CN102875694A - Method for preparing water soluble chitosan oligosaccharide from squid sheath and bioactivity of chitosan oligosaccharide - Google Patents
Method for preparing water soluble chitosan oligosaccharide from squid sheath and bioactivity of chitosan oligosaccharide Download PDFInfo
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- CN102875694A CN102875694A CN2011101991347A CN201110199134A CN102875694A CN 102875694 A CN102875694 A CN 102875694A CN 2011101991347 A CN2011101991347 A CN 2011101991347A CN 201110199134 A CN201110199134 A CN 201110199134A CN 102875694 A CN102875694 A CN 102875694A
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Abstract
The invention discloses a method for preparing water soluble chitosan oligosaccharide from squid sheath. The method is characterized by comprising the following steps of: crushing the squid sheath, adding 6 to 9 percent HCl solution, ensuring that a material/solution (w/v) ratio is 1:14, decalcifying for 20 to 60 minutes, washing to neutrality, adding 10 percent NaOH solution, ensuring that a material/solution (w/v) ratio is 1:10, removing protein, filtering and washing to neutrality, and drying to obtain beta-chitin; adding 30 to 50 percent NaOH solution into the beta-chitin, ensuring that a material/solution ratio (w/v) is 1:10, performing ultrasonic bath reaction at the temperature of between 70 and 100DEG C for 2 to 4 hours, filtering, washing to neutrality, and drying to obtain chitosan; and adding 3 to 12 percent H2O2 into the chitosan, ensuring that a material/solution ratio (m/v) is 1:15-1:25, reacting at the temperature of between 45 and 75DEG C for 2 to 5 hours in the assistance of ultrasonic waves, concentrating filtrate, adding ethanol in an amount which is 3 to 5 times volume of the concentrated filtrate, precipitating and filtering a product, and drying to obtain the chitosan oligosaccharide, wherein the chitosan oligosaccharide has the molecular weight of less than 1*10<4>Da, the DPPH free radical removing capacity (EC50) of between 0.3 and 1.3mg/mL and the cholic acid combining capacity of between 20.47 and 63.53mg/g. The invention provides technical support for comprehensively utilizing leftovers, namely the squid sheath in aquatic product processing and industrially producing the water soluble chitosan oligosaccharide.
Description
Technical field
The present invention relates to a kind of squid theca that utilizes and prepare the method for water-soluble chitosan oligosaccharide and the biological activity of oligochitosan thereof.
Background technology
Chitin (chitin) has another name called chitin, chitin etc., be widespread in nature in arthropods (such as shrimp, crab, squid etc.) shell, also being present in zooticly as in mushroom, insects, the alga cells wall, is the nitrogenous natural high moleculer eompound that is only second to cellulosic quantity maximum on the earth.The chitin formal name used at school is β-Isosorbide-5-Nitrae-poly-n-acetyl-D-glucosamine, and molecular formula is (C
8H
13NO
5) n.Chitosan (chitosan) is the product after chitin removes ethanoyl, also claims chitosan, chitosan etc., and formal name used at school is β-Isosorbide-5-Nitrae-poly--D-glucosamine, and molecular formula is (C
6H
11NO
4) n.Chitosan is white or canescence, amorphous, translucent, sheet or powdery solid that the pearly-lustre color is arranged, and water insoluble and alkaline solution dissolves in most of organic acids such as the mineral acids such as dilute hydrochloric acid, nitric acid and acetic acid.The lot of documents report, chitosan has important application in fields such as sewage disposal, foodstuffs industry, medical and health, detergents and cosmetics.Yet, the water insoluble and basic solution of chitosan, its acid solution viscosity is very high, has limited the application of chitosan, therefore prepares water-soluble chitosan significant.Studies show that, molecular weight is 1 * 10 after degraded
4Following low-molecular weight chitoglycan (oligochitosan), not only water-soluble, also have unique physiologically active and physico-chemical property, thereby range of application is widened greatly.Oligochitosan has following physiological function: activation and proliferation bifidus bacillus in human intestinal, suppress colibacillary growth; Promote spleen antibody to generate the growth of inhibition tumor cell; Strengthen liver function, prevent apoplexy and stomach ulcer; Reduce blood pressure, blood sugar, blood fat, the functions such as absorption cholesterol.Therefore, water miscible oligochitosan has very widely range of application and development prospect, receives investigator's concern.
Oligochitosan utilizes chitosan to make through backbone breaking usually.H
2O
2The oxidative degradation chitosan has the advantages such as speed of response is fast, productive rate is high, reactant is nontoxic, is a kind of desirable chemical degradation method, is just receiving increasing concern.Along with the development of medicine, foodstuffs industry, the demand of oligochitosan increases day by day, and tradition needs to consume a large amount of acid, hydrogen peroxide and ethanol etc. by the technique of hydrolyzing chitosan oligochitosan processed, and etching apparatus causes environmental pollution, and productive rate is relatively not high.The present invention has reported by squid theca and has produced β-chitosan and the further method for preparing oligochitosan, by the effective Reaction time shorten of ultrasonic wave auxiliary energy, reduce production costs, improve the productive rate of oligochitosan, environmental contamination reduction, coastland squid theca resource is very abundant, and the present invention can take full advantage of the tankage of processing of aquatic products, for the preparation of industrialization oligochitosan provides scientific basis, has good economic and social benefit.
Summary of the invention
The purpose of this invention is to provide a kind of squid theca that utilizes prepares the method for water-soluble chitosan oligosaccharide and has studied its anti-oxidant activity and the cholic acid binding ability for raw material.The present invention at first extracts β-chitin from squid theca, β-chitin deacetylase generating chitosan, then by the low molecule oligochitosan of chitosan preparation, this process is in conjunction with ultrasonic-assisted extraction technique, and optimize hydrogen peroxide concentration and the factors such as usage quantity, temperature of reaction and reaction times, for tankage squid theca and the suitability for industrialized production oligochitosan that fully utilizes processing of aquatic products provides technical support.
The technical process that is prepared oligochitosan by squid theca:
Squid theca → pulverizing → acid treatment → be washed to neutrality → alkaline purification → be washed to neutrality → drying → ultrasonic auxiliary preparation chitosan → be washed to neutrality → drying → pulverizing → ultrasonic auxiliary preparation oligochitosan → concentrating under reduced pressure → add ethanol precipitation → filtration → drying → oligochitosan finished product
The major technique step that the present invention includes is as follows:
(1) acid treatment is ground into powdery with squid theca, takes by weighing a certain amount of squid theca powder, adds 6～9% HCl solution, solid-to-liquid ratio (w/v) is 1: 14, and room temperature decalcification 20～60min observes without Bubble formation, but the pH value still maintains slightly acidic and gets final product, and then washing is to neutral.
(2) alkaline purification adds 10%NaOH solution, and solid-liquid ratio (w/v) is 1: 8～1: 12,80～100 ℃ of water-bath deproteinated 2h, and rear filtration washing is to neutral, and 60 ℃ of dryings just are β-chitin, pulverize stand-by.
(3) ultrasonic auxiliary preparation chitosan is got a certain amount of chitosan, adds 30～50%NaOH solution, and solid-liquid ratio (w/v) is 1: 10,70～100 ℃ of ultrasonic water bath reaction 2～4h, and frequency is 25～71KHz.
(4) ultrasonic auxiliary preparation oligochitosan is got the 40g chitosan, adds 3%～12% H
2O
2, solid-liquid ratio (m/v) is 1: 15～1: 25, at the auxiliary lower 45-75 ℃ of reaction 2～5h of ultrasonic wave, frequency is 25～71KHz.。
(5) is adjusted to neutrality with NaOH after crystallization reaction finishes, underpressure distillation so that volume is concentrated into is original 1/10, adds 3～5 times of volume ethanol, and filtration is 60 ℃ of lower vacuum-dryings.Preparing the oligochitosan productive rate by β-chitosan reaches more than 65%.
(6) mensuration of oligochitosan molecular weight adopts viscosimetry to measure the viscosity-average molecular weight of oligochitosan.
(7) different molecular weight oligochitosan scavenging ability of DPPH free radical is got oligochitosan solution (0-2.0mg/mL) adding 2.5mL DPPH ethanolic soln (50mg/L) mixing of 2.5mL different concns, at room temperature react 30min, measure absorbancy at last under 517nm, xitix is product in contrast.Calculate its scavenging ability of DPPH free radical by following formula:
Removing ability (%)=(1-A
1/ A
0) * 100
Wherein A1 is the absorbancy behind the adding sample oligochitosan, and A0 is the absorbancy that replaces sample with distilled water.
EC
50(removing the concentration of the required sample of 50%DPPH free radical) assess sample is removed the ability of free radical.
(8) the cholic acid binding ability is measured and is got 0.05g oligochitosan sample, adds 2mL cholic acid (5mg/mL), regulates cumulative volume to 25mL with distilled water, mixing, mixture filters at 37 ℃ of lower reaction 2h, gets 1mL filtrate, add 1mL furfural solution (1%, w/v) and the 13mL sulphuric acid soln (45%, v/v), mixture is at 70 ℃ of lower reaction 20mmin, under 605nm, measure absorbancy, obtain cholic acid content according to the cholic acid typical curve.
Description of drawings
The oligochitosan product infrared spectrogram of Fig. 1 preparation
Embodiment
Squid theca is ground into powdery, the HCl solution of adding 8%, solid-liquid ratio (w/v) is 1: 14, room temperature decalcification 20～60min, and washing is to neutral, add again 10%NaOH solution, solid-liquid ratio (w/v) is 1: 10,100 ℃ of water-bath deproteinated 2h, and then filtration washing is to neutral, 60 ℃ of dryings namely get β-chitin; Add 50%NaOH solution in β-chitin, solid-liquid ratio (w/v) is 1: 10,80 ℃ of ultrasonic water bath reaction 2h, and ultrasonic frequency 25KHz, suction filtration after the reaction, filter cake washing is to neutral chitosan.Get the 40g chitosan, add certain volume 30%H
2O
2, then adding the water of certain volume, cumulative volume reaches 900mL, so that H
2O
2Concentration is 3%-12%, at the auxiliary lower 45-75 ℃ of reaction 2-5h of ultrasonic wave.
Is adjusted to neutrality with NaOH after reaction finishes, underpressure distillation so that volume is concentrated into is original 1/10, adds 3-5 times of volume ethanol, and filtration is 60 ℃ of lower vacuum-dryings.Preparing the oligochitosan productive rate by β-chitosan reaches more than 65%.Then different molecular weight oligochitosan scavenging ability of DPPH free radical and cholic acid binding ability are studied, the result shows prepared oligochitosan EC
50Between 0.3-1.3mg/mL, the cholic acid binding ability is between the 20.47-63.53mg/g.
Embodiment 1
Squid theca is ground into powdery, takes by weighing the squid theca powder of 400g, add the HCl solution 5600mL of 8% concentration, room temperature decalcification 30mmin observes without Bubble formation, and washing is to neutral.Add again 10%NaOH solution 4000mL, 100 ℃ of water-bath deproteinated 1h, rear filtration washing is to neutral, and 60 ℃ of dryings just are β-chitin, pulverize stand-by.Get β-chitin 200g and add 50%HCl solution 2000mL, 80 ℃ of ultrasonic reaction 2h, ultrasonic frequency 25KHz.Suction filtration after the reaction, filter cake washing is to neutral chitosan.Get the 40g chitosan, add 90mL 30%H
2O
2, then add 810mL distilled water, make H
2O
2Concentration is 3%, and at the auxiliary lower 65 ℃ of reaction 4h of ultrasonic wave, frequency is 40KHz, and reaction is adjusted to neutrality with NaOH after finishing, and underpressure distillation so that volume is concentrated into is original 1/10, adds 3-5 times of volume ethanol, and filtration is 60 ℃ of lower vacuum-dryings.Preparing the oligochitosan productive rate by β-chitosan reaches more than 65%.Preparation oligochitosan molecular weight is 2242Da, and it removes DPPHH free radical ability EC
50Be 0.31mmg/mL, the cholic acid binding ability is 54.51mg/g.
Embodiment 2
Squid theca is ground into powdery, takes by weighing the squid theca powder of 400g, add the HCl solution 5600mL of 8% concentration, room temperature decalcification 30min observes without Bubble formation, and washing is to neutral.Add again 10%NaOH solution 4000mL, 100 ℃ of water-bath deproteinated 1h, rear filtration washing is to neutral, and 60 ℃ of dryings just are β-chitin, pulverize stand-by.Get β-chitin 200g and add 50%HCl solution 2000mL, 80 ℃ of ultrasonic reaction 2h, ultrasonic frequency 25KHz.Suction filtration after the reaction, filter cake washing is to neutral chitosan.Get the 40g chitosan, add 270mL 30%H
2O
2, then add 630mL distilled water, so that H
2O
2Concentration is 9%, and at the auxiliary lower 65 ℃ of reaction 4h of ultrasonic wave, frequency is 40KHz, and reaction is adjusted to neutrality with NaOH after finishing, and underpressure distillation so that volume is concentrated into is original 1/10, adds 3-5 times of volume ethanol, and filtration is 60 ℃ of lower vacuum-dryings.Preparing the oligochitosan productive rate by β-chitosan reaches more than 65%.Preparation oligochitosan molecular weight is 4169Da, its scavenging ability of DPPH free radical EC
50Be 0.87mg/mL, the cholic acid binding ability is 63.53mg/g
Embodiment 3
Squid theca is ground into powdery, takes by weighing the squid theca powder of 400g, add the HCl solution 5600mL of 8% concentration, room temperature decalcification 30mmin observes without Bubble formation, and washing is to neutral.Add again 10%NaOH solution 4000mL, 100 ℃ of water-bath deproteinated 1h, rear filtration washing is to neutral, and 60 ℃ of dryings just are β-chitin, pulverize stand-by.Get β-chitin 200g and add 50%HCl solution 2000mL, 80 ℃ of ultrasonic reaction 2h, ultrasonic frequency 25KHz.Suction filtration after the reaction, filter cake washing is to neutral chitosan.Get the 40g chitosan, add 180mL 30%H
2O
2, then add 720mL distilled water, so that H
2O
2Concentration is 6%, and at the auxiliary lower 45 ℃ of reaction 3h of ultrasonic wave, frequency is 40KHz, and reaction is adjusted to neutrality with NaOH after finishing, and underpressure distillation so that volume is concentrated into is original 1/10, adds 3-5 times of volume ethanol, and filtration is 60 ℃ of lower vacuum-dryings.Preparing the oligochitosan productive rate by β-chitosan reaches more than 65%.Preparation oligochitosan molecular weight is 7011Da, its scavenging ability of DPPH free radical EC
50Be 1.09mg/mL, the cholic acid binding ability is 29.25mg/g.
Claims (3)
1. a method of utilizing squid theca to prepare water-soluble chitosan oligosaccharide is characterized in that, utilizes the tankage-squid theca of processing of aquatic products to be raw material, prepares β-chitin by the auxiliary deproteinated of ultrasonic wave, decalcification technique; Add certain density NaOH solution in β-chitin, take off acetyl and prepare chitosan by ultrasonic wave is auxiliary; Chitosan adds certain density H
2O
2Solution, the ultrasonic wave assisting hydrolyzing prepares water-soluble chitosan oligosaccharide.
2. a kind of method of utilizing squid theca to prepare water-soluble chitosan oligosaccharide according to claim 1 is characterized in that:
(1) with the HCl solution that adds 6～9% after the squid theca pulverizing, solid-liquid ratio (w/v) is 1: 14, decalcification 20～60min, washing adds 10%NaOH solution again to neutral, and solid-liquid ratio (w/v) is 1: 10, water-bath deproteinated 2h, filtration washing are extremely neutral, the dry β-chitin that gets.
(2) add 30～50%NaOH solution in β-chitin, solid-liquid ratio (w/v) is 1: 10,70～100 ℃ of ultrasonic water bath reaction 2～4h, and frequency is 25～71KHz, washing is to the neutral dry chitosan that gets after the filtration.
(3) get chitosan and add 3%～12% H
2O
2, solid-liquid ratio (m/v) is 1: 15～1: 25, and 45～75 ℃ of reaction 2～5h under ultrasonic wave is auxiliary add 3～5 times of volume ethanol after filtrate is concentrated, and product is separated out, is filtered, the dry water Ji property oligochitosan that gets.
3. the oligochitosan product that makes of method as claimed in claim 2 is characterized in that molecular weight is 1 * 10
4Below the Da, scavenging ability of DPPH free radical EC
50Between 0.3～1.3mg/ml, the cholic acid binding ability is between 20.47～63.53mg/g.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103435717A (en) * | 2013-08-16 | 2013-12-11 | 江苏顺心德生物科技有限公司 | Chitosan oligosaccharide |
| CN104693318A (en) * | 2015-03-24 | 2015-06-10 | 中国科学院海洋研究所 | Beta-chitosan preparation method |
| CN105348411A (en) * | 2015-11-24 | 2016-02-24 | 荣成广润水产食品有限公司 | Method for preparing beta-chitin from sector bone of squid |
| CN105732842A (en) * | 2016-02-23 | 2016-07-06 | 李海洋 | High-bioactivity chitin and preparing method thereof |
| CN106190768A (en) * | 2016-07-22 | 2016-12-07 | 重庆修贤生物科技有限公司 | Oligofructose and oligochitosan application in vinegar goods |
| TWI718844B (en) * | 2020-01-15 | 2021-02-11 | 國立高雄科技大學 | Method for extracting chitosan |
| CN117510680A (en) * | 2023-12-07 | 2024-02-06 | 中国海洋大学 | Beta-type chitosan and rapid extraction method and application thereof |
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2011
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| 乌德等: "鱿鱼软骨壳聚糖的制备及其理化特性研究", 《食品与机械》 * |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103435717A (en) * | 2013-08-16 | 2013-12-11 | 江苏顺心德生物科技有限公司 | Chitosan oligosaccharide |
| CN103435717B (en) * | 2013-08-16 | 2014-06-25 | 江苏顺心德生物科技有限公司 | Chitosan oligosaccharide |
| CN104693318A (en) * | 2015-03-24 | 2015-06-10 | 中国科学院海洋研究所 | Beta-chitosan preparation method |
| CN105348411A (en) * | 2015-11-24 | 2016-02-24 | 荣成广润水产食品有限公司 | Method for preparing beta-chitin from sector bone of squid |
| CN105732842A (en) * | 2016-02-23 | 2016-07-06 | 李海洋 | High-bioactivity chitin and preparing method thereof |
| CN105732842B (en) * | 2016-02-23 | 2018-01-23 | 李海洋 | A kind of high bioactivity chitin and preparation method thereof |
| CN106190768A (en) * | 2016-07-22 | 2016-12-07 | 重庆修贤生物科技有限公司 | Oligofructose and oligochitosan application in vinegar goods |
| TWI718844B (en) * | 2020-01-15 | 2021-02-11 | 國立高雄科技大學 | Method for extracting chitosan |
| CN117510680A (en) * | 2023-12-07 | 2024-02-06 | 中国海洋大学 | Beta-type chitosan and rapid extraction method and application thereof |
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Application publication date: 20130116 |