Cassava polysaccharide copper as well as synthesis method and analysis method thereof

Abstract

The invention discloses cassava polysaccharide copper as well as a synthesis method and an analysis method thereof, the synthesis method comprises the following steps: S11, preparation of cassava starch hydrolysate: adding cassava starch into water, uniformly stirring, adding hydrochloric acid, continuously stirring for reaction at 65-75 DEG C, and after the reaction is finished, adding a sodium hydroxide solution for continuous reaction to obtain hydrolysate; s12, preparation of polysaccharide copper: taking the hydrolysate, adjusting the pH value to 9-13, adding a copper chloride solid under stirring, and carrying out a reaction to obtain a reaction complexing solution; and S13, purification of polysaccharide copper: dialyzing the reaction liquid prepared in the step S12, taking out the reaction liquid after dialysis, and freeze-drying the reaction liquid to obtain the polysaccharide copper. The prepared cassava polysaccharide copper can be used as a feed additive, has a copper supplementing effect on livestock and poultry, has no stimulation to kidneys and gastrointestinal tracts of organisms, and is better in absorption efficiency, low in cost, stable in structure, non-toxic, harmless and environment-friendly.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical materials, and in particular to cassava polysaccharide copper and a synthesis method and an analysis method thereof. Background Art

[0002] Polysaccharides are polymeric carbohydrates composed of more than ten monosaccharides linked by glycosidic bonds. They participate in various physiological activities in the body and play an important role in maintaining normal life activities. They have a wide range of physiological activities, such as lowering blood sugar, anti-oxidation, lowering blood lipids, and anti-virus, and have become another hot spot in functional food research after protein and nucleic acid research. Cassava polysaccharide is a natural polysaccharide extracted from cassava, which is biodegradable, biocompatible and has multiple biological activities. Copper is an important trace element that participates in the active center of multiple enzymes in the body and has physiological functions and biological activities such as anti-oxidation, antibacterial, anti-tumor, and immune regulation.

[0003] Copper is one of the essential trace elements for organisms. A large number of studies have shown that copper has a variety of important biological functions. It is a component of some important enzymes in animals and is related to the activity of enzymes. Copper deficiency in animals can lead to reduced antibody titer and antibody synthesis disorders, and can also affect the development of their immune organs. Modern pharmacological studies have found that metal ions can bind to polysaccharides and their derivatives through covalent bonds and ionic bonds to form complexes or chelates. Polysaccharide metal complexes such as chitosan copper, chitosan zinc and chitosan iron can significantly improve the production performance, immune performance, intestinal health and intestinal flora balance of animals, and have a certain role in replacing antibiotics. In addition, an appropriate concentration of copper ions can also activate pepsin and improve the digestive function of livestock and poultry. However, compared with complex copper, inorganic copper salts can irritate the gastrointestinal tract of the body and have lower absorption efficiency.

[0004] At present, there is no report on the synthesis method of cassava polysaccharide copper complex. Combining cassava polysaccharide with copper ions can form a new functional complex, which may improve the biological metabolic rate, growth performance and immune organ index to a certain extent. Therefore, it is urgent to develop a cassava polysaccharide copper complex that can be used as a feed additive to supplement copper for livestock and poultry, without any stimulation to the gastrointestinal tract of the body, with better absorption efficiency, low cost, stable structure, non-toxic and harmless, and environmentally friendly. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides a synthesis and analysis method of cassava polysaccharide copper. The prepared cassava polysaccharide copper can be used as a feed additive to supplement copper for livestock and poultry, and has no stimulation to the kidneys and gastrointestinal tract of the body. It has better absorption efficiency, low cost, stable structure, non-toxicity, and environmental friendliness. The specific technical scheme is as follows:

[0006] The present invention provides a method for synthesizing cassava polysaccharide copper, comprising the following steps:

[0007] S11. Preparation of cassava starch hydrolysate: Under continuous stirring, slowly add cassava starch to a certain amount of water. After stirring evenly, add hydrochloric acid, and continuously stir and react at 65 - 75 °C. After the reaction ends, add sodium hydroxide solution and continue the reaction. After the reaction ends, the hydrolysate is obtained.

[0008] S12. Preparation of polysaccharide copper: Take the hydrolysate and a certain amount of water, add them to a flask, adjust the pH to 9 - 13, continuously stir and react, add copper chloride solid, and react for 40 - 60 min to obtain a reaction complex solution.

[0009] S13. Purification of polysaccharide copper: Take the reaction solution prepared in step S12 for dialysis treatment. After dialysis is completed, take it out and freeze-dry to obtain the polysaccharide copper.

[0010] Preferably, in step S11, the mass fraction of sodium hydroxide is 10 - 25%, and the addition amount ratio to the mass of cassava starch is 1 - 2.4:1; the mass fraction of hydrochloric acid is 9 - 25%, and the addition amount ratio to the mass of cassava starch is 0.1 - 0.2:1.

[0011] Preferably, in step S11, the reaction temperature is 45 - 75 °C, and the reaction time is 10 - 20 min.

[0012] Preferably, in step S12, a buffer aid is further added.

[0013] Preferably, the buffer aid includes, by mass: 1 - 4 parts of triethanolamine, 0.5 - 2 parts of glycine, 3 - 8 parts of sodium tartrate, and 40 - 80 parts of water.

[0014] Preferably, the added mass of the buffer aid is 5 times the mass of the hydrolysate.

[0015] Preferably, in step S12, the mass ratio of the hydrolysate to the copper chloride solid is 20 - 35:3 - 5.

[0016] Preferably, in step S13, the dialysis treatment is divided into primary dialysis and secondary dialysis. The primary dialysis treatment is to dialyze at 4 - 6 °C for 12 - 24 h in a dialysis bag with a molecular weight of 1 - 2 kDa, and the secondary dialysis treatment is to dialyze at 4 - 6 °C for 12 - 24 h in a dialysis bag with a molecular weight of 0.5 kDa; before use, boil the dialysis bag in deionized water for 5 - 10 min, and change the dialysis fluid 2 - 3 times during dialysis. The volume of the dialysis fluid is 300 times the volume of the polysaccharide copper.

[0017] The present invention provides a cassava polysaccharide copper, which is synthesized by the above synthesis method.

[0018] The present invention also provides an analytical method for cassava polysaccharide copper, comprising the following steps:

[0019] S21. Sample pretreatment: Weigh the polysaccharide copper prepared in step S13, add 1+1 hydrochloric acid solution and nitric acid solution, boil, cool, add water, dissolve by gentle boiling, cool again, dilute with water, shake well, filter, and the obtained filtrate is the pretreated sample.

[0020] S22. Determination of copper content: Take the pretreated sample obtained in step S21, add potassium fluoride solution, heat to boiling, cool, add 1 drop of bromocresol green indicator, adjust the solution to just turn blue with sodium hydroxide solution, add acetic acid-sodium acetate buffer solution, 2 drops of xylenol orange indicator, shake well, and titrate with disodium ethylenediaminetetraacetate standard titration solution until the solution turns bright yellow as the end point. Record the volume of the standard titration solution consumed as V2. The calculation formula for copper content is as follows:

[0021]

[0022] C1 - Concentration of disodium ethylenediaminetetraacetate standard titration solution, mol·L-1; V1 - Volume of disodium ethylenediaminetetraacetate standard titration solution consumed in titration (blank sample without adding filtrate), mL; V2 - Volume of disodium ethylenediaminetetraacetate standard titration solution consumed in titrating copper, mL; m - Mass of the polysaccharide copper sample to be measured, g; x - Copper content, %.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. The present invention provides a synthesis method for cassava polysaccharide copper. Using raw materials such as cassava starch, copper chloride, sodium hydroxide, hydrochloric acid, and water, cassava polysaccharide copper is synthesized, and the copper content can be as high as 16%. The polysaccharide copper synthesized in this application can be used as a feed additive to supplement copper for livestock and poultry, and it has the characteristics of low cost, stable structure, non-toxic and harmless, and environmentally friendly.

[0025] 2. The buffer aid of the present invention is composed of triethanolamine, glycine, and sodium tartrate. It has strong complexing ability under alkaline conditions, can form a very stable complex with copper ions, can improve the solubility of copper ions, promote their combination with cassava polysaccharide, and can provide a certain buffering capacity. At the same time, the present invention also controls the dosage ratio of triethanolamine, glycine, and sodium tartrate to avoid the too strong complexing ability of triethanolamine, which may completely wrap copper ions and inhibit their combination with polysaccharide.

[0026] 3. The two-stage dialysis method of the present invention realizes step-by-step purification, significantly reduces the impurity content in the polysaccharide copper, and the purity of the product is higher, effectively removing unreacted copper ions and small molecule impurities.

[0027] 4. The analysis method of the present invention is an improvement based on the ethylenediaminetetraacetic acid (EDTA) complexometric titration method. The acetic acid - sodium acetate buffer solution is used to mask non - determined elements, and then the element content is determined by the complexation of ethylenediaminetetraacetic acid (EDTA) with an indicator. This analysis method can quickly determine the copper content of the product, is simpler, and has lower costs. Detailed implementation manners

[0028] The following is a detailed description of the specific implementation manners of the present invention. However, it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention. Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be prepared by existing methods.

[0029] Example 1

[0030] The synthesis of cassava polysaccharide copper in this example includes the following steps:

[0031] S11. Preparation of cassava starch hydrolysate: Slowly add 25 g of cassava starch to 80 mL of continuously stirred water. After stirring evenly, add it to a 250 - mL flask, add 3 g of 10% hydrochloric acid, and continuously stir and react at 65 °C for 1 h. After the reaction, add 60 g of 10% sodium hydroxide solution and react at 45 °C for 10 min. After the reaction, the hydrolysate is obtained.

[0032] S12. Preparation of polysaccharide copper: Take 20 g of the hydrolysate and 120 mL of water, add them to a 500 - mL flask, adjust the pH to 9, continuously stir and react, add 100 g of buffer assistant, add 3 g of copper chloride solid, and react for 40 min to obtain a reaction solution.

[0033] S13. Purification of polysaccharide copper: Take 50 mL of the reaction solution prepared in step S12 for dialysis treatment. The dialysis treatment is divided into primary dialysis and secondary dialysis. The primary dialysis treatment is to dialyze at 4 °C for 12 h in a dialysis bag with a molecular weight of 1 kDa, and the secondary dialysis treatment is to dialyze at 4 °C for 12 h in a dialysis bag with a molecular weight of 0.5 kDa; Before use, boil the dialysis bag in deionized water for 5 min, change the dialysis solution 2 times during dialysis, the volume of the dialysis solution is 300 times the volume of the polysaccharide copper, and after dialysis is completed, take it out and freeze - dry to obtain the polysaccharide copper.

[0034] The buffer assistant in this example includes, by mass: 1 part of triethanolamine, 0.5 part of glycine, 3 parts of sodium tartrate, and 40 parts of water.

[0035] Example 2

[0036] S11. Preparation of cassava starch hydrolyzate: Slowly add 35 g of cassava starch into 150 mL of continuously stirred water. After stirring evenly, transfer it to a 250 mL flask, add 4 g of 25% hydrochloric acid, and continuously stir and react at 75 °C for 2 h. After the reaction is completed, add 40 g of 10% sodium hydroxide solution for reaction and react at 75 °C for 20 min. After the reaction is completed, the hydrolyzate is obtained.

[0037] S12. Preparation of copper polysaccharide: Take 35 g of hydrolyzate and 150 mL of water, add them into a 500 mL flask, adjust the pH to 13, continuously stir and react, add 175 g of buffer aid, add 5 g of copper chloride solid, and react for 60 min to obtain a reaction solution.

[0038] S13. Purification of copper polysaccharide: Take 0 mL of the reaction solution prepared in step S12 for dialysis treatment. The dialysis treatment is divided into primary dialysis and secondary dialysis. The primary dialysis treatment is to dialyze at 6 °C for 24 h in a dialysis bag with a molecular weight of 2 kDa, and the secondary dialysis treatment is to dialyze at 6 °C for 24 h in a dialysis bag with a molecular weight of 0.5 kDa; Before use, boil the dialysis bag in deionized water for 10 min, change the dialysis solution 3 times during dialysis, and the volume of the dialysis solution is 300 times the volume of the copper polysaccharide. After dialysis is completed, take it out and freeze-dry to obtain the copper polysaccharide.

[0039] The buffer aid in this example includes, by mass: 4 parts of triethanolamine, 2 parts of glycine, 8 parts of sodium tartrate, and 80 parts of water.

[0040] Example 3

[0041] S11. Preparation of cassava starch hydrolyzate: Slowly add 30 g of cassava starch into 120 mL of continuously stirred water. After stirring evenly, transfer it to a 250 mL flask, add 5 g of 18% hydrochloric acid, and continuously stir and react at 70 °C for 1.5 h. After the reaction is completed, add 50 g of 18% sodium hydroxide solution for reaction and react at 60 °C for 15 min. After the reaction is completed, the hydrolyzate is obtained.

[0042] S12. Preparation of copper polysaccharide: Take 28 g of hydrolyzate and 135 mL of water, add them into a 500 mL flask, adjust the pH to 11, continuously stir and react, add 140 g of buffer aid, add 4 g of copper chloride solid, and react for 50 min to obtain a reaction solution.

[0043] S13. Purification of copper polysaccharide: Take 60 mL of the reaction solution obtained in step S12 for dialysis treatment. The dialysis treatment is divided into primary dialysis and secondary dialysis. The primary dialysis treatment is to dialyze at 5 °C for 18 h in a dialysis bag with a molecular weight cut-off of 1 kDa, and the secondary dialysis treatment is to dialyze at 5 °C for 18 h in a dialysis bag with a molecular weight cut-off of 0.5 kDa. Before use, boil the dialysis bag in deionized water for 7 min, and change the dialysis fluid 3 times during dialysis. The volume of the dialysis fluid is 300 times the volume of the copper polysaccharide. After dialysis, take out and freeze-dry to obtain the copper polysaccharide.

[0044] The buffer aid in this example includes, by mass: 3 parts of triethanolamine, 1 part of glycine, 5 parts of sodium tartrate, and 60 parts of water.

[0045] Example 4

[0046] An analysis method for cassava copper polysaccharide in this example includes the following steps:

[0047] S21. Sample pretreatment: Weigh 0.5 g of the copper polysaccharide prepared in Examples 1 - 3 (accurate to ±0.0002 g), place it in a 250 mL beaker, add 20 mL of 1+1 hydrochloric acid solution and 1 mL of nitric acid solution, boil on an electric furnace for 10 min, cool, add 50 mL of water, dissolve by gentle boiling, cool again, transfer to a 250 mL volumetric flask, dilute to the mark with water, shake well, filter, and the obtained filtrate is the pretreated sample.

[0048] S22. Determination of copper content: Pipette 25 mL of the pretreated sample obtained in step S21 into a 250 mL volumetric flask, add 2 mL of potassium fluoride solution, heat and boil for 5 mL, cool, add 1 drop of bromocresol green indicator, adjust the solution to just turn blue with sodium hydroxide solution, add 10 mL of acetic acid - sodium acetate buffer solution, 2 drops of xylenol orange indicator, shake well, and titrate with a standard titration solution of disodium ethylenediaminetetraacetate until the solution turns bright yellow as the end point. Record the volume of the standard titration solution consumed as V2. The calculation formula for copper content is as follows:

[0049]

[0050] C1 - Concentration of the standard titration solution of disodium ethylenediaminetetraacetate, mol·L -1 ; V1 - Volume of the standard titration solution of disodium ethylenediaminetetraacetate consumed in titration (blank sample without adding filtrate), mL; V2 - Volume of the standard titration solution of disodium ethylenediaminetetraacetate consumed in titrating copper, mL; m - Mass of the copper polysaccharide sample to be measured, g; x - Copper content, %.

[0051] Comparative Example 1

[0052] The difference between this comparative example and Example 3 is that triethanolamine is not added to the buffer aid.

[0053] Comparative Example 2

[0054] The difference between this comparative example and Example 3 is that glycine is not added to the buffering aid.

[0055] Comparative Example 3

[0056] The difference between this comparative example and Example 3 is that sodium tartrate is not added to the buffering aid.

[0057] Comparative Example 4

[0058] The difference between this comparative example and Example 3 is that the buffering aid is not added.

[0059] 1. Component Detection

[0060] The total polysaccharide content was determined by the phenol-sulfuric acid method, the protein content was determined by a BCA protein concentration assay kit, the uronic acid content was determined by the carbazole-sulfuric acid method, and combined with the analysis method of the copper content of the present invention, the test results of the product components obtained in Examples 1-3 and Comparative Examples 1-3 are shown in the following table.

[0061] Polysaccharide (%) Protein (%) Uronic acid (%) Copper (%) Example 1 70.26 2.15 3.21 16.35 Example 2 69.58 2.63 3.26 16.20 Example 3 70.08 2.48 3.18 16.48 Comparative Example 1 67.04 2.56 10.52 5.23 Comparative Example 2 63.56 2.30 7.02 10.64 Comparative Example 3 66.93 2.84 12.30 6.93 Comparative Example 4 68.32 2.99 14.85 2.14

[0062] 2. Detection of Gastrointestinal Adaptability of the Organism

[0063] After 30 mice were fed adaptively for 1 week, they were administered polysaccharide copper at 200 mg / kg once a day for 14 consecutive days by gavage. On the 14th day of administration, the livers and kidneys of the mice were collected, and on the 28th day, the colons of the mice were collected to observe the pathological changes of each tissue. The observation showed that the liver structure of the mice was clear and normal, desmosomes were visible, the hepatocytes were radially arranged around the central vein, the hepatocyte and hepatic lobule structures were both intact, the cells were closely and orderly distributed, and there were no lesions; the nephron structure of the mice was clear, the glomerulus and each level of renal tubule tissue structures were intact, and there were no lesions; the intestinal mucosa layer, submucosa layer, muscular layer, and serosa layer of the mice had normal tissue structures, the intestinal glands were rich and arranged regularly, the villi were neatly arranged and had a high density, and the structures and morphologies of the intestinal villi and crypts were normal.

[0064] In summary, a method for synthesizing cassava polysaccharide copper of the present invention is provided. Cassava polysaccharide copper is synthesized from several raw materials such as cassava starch, copper chloride, sodium hydroxide, hydrochloric acid, and water, and the copper content is as high as 16%. The polysaccharide copper synthesized in this application can be used as a feed additive to supplement copper for livestock and poultry, has no irritation to the kidneys and gastrointestinal tract of the body, has a better absorption efficiency, has a low cost of cassava raw materials, and the prepared polysaccharide copper has a stable structure, is non-toxic and harmless, and is environmentally friendly. The triethanolamine, glycine, and sodium tartrate of the present invention have a synergistic effect and have strong coordination ability under alkaline conditions, can form a very stable complex with copper ions, can increase the solubility of copper ions, promote their combination with cassava polysaccharide, and can provide a certain buffering capacity. At the same time, the present invention also controls the dosage ratio of triethanolamine, glycine, and sodium tartrate to avoid the over-strong coordination ability of triethanolamine, which may completely wrap copper ions and inhibit their combination with polysaccharide. The two-stage dialysis method of the present invention realizes step-by-step purification, significantly reduces the impurity content in the polysaccharide copper, and the product has a higher purity, effectively removing unreacted copper ions and small molecule impurities. The analysis method of the present invention is improved on the basis of the ethylenediaminetetraacetic acid (EDTA) complexometric titration method. The acetic acid-sodium acetate buffer solution is used to mask non-determined elements, and then the element content is determined by complexing with ethylenediaminetetraacetic acid (EDTA) through an indicator. This analysis method can quickly determine the copper content of the product, is simpler, and has a lower cost. As shown by the test results of the present invention, the cassava polysaccharide copper of the present invention does not damage the liver, kidneys and intestinal tissues of mice, showing good safety.

[0065] The foregoing description of the specific exemplary embodiments of the invention has been presented for purposes of illustration and example. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many modifications and variations are possible in light of the above teaching. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the invention, as well as various different selections and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for synthesizing cassava polysaccharide copper, characterized in that, It includes the following steps: S11. Preparation of cassava starch hydrolyzate: Under continuous stirring, slowly add cassava starch into a certain amount of water. After stirring evenly, add hydrochloric acid, and continuously stir and react at 65 - 75 °C. After the reaction ends, add sodium hydroxide solution to continue the reaction. After the reaction ends, the hydrolyzate is obtained. S12. Preparation of polysaccharide copper: Take the hydrolyzate and a certain amount of water, add them into a flask, adjust the pH to 9 - 13, and slowly add solid copper chloride under stirring. React for 40 - 60 min to obtain a reaction complex solution. S13. Purification of polysaccharide copper: Take the reaction complex solution obtained in step S12 for dialysis treatment. After dialysis is completed, take it out and freeze-dry to obtain the polysaccharide copper.

2. The synthesis method of copper cassava polysaccharide according to claim 1, wherein, In step S11, the mass fraction of sodium hydroxide in the sodium hydroxide solution is 10 - 25%, and the addition ratio of the sodium hydroxide solution to the mass of cassava starch is 1 - 2.4:1; the mass fraction of hydrochloric acid is 9 - 25%, and the addition ratio to the mass of cassava starch is 0.1 - 0.2:

1.

3. The synthesis method of cassava polysaccharide copper according to claim 1, characterized in that, In step S11, the reaction temperature is 45 - 75 °C, and the reaction time is 10 - 20 min.

4. A method for synthesizing cassava polysaccharide copper according to claim 1, characterized in that, Step S12 also includes adding a buffering aid.

5. The synthesis method of cassava polysaccharide copper according to claim 4, characterized in that, The buffering aid includes, by mass parts: 1 - 4 parts of triethanolamine, 0.5 - 2 parts of glycine, 3 - 8 parts of sodium tartrate, and 40 - 80 parts of water.

6. A method for synthesizing cassava polysaccharide copper according to claim 4, characterized in that, The added mass of the buffering aid is 5 times the mass of the hydrolyzate.

7. A method for synthesizing cassava polysaccharide copper according to claim 1, characterized in that, In step S12, the mass ratio of the hydrolyzate to the solid copper chloride is 20 - 35:3 - 5.

8. A method for synthesizing cassava polysaccharide copper according to claim 1, characterized in that, In step S13, the dialysis treatment is divided into primary dialysis and secondary dialysis. The primary dialysis treatment is to dialyze at 4 - 6 °C for 12 - 24 h in a dialysis bag with a molecular weight of 1 - 2 kDa, and the secondary dialysis treatment is to dialyze at 4 - 6 °C for 12 - 24 h in a dialysis bag with a molecular weight of 0.5 kDa.

9. A cassava polysaccharide copper, characterized in that, The cassava polysaccharide copper is synthesized by the synthesis method described in any one of claims 1 - 8.

10. An analytical method for cassava polysaccharide copper as described in claim 9, characterized in that, It includes the following steps: S21. Sample pretreatment: Weigh the polysaccharide copper prepared in step S13, add 1 + 1 hydrochloric acid solution and nitric acid solution, boil, cool, add water, dissolve by gentle boiling, cool again, dilute with water, shake well, and filter. The obtained filtrate is the pretreated sample. S22. Determination of copper content: Take the pretreated sample obtained in step S21, add potassium fluoride solution, heat to boiling, cool, add bromocresol green indicator, adjust the solution to just turn blue with sodium hydroxide solution, add acetic acid - sodium acetate buffer solution and xylenol orange indicator, shake well, and titrate with ethylenediaminetetraacetic acid disodium standard titration solution until the solution turns bright yellow as the end point. The calculation formula for copper content is as follows: Where, C1 - Concentration of ethylenediaminetetraacetic acid disodium standard titration solution, mol·L-1; V1 - Volume of ethylenediaminetetraacetic acid disodium standard titration solution consumed in titration (blank sample without adding filtrate), mL; V2 - Volume of ethylenediaminetetraacetic acid disodium standard titration solution consumed in titrating copper, mL; m - Mass of the polysaccharide copper sample to be measured, g; x - Copper content, %.

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