AU2021103306A4 - A method for preparing chito-oligosaccharide - Google Patents
A method for preparing chito-oligosaccharide Download PDFInfo
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- AU2021103306A4 AU2021103306A4 AU2021103306A AU2021103306A AU2021103306A4 AU 2021103306 A4 AU2021103306 A4 AU 2021103306A4 AU 2021103306 A AU2021103306 A AU 2021103306A AU 2021103306 A AU2021103306 A AU 2021103306A AU 2021103306 A4 AU2021103306 A4 AU 2021103306A4
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- oligosaccharide
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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
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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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
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
The invention discloses a method for preparing chito-oligosaccharide, which belongs to the
preparing chito-oligosaccharide field. The method comprises: (1) Colloidization of chitosan:
dissolving chitosan in acid solution to obtain colloidal chitosan, heating;(2) Enzymolysis: adding
compound enzyme A to colloidal chitosan, heating and stirring, adding compound enzyme B.
stirring at constant temperature, heating again, adding chitosanase and obtaining chitosan
enzymolysis liquid after stirring continuingly; (3) Separation: adopting the chitosan enzymolysis
liquid, centrifuging and membrane concentrating, and obtaining the chito-oligosaccharide. The
method obtains a chitosan oligosaccharide with a narrow molecular weight distribution and a
high content, which can be applied to the fields of fertilizers, feeds, pesticides and the like more
effectively.
Description
Technical Field
[0001] The invention relates to the preparation of chito-oligosaccharide, in particular to a method for preparing chito-oligosaccharide.
Background technique
[0002] Chito-oligosaccharide is an oligosaccharide product with a degree of polymerization between 2-20, which is obtained by degrading chitosan with a special biological enzyme or acid. It has high solubility and is easily absorbed by organisms, and can be widely used in many fields such as medicine, cosmetics, beverages, agriculture, and printing. In field of agriculture, chito oligosaccharide is an amino polysaccharide polymer rich in carbon and nitrogen, which can change the soil flora and promote the growth of beneficial microorganisms. Enzymatic degradation is a common method for preparing chito-oligosaccharides. Enzymatic degradation is generally divided into non-specific enzymatic hydrolysis and specific enzymatic hydrolysis. The enzymes in non-specific enzymatic hydrolysis usually include lipase and cellulase. However, non-specific enzymatic hydrolysis consumes a lot of enzymes, and there are problems such as insufficient degradation of chitosan and difficult separation of products. Specific enzymatic hydrolysis is achieved by chitosanase, etc., which is more targeted.
[0003] Chinese patent NO. CN111995456A discloses an agricultural chito-oligosaccharide, a fertilizer and a preparation method thereof. The method specifically includes enzymatic hydrolysis of chitosan with lysozyme in an acid environment to obtain an agricultural chitosan enzymolysis liquid; or drying the agricultural chitosan enzymolysis liquid to obtain the enzymatically hydrolyzed agricultural chito-oligosaccharide. The prepared chito-oligosaccharide has stable quality and good compatibility. When added to fertilizers, the chito-oligosaccharide fertilizer is obtained, and it will not cause side effects when applied to crops. However, the content of chito-oligosaccharide in this invention is relatively low, and at the same time, due to the relatively single selection of enzymes, the degradation efficiency of chito-oligosaccharide will be stratified in consideration of their different degrees of polymerization, which lead to incompletely degradation, and then affects the efficiency and product quality. Chinese patent
NO. CN102492664B discloses a method for preparing chito-oligosaccharides by using compound enzymes. The method includes adding compound enzymes to the reaction buffer of chitosan and the mass ratio of compound enzymes to chitosan is 1-20:100. The chito oligosaccharides have uniform molecular weight distribution and high yield. However, in this invention, the average molecular weight of chito-oligosaccharides is mainly in the range of 1000-5000, and the relative molecular weight is still relatively high overall.
[0004] The current preparation methods of chito-oligosaccharides have problems such as high molecular weight and low content. To solve the above problems, it is necessary to find a chito oligosaccharide with narrow molecular weight distribution and high content, which will be used in fertilizers, feeds, pesticides and other fields more effectively.
Summary of the Invention
[0005] In view of the problems in the prior art, the invention provides a method for preparing chito-oligosaccharides. The method optimizes the type, amount and time of addition of enzymes, and obtain chito-oligosaccharides with narrow molecular weight distribution and high content quickly and effectively. Adding it to fertilizer can improve yield and quality of crops.
[0006] The technical scheme of the present invention is as follows. The first object of the invention is to disclose a method for preparing chito-oligosaccharide, comprising: (1) Colloidization of chitosan: dissolving chitosan in acid solution to obtain colloidal chitosan, heating; (2) Enzymolysis: adding compound enzyme A to the colloidal chitosan, heating up, stirring and adding compound enzyme B, stirring at constant temperature, heating up again, adding chitosanase and obtaining chitosan enzymolysis liquid after stirring continuingly; (3) Separation: adopting the chitosan enzymolysis liquid, centrifuging and membrane concentrating, and obtaining the chito-oligosaccharide.
Preferably, in step (1), the mass fraction of the colloidal chitosan is 5% to 10%. Preferably, in step (1), the method of the heating is water bath and the temperature of the heating is 40°C to 48°C.
Preferably, in step (2), the temperature of the heating up is 50°C to 52°C, the temperature of the heating up again is 53°C to 55°C. Preferably, in step (1), the acid solution is selected from acetic acid, hydrochloric acid or a mixture of acetic acid and hydrochloric acid. Preferably, in step (2), the compound enzyme A accounts for 0.1% to 0.2% of the weight of the colloidal chitosan, the compound enzyme B accounts for 0.08% to 0.3% of the weight of the colloidal chitosan, the chitosanase accounts for 0.1% of the weight of the colloidal chitosan. More preferably, the compound enzyme A accounts for 0.15% of the weight of the colloidal chitosan, the compound enzyme B accounts for 0.08% to 0.2% of the weight of the colloidal chitosan, the chitosanase accounts for 0.1% of the weight of the colloidal chitosan. Preferably, the compound enzyme A includes chitosanase and bromelain, and the compound enzyme B includes chitosanase and lipase. Preferably, the weight ratio of the chitosanase and the bromelain in the compound enzyme A is 2-5:1, more preferably 3:1. Preferably, the weight ratio of the chitosanase and the lipase in the compound enzyme B is 5-8:2 3, more preferably 3:1. Preferably, in step (2), the time of the stirring is 2 min to 3 min, the time of the stirring at constant temperature is 30 min to 40 min, and the time of the stirring continuingly is 10 min to min. Preferably, in step (2), the time of the stirring is 2.5 min, the time of the stirring at constant temperature is 36 min, and the time of the stirring continuingly is 18 min. The second object of the present invention is to provide a chito-oligosaccharide that is prepared according to the method mentioned above.
[0007] The beneficial technical effects brought by the invention: The present invention firstly optimizes the type and content of the enzyme, especially adding a special chitosanase (the chitosanase has high activity, small addition amount, mild and rapid action). Secondly, the present invention optimizes the time for adding various enzymes, and uses programmed heating and stirring to affect enzyme activity, which can promote the enzymatic hydrolysis of chitosan, and obtain chito-oligosaccharides with narrow average molecular weight distribution and high content finally. The chito-oligosaccharide can be used in the fields of feed, pesticides, fertilizers and the like. Using the chito-oligosaccharide of the present invention in high-efficiency biological fertilizers can improve yields and quality of crops effectively, especially for fruits and vegetables. It can increase the plant height, fresh weight per plant, and Vc content effectively.
Detailed ways
[0008] It is worth noting that the chitosanase used in the invention is CGMCCNO.7187, provided by Ludong University (for details, please refer to Chinese patent NO. CN108998435A), and the other raw materials (including bromelain and lipase) are common commercial products, so their sources are not specifically limited.
[0009] Example 1: A method for preparing chito-oligosaccharide, comprising: (1) Colloidization of chitosan: dissolving chitosan in acetic acid solution to obtain colloidal chitosan with a mass fraction of 5%, heating in a water bath and the heating temperature is 40°C; (2) Enzymolysis: adding compound enzyme A to the colloidal chitosan, heating up to 50°C, stirring for 3 min and adding compound enzyme B, stirring at constant temperature for 30 min, heating up to 53°C, adding chitosanase and obtaining chitosan enzymolysis liquid after stirring continuingly for 20 min. Specifically, the compound enzyme A accounts for 0.1% of the weight of the colloidal chitosan, the compound enzyme B accounts for 0.3% of the weight of the colloidal chitosan, the chitosanase accounts for 0.1% of the weight of the colloidal chitosan. Compound enzyme A is composed of chitosanase and bromelain, and the weight ratio of the chitosanase and the bromelain in the compound enzyme A is 2:1. Compound enzyme B is composed of chitosanase and lipase, and the weight ratio of the chitosanase and the lipase in the compound enzyme B is 5:2. (3) Separation: adopting the chitosan enzymolysis liquid, centrifuging and membrane concentrating, obtaining the chito-oligosaccharide.
[0010] Example 2: A method for preparing chito-oligosaccharide, comprising: (1) Colloidization of chitosan: dissolving chitosan in acetic acid solution to obtain colloidal chitosan with a mass fraction of 10%, heating in a water bath and the heating temperature is 48°C;
(2) Enzymolysis: adding compound enzyme A to the colloidal chitosan, heating up to 52°C, stirring for 2 min and adding compound enzyme B, stirring at constant temperature for 40 min, heating up to 55°C, adding chitosanase and obtaining chitosan enzymolysis liquid after stirring continuingly for 10 min. Specifically, the compound enzyme A accounts for 0.2% of the weight of the colloidal chitosan, the compound enzyme B accounts for 0.08% of the weight of the colloidal chitosan, the chitosanase accounts for 0.1% of the weight of the colloidal chitosan. Compound enzyme A is composed of chitosanase and bromelain, and the weight ratio of the chitosanase and the bromelain in the compound enzyme A is 5:1. Compound enzyme B is composed of chitosanase and lipase, and the weight ratio of the chitosanase and the lipase in the compound enzyme B is 8:3. (3) Separation: adopting the chitosan enzymolysis liquid, centrifuging and membrane concentrating, obtaining the chito-oligosaccharide.
[0011] Example 3: A method for preparing chito-oligosaccharide, comprising: (1) Colloidization of chitosan: dissolving chitosan in acetic acid solution to obtain colloidal chitosan with a mass fraction of 8%, heating in a water bath and the heating temperature is 45°C; (2) Enzymolysis: adding compound enzyme A to the colloidal chitosan, heating up to 51°C, stirring for 2.5 min and adding compound enzyme B, stirring at constant temperature for 36 min, heating up to 54°C, adding chitosanase and obtaining chitosan enzymolysis liquid after stirring continuingly for 18 min. Specifically, the compound enzyme A accounts for 0.15% of the weight of the colloidal chitosan, the compound enzyme B accounts for 0.2% of the weight of the colloidal chitosan, the chitosanase accounts for 0.1% of the weight of the colloidal chitosan. Compound enzyme A is composed of chitosanase and bromelain, and the weight ratio of the chitosanase and the bromelain in the compound enzyme A is 3:1. Compound enzyme B is composed of chitosanase and lipase, and the weight ratio of the chitosanase and the lipase in the compound enzyme B is 3:1. (3) Separation: adopting the chitosan enzymolysis liquid, centrifuging and membrane concentrating, obtaining the chito-oligosaccharide.
[0012] Comparative Example 1:
The only difference between Example 1 and the present invention is that in the step (2), the weight fraction of the compound enzyme A in the colloidal chitosan is 0.08%, and the weight fraction of the compound enzyme B in the colloidal chitosan is 0.32%. The weight ratio of different enzymes in the compound enzyme A or compound enzyme B is as same as that in Example 3.
[0013] Comparative Example 2: The only difference between Example 1 and the present invention is that in the step (2), the weight ratio of the chitosanase and the bromelain in the compound enzyme A is 1:1, and the weight ratio of the chitosanase and the lipase in the compound enzyme B is 9:1. The weight fraction of the compound enzyme A or compound enzyme B in the colloidal chitosan is as same as that in Example 1.
[0014] Comparative Example 3: The only difference from Example 1 is that the step (2) is adding a mixture of compound enzyme A, compound enzyme B and chitosanase to the colloidal chitosan (the addition amounts of enzymes are as same as those in Example 1), heating up to 54°C, stirring for 53 min(means 3min+30min+20min), and obtaining chitosan enzymolysis liquid.
[0015] Comparative Example 4: The only difference from Example 1 is that no bromelain and lipase are added.
[0016] Test of chito-oligosaccharide properties The chito-oligosaccharides from the examples and comparative examples of the present invention were dried into powdery chito-oligosaccharides, and test their average molecular weight and content. The average molecular weight and molecular weight were tested according to QB/T 5503-2020. The results are summarized in Table 1. Table 1
Average molecular M> 340<M< weight, Mw 1610 1610 (%) (%)
Example 1 1015 10.2 87.6 Example 2 1050 9.7 88.5 Example 3 1029 8.6 90.7 Comparative Example 1038 13.0 85.2 1 Comparative Example 1054 14.8 83.6 2 Comparative Example 1205 15.6 78.5 3 Comparative Example 1103 14.3 81.3 4 It can be known from Table 1 that on the whole, the average molecular weight distribution of chito-oligosaccharides of the examples of the present invention is relatively narrow, and those with a molecular weight between 340 and 1610 are above 87.6%, while the molecular weight distribution of the comparative examples is relatively wide, so the technical effect of the comparative examples is difficult to guarantee.
[0017] 2. Test of Fertilizer effect of chito-oligosaccharide Designing fertilizer groups: The basic fertilizer is the "black and white horse macroelement 20-10-20" produced by Yantai Hongyuan Biological Fertilizer Co., Ltd. diluted 200 times, and "a lot of fish" diluted 100 times. 400mL fertilizer is applied to each pot along with water. The basic fertilizer group was regarded as the control group. The chito-oligosaccharides from Examples or Comparative Examples were added to the basic fertilizer respectively, and the addition amount of the chito-oligosaccharides was 0.5% of the total weight of the fertilizer, which was regarded as example groups. Test method: The rape seedlings were cultivated and transplanted. The rape seedlings were divided into 8 groups randomly, about 50 plants in each group. They were sprayed with different fertilizers on the 5th day after transplanting, and were sprayed every 7 days. After spraying three times, the average plant height, average single plant fresh weight and the content of Vc of rapeseeds of each group were counted on the 35th day, and the test results were summarized in Table 2. Table 2 average plant average single content of Vc height plant fresh weight (mg/100g) (cm) (g) Control 11.8 30.5 49.8 Example 1 13.6 32.0 54.7 Example 2 14.1 32.6 54.2 Example 3 14.5 32.8 55.3 Comparative Example 1 13.3 31.1 53.4 Comparative Example 2 13.0 31.0 51.1 Comparative Example 3 12.2 30.6 50.1 Comparative Example 4 12.5 30.7 50.9 It can be known from Table 2 that compared with the control group applied with basic fertilizer, the average plant height, average single plant fresh weight, and Vc content of rapeseeds in Examples of the present invention are improved significantly. Although the result of Comparative Examples has also been improved compared to the control group, the degree of improvement is less obvious than the Examples. It will be apparent to those skilled in the art that modifications and variations can be made without departing from the scope and spirit disclosed by the appended claims of the present disclosure, and such modifications and variations all fall in the protection extent of the claims of the present disclosure.
Claims (10)
1. A method for preparing chito-oligosaccharide, comprising: (1)Colloidization of chitosan: dissolving chitosan in acid solution to obtain colloidal chitosan, heating; (2)Enzymolysis: adding compound enzyme A to the colloidal chitosan, heating up, stirring and adding compound enzyme B, stirring at constant temperature, heating up again, adding chitosanase and obtaining chitosan enzymolysis liquid after stirring continuingly ; (3)Separation: adopting the chitosan enzymolysis liquid, centrifuging and membrane concentrating, obtaining the chito-oligosaccharide.
2. The method according to claim 1, wherein in step (1), the mass fraction of the colloidal chitosan is 5% to 10%.
3. The method according to claim 1, wherein in step (1), the temperature of the heating is °C to 48°C.
4. The method according to claim 1, wherein in step (1), the acid solution is selected from acetic acid, hydrochloric acid or a mixture of acetic acid and hydrochloric acid.
5. The method according to claim 1, wherein in step (2), the compound enzyme A accounts for 0.1% to 0.2% of the weight of the colloidal chitosan, and the compound enzyme B accounts for 0.08% to 0.3% of the weight of the colloidal chitosan.
6. The method according to claim 1, the compound enzyme A includes chitosanase and bromelain, and the compound enzyme B includes chitosanase and lipase.
7. The method according to claim 6, the weight ratio of the chitosanase and the bromelain in the compound enzyme A is 2-5:1.
8. The method according to claim 1, the weight ratio of the chitosanase and the lipase in the compound enzyme B is 5-8:2-3.
9. The method according to claim 1, wherein in step (2), the temperature of the heating up is °C to 52°C, the temperature of the heating up again is 53°C to 55°C.
10. A chito-oligosaccharide that is prepared according to the method as in any one of claims 1-9.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110262992.5A CN112980905B (en) | 2021-03-11 | 2021-03-11 | Preparation method of chitosan oligosaccharide |
| CN202110262992.5 | 2021-03-11 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1126756C (en) * | 2000-01-05 | 2003-11-05 | 中国科学院大连化学物理研究所 | Process for preparing chitoligose by coupling enzyme degradation of chitosan with membrane separation |
| CN1327852C (en) * | 2002-06-07 | 2007-07-25 | 中国科学院大连化学物理研究所 | A health food possessing immunity regulating function and method for preparing the same |
| CN1287687C (en) * | 2002-06-28 | 2006-12-06 | 中国科学院大连化学物理研究所 | Lead-removing health care food and preparation method thereof |
| CN1608487A (en) * | 2003-10-23 | 2005-04-27 | 济南海得贝海洋生物工程有限公司 | Feed additive containing chitin oligose and its prepn and use |
| CN1321191C (en) * | 2004-11-10 | 2007-06-13 | 中国科学院大连化学物理研究所 | Chitosn producing process with immobilized enzyme |
| IT1391095B1 (en) * | 2008-08-04 | 2011-11-18 | Valagro Spa | A COMPOSITION FOR USE IN AGRICULTURE, METHOD FOR ITS PREPARATION AND ITS USE IN TREATMENT OF CROPS |
| CN101381752A (en) * | 2008-10-17 | 2009-03-11 | 扬州日兴生物科技股份有限公司 | Technique for preparing low chitose and chitosan oligosaccharide |
| CN101736056A (en) * | 2008-11-27 | 2010-06-16 | 许乐 | Biological preparation method of chitooligosaccharide |
| CN108912247B (en) * | 2018-08-28 | 2021-01-22 | 郑州中科新兴产业技术研究院 | Oligomeric chitin prepared by acid enzyme compounding method and preparation method |
| CN109609571A (en) * | 2018-12-21 | 2019-04-12 | 青岛颂田生物技术有限公司 | A kind of preparation method of anacidity chitosan oligosaccharide |
| CN111718972B (en) * | 2020-07-27 | 2023-07-04 | 安亦臣(武汉)健康科技有限公司 | Preparation method of chitosan oligosaccharide with specific polymerization degree |
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