Chitosan oligosaccharide sulfate and preparation method therefor

Abstract

Chitosan oligosaccharide sulfate and a preparation method therefor. The preparation method comprises: firstly, adding an equal volume of 1.0%-2.0% of a potassium sulfate solution into a chitosan oligosaccharide hydrochloride solution having a mass percentage concentration of 2.0%-4.0%, uniformly mixing to obtain a mixed solution, enabling a molar ratio of amino to sulfate ions in the mixed solution to be 2:1, then adding absolute ethyl alcohol to obtain a chitosan oligosaccharide sulfate suspension, standing, filtering, collecting precipitates, washing, drying and crushing to finally obtain the chitosan oligosaccharide sulfate. According to the preparation method, by ingeniously utilizing the characteristics that the chitosan oligosaccharide sulfate is hardly soluble in a 75% ethanol aqueous solution and potassium chloride is slightly soluble in the 75% ethanol aqueous solution, the chitosan oligosaccharide sulfate is obtained by means of a precipitation method, and the preparation method is simple to operate, economical and environment-friendly, does not need to use highly corrosive sulfuric acid, and is friendly to operators, environment and production devices.

Description

Chitosan oligosaccharide sulfate and preparation method thereof Technical Field

[0001] The invention belongs to the field of chitosan oligosaccharide preparation, and particularly relates to chitosan oligosaccharide sulfate and a preparation method thereof. Background Art

[0002] Chitosan oligosaccharides are the only naturally occurring, positively charged, cationic, alkaline amino oligosaccharides. Chitosan oligosaccharides are small, amino-containing oligosaccharides produced by the partial hydrolysis of chitosan. They are sugar chains with a degree of polymerization of 2-10. Chitosan oligosaccharides have a wide range of applications in the biopharmaceutical field, serving as a nutrient source for probiotics and exhibiting antibacterial and antioxidant activities.

[0003] The Chinese patent "Chitosan oligosaccharide sulfate and its preparation method" (Patent No.: ZL 200610020011.1) provides a method for preparing chitosan oligosaccharide sulfate. The method comprises adding chitosan to a weak organic acid solution, dissolving and enzymolyzing it to obtain chitosan oligosaccharides with a degree of polymerization of 2-30, adding sulfuric acid, and then settling and washing with an organic solvent to obtain chitosan oligosaccharide sulfate. This patent requires the use of sulfuric acid, a highly corrosive raw material, which is very unfriendly to operators, the environment, and production equipment. Therefore, it is very necessary to develop a new method for preparing chitosan oligosaccharide sulfate.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art and provide a chitosan oligosaccharide sulfate and a preparation method thereof, specifically adopting the following technical solutions:

[0006] The first aspect of the present invention provides a method for preparing chitosan oligosaccharide sulfate, comprising the following steps:

[0007] First, an equal volume of 1.0%-2.0% potassium sulfate solution is added to a chitosan oligosaccharide hydrochloride solution with a mass percentage concentration of 2.0%-4.0%, and the mixture is evenly mixed to obtain a mixed solution, wherein the molar ratio of amino groups to sulfate ions in the mixed solution is 2:1, and then anhydrous ethanol is added to obtain a chitosan oligosaccharide sulfate suspension, which is allowed to stand, filtered, and the precipitate is collected, washed, dried, and crushed to finally obtain chitosan oligosaccharide sulfate.

[0008] The inventors of the present invention provide a method for preparing chitosan oligosaccharide sulfate. By rationally setting the molar ratio of amino groups in chitosan oligosaccharide hydrochloride to sulfate ions in potassium sulfate, a 2:1 match is achieved between amino groups with a single positive charge on the chitosan oligosaccharide and sulfate ions with two negative charges. Chitosan oligosaccharide sulfate precipitates are formed in a 75% aqueous ethanol solution, while low concentrations of chloride ions and potassium ions dissolve in the 75% aqueous ethanol solution, remaining in an ionic state and not forming a precipitate. Therefore, the present invention can prepare chitosan oligosaccharide sulfate by precipitation, and washing the precipitate with a 75% aqueous ethanol solution can remove a small amount of potassium chloride adsorbed on the surface of the precipitate, thereby improving the purity of the chitosan oligosaccharide sulfate. This preparation method is simple to operate, has low risk, and uses potassium sulfate that is inexpensive, safe, and readily available. Compared to sulfuric acid, it is more suitable for large-scale industrial production applications. Furthermore, it avoids the disadvantage of the prior art of requiring sulfuric acid, a highly corrosive raw material, which is unfriendly to operators, the environment, and production equipment.

[0009] The present invention uses chitosan hydrochloride instead of various chitosan organic acid salts because various organic acids contain carbonyl groups and are prone to Maillard reaction with the amino groups of chitosan. Other chitosan inorganic acid salts are not used because, excluding the target product chitosan sulfate, the only commercially available chitosan inorganic acid salt is chitosan hydrochloride. In fact, chitosan is produced by the hydrolysis of chitosan, which is insoluble in water and needs to be dissolved in organic acids or hydrochloric acid to be hydrolyzed under the action of enzymes or chemical catalysts. Salts formed by chitosan and divalent or higher-valent inorganic acids (including chitosan sulfate) are generally insoluble in water and cannot serve as suitable hydrolysis substrates. Therefore, chitosan hydrochloride is selected as the raw material. The molar ratio of amino groups in chitosan to sulfate ions in potassium sulfate in the mixed solution is controlled at 2:1, which allows sulfate ions to ionize with amino groups in chitosan to form chitosan sulfate, which can be fully precipitated in a 75% ethanol aqueous solution system. If the concentration of chitosan oligosaccharide hydrochloride is too high, chitosan oligosaccharide hydrochloride will be mixed in the precipitate, reducing the quality of chitosan oligosaccharide sulfate; if the concentration of chitosan oligosaccharide hydrochloride is too low, the precipitation of chitosan oligosaccharide sulfate will be insufficient, reducing the yield of chitosan oligosaccharide sulfate; if the concentration of potassium sulfate is too high, the precipitate will be mixed with excess potassium sulfate, reducing the quality of chitosan oligosaccharide sulfate; if the concentration of potassium sulfate is too low, it will not be possible to convert all chitosan oligosaccharide hydrochloride into chitosan oligosaccharide sulfate, and the precipitate will be mixed with chitosan oligosaccharide hydrochloride, reducing the quality of chitosan oligosaccharide sulfate. Therefore, the chitosan oligosaccharide hydrochloride solution mass percentage concentration of 2.0%-4.0% and the potassium sulfate solution concentration of 1.0%-2.0% used in the present invention are both the optimal concentrations of this experiment. This concentration can not only improve the yield of chitosan oligosaccharide sulfate, but also improve the quality of chitosan oligosaccharide sulfate.

[0010] The present invention uses potassium sulfate as a raw material because potassium sulfate, as a neutral salt, has many advantages over sulfuric acid, as a strong acid, including being non-toxic, harmless, and non-corrosive, and having no harmful effects on the human body and the environment. In addition, the present invention uses potassium sulfate rather than sodium sulfate or other neutral sulfates because after potassium sulfate is mixed with chitosan oligosaccharide hydrochloride and the chitosan oligosaccharide sulfate is precipitated with a 75% ethanol aqueous solution, the small amount of water contained in the ethanol solution can act as a solvent to ionize the solute. Potassium chloride has a greater solubility than sodium chloride. Therefore, by controlling the concentration of the chitosan oligosaccharide hydrochloride solution, the concentration of the potassium sulfate solution, and the ratio of the two, a process can be achieved in which chitosan oligosaccharide sulfate is fully precipitated and potassium chloride is completely dissolved in the 75% ethanol aqueous solution. If sodium sulfate is used instead of potassium sulfate, the solubility of sodium chloride in the 75% ethanol aqueous solution is low, making it difficult to effectively separate chitosan oligosaccharide sulfate from sodium chloride. Among other neutral sulfates, calcium sulfate and barium sulfate have too low solubility, ferrous sulfate and ammonium sulfate are irritating, and copper sulfate, potassium aluminum sulfate, and aluminum sulfate are toxic, making them unsuitable for use.

[0011] As a further preferred embodiment, the degree of polymerization of the chitosan oligosaccharide hydrochloride is 2-10. Compared with the prior art degree of polymerization of 2-30, its degree of polymerization is lower and the proportion of active ingredients is higher. Many physiological and biochemical activities of chitosan oligosaccharides are closely related to the distribution range of the degree of polymerization of chitosan oligosaccharides. For example, in terms of enhancing immunity, chitosan oligosaccharides with a degree of polymerization of 6-8 (or 4-7, 5-7) have good antibacterial, immunity-enhancing and anti-tumor activities, among which chitosan hexaose shows a higher tumor inhibitory activity; in terms of inducing plant disease resistance, chitosan oligosaccharides with a degree of polymerization of 4-6 can regulate the generation of plant rhizobia.

[0012] As a further preferred embodiment, the molecular weight of the chitosan oligosaccharide hydrochloride is less than or equal to 2000 Da. The higher the molecular weight, the higher the proportion of active ingredients. In terms of blood sugar regulation, chitosan oligosaccharides with a molecular weight of approximately 1700 Da are suitable for patients with hyperlipidemia, lowering blood sugar and improving antioxidant capacity, which has a health-promoting effect. In terms of antibacterial function, chitosan oligosaccharides with an average molecular weight of 610 Da have a certain antibacterial effect on raw milk and significantly inhibit the post-acidification of yogurt.

[0013] As a further preferred embodiment, the volume ratio of the anhydrous ethanol to the mixed solution is 3:1. When the volume ratio of the anhydrous ethanol to the mixed solution is 3:1, the concentration of the ethanol aqueous solution is 75%, which is exactly the most effective disinfection concentration of ethanol. It can prevent microbial contamination and reproduction during the long-term static precipitation of chitosan oligosaccharide sulfate, thereby improving the quality of the product.

[0014] As a further preferred embodiment, the standing time is 10 hours.

[0015] In a further preferred embodiment, the washing step comprises washing the collected precipitate three times with a solvent. The solvent is a 75% aqueous ethanol solution. Each wash lasts 10 minutes. Washing the precipitate with a 75% aqueous ethanol solution removes a small amount of potassium chloride adsorbed on the surface of the precipitate, thereby improving the purity of the chitosan oligosaccharide sulfate. Silver acetate solution is added to the washing solution after each wash until no white precipitate is formed in the washing solution, indicating that the washing solution is free of chloride ions and the precipitate has been cleaned.

[0016] As a further preferred embodiment, the drying temperature is 50-60° C. If the temperature is too high, the amino groups of chitosan oligosaccharides and the aldehyde groups at the reducing ends of chitosan oligosaccharides are prone to Maillard reaction, which reduces the quality of the product; if the temperature is too low, it is difficult to effectively dry the product.

[0017] Another aspect of the present invention provides chitosan oligosaccharide sulfate obtained by the above preparation method, which has a yield of more than 96% and is a non-toxic, harmless and high-quality chitosan oligosaccharide product.

[0018] The present invention has the following beneficial effects: the preparation method adopted in the present invention is simple to operate, economical, environmentally friendly, does not require the use of highly corrosive sulfuric acid, and is friendly to operators, the environment, and production equipment. The method cleverly utilizes the characteristics that chitosan oligosaccharide sulfate is poorly soluble in a 75% ethanol aqueous solution, while potassium chloride is slightly soluble in a 75% ethanol aqueous solution. The chitosan oligosaccharide sulfate is obtained by a precipitation method, avoiding the disadvantage of the prior art of requiring the use of sulfuric acid, a highly corrosive raw material. DETAILED DESCRIPTION

[0019] The following will be combined with the embodiments to clearly and completely describe the concept, specific structure and technical effects of the present invention so as to fully understand the purpose, scheme and effect of the present invention. It should be noted that the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict.

[0020] Example 1

[0021] A method for preparing chitosan oligosaccharide sulfate specifically comprises the following steps:

[0022] Step 1: Weigh 10 g of chitosan oligosaccharide hydrochloride, add 500 mL of deionized water, and stir until the chitosan oligosaccharide is completely dissolved to obtain a 2.0% chitosan oligosaccharide hydrochloride solution;

[0023] Step 2: Measure 500 mL of 1.0% potassium sulfate solution and add it to the chitosan oligosaccharide solution obtained in step 1 to obtain a mixed solution of chitosan oligosaccharide hydrochloride and potassium sulfate;

[0024] Step 3: adding 3000 mL of anhydrous ethanol to the mixed solution of chitosan oligosaccharide hydrochloride and potassium sulfate obtained in step 2 to obtain a chitosan oligosaccharide sulfate suspension;

[0025] Step 4: The chitosan oligosaccharide sulfate suspension obtained in step 3 is allowed to settle for 10 hours;

[0026] Step 5: After step 4 is completed, filter and collect the precipitate;

[0027] Step 6: the precipitate collected in step 5 was washed 3 times with 75% ethanol water to remove residual potassium chloride. Each wash time was 10 min, and the volume of 75% ethanol water consumed in each washing was 150 mL. Silver acetate solution was added to the washings after each washing. It was found that white precipitate was generated in the washings of the first two times, while no white precipitate was generated in the washings of the third time. This indicated that no chloride ion was present in the washings of the third time. The precipitation was cleaned and then dried at 55 ° C. Finally, pulverized to obtain 9.67 g chitosan oligosaccharide sulfate with a yield of 96.7%.

[0028] Example 2

[0029] A method for preparing chitosan oligosaccharide sulfate specifically comprises the following steps:

[0030] Step 1: Weigh 15 g of chitosan oligosaccharide hydrochloride, add 500 mL of deionized water, and stir until the chitosan oligosaccharide hydrochloride is completely dissolved to obtain a 3.0% chitosan oligosaccharide solution;

[0031] Step 2: Measure 500 mL of 1.5% potassium sulfate solution and add it to the chitosan oligosaccharide solution obtained in step 1 to obtain a mixed solution of chitosan oligosaccharide hydrochloride and potassium sulfate;

[0032] Step 3: adding 3000 mL of anhydrous ethanol to the mixed solution of chitosan oligosaccharide and potassium sulfate obtained in step 2 to obtain a chitosan oligosaccharide sulfate suspension;

[0033] Step 4: The chitosan oligosaccharide sulfate suspension obtained in step 3 was allowed to settle for 10 hours;

[0034] Step 5: After step 4 is completed, filter and collect the precipitate;

[0035] Step 6: the precipitation collected in step 5 was washed 3 times with 75% ethanol water to remove residual potassium chloride. Each washing time was 10 min, and the volume of 75% ethanol water consumed in each washing was 150 mL. Silver acetate solution was added to the washings after each washing. It was found that white precipitate was generated in the washings of the first two times, while no white precipitate was generated in the washings of the third time. This indicated that no chloride ion was present in the washings of the third time. The precipitation was washed clean, then dried at 55 ° C. and finally pulverized to obtain 14.62 g chitosan oligosaccharide sulfate with a yield of 97.5%.

[0036] Example 3

[0037] A method for preparing chitosan oligosaccharide sulfate specifically comprises the following steps:

[0038] Step 1: Weigh 20 g of chitosan oligosaccharide hydrochloride, add 500 mL of deionized water, and stir until the chitosan oligosaccharide hydrochloride is completely dissolved to obtain a 4.0% chitosan oligosaccharide solution;

[0039] Step 2: Measure 500 mL of 2.0% potassium sulfate solution and add it to the chitosan oligosaccharide solution obtained in step 1 to obtain a mixed solution of chitosan oligosaccharide hydrochloride and potassium sulfate;

[0040] Step 3: adding 3000 mL of anhydrous ethanol to the mixed solution of chitosan oligosaccharide and potassium sulfate obtained in step 2 to obtain a chitosan oligosaccharide sulfate suspension;

[0041] Step 4: The chitosan oligosaccharide sulfate suspension obtained in step 3 is allowed to settle for 10 hours;

[0042] Step 5: After step 4 is completed, filter and collect the precipitate;

[0043] Step 6: the precipitation collected in step 5 was washed 3 times with 75% ethanol water to remove residual potassium chloride. Each washing time was 10 min, and the volume of 75% ethanol water consumed in each washing was 150 mL. Silver acetate solution was added to the washings after each washing. It was found that white precipitate was generated in the washings of the first two times, while no white precipitate was generated in the washings of the third time. This indicated that no chloride ion was present in the washings of the third time. The precipitation was washed clean, then dried at 55 ° C. and finally pulverized to obtain 19.56 g chitosan oligosaccharide sulfate with a yield of 97.8%.

[0044] Comparative Example 1

[0045] A method for preparing chitosan oligosaccharide sulfate specifically comprises the following steps:

[0046] Step 1: Weigh 10 g of chitosan oligosaccharide hydrochloride, add 500 mL of deionized water, and stir until the chitosan oligosaccharide is completely dissolved to obtain a 2.0% chitosan oligosaccharide hydrochloride solution;

[0047] Step 2: Measure 500 mL of 1.0% sodium sulfate solution and add it to the chitosan oligosaccharide solution obtained in step 1 to obtain a mixed solution of chitosan oligosaccharide hydrochloride and sodium sulfate;

[0048] Step 3: adding 3000 mL of anhydrous ethanol to the mixed solution of chitosan oligosaccharide hydrochloride and sodium sulfate obtained in step 2 to obtain a chitosan oligosaccharide sulfate suspension;

[0049] Step 4: The chitosan oligosaccharide sulfate suspension obtained in step 3 is allowed to settle for 10 hours;

[0050] Step 5: After step 4 is completed, filter and collect the precipitate;

[0051] Step 6: the precipitation that step 5 is collected is washed 3 times with 75% ethanolic water, so that remove residual sodium-chlor, each washing time is 10min, and each washing consumes 75% ethanolic water volume and is 150mL, in the washings after each washing finishes, add silver acetate solution, find that white precipitate is arranged to generate in three washings, illustrate all containing chloride ion in three washings, precipitation is not washed clean, follow-up increases washing times, find not containing chloride ion in the washings of the 11th time, precipitation is washed clean, then at 55 ℃ of lower oven dry, pulverize at last, obtain 9.08g chitosan oligosaccharide sulfate, yield is 90.8%; Be that embodiment 1 of raw material that 10g chitosan oligosaccharide hydrochloride is also compared, Comparative Example 1 has two obvious deficiencies: the one, washing times significantly increases, increases to 11 times by 3 times, consumes more time and reagent; The 2nd, yield significantly reduces, is reduced to 90.8% by 96.7%, and increasing washing times has caused the loss of product.

[0052] Comparative Example 2

[0053] A method for preparing chitosan oligosaccharide sulfate specifically comprises the following steps:

[0054] Step 1: Weigh 15 g of chitosan oligosaccharide hydrochloride, add 500 mL of deionized water, and stir until the chitosan oligosaccharide hydrochloride is completely dissolved to obtain a 3.0% chitosan oligosaccharide solution;

[0055] Step 2: Measure 500 mL of 1.5% sodium sulfate solution and add it to the chitosan oligosaccharide solution obtained in step 1 to obtain a mixed solution of chitosan oligosaccharide hydrochloride and sodium sulfate;

[0056] Step 3: adding 3000 mL of anhydrous ethanol to the mixed solution of chitosan oligosaccharide and sodium sulfate obtained in step 2 to obtain a chitosan oligosaccharide sulfate suspension;

[0057] Step 4: The chitosan oligosaccharide sulfate suspension obtained in step 3 was allowed to settle for 10 hours;

[0058] Step 5: After step 4 is completed, filter and collect the precipitate;

[0059] Step 6: the precipitation that step 5 is collected is washed 3 times with 75% ethanolic water, so that remove residual sodium-chlor, each washing time is 10min, and each washing consumes 75% ethanolic water volume and is 150mL, in the washings after each washing finishes, add silver acetate solution, find that white precipitate is arranged to generate in three washings, illustrate all containing chloride ion in three washings, precipitation is not washed clean, follow-up increases washing times, find not containing chloride ion in the washings of the 15th time, precipitation is washed clean, then at 55 ℃ of lower oven dry, pulverize at last, obtain 13.06g chitosan oligosaccharide sulfate, yield is 87.1%; Be that embodiment 2 of raw material that 15g chitosan oligosaccharide hydrochloride is also compared, Comparative Example 2 has two obvious deficiencies: the one, washing times significantly increases, increases to 15 times by 3 times, consumes more time and reagent; The 2nd, yield significantly reduces, is reduced to 87.1% by 97.5%, and increasing washing times has caused the loss of product.

[0060] Comparative Example 3

[0061] A method for preparing chitosan oligosaccharide sulfate specifically comprises the following steps:

[0062] Step 1: Weigh 20 g of chitosan oligosaccharide hydrochloride, add 500 mL of deionized water, and stir until the chitosan oligosaccharide hydrochloride is completely dissolved to obtain a 4.0% chitosan oligosaccharide solution;

[0063] Step 2: Measure 500 mL of 2.0% sodium sulfate solution and add it to the chitosan oligosaccharide solution obtained in step 1 to obtain a mixed solution of chitosan oligosaccharide hydrochloride and sodium sulfate;

[0064] Step 3: adding 3000 mL of anhydrous ethanol to the mixed solution of chitosan oligosaccharide and sodium sulfate obtained in step 2 to obtain a chitosan oligosaccharide sulfate suspension;

[0065] Step 4: The chitosan oligosaccharide sulfate suspension obtained in step 3 is allowed to settle for 10 hours;

[0066] Step 5: After step 4 is completed, filter and collect the precipitate;

[0067] Step 6: the precipitation that step 5 is collected is washed 3 times with 75% ethanolic water, so that remove residual sodium-chlor, each washing time is 10min, and each washing consumes 75% ethanolic water volume and is 150mL, in the washings after each washing finishes, add silver acetate solution, find that white precipitate is arranged to generate in three washings, illustrate all containing chloride ion in three washings, precipitation is not washed clean, follow-up increases washing times, find not containing chloride ion in the washings of the 19th time, precipitation is washed clean, then at 55 ℃ of lower oven dry, pulverize at last, obtain 17.06g chitosan oligosaccharide sulfate, yield is 85.3%; Be that embodiment 3 of raw material that 20g chitosan oligosaccharide hydrochloride is equally compared, Comparative Example 3 has two obvious deficiencies: the one, washing times significantly increases, increases to 19 times by 3 times, consumes more time and reagent; The 2nd, yield significantly reduces, is reduced to 85.3% by 97.8%, and increasing washing times has caused the loss of product.

[0068] Obviously, the described embodiments are only some of the embodiments of the present application, rather than all of the embodiments. Mentioning "embodiment" in this article means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present embodiment application. The appearance of this phrase in various positions in the specification does not necessarily mean that they are all the same embodiments, nor are they independent or alternative embodiments that are mutually exclusive with other embodiments. It can be understood explicitly and implicitly by those skilled in the art that the embodiments described herein can be combined with other embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of this application.

[0069] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the claims and their equivalents.

Claims

1. A method for preparing chitosan oligosaccharide sulfate, characterized in that: The following steps are involved: First, an equal volume of 1.0%-2.0% potassium sulfate solution is added to a chitosan oligosaccharide hydrochloride solution with a mass percentage concentration of 2.0%-4.0%, and the mixture is evenly mixed to obtain a mixed solution, so that the molar ratio of amino groups to sulfate ions in the mixed solution is 2:1, and then anhydrous ethanol is added to obtain a chitosan oligosaccharide sulfate suspension, which is allowed to stand, filtered, precipitated, washed, dried, and crushed to finally obtain chitosan oligosaccharide sulfate; the washing solvent is a 75% ethanol aqueous solution.

2. The preparation method according to claim 1, characterized in that: The polymerization degree of the chitosan oligosaccharide hydrochloride is 2-10.

3. The preparation method according to claim 1, characterized in that: The molecular weight of the chitosan oligosaccharide hydrochloride is less than or equal to 2000Da.

4. The preparation method according to claim 1, characterized in that: The volume ratio of the anhydrous ethanol to the mixed solution is 3:

1.

5. The preparation method according to claim 1, characterized in that: The standing time is 10 hours.

6. The preparation method according to claim 1, characterized in that: The washing is washing the collected precipitate with a solvent for 3 times.

7. The preparation method according to claim 6, characterized in that: The washing time is 10 min / time.

8. The preparation method according to claim 1, characterized in that: The drying temperature is 50°C-60°C.