阅读说明：本技术 一种用甲壳类动物外壳制备壳聚糖的方法 (Method for preparing chitosan from shells of crustaceans ) 是由 范芳芳 刘北辰 刘运江 张香梅 赵继东 李兵 于 2020-12-23 设计创作，主要内容包括：本发明公开了一种用甲壳类动物外壳制备壳聚糖的方法,包括如下步骤：S1：脱蛋白：以1-12ml：1g(w/v)的比例添加0.5-2mol/L NaOH和虾壳粉,混匀,在60-80℃下反应10-30min,进行去蛋白作用,收集固体产物,并用蒸馏水洗涤,将固体产物真空干燥,并用分析天平进行称重；S2：脱盐：将S1干燥后的固体产物在室温下,以14ml：1g(w/v)的比例,用1-10％HCl溶液在500ml烧杯中进行脱盐处理,抽滤收集固体产物,并用蒸馏水对固体产物洗涤,然后对固体产物进行干燥；S3：脱乙酰：在室温下用比例为14ml：1g(w/v)的20-40％NaOH处理S2得到产品20-80h。本发明制备的壳聚糖止血材料具有优异的抗菌、止血、止痛功效,且能促进缺损组织修复,防粘连效果好,满足生物相容性、无抗原性,使用安全。(The invention discloses a method for preparing chitosan by using crustacean shells, which comprises the following steps: s1: deproteinization: mixing 1-12 ml: adding 0.5-2mol/L NaOH and shrimp shell powder into 1g (w/v), mixing uniformly, reacting at 60-80 ℃ for 10-30min, removing protein, collecting solid product, washing with distilled water, vacuum drying the solid product, and weighing with an analytical balance; s2: desalting: the dried solid product of S1 was mixed at room temperature with 14 ml: desalting with 1-10% HCl solution at a ratio of 1g (w/v) in 500ml beaker, collecting solid product by suction filtration, washing the solid product with distilled water, and drying the solid product; s3: deacetylation: at room temperature with a ratio of 14 ml: 1g (w/v) of 20-40% NaOH is used for treating S2 to obtain the product for 20-80 h. The chitosan hemostatic material prepared by the invention has excellent antibacterial, hemostatic and analgesic effects, can promote the repair of defective tissues, has a good anti-adhesion effect, meets biocompatibility, has no antigenicity, and is safe to use.)

1. A method for preparing chitosan from shells of crustaceans is characterized in that: the method comprises the following steps:

s1: deproteinization: mixing 1-12 ml: adding 0.5-2mol/L NaOH and shrimp shell powder into 1g (w/v), mixing uniformly, reacting at 60-80 ℃ for 10-30min, removing protein, collecting solid product, washing with distilled water, vacuum drying the solid product, and weighing with an analytical balance;

s2: desalting: the dried solid product of S1 was mixed at room temperature with 14 ml: desalting with 1-10% HCl solution at a ratio of 1g (w/v) in 500ml beaker, collecting solid product by suction filtration, washing the solid product with distilled water, and drying the solid product;

s3: deacetylation: at room temperature with a ratio of 14 ml: treating S2 with 20-40% NaOH 1g (w/v) to obtain product 20-80h, filtering to remove acetylated solid, collecting product, washing with distilled water, and vacuum drying to obtain chitosan hemostatic material.

2. A method for preparing chitosan from crustacean shells according to claim 1, wherein: and in the S2, the desalting treatment time is 4h, and the shrimp shells are soaked in HCl until the shrimp shells are completely softened.

3. A method for preparing chitosan from crustacean shells according to claim 1, wherein: the desalting treatment in S2 adopts 3% HCl, and the desalting temperature and time are respectively 30 ℃ and 18 h.

4. A method for preparing chitosan from crustacean shells according to claim 1, wherein: and (3) sterilizing the chitosan hemostatic material in the S3 by using an ultraviolet lamp for 20min, and packaging the chitosan hemostatic material by using a polyethylene bag and storing the chitosan hemostatic material at normal temperature.

5. A method for preparing chitosan from crustacean shells according to claim 1, wherein: and when the protein of the S1 is removed, the protein is removed by adopting 5% NaOH solution, and the immersion temperature and the immersion time are respectively 85 ℃ and 4 h.

6. A method for preparing chitosan from crustacean shells according to claim 1, wherein: the temperature of the distilled water washing is 80 ℃, the washing time is 2 hours, and the bath ratio is 1: 40.

Technical Field

The invention relates to the technical field of medical pharmacy, in particular to a method for preparing chitosan by using shells of crustaceans.

Background

The shell of crustacean is rich in chitin, chitosan is produced by deacetylation of chitin, is a copolymer of D glucose-pyranose units, can be degraded in human body, has the functions of immunity, antibiosis and promotion of wound healing, and can be widely applied to the fields of food, medical pharmacy and the like.

The exoskeletons of crabs, shrimps and lobsters, as well as marine phytoplankton, including coral and jellyfish, all contain abundant chitin, and various sources of chitin can be extracted using NaOH in various concentrations and converted into chitosan by deacetylation in various degrees, and can find various applications in the industrial field because chitosan has good solubility in acidic aqueous media, the solubility of which is due to the degree of acetylation, molecular weight, and distribution of acetyl and amino groups on the chain, and also, when the amino groups are in the form of cations, chitosan has antibacterial activity, which means that the antibacterial activity of chitosan is higher at low pH values, and chitosan has broad-spectrum antibacterial activity against both gram-positive bacteria and gram-negative bacteria, the objective of the present study is to prepare low-shore chitosan with various deacetylation degrees from shrimp shell waste, and used as a key material for many applications.

The traumatic hemorrhage is hard to avoid in daily life, serious hemorrhage caused by traffic accidents, natural disasters, war and the like is more likely to harm life, if bleeding occurs within 30 minutes, effective hemostasis can be achieved, about 40% of unnecessary death can be avoided, but the traditional hemostatic material has certain defects in the aspect of rapid hemostasis, so that people pay more and more attention to finding a hemostatic material with rapid hemostasis, high yield and good biocompatibility, chitosan has the function of bacteriostasis and hemostasis, the healing promotion capability and the degradability of chitosan are very good, and the preparation of chitosan from various biomasses gradually becomes a research hotspot, so technical personnel in the field provide a method for preparing chitosan from shells of crustaceans.

Disclosure of Invention

The present invention is directed to a method for preparing chitosan from crustacean shells, which solves the problems set forth in the background art.

In order to achieve the purpose, the invention provides the following technical scheme: a method for preparing chitosan from crustacean shells comprises the following steps:

s1: deproteinization: mixing 1-12 ml: adding 0.5-2mol/L NaOH and shrimp shell powder into 1g (w/v), mixing uniformly, reacting at 60-80 ℃ for 10-30min, removing protein, collecting solid product, washing with distilled water, vacuum drying the solid product, and weighing with an analytical balance;

s2: desalting: the dried solid product of S1 was mixed at room temperature with 14 ml: desalting with 1-10% HCl solution at a ratio of 1g (w/v) in 500ml beaker, collecting solid product by suction filtration, washing the solid product with distilled water, and drying the solid product;

s3: deacetylation: at room temperature with a ratio of 14 ml: treating S2 with 20-40% NaOH 1g (w/v) to obtain product 20-80h, filtering to remove acetylated solid, collecting product, washing with distilled water, and vacuum drying to obtain chitosan hemostatic material.

Preferably, the desalting treatment time in S2 is 4h, and the shrimp shells are soaked in HCl until they are completely softened.

Preferably, 3% HCl is used for the desalting treatment in S2, and the desalting temperature and time are 30 ℃ and 18h respectively.

Preferably, the chitosan hemostatic material in S3 is sterilized by ultraviolet irradiation for 20min, and then sealed by a polyethylene bag and stored at room temperature.

Preferably, the S1 is deproteinized by leaching with 5% NaOH solution at 85 deg.C for 4 h.

Preferably, the temperature of the distilled water washing is 80 ℃, the washing time is 2 hours, and the bath ratio is 1: 40.

Compared with the prior art, the invention has the following beneficial effects: the chitosan hemostatic material prepared by the invention has excellent antibacterial, hemostatic and analgesic effects, can promote the repair of defective tissues, has a good anti-adhesion effect, meets biocompatibility, has no antigenicity, is safe to use, and is simple in preparation method.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A method for preparing chitosan from crustacean shells comprises the following steps: s1: deproteinization: the weight ratio of 5 ml: adding 0.5mol/L NaOH and shrimp shell powder into 1g (w/v), uniformly mixing, reacting at 60 ℃ for 10min, performing deproteinization, collecting a solid product, washing with distilled water, performing vacuum drying on the solid product, and weighing with an analytical balance; s2: desalting: the dried solid product of S1 was mixed at room temperature with 14 ml: desalting with 3% HCl solution at a ratio of 1g (w/v) in a 500ml beaker, collecting the solid product by suction filtration, washing the solid product with distilled water, and drying the solid product; s3: deacetylation: at room temperature with a ratio of 14 ml: treating S2 with 1g (w/v) of 20% NaOH to obtain a product for 20h, filtering the deacetylated solid, collecting the product, washing with distilled water, and vacuum drying the product to obtain the chitosan hemostatic material.

Example 2

A method for preparing chitosan from crustacean shells comprises the following steps: s1 desalting: at room temperature, 2% HCl and shrimp shell meal were mixed at 14 ml: desalting at a ratio of 1g (w/v) for 12h, washing the product under tap water to neutrality, collecting the solid, washing with distilled water, and vacuum drying; s2 deproteinization: at 90 ℃, 5% NaOH was added as 12 ml: mixing the solid collected in S1 with the ratio of 1g (w/v), deproteinizing for 12h, collecting deproteinized product, and washing with distilled water; s3 deacetylation: the product was purified at room temperature using 60% NaOH and the product collected in step 2 as 14 ml: deacetylation was carried out for 50h with mixing at a ratio of 1g (w/v), the solid was collected and washed with distilled water, and the deacetylated product was then dried in vacuo.

Example 3

A method for preparing chitosan from crustacean shells comprises the following steps: s1: deproteinization: the weight ratio of 5 ml: adding 2mol/L NaOH and shrimp shell powder into 1g (w/v), uniformly mixing, reacting at 80 ℃ for 30min, performing deproteinization, collecting a solid product, washing with distilled water, performing vacuum drying on the solid product, and weighing with an analytical balance; s2: desalting: the dried solid product of S1 was mixed at room temperature with 14 ml: desalting with 5% HCl solution at a ratio of 1g (w/v) in 500ml beaker, collecting solid product by suction filtration, washing the solid product with distilled water, and drying the solid product; s3: deacetylation: at room temperature with a ratio of 14 ml: treating S2 with 1g (w/v) of 30% NaOH to obtain a product for 50h, filtering the deacetylated solid, collecting the product, washing with distilled water, and vacuum drying the product to obtain the chitosan hemostatic material.

Example 4

A method for preparing chitosan from crustacean shells comprises the following steps: s1 desalting: at room temperature, 10% HCl and shrimp shell meal were mixed at 14 ml: desalting at a ratio of 1g (w/v) for 16h, washing the product under tap water to neutrality, collecting the solid, washing with distilled water, and vacuum drying; s2 deproteinization: at 90 ℃, 9% NaOH was added as 12 ml: mixing the solid collected in the first step with the solid at the ratio of 1g (w/v), performing deproteinization treatment for 18h, collecting a deproteinized product, and washing the deproteinized product with distilled water; s3 deacetylation: the product was purified at room temperature using a mixture of 70% NaOH and product collected in step 2 as 14 ml: deacetylation was carried out for 80h with mixing at a ratio of 1g (w/v), the solid was collected and washed with distilled water, and the deacetylated product was then dried in vacuo.

Example 5

A method for preparing chitosan from crustacean shells comprises the following steps: s1: deproteinization: the ratio of 8 ml: adding 1mol/L NaOH and shrimp shell powder into 1g (w/v), uniformly mixing, reacting at 60 ℃ for 10min, performing deproteinization, collecting a solid product, washing with distilled water, performing vacuum drying on the solid product, and weighing with an analytical balance; s2: desalting: the dried solid product of S1 was mixed at room temperature with 14 ml: desalting with 8% HCl solution at a ratio of 1g (w/v) in 500ml beaker, collecting solid product by suction filtration, washing the solid product with distilled water, and drying the solid product; s3: deacetylation: at room temperature with a ratio of 14 ml: treating S2 with 1g (w/v) of 35% NaOH to obtain a product for 40h, filtering the deacetylated solid, collecting the product, washing with distilled water, and vacuum drying the product to obtain the chitosan hemostatic material.

Example 6

A method for preparing chitosan from crustacean shells comprises the following steps: s1 desalting: at room temperature, 2% HCl and shrimp shell meal were mixed at 14 ml: desalting at a ratio of 1g (w/v) for 15h, washing the product under tap water to neutrality, collecting the solid, washing with distilled water, and vacuum drying; s2 deproteinization: at 90 ℃, 3% NaOH was added as 12 ml: mixing the solid collected in the first step with the solid at the ratio of 1g (w/v), performing deproteinization treatment for 18h, collecting a deproteinized product, and washing the deproteinized product with distilled water; s3 deacetylation: the product was purified at room temperature using a mixture of 75% NaOH and product collected in step 2 as 14 ml: deacetylation was carried out for 80h with mixing at a ratio of 1g (w/v), the solid was collected and washed with distilled water, and the deacetylated product was then dried in vacuo.

Example 7

A method for preparing chitosan from crustacean shells comprises the following steps: s1: deproteinization: the weight ratio of 10 ml: adding 02mol/L NaOH and shrimp shell powder into 1g (w/v), uniformly mixing, reacting at 65 ℃ for 15min, performing deproteinization, collecting a solid product, washing with distilled water, performing vacuum drying on the solid product, and weighing with an analytical balance; s2: desalting: the dried solid product of S1 was mixed at room temperature with 14 ml: desalting with 4% HCl solution at a ratio of 1g (w/v) in a 500ml beaker, collecting the solid product by suction filtration, washing the solid product with distilled water, and drying the solid product; s3: deacetylation: at room temperature with a ratio of 14 ml: treating S2 with 1g (w/v) of 30% NaOH to obtain a product for 70h, filtering the deacetylated solid, collecting the product, washing with distilled water, and vacuum drying the product to obtain the chitosan hemostatic material.

Example 8

A method for preparing chitosan from crustacean shells comprises the following steps: s1 desalting: at room temperature, 6% HCl and shrimp shell meal were mixed at 14 ml: desalting at a ratio of 1g (w/v) for 20h, washing the product under tap water to neutrality, collecting the solid, washing with distilled water, and vacuum drying; s2 deproteinization: at 90 ℃, 7% NaOH was added as 12 ml: mixing the solid collected in the first step with the solid at the ratio of 1g (w/v), performing deproteinization treatment for 23h, collecting a deproteinized product, and washing the deproteinized product with distilled water; s3 deacetylation: the product was purified at room temperature using 60% NaOH and the product collected in step 2 as 14 ml: deacetylation was carried out for 50h with mixing at a ratio of 1g (w/v), the solid was collected and washed with distilled water, and the deacetylated product was then dried in vacuo.

Example 9

A method for preparing chitosan from shells of crustaceans is characterized in that: the method comprises the following steps: s1: deproteinization: mixing 1-12 ml: adding 0.5-2mol/L NaOH and shrimp shell powder into 1g (w/v), mixing uniformly, reacting at 60-80 ℃ for 10-30min, removing protein, collecting solid product, washing with distilled water, vacuum drying the solid product, and weighing with an analytical balance; s2: desalting: the dried solid product of S1 was mixed at room temperature with 14 ml: desalting with 1-10% HCl solution at a ratio of 1g (w/v) in 500ml beaker, collecting solid product by suction filtration, washing the solid product with distilled water, and drying the solid product; s3: deacetylation: at room temperature with a ratio of 14 ml: treating S2 with 20-40% NaOH 1g (w/v) to obtain product 20-80h, filtering to remove acetylated solid, collecting product, washing with distilled water, and vacuum drying to obtain chitosan hemostatic material.

Example 10

A method for preparing chitosan from crustacean shells comprises the following steps: s1 desalting: at room temperature, 2% HCl and shrimp shell meal were mixed at 14 ml: desalting at a ratio of 1g (w/v) for 16h, washing the product under tap water to neutrality, collecting the solid, washing with distilled water, and vacuum drying; s2 deproteinization: at 90 ℃, 9% NaOH was added as 12 ml: mixing the solid collected in the first step with the solid at the ratio of 1g (w/v), performing deproteinization treatment for 12 hours, collecting a deproteinized product, and washing the deproteinized product with distilled water; s3 deacetylation: the product was purified at room temperature using a mixture of 65% NaOH and product collected in step 2 as 14 ml: deacetylation was carried out for 50h with mixing at a ratio of 1g (w/v), the solid was collected and washed with distilled water, and the deacetylated product was then dried in vacuo.

Cpd.No	％C	％H	％N	％DD	％NaoH
						Cs1	40.66	5.75	6.65	6.917	35
Cs1	28.90	3.70	5.51	90.4	50

TABLE 1 elemental analysis, degree of deacetylation and Degree of Deacetylation (DD) of Chitosan

Wound healing test of chitosan hemostatic materials

The experimental raw materials are as follows: chitosan hemostatic material, medical gauze and mouse.

The experimental procedure was as follows:

(1) selecting 6 adult healthy female mice, depilating two sides of the spine of the back of the mice 24h before a test to completely expose the skin, paying attention to not hurt the skin in the process of shaving, respectively selecting 2 test areas with the area of 3cm by 3cm on the left side and the right side of the spine of a depilating area as a test area of a chitosan hemostatic material group and a gelatin sponge control group, disinfecting by using 70% ethanol, and shearing a sample into corresponding sizes for standby: 2cm by 2cm of chitosan composite hemostatic material; 3cm for medical gauze 3 cm;

(2) before scalding, anesthetizing a tested mouse by adopting an open inhalation method, covering 5 layers of medical gauze on the mouth and nose of the mouse, slowly and gradually dripping a small amount of diethyl ether on the gauze until the mouse does not struggle and breathes stably, putting a flat-bottomed test tube with a certain bottom area into a constant-temperature water bath kettle at 99 ℃ for heating, taking out after l0min, immediately attaching to test areas on two sides of a spine of the mouse for scalding, paying attention to avoid iliac bones and shoulder and foot bones, starting timing by contacting the bottom of the test tube with the skin of the mouse, wherein the scalding time is 30s, and preparing two scalding wound surfaces on one side of each mouse;

(3) randomly dividing each side of the mouse into a chitosan hemostatic material test group and a blank control group, applying a chitosan hemostatic material on the scald wound surface of the test group, covering medical gauze on the chitosan hemostatic material for fixation, directly covering the medical gauze on the scald wound surface of the blank control group for fixation, and replacing the applied sample gauze every other day.

Experimental results and analysis:

TABLE 2 wound area and wound healing Rate at different times

From table 2, the change conditions of the wound surface area and the healing rate of two groups of scald wound surfaces with the chitosan hemostatic material and without samples at 3d, 7d, 14d and 21d can be seen, the original wound surface area is the area of the bottom of the test tube in the scald test, after the healing rate is 0.21d, the wound surface area with the chitosan hemostatic material is reduced to about 19 square millimeters, and the healing rate reaches 96.07%; the wound area of the blank group without the sample is only reduced to about 163 square millimeters, the healing rate is only 67.34%, and the chitosan hemostatic material has obvious effect on promoting the healing of the wound of a mouse scald model.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.