Preparation method of algin oligomer

Abstract

The invention discloses a preparation method of an algin oligomer, which realizes efficient degradation of sodium alginate under specific reaction conditions by controlling the ratio of TEMPO (Tetramethylpiperidinooxy), sodium bromide and sodium hypochlorite. According to the specific technical scheme, the mass ratio of TEMPO to sodium alginate is 1-7%, the mass ratio of sodium bromide to sodium alginate is 12-64%, and the ratio of the dosage of sodium hypochlorite to the dosage of sodium alginate is 1 g: 3-24 mmol. Within the temperature range of 40-50 DEG C, the algin oligomer with the average molecular weight of 4-6.5 kDa can be obtained after the reaction. Acid-base, enzyme preparations or other complex physical means are not needed, and the product recovery rate reaches 75-95%. Experiments show that compared with a clear water control group, the rotting rate and the weight loss rate of strawberries treated by the algin oligomer aqueous solution with the concentration of 100 mg / L are remarkably reduced by 51.5% and 45.6% respectively, and the fruit hardness reduction rate is reduced by about 30%.

Description

Technical Field

[0001] The present invention belongs to the field of deep processing of fucoidan, and particularly relates to a method for preparing alginate oligomers. Background Art

[0002] Alginate is a natural polysaccharide extracted from large brown algae, mainly composed of two units of α-L-guluronic acid and β-D-mannuronic acid, and has biodegradability and low toxicity. Sodium alginate is a typical form of alginate and has been applied in the fields of food additives, drug sustained release, wound repair, and skin care. However, due to its large molecular weight, poor solubility, and high viscosity, its application range is limited. In contrast, alginate oligomers prepared by degrading sodium alginate have gradually become a research hotspot due to their low viscosity, good bioabsorbability, and strong biological activity. At present, the preparation of alginate oligomers mainly uses technical means such as chemical oxidation method, physical ultrasound method, and biological enzymatic hydrolysis method.

[0003] The Chinese invention patent application with the patent publication number CN105315384A discloses a method for preparing alginate oligosaccharides by gradient dilution oxidation, which is to carry out a preliminary reaction with 30% hydrogen peroxide for 5 to 20 minutes at 95°C, then dilute the solution by one time, and continue the reaction for 10 to 60 minutes at 70°C. Through alcohol precipitation treatment, alginate oligosaccharides with a degree of polymerization of 4 to 9 are finally obtained, and the yield can reach 91.27%. The Chinese invention patent with the patent announcement number CN107474155B discloses a method for preparing alginate oligosaccharides by using ultrasound and spectroscopy assistance, which is to place the hydrochloric acid acidified and dehydrated alginate block in an ultrasonic reactor with a power of 500 to 1500W for reaction for 0.5 to 1 hour, and then transfer it to a light wave reactor, set the light wave power to 300 to 350W, and carry out light wave radiation for 30 minutes. This method can obtain alginate oligomers with a degree of polymerization lower than 10, and the yield exceeds 95%. The Chinese invention patent application with the patent publication number CN104099386A discloses a method for preparing alginate oligosaccharides by enzymatic hydrolysis. This method uses the alginate lyase produced by Streptomyces violaceus to react for 8 to 10 hours at 45 to 50°C to prepare alginate oligosaccharides with a degree of polymerization of 2 to 6.

[0004] Bose et al. (Alginate Oligosaccharide Postharvest Treatment Preserve Fruit Quality and Increase Storage Life Via Abscisic Acid Signaling in Strawberry[J]. Food Chemistry, 2019, 283 665-674.) publicly reported an alginate oligosaccharide with a degree of polymerization of 2-7 prepared by an enzymatic method and applied it to strawberry preservation. The research results showed that strawberries soaked in an alginate oligosaccharide solution for 1 minute and air-dried, when stored at 20±2°C and a relative humidity of 80±2% for one week, compared with the control group of clear water, the hardness reduction rate was significantly reduced by 28%, and the decay rate was reduced by about 50%, showing a certain preservation effect.

[0005] TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) is a stable free radical compound widely used in organic chemistry and materials science, and can act as an efficient oxidant or catalyst in chemical reactions. TEMPO exhibits excellent catalytic performance in oxidation reactions, especially when oxidizing sugars, alcohols and other reducing compounds, with potential advantages such as mildness, strong selectivity and loose reaction conditions. So far, there has been no report on the precise modification of alginate based on the TEMPO oxidation method, especially the related reports on obtaining low molecular weight products by inducing glycosidic bond cleavage. Summary of the Invention

[0006] The present invention is to solve the above technical problems existing in the prior art and provide a method for preparing alginate oligomers.

[0007] The technical solution of the present invention is: a method for preparing alginate oligomers, which is carried out in sequence according to the following steps: Step 1. Dissolve sodium alginate in water to obtain Solution I; Step 2. Add TEMPO and sodium bromide to Solution I and stir evenly to obtain Solution II; Step 3. Add sodium hypochlorite to Solution II and stir evenly to obtain Solution III; Step 4. Add sodium hydroxide dropwise to Solution III to adjust the pH value to weakly alkaline, and obtain Solution IV after reaction; Step 5. Adjust the pH value of Solution IV to neutral to obtain Solution V; Step 6. Add absolute ethanol to Solution V to obtain Solution VI; Step 7. Solution VI is dialyzed and freeze-dried to obtain alginate oligomers.

[0008] Preferably, in step 1, sodium alginate and water are mixed at a mass-to-volume ratio of 5-25 mg:1 ml and continuously stirred evenly at 60-80 °C.

[0009] Preferably, in step 2, after solution I is cooled to 40-50 °C, TEMPO and sodium bromide are added. The mass ratio of TEMPO to sodium alginate in solution II is 1-7%, and the mass ratio of sodium bromide to sodium alginate is 12-64%.

[0010] Preferably, in step 3, the dosage ratio of the added sodium alginate to sodium hypochlorite is 1 g:3-24 mmol.

[0011] Preferably, in step 4, a 0.5 mol / mL sodium hydroxide solution is used to adjust the pH to 9-11, and continuously stirred for 8-10 h to obtain solution IV.

[0012] Preferably, in step 5, a 1 mol / mL hydrochloric acid solution is used to adjust the pH of solution V to 7 to obtain solution V.

[0013] Preferably, in step 6, the volume ratio of the added absolute ethanol to solution V is 1-20%.

[0014] Preferably, the dialysis cut-off molecular weight in step 7 is 300-500 Da.

[0015] The present invention utilizes the TEMPO / sodium bromide / sodium hypochlorite oxidation system to oxidize the hydroxyl groups of sodium alginate into carboxyl groups, induce the cleavage of the glycosidic bonds in the molecular chain, thereby realizing the directional degradation of polysaccharides and further obtaining oligomers. Compared with the prior art, it has the following advantages: 1. Mild reaction conditions: There is no need to add strong acids and enzyme preparations, nor rely on auxiliary means such as microwave or ultrasound, avoiding side reactions or over-oxidation. 2. Few reaction by-products: Only a small amount of sodium chloride is produced during the reaction, the product is easy to separate, the recovery rate reaches 55-75%, and the raw material utilization rate, product purity, production efficiency and product quality are all significantly improved. 3. The reaction product has a preservation function: The prepared sodium alginate oligomer, as an environmentally friendly water-soluble non-toxic plant biostimulant, exhibits a significant preservation effect and can effectively extend the shelf life of fruits and vegetables. Description of the Drawings

[0016] Figure 1 is the flow chart of the sodium alginate oligomer prepared in Example 1 of the present invention.

[0017] Figure 2 is the relative molecular weight distribution diagram of the sodium alginate oligomer and sodium alginate prepared in Example 1 of the present invention.

[0018] Figure 3It is the infrared spectrogram of the alginate oligomer and sodium alginate prepared in Example 1 of the present invention.

[0019] Figure 4 It is a schematic diagram of the strawberry decay rate within one week after the strawberry is preserved with the alginate oligomer, sodium alginate and distilled water prepared in Example 1 of the present invention.

[0020] Figure 5 It is a schematic diagram of the strawberry weight loss rate within one week after the strawberry is preserved with the alginate oligomer, sodium alginate and distilled water prepared in Example 1 of the present invention.

[0021] Figure 6 It is a schematic diagram of the strawberry hardness change within one week after the strawberry is preserved with the alginate oligomer, sodium alginate and distilled water prepared in Example 1 of the present invention. Detailed implementation manners

[0022] The sodium alginate used in the examples of the present invention was purchased from Qingdao Mingyue Seaweed Group Co., Ltd. and meets the requirements of the national standard GB 1886.243-2016 "National Food Safety Standard Food Additive Sodium Alginate (also known as sodium alginate)". Examples 1-8 are all carried out according to Figure 1 the shown process. Example 1

[0023] Step 1. Dissolve sodium alginate in water at a concentration of 10 mg / mL and continuously stir and dissolve at 80 °C to obtain Solution I; Step 2. Take 100 mL of Solution I and cool it to 45 °C, add 0.032 g of TEMPO and 0.32 g of sodium bromide, and stir evenly to obtain Solution II; Step 3. Add 4 mmol of sodium hypochlorite to Solution II and stir evenly to obtain Solution III; Step 4. Add 0.5 mol / L sodium hydroxide solution to Solution III to adjust the pH to 10 ± 0.5, and continuously stir for 8 h to obtain Solution IV; Step 5. Adjust the pH of Solution IV to 7 with 1 mol / L hydrochloric acid solution to obtain Solution V; Step 6. Add absolute ethanol to Solution V, and the volume ratio of absolute ethanol to Solution V is 1% to obtain Solution VI; Step 7. Solution VI is dialyzed (cut-off amount 300 Da) and freeze-dried to obtain alginate oligomer.

[0024] The alginate oligomer prepared in Example 1 is in the form of a white powder, and the product yield is 91.73%. The relative molecular mass distribution of the alginate oligomer and sodium alginate prepared in Example 1 is as Figure 2As shown, it can be seen that the relative molecular weight of the raw material sodium alginate is 25 kDa, while the relative molecular weight of the alginate oligomer is less than 6 kDa. The Fourier transform infrared spectrum (FTIR) of the alginate oligomer prepared in Example 1 is as Figure 3 shown. It can be seen that compared with the spectrum of the raw material sodium alginate, the peak at 2928.12 cm -1 (attributed to the -CH- stretching vibration) has no obvious change, indicating that the basic structure remains unchanged during the degradation process. However, the peak at 3345.72 - 3386.29 cm -1 (attributed to the -OH stretching vibration) shows a blue shift, and the peak at 1607.37 - 1613.3 cm -1 (attributed to the -COO - stretching vibration) is enhanced and shows a red shift, indicating that the hydroxyl group in the uronic acid unit is partially oxidized to a carboxyl group. Conductometric titration analysis shows that the carboxyl group content of the product increases from 2.05 ± 0.045 mmol / g before the reaction to 2.77 ± 0.025 mmol / g, creating a structural basis for inducing the cleavage of the glycosidic bond in the molecular chain. The alginate oligomer prepared in Example 1 of the present invention and the raw material sodium alginate were respectively made into solutions with a concentration of 100 mg / L. After soaking three groups of fresh strawberries of the same batch for 1 minute and then air-drying, during the one-week storage at 20 ± 2°C and 80 ± 2% relative humidity, the decay rate, weight loss rate, and hardness change of each group of strawberries are respectively as Figure 4 , 5 , and 6 shown. The experimental results show that during the one-week storage period, the fresh-keeping activity of the untreated sodium alginate is relatively limited and the effect is not obvious, while the prepared alginate oligomer exhibits significant postharvest fresh-keeping activity. Compared with the distilled water control group, the decay rate of strawberry fruits in the alginate oligomer treatment group decreased by 51.5% on the 6th day of storage, and the weight loss rate and the rate of hardness reduction slowed down by 46.5% and 30% respectively. Thus, it can be seen that the alginate oligomer prepared in the present invention can function as a green natural fruit and vegetable fresh-keeping agent.

[0025] Example 2: Step 1. Dissolve sodium alginate in water at a concentration of 10 mg / mL, and continuously stir and dissolve at 80°C to obtain Solution I; Step 2. Take 100 mL of Solution I and cool it to 45°C, add 0.03 g of TEMPO and 0.31 g of sodium bromide, and stir evenly to obtain Solution II; Step 3. Add 6 mmol of sodium hypochlorite to Solution II and stir evenly to obtain Solution III; Step 4. Add 0.5 mol / L sodium hydroxide solution to Solution III to adjust the pH to 10 ± 0.5, and continuously stir for 8.15 h to obtain Solution IV; Step 5. Adjust the pH of Solution Ⅳ to 7 with 1 mol / L hydrochloric acid solution to obtain Solution Ⅴ; Step 6. Add absolute ethanol to Solution Ⅴ, and the volume ratio of absolute ethanol to Solution Ⅴ is 1% to obtain Solution Ⅵ; Step 7. Dialyze Solution Ⅵ (cut-off molecular weight 300 Da), and then freeze-dry to obtain alginate oligomers.

[0026] It was determined that the relative molecular weight of the alginate oligomers prepared in Example 2 was about 5.5 kDa, and the product yield was 90.82%.

[0027] Example 3: Step 1. Dissolve sodium alginate in water at a concentration of 10 mg / mL, and continuously stir and dissolve at 80 °C to obtain Solution Ⅰ; Step 2. Take 100 mL of Solution Ⅰ and cool it to 45 °C, add 0.025 g of TEMPO and 0.28 g of sodium bromide, and stir evenly to obtain Solution Ⅱ; Step 3. Add 8 mmol of sodium hypochlorite to Solution Ⅱ and stir evenly to obtain Solution Ⅲ; Step 4. Add 0.5 mol / L sodium hydroxide solution dropwise to Solution Ⅲ to adjust the pH to 10 ± 0.5, and continuously stir for 8.23 h to obtain Solution Ⅳ; Step 5. Adjust the pH of Solution Ⅳ to 7 with 1 mol / L hydrochloric acid solution to obtain Solution Ⅴ; Step 6. Add absolute ethanol to Solution Ⅴ, and the volume ratio of absolute ethanol to Solution Ⅴ is 1% to obtain Solution Ⅵ; Step 7. Dialyze Solution Ⅵ (cut-off molecular weight 300 Da), and then freeze-dry to obtain alginate oligomers.

[0028] It was determined that the relative molecular weight of the alginate oligomers prepared in Example 3 was about 5.1 kDa, and the product yield was 86.35%.

[0029] Example 4: Step 1. Dissolve sodium alginate in water at a concentration of 10 mg / mL, and continuously stir and dissolve at 80 °C to obtain Solution Ⅰ; Step 2. Take 100 mL of Solution Ⅰ and cool it to 45 °C, add 0.03 g of TEMPO and 0.29 g of sodium bromide, and stir evenly to obtain Solution Ⅱ; Step 3. Add 10 mmol of sodium hypochlorite to Solution Ⅱ and stir evenly to obtain Solution Ⅲ; Step 4. Add 0.5 mol / L sodium hydroxide solution dropwise to Solution Ⅲ to adjust the pH to 10 ± 0.5, and continuously stir for 8.3 h to obtain Solution Ⅳ; Step 5. Adjust the pH of Solution Ⅳ to 7 with 1 mol / L hydrochloric acid solution to obtain Solution Ⅴ; Step 6. Add absolute ethanol to Solution Ⅴ, and the volume ratio of absolute ethanol to Solution Ⅴ is 1% to obtain Solution Ⅵ; Step 7. Dialyze Solution Ⅵ (cut-off molecular weight 300 Da) and freeze-dry to obtain alginate oligomers.

[0030] It was determined that the relative molecular weight of the alginate oligomers prepared in Example 4 was about 4.8 kDa, and the product yield was 83.72%.

[0031] Example 5: Step 1. Dissolve sodium alginate in water at a concentration of 10 mg / mL and continuously stir and dissolve at 80 °C to obtain Solution Ⅰ; Step 2. Take 100 mL of Solution Ⅰ and cool it to 45 °C, add 0.023 g of TEMPO and 0.27 g of sodium bromide, and stir evenly to obtain Solution Ⅱ; Step 3. Add 12 mmoL of sodium hypochlorite to Solution Ⅱ and stir evenly to obtain Solution Ⅲ; Step 4. Add 0.5 mol / L sodium hydroxide solution dropwise to Solution Ⅲ to adjust the pH to 10 ± 0.5, and continuously stir for 8.43 h to obtain Solution Ⅳ; Step 5. Adjust the pH of Solution Ⅳ to 7 with 1 mol / L hydrochloric acid solution to obtain Solution Ⅴ; Step 6. Add absolute ethanol to Solution Ⅴ, and the volume ratio of absolute ethanol to Solution Ⅴ is 1% to obtain Solution Ⅵ; Step 7. Dialyze Solution Ⅵ (cut-off molecular weight 300 Da) and freeze-dry to obtain alginate oligomers.

[0032] It was determined that the relative molecular weight of the alginate oligomers prepared in Example 5 was about 4 kDa, and the product yield was 78.74%.

[0033] Example 6: Step 1. Dissolve sodium alginate in water at a concentration of 5 mg / mL and continuously stir and dissolve at 80 °C to obtain Solution Ⅰ; Step 2. Take 100 mL of Solution Ⅰ and cool it to 45 °C, add 0.032 g of TEMPO and 0.32 g of sodium bromide, and stir evenly to obtain Solution Ⅱ; Step 3. Add 12 mmoL of sodium hypochlorite to Solution Ⅱ and stir evenly to obtain Solution Ⅲ; Step 4. Add 0.5 mol / L sodium hydroxide solution dropwise to Solution Ⅲ to adjust the pH to 10 ± 0.5, and continuously stir for 9.53 h to obtain Solution Ⅳ; Step 5. Adjust the pH of Solution Ⅳ to 7 with 1 mol / L hydrochloric acid solution to obtain Solution Ⅴ; Step 6. Add absolute ethanol to Solution Ⅴ, and the volume ratio of absolute ethanol to Solution Ⅴ is 1% to obtain Solution Ⅵ; Step 7. Solution Ⅵ is dialyzed (cut-off molecular weight 300 Da) and freeze-dried to obtain alginate oligomers.

[0034] It was determined that the relative molecular weight of the alginate oligomers prepared in Example 6 was about 4.35 kDa, and the product yield was 80.19%.

[0035] Example 7: Step 1. Dissolve sodium alginate in water at a concentration of 20 mg / mL, and continuously stir and dissolve at 80 °C to obtain Solution Ⅰ; Step 2. Take 100 mL of Solution Ⅰ and cool it to 45 °C, add 0.032 g of TEMPO and 0.32 g of sodium bromide, and stir evenly to obtain Solution Ⅱ; Step 3. Add 12 mmol of sodium hypochlorite to Solution Ⅱ and stir evenly to obtain Solution Ⅲ; Step 4. Add 0.5 mol / L sodium hydroxide solution dropwise to Solution Ⅲ to adjust the pH to 10 ± 0.5, and continuously stir for 8.1 h to obtain Solution Ⅳ; Step 5. Adjust the pH of Solution Ⅳ to 7 with 1 mol / L hydrochloric acid solution to obtain Solution Ⅴ; Step 6. Add absolute ethanol to Solution Ⅴ, and the volume ratio of absolute ethanol to Solution Ⅴ is 1% to obtain Solution Ⅵ; Step 7. Solution Ⅵ is dialyzed (cut-off molecular weight 300 Da) and freeze-dried to obtain alginate oligomers.

[0036] It was determined that the relative molecular weight of the alginate oligomers prepared in Example 7 was about 5.3 kDa, and the product yield was 88.28%.

[0037] Example 8: Step 1. Dissolve sodium alginate in water at a concentration of 10 mg / mL, and continuously stir and dissolve at 80 °C to obtain Solution Ⅰ; Step 2. Take 100 mL of Solution Ⅰ and cool it to 40 °C, add 0.032 g of TEMPO and 0.32 g of sodium bromide, and stir evenly to obtain Solution Ⅱ; Step 3. Add 12 mmol of sodium hypochlorite to Solution Ⅱ and stir evenly to obtain Solution Ⅲ; Step 4. Add 0.5 mol / L sodium hydroxide solution dropwise to Solution Ⅲ to adjust the pH to 10 ± 0.5, and continuously stir for 8.7 h to obtain Solution Ⅳ; Step 5. Adjust the pH of Solution Ⅳ to 7 with 1 mol / L hydrochloric acid solution to obtain Solution Ⅴ; Step 6. Add absolute ethanol to Solution Ⅴ, and the volume ratio of absolute ethanol to Solution Ⅴ is 1% to obtain Solution Ⅵ; Step 7. Dialyze Solution Ⅵ (cut-off molecular weight 300 Da) and freeze-dry to obtain alginate oligomers.

[0038] It was determined that the relative molecular weight of the alginate oligomers prepared in Example 8 was about 6.3 kDa, and the product yield was 93.14%.

Claims

1. A method for preparing alginate oligomers, characterized in that Follow these steps in order: Step 1. Dissolve sodium alginate in water to prepare solution I; Step 2. Add TEMPO and sodium bromide to solution I and stir to obtain solution II; Step 3. Add sodium hypochlorite to solution II and stir evenly to obtain solution III; Step 4. Add sodium hydroxide dropwise to solution III to adjust the pH value to be weakly alkaline, and obtain solution IV after reaction; Step 5. Adjusting the pH value of solution IV to neutral to obtain solution V; Step 6. Add anhydrous ethanol to solution V to obtain solution VI; Step 7. Solution VI is dialyzed and freeze-dried to obtain alginate oligomers.

2. The method for preparing alginate oligomer according to claim 1, characterized in that The step 1 is to mix sodium alginate and water in a mass volume ratio of 5-25 mg:1 ml and continuously stir at 60-80° C. to uniformly mix.

3. The method for preparing alginate oligomer according to claim 2, characterized in that The step 2 is to cool the solution I to 40-50° C. and then add TEMPO and sodium bromide, wherein the mass ratio of TEMPO to sodium alginate in the solution II is 1-7%, and the mass ratio of sodium bromide to sodium alginate is 12-64%.

4. The method for preparing alginate oligomer according to claim 3, characterized in that In step 3, the ratio of sodium alginate to sodium hypochlorite added is 1 g:3-24 mmoL.

5. The method for preparing alginate oligomer according to claim 4, characterized in that The step 4 is to adjust the pH to 9-11 using 0.5 mol / mL sodium hydroxide solution, and continue stirring for 8-10 h to obtain solution IV.

6. The method for preparing alginate oligomer according to claim 5, characterized in that The step 5 is to adjust the pH of solution V to 7 using 1 mol / mL hydrochloric acid solution to obtain solution V.

7. The method for preparing alginate oligomer according to claim 6, characterized in that The volume ratio of the anhydrous ethanol added in step 6 to the solution V is 1-20%.

8. The method for preparing alginate oligomer according to claim 7, characterized in that The dialysis cut-off molecular weight in step 7 is 300-500 Da.

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