyces.
  • the protease-producing microorganism is characterized in that it is a microorganism of the genus Bacillus, the genus Pseudomonas, the genus Pseudomonas, the genus Streptomyces, the genus Brevibacterium, or the genus Micrococcus.
  • the microbial fermentation process is characterized in that it is carried out for 5 to 7 days at 30 ⁇ 40 °C.
  • the deacetylation reaction process is characterized in that it is carried out for 4 to 24 hours at 60 to 100 °C with a sodium hydroxide solution having a concentration of 20 to 70% by weight.
  • Chitin prepared according to the present invention may have a purity of 80%, preferably a purity of 85% or more.
  • chitin prepared according to the present invention may have a degree of deacetylation of 90%, preferably 95% or more.
  • chitosan prepared according to the present invention may be ⁇ , ⁇ , ⁇ chitosan, preferably ⁇ chitosan.
  • a method for producing chitosan having high purity and deacetylation degree by extracting chitin by a biological process and deacetylating it after undergoing pretreatment processes such as slaughtering, crushing and pressing from larvae to Dongae et al. is provided. can do.
  • 1 is a schematic process diagram showing step-by-step a method for producing chitosan from larvae of Dongae et al. according to the present invention.
  • the present invention comprises the steps of: I) slaughtering the larvae in Dongae, etc. while heating and drying in microwave while alive;
  • Dongae-deung is a generic term for insects belonging to the family (Stratiomyidae) of the order of the fly (Diptera) and (Stratiomyidae). About 1,500 species are known around the world, and in Korea, 14 species of 9 genera are reported. In particular, the larvae of Dongae et al. are carnivorous or herbivorous and live in various habitats (water, rotten organic matter, vegetables, etc.).
  • larvae of Dongae et al. are used in various fields such as feed additives, fertilizers, biodiesel, etc., because mass breeding and freezing storage are easy.
  • these larvae of Dongae et al. contain chitin components in their outer shells, and in recent years, research on the process of extracting chitin from the larvae of Dongae et al. has been actively conducted.
  • steps III) related to the process of extracting chitin and IV) related to the process of converting chitin to chitosan It will be described in detail for each step shown in the accompanying Figure 1 with respect to the method for producing chitosan derived from Dongae et al. including the steps sequentially.
  • the present invention is characterized in that the larvae are killed while heating and drying in a microwave while still alive.
  • the heating drying process in the microwave is preferably performed at 70 to 80° C. for 0.5 to 1 hour, and when the above conditions are out, some larvae may not be completely slaughtered or browning may occur.
  • the larvae are pulverized and compressed on the larvae slaughtered in step I).
  • This pulverization and compression process is characterized in that the larva particles are adjusted to a size of 150 to 200 ⁇ m in Dongae, etc. using a pulverizer, and then compressed with a double screw compactor.
  • the larvae on the slaughtered Dongae, etc. are adjusted to a size of 150 to 200 ⁇ m using a blade grinder, and a certain amount of water is added to facilitate pulverization. It is difficult to mechanically control the particles of larvae in Dongae et al. to a size of less than 150 ⁇ m using a grinder, and if the size exceeds 200 ⁇ m, the purity of chitin obtained from the chitin extraction process in step III) decreases, and IV) In the step of chitosan conversion process, the degree of deacetylation may not be high because the surface area is relatively small and the reactivity is low.
  • the compression process is carried out by compressing the pulverized wet larva particles with a double-screw compactor to increase the compression efficiency, thereby adjusting the oil content in the larvae to less than 10% by weight and the dry matter content to less than 30 to 50% by weight. You can get one squeezed cake.
  • the chitin component is extracted by enzymatic hydrolysis of the pressed cake of the larva on the pulverized and compressed copper ae.
  • the pressed cake is put into a reactor containing an enzyme together with water, and enzyme hydrolysis is performed for 4 to 8 hours at 40 to 60° C. in an atmosphere of pH 6.5 to 8.5.
  • the enzyme used in the enzymatic hydrolysis process may be a single protease, or may be a mixture of various proteases.
  • the protease is aminopeptidase, metallocarboxypeptidase, serine endopeptidase, cysteine endopeptidase, aspartic endopeptidase. ) And at least one selected from the group consisting of metalloendopeptidase.
  • the enzyme hydrolysis process may use an enzyme having an activity in an acidic medium.
  • Phosphoric acid as an acidic medium and pepsin as an enzyme are preferably used, and 5 to 40° C. in an acidic medium of pH 1.5 to 2.5 Run for 10 hours.
  • the microorganism used in the microbial fermentation process may be a mixture of a lactic acid-producing microorganism and a protease-producing microorganism.
  • the lactic acid-producing microorganism is preferably a microorganism of the genus Lactobacillus or Saccharomyces
  • the protease-producing microorganism is of the genus Bacillus, Pseudomonas, Pseudoalteromonas. It is preferably a microorganism of the genus, Streptomyces genus, Brevibacterium genus or Micrococcus genus.
  • the microbial fermentation process may be performed at 30 to 40° C. for 5 to 7 days to extract chitin from larvae such as Dongae.
  • Chitosan is obtained by deacetylating the chitin extracted from step III), and the deacetylation reaction is performed at 60 to 100°C for 4 to 24 hours with a sodium hydroxide solution having a concentration of 20 to 70% by weight.
  • chitosan having various molecular weight ranges such as high molecular weight chitosan having a number average molecular weight of 100,000 or more and low molecular weight chitosan having a number average molecular weight of 10,000 or less can be obtained.
  • the number average molecular weight is in the range of 1,000 to 5,000, which greatly improves water solubility and is applied to various fields. It can be used as possible material.
  • Chitosan obtained through the deacetylation process may be naturally dried or dried in a vacuum oven to prepare chitosan in powder form.
  • a vacuum oven it is not limited, but is preferably dried in a vacuum oven at 50 to 80° C. to prepare chitosan in the form of a powder.
  • the larvae of Dongae et al. were killed by heating and drying in microwave at 70°C for 0.5 hours while still alive.
  • the larvae were mixed with water on the slaughtered Dongae et al., and the larva particles were adjusted to a size of 170 ⁇ m using a grinder, and the pulverized wet larva particles were compressed with a double-screw press to obtain a compressed cake.
  • the pressed cake was put into a reactor containing an enzyme [papain: cysteine endopeptidase series] with water, and then subjected to enzymatic hydrolysis at 40° C. for 6 hours in an atmosphere of pH 8.0 to extract chitin.
  • Chitosan was obtained by deacetylating the extracted chitin with sodium hydroxide solution at a concentration of 30% by weight for 10 hours at 100° C., and dried naturally to prepare chitosan in the form of a powder.
  • Chitosan was prepared in the same manner as in Example 1, except that an enzyme having activity [pepsin] was used for 6 hours at 35°C in a phosphoric acid solution of pH 2.0 as an enzymatic hydrolysis process.
  • the pressurized cake according to Example 1 was introduced into a reactor containing a mixture of microorganisms of Lactobacillus genus and Pseudomonas genus, except that the microbial fermentation process was performed at 30° C. for 7 days. Chitosan was prepared in the same manner as in 1.
  • Chitosan was prepared in the same manner as in Example 1, except that the pretreatment process of the slaughtering, crushing, and pressing steps according to Example 1 was not performed.
  • Table 1 shows the purity and deacetylation of chitin prepared from Examples 1 to 3 and Comparative Examples.
  • the purity of chitin was calculated by weight measurement, that is, by measuring the mass of the insoluble sample before and after treating the insoluble portion with 1.25 mol/L sodium hydroxide solution at 90° C. for 1 hour.
  • the degree of deacetylation (%) of chitin was quantified by performing elemental analysis of the obtained chitosan, acid-base titration and infrared spectroscopy (FT-IR spectroscopy) in parallel.
  • chitosan prepared according to the present invention is expected to be a material that can be applied to various industrial fields such as bio-pharmaceuticals, food, agriculture, feed additives, etc.
  • chitosan according to the present invention has an ⁇ structure.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

La présente invention concerne un procédé de préparation de chitosane dérivé de mouche soldat noire consistant I) à tuer les larves de mouche soldat noire par chauffage et séchage au moyen de micro-ondes, II) à broyer et comprimer les larves de mouche soldat de noir morte ; III) à extraire la chitine du sol et des larves de mouche soldat noires comprimées au moyen d'une hydrolyse enzymatique ou d'une fermentation microbienne ; et IV) à obtenir du chitosane par désacétylation de la chitine extraite. Selon la présente invention, le chitosane présentant une pureté et un niveau de désacétylation élevés peut être préparé par la mise en oeuvre des procédés de prétraitement de destruction, de broyage, de compression et analogues, par l'extraction de la chitine des larves de mouche soldat noire au moyen d'un procédé biologique, et par la désacétylation de cette dernière.
PCT/KR2019/015019 2019-11-07 2019-11-07 Procédé de préparation de chitosane dérivé de mouche soldat noire WO2021090982A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120315668A1 (en) * 2009-12-23 2012-12-13 Agrinos AS Biodegradation Process and Composition
US20140100361A1 (en) * 2011-05-26 2014-04-10 Ifremer (Institut Francais De Recherche Pour L'exploitation De La Mer) Extraction of chitins in a single step by enzymatic hydrolysis in an acid medium
CN104878057A (zh) * 2015-04-22 2015-09-02 山西农业大学 一种黄粉虫低聚壳聚糖规模化生产的方法
US20180002452A1 (en) * 2014-12-31 2018-01-04 Ynsect Chitin, hydrolysate and method for the production of one or more desired products by means of enzymatic hydrolysis, including pre-treatment with an oxidising agent

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120315668A1 (en) * 2009-12-23 2012-12-13 Agrinos AS Biodegradation Process and Composition
US20140100361A1 (en) * 2011-05-26 2014-04-10 Ifremer (Institut Francais De Recherche Pour L'exploitation De La Mer) Extraction of chitins in a single step by enzymatic hydrolysis in an acid medium
US20180002452A1 (en) * 2014-12-31 2018-01-04 Ynsect Chitin, hydrolysate and method for the production of one or more desired products by means of enzymatic hydrolysis, including pre-treatment with an oxidising agent
CN104878057A (zh) * 2015-04-22 2015-09-02 山西农业大学 一种黄粉虫低聚壳聚糖规模化生产的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KIM , HYEONGGZU: "Insect snack for companion Animals... 1 billion won in sales in two zears appealing to pet owners", THE KOREAN ECONOMIC DAILY, 20 September 2019 (2019-09-20), pages 1 - 14, XP055821861, Retrieved from the Internet <URL:https://www.hankyung.com/life/article/2019092037841> *

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