A method for preparing high-purity brown algae oligosaccharide

Abstract

The invention discloses a method for preparing high-purity brown algae oligosaccharide, and belongs to the field of marine chemical technology. The method for preparing brown algae oligosaccharide of the invention comprises the following steps: after sodium alginate is divided into three times for enzymatic hydrolysis, the enzymatic hydrolysis liquid is ultrafiltered to obtain a brown algae oligosaccharide clear liquid; the brown algae oligosaccharide clear liquid is spray-dried to obtain the brown algae oligosaccharide. In the invention, the sodium alginate has a high degree of degradation, the obtained brown algae oligosaccharide has a high purity, and the production process is safe and efficient.

Description

Technical Field

[0001] The invention relates to the technical field of marine chemical industry, and in particular to a method for preparing high-purity brown algae oligosaccharide. Background Art

[0002] Brown algae oligosaccharide (AOS) is an important signal molecule for plants. Brown algae oligosaccharide can not only promote plant growth, but also act as an inducer to induce plants to produce stress resistance. Therefore, brown algae oligosaccharide has received extensive attention in the field of agricultural production. Due to its unique composition and biological activity, it has become a hot spot in the current research on plant growth regulation and induced stress resistance.

[0003] Brown algae oligosaccharides are degradation products of macromolecular alginic acid with a degree of polymerization of 2-10. They can be prepared by chemical, physical, bioenzymatic and other methods. Compared with high molecular weight polysaccharides, brown algae oligosaccharides have the advantages of stable properties, easy solubility in water, safety and non-toxicity. However, the current market price of high-purity brown algae oligosaccharides is relatively high, which seriously affects its application in fertilizers. Therefore, how to optimize the production process of brown algae oligosaccharides to improve its output efficiency and reduce production costs has become a major difficulty in realizing the industrial application of brown algae oligosaccharides in fertilizers.

[0004] Chinese patent application with application number 202410136218.3 discloses a method for preparing brown algae oligosaccharides, which uses sodium alginate as raw material and obtains brown algae oligosaccharides through enzymatic hydrolysis, filtration, and freeze-drying. Although this method uses an enzymatic hydrolysis method, the process of preparing "polyacrylonitrile-polyurethane blend film" is relatively complicated and time-consuming, and freeze-drying is selected as the drying method. Both post-processing methods consume high energy and cannot be applied to large-scale production. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing high-purity brown algae oligosaccharides, which obtains high-purity brown algae oligosaccharides through three steps of batch feeding enzymatic hydrolysis, ultrafiltration and spray drying; the method has the advantages of high degradation degree, high yield, simple production process, safety, environmental protection, high efficiency and low production cost, and the prepared brown algae oligosaccharide powder has high purity and is more stable, and has good development and application prospects.

[0006] The present invention first provides a method for preparing brown algae oligosaccharides, comprising the following steps:

[0007] After sodium alginate is added three times for enzymolysis, the enzymolysis solution is ultrafiltered to obtain a brown algae oligosaccharide clear solution; the brown algae oligosaccharide clear solution is spray-dried to obtain the brown algae oligosaccharide.

[0008] The above preparation method specifically comprises the following steps:

[0009] (1) Mix sodium alginate, alginate lyase and water evenly, and perform enzymatic hydrolysis at 25-50° C. for 6-9 hours;

[0010] The sodium alginate and alginate lyase are added three times, the first addition is followed by enzymatic hydrolysis for 1-2 hours, and then the second addition is followed by enzymatic hydrolysis for 1-2 hours and the third addition is followed by enzymatic hydrolysis for 4-5 hours;

[0011] (2) subjecting the enzymatic hydrolysis mixture obtained in step (1) to ceramic membrane ultrafiltration to obtain a brown algae oligosaccharide clear solution;

[0012] (3) spray-drying the brown algae oligosaccharide solution to obtain the brown algae oligosaccharide.

[0013] In the above preparation method, in step (1), the temperature of the enzymatic hydrolysis is 35-40°C.

[0014] In the above preparation method, the sodium alginate is in granular form and has a purity greater than 90%;

[0015] The enzyme activity of the alginate lyase is greater than 100,000 U / g.

[0016] In the above-mentioned preparation method, in step (1), the mass ratio of sodium alginate to water is 1:3-4;

[0017] The added amount of the alginate lyase is 2%-4% of the mass of the sodium alginate.

[0018] In the above preparation method, in step (1), the sodium alginate and alginate lyase are added three times, and each addition is 1 / 3 of the total mass.

[0019] In the above-mentioned preparation method, in step (2), the pore size of the ceramic membrane is 300-600nm.

[0020] In the above-mentioned preparation method, in step (3), the inlet air temperature of the spray drying is 130-170°C, and the outlet air temperature is 80-110°C.

[0021] The present invention also provides brown algae oligosaccharide prepared by the above preparation method.

[0022] The purity of the brown algae oligosaccharide is greater than 90%.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) The present invention divides the enzymatic hydrolysis into three steps, thereby improving the enzymatic hydrolysis efficiency and reducing the possibility of sodium alginate agglomerating or failing to completely dissolve in water when it is fed once, and causing the agitator motor to become sticky and break;

[0025] (2) The method of the present invention adopts a biological enzymatic hydrolysis method, which can fully decompose the macromolecular sugars of sodium alginate into oligosaccharides, and cooperates with a nanofiltration membrane to further filter out the undegraded macromolecular sugar molecules, retaining brown algae oligosaccharides with a polymerization degree of 2-10, which is more conducive to plant absorption;

[0026] (3) The alginate content of the brown algae oligosaccharide prepared by the method of the present invention is above 90%, the process is simple, and it is suitable for industrial mass production. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0028] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0029] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] The ceramic membranes used in the following examples are 0.5 m2 experimental machines (Example 1, Example 2, Example 3, Comparative Example 2, Comparative Example 3, Comparative Example 1) and 32 m2 industrial machines (Example 4) of Suzhou Juwei Environmental Protection Technology Co., Ltd., and the membrane pore diameters are all 500 nm.

[0032] The spray drying equipment used in the following examples is the LPG-5 experimental machine (Example 1, Example 2, Example 3) and the LPG-1000 industrial machine (Example 4) produced by Changzhou Qianjiang Drying Equipment Engineering Co., Ltd.

[0033] The freeze dryer used below is a BFD-5LF2 experimental machine manufactured by Qingdao Borui (Comparative Example 1).

[0034] The enzyme activity of the alginate lyase used in the following examples and comparative examples is 106,000 U / g. Definition of enzyme activity: 1U is defined as the amount of enzyme required to produce 1 μg of reducing sugar per minute at 35°C.

[0035] The purity of the sodium alginate particles used in the following examples is 91.7%.

[0036] Example 1

[0037] (1) 30 kg of water and 120 g of alginate lyase were mixed evenly and heated to 35 °C, then 3.2 kg of sodium alginate particles were added and stirred evenly for enzymatic hydrolysis for 1 h;

[0038] (2) adding 120 g of alginate lyase to the enzymatic hydrolysis mixture of step (1), followed by adding 3.2 kg of sodium alginate particles, stirring evenly and enzymatic hydrolysis again for 1 h;

[0039] (3) Add 120 g of alginate lyase to the enzymatic hydrolysis mixture of step (2), then add 3.2 kg of sodium alginate particles, stir evenly, and enzymatic hydrolyze again for 4 h;

[0040] (4) The enzymatic hydrolysis mixture of step (3) was subjected to ceramic membrane ultrafiltration to obtain 26.5 L of a high-content brown algae oligosaccharide clear solution with a mass percentage of 29.87%.

[0041] (5) In order to compare with Comparative Example 1, only 25 L of brown algae oligosaccharide liquid was spray dried, the inlet air temperature of the spray drying tower was set to 150°C, and the outlet air temperature was controlled at 100°C. 25 L of brown algae oligosaccharide liquid with a content of 29.87% was spray dried by atomization for 5.2 h to obtain 7.34 kg of brown algae oligosaccharide powder with a mass percentage of 95.17%.

[0042] Comparative Example 1

[0043] Brown algae oligosaccharides are prepared according to a method similar to that of Chinese patent application number 202410136218.3 "A method for preparing brown algae oligosaccharides", the steps are as follows:

[0044] (1) 30 kg of water and 60 g of alginate lyase were mixed evenly and heated to 35 °C, then 1.6 kg of sodium alginate particles were added and stirred evenly for 8 h;

[0045] (2) The enzymatic hydrolysis mixture was subjected to ceramic membrane ultrafiltration to obtain 27.5 L of brown algae oligosaccharide clear solution with a mass percentage of 5.05%.

[0046] (3) Since the freeze drying equipment can only process 5L of brown algae oligosaccharide liquid in a single batch, 25L of the brown algae oligosaccharide liquid was freeze-dried in 5 batches, each batch taking 21 hours. The heat source inlet temperature was controlled at 32°C and the outlet temperature was controlled at 30°C throughout the process. 25L of brown algae oligosaccharide liquid with a content of 5.03% was freeze-dried to obtain 1.18kg of brown algae oligosaccharide powder with a mass percentage of 95.33%.

[0047] (Since there is no preparation method for the patented enzyme preparation, the enzyme preparation used in the present invention is used; the preparation method of the filtration membrane is relatively complicated and cannot be replicated, so the ceramic membrane ultrafiltration in the present invention is used; the energy consumption of the two methods is compared.)

[0048] In Example 1, the spray drying tower model is LPG-5, the comprehensive power is 12kw / h (including electric heater, blower and atomizer), and it takes 5.2h to spray dry 25L of brown algae oligosaccharide liquid, and the total energy consumption is 12kw / h×5.2h=62.4kw. The total amount of powder prepared is 7.34kg, and the energy consumption per unit mass of powder is 62.4kw÷7.34=8.50kw / kg (Table 1).

[0049] In comparative example 1, the spray drying tower model is BFD-5LF2, the comprehensive power is 4kw / h (including heating plate, vacuum pump and refrigerator), and a single batch of 5L brown algae oligosaccharide liquid is processed in 21h. 25L brown algae oligosaccharide liquid is freeze-dried in 5 batches, and the total time is 105h. The total energy consumption is 4kw / h×105h=420kw, and the total amount of powder prepared is 1.18kg, and the energy consumption per unit mass of powder is 420kw÷1.18=355.93kw / kg (Table 1).

[0050] Table 1 Data summary of Example 1 and Comparative Example 1

[0051] Brown algae oligosaccharide serum content Brown algae oligosaccharide powder content Weight of prepared powder Energy consumption Energy consumption per unit mass of powder Example 1 29.87％ 95.17％ 7.34kg 62.4kw 8.50kw / kg Comparative Example 1 5.05％ 95.33％ 1.18kg 420kw 355.93kw / kg

[0052] As can be seen from Table 1, a batch of brown algae oligosaccharide powders were prepared, and there was no difference in recovery rate and powder content, but 25L of brown algae oligosaccharide clear liquid was dried, and the mass of brown algae oligosaccharide powder obtained by the present invention was 6.22 times that of comparative example 1, and the energy consumption per kilogram for preparing brown algae oligosaccharide powder was less than 1 / 40 of that of comparative example 1. Comparative example 1 uses freeze drying and needs to be processed in batches, and continuous feeding is impossible. The batch processing capacity is limited, the processing time is long, the energy consumption is high, and it is not suitable for large-scale production. The present invention not only has a high preparation concentration, but also has low energy consumption and convenient operation, and is completely suitable for use in large-scale production.

[0053] Example 2

[0054] (1) 37.54 kg of water and 120 g of alginate lyase were mixed evenly and heated to 40 °C, then 4 kg of sodium alginate particles were added and stirred evenly for enzymatic hydrolysis for 1 h;

[0055] (2) adding 120 g of alginate lyase to the enzymatic hydrolysis mixture of step (1), then adding 4 kg of sodium alginate particles, stirring evenly and enzymatic hydrolysis again for 1 h;

[0056] (3) Add 120 g of alginate lyase to the enzymatic hydrolysis mixture of step (2), then add 4 kg of sodium alginate particles, stir evenly, and enzymatic hydrolyze again for 4 h;

[0057] (4) The enzymatic hydrolysis mixture of step (3) was subjected to ceramic membrane ultrafiltration to obtain 36.4 L of a high-content brown algae oligosaccharide clear solution with a mass percentage of 28.7%.

[0058] (5) The inlet air temperature of the spray drying tower was set to 165° C., and the outlet air temperature was controlled at 95° C. The 36.4 L brown algae oligosaccharide solution in step (4) was spray dried for 7.5 h to obtain 9.7 kg of brown algae oligosaccharide powder with a mass percentage of 97.73%.

[0059] Example 3

[0060] (1) 35 kg of water and 100 g of alginate lyase were mixed evenly and heated to 40 °C, then 3.7 kg of sodium alginate particles were added, stirred evenly and enzymolyzed for 1 h;

[0061] (2) adding 100 g of alginate lyase to the enzymatic hydrolysis mixture of step (1), then adding 3.7 kg of sodium alginate particles, stirring evenly and enzymatic hydrolysis again for 1 h;

[0062] (3) adding 100 g of alginate lyase to the enzymatic hydrolysis mixture of step (2), then adding 3.7 kg of sodium alginate particles, stirring evenly, and enzymolyzing again for 4.5 h;

[0063] (4) The enzymatic hydrolysis mixture of step (3) was subjected to ceramic membrane ultrafiltration to obtain 31.75 L of a high-content brown algae oligosaccharide clear solution with a mass percentage of 28.88%.

[0064] (5) The inlet air temperature of the spray drying tower was set to 160° C., and the outlet air temperature was controlled at 95° C. The 31.75 L brown algae oligosaccharide solution in step (4) was spray dried by atomization for 6.5 h to obtain 8.97 kg of brown algae oligosaccharide powder with a mass percentage of 96.63%.

[0065] Example 4

[0066] (1) 1.564t of water and 4.2kg of alginate lyase were mixed evenly and heated to 40°C, then 150kg of sodium alginate particles were added and stirred evenly for enzymatic hydrolysis for 1h;

[0067] (2) adding 4.2 kg of alginate lyase to the enzymatic hydrolysis mixture of step (1), then adding 150 kg of sodium alginate particles, stirring evenly and enzymatic hydrolysis again for 1 hour;

[0068] (3) adding 4.2 kg of alginate lyase to the enzymatic hydrolysis mixture of step (2), followed by adding 150 kg of sodium alginate particles, stirring evenly, and performing enzymatic hydrolysis again for 4 h;

[0069] (4) The enzymatic hydrolysis mixture of step (3) was subjected to ceramic membrane ultrafiltration to obtain 1.75 tons of high-content brown algae oligosaccharide clear liquid with a mass percentage of 21.4%.

[0070] (5) The inlet air temperature of the spray drying tower was set to 160° C., and the outlet air temperature was controlled at 90° C. 1.75 t of the brown algae oligosaccharide liquid in step (4) was spray-dried for 2.5 h to obtain 368 kg of brown algae oligosaccharide powder with a content of 95.77%.

[0071] Comparative Example 2

[0072] (1) Mix 10L of water and 31.5g of alginate lyase and heat to 65°C, then add 0.9kg of sodium alginate particles and stir evenly for enzymatic hydrolysis for 1h;

[0073] (2) Add 31.5 g of alginate lyase to the enzymatic hydrolysis mixture of step (1), then add 0.9 kg of sodium alginate particles, stir evenly and enzymatically hydrolyze again for 1 hour;

[0074] (3) Add 31.5 g of alginate lyase to the enzymatic hydrolysis mixture of step (2), then add 0.9 kg of sodium alginate particles, stir evenly, and enzymatic hydrolyze again for 4 h;

[0075] (4) The enzymatic hydrolysis mixture of step (3) was subjected to ceramic membrane ultrafiltration to obtain 2.5 L of brown algae oligosaccharide clear solution with a mass percentage of 20.1%.

[0076] Since the enzymatic hydrolysis mixture was too viscous and contained a lot of colloids, ceramic membrane filtration was difficult, the yield was low, and the alginate content in the clear liquid was low, so it was chosen not to perform atomization spray drying.

[0077] Since the temperature during enzymatic hydrolysis was too high, the enzymatic activity of alginate lyase was ineffective and the enzymatic hydrolysis failed.

[0078] Comparative Example 3

[0079] (1) Mix 12L of water and 10g of alginate lyase and heat to 45°C, then add 1kg of sodium alginate particles and stir evenly for enzymatic hydrolysis for 1h;

[0080] (2) adding 10 g of alginate lyase to the enzymatic hydrolysis mixture of step (1), then adding 1 kg of sodium alginate particles, stirring evenly and enzymatic hydrolysis again for 1 h;

[0081] (3) adding 10 g of alginate lyase to the enzymatic hydrolysis mixture of step (2), then adding 1 kg of sodium alginate particles, stirring evenly, and enzymolyzing again for 4 h;

[0082] (4) The enzymatic hydrolysis mixture of step (3) was subjected to ceramic membrane ultrafiltration to obtain 1.8 L of brown algae oligosaccharide clear solution with a mass percentage of 18.87%.

[0083] Since the enzymatic hydrolysis mixture was too viscous and contained a lot of colloids, ceramic membrane filtration was difficult, the yield was low, and the alginate content in the clear liquid was low, so it was chosen not to perform atomization spray drying.

[0084] The enzymatic hydrolysis failed because too little alginate lyase was added.

[0085] In summary, compared with the existing traditional technology, the advantages of the present invention are: (1) the purity of brown algae oligosaccharides is higher; (2) the production process is simple, safe and environmentally friendly; (3) the preparation process is more efficient, the overall energy consumption is lower, and the yield is high.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing brown algae oligosaccharide, comprising the following steps: After sodium alginate is added three times for enzymolysis, the enzymolysis solution is ultrafiltered to obtain a brown algae oligosaccharide clear solution; the brown algae oligosaccharide clear solution is spray-dried to obtain the brown algae oligosaccharide.

2. The preparation method according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Mix sodium alginate, alginate lyase and water evenly, and perform enzymatic hydrolysis at 25-50° C. for 6-9 hours; The sodium alginate and alginate lyase are added three times, the first addition is followed by enzymatic hydrolysis for 1-2 hours, and then the second addition is followed by enzymatic hydrolysis for 1-2 hours and the third addition is followed by enzymatic hydrolysis for 4-5 hours; (2) subjecting the enzymatic hydrolysis mixture obtained in step (1) to ceramic membrane ultrafiltration to obtain a brown algae oligosaccharide clear solution; (3) spray-drying the brown algae oligosaccharide solution to obtain the brown algae oligosaccharide.

3. The preparation method according to claim 2, characterized in that: The sodium alginate is in granular form and has a purity greater than 90%; The enzyme activity of the alginate lyase is greater than 100,000 U / g.

4. The preparation method according to claim 2 or 3, characterized in that: In step (1), the mass ratio of sodium alginate to water is 1:3-4; The added amount of the alginate lyase is 2%-4% of the mass of the sodium alginate.

5. The preparation method according to any one of claims 2 to 4, characterized in that: In step (1), the sodium alginate and alginate lyase are added in three times, with each addition accounting for 1 / 3 of their total mass.

6. The preparation method according to any one of claims 2 to 5, characterized in that: In step (2), the pore size of the ceramic membrane is 300-600nm.

7. The preparation method according to any one of claims 2 to 6, characterized in that: In step (3), the inlet air temperature of the spray drying is 130-170°C, and the outlet air temperature is 80-110°C.

8. Brown algae oligosaccharide prepared by the preparation method according to any one of claims 1 to 7.

9. The brown algae oligosaccharide according to claim 8, characterized in that: The purity of the brown algae oligosaccharide is greater than 90%.