Preparation method and application of seaweed enzymatic hydrolysate with high content of phloro-disaccharide and phloro-trisaccharide

Abstract

The application discloses a preparation method of seaweed enzymolysis liquid with high contents of phlorodextrin and phlorotriose and application thereof. The hydrogen bond network of fucoidan is destroyed by pretreatment with a choline chloride-lactic acid low eutectic solvent, and then the fucoidan is degraded in a targeted manner by using a fucoidan lyase, so that the content of phlorodextrin (DP2) in the product is 42-48%, the content of phlorotriose (DP3) is 35-40%, and the total content of DP2+DP3 is greater than or equal to 77%. The multiple defects of low efficiency of traditional single enzymolysis, uneven oligosaccharide polymerization degree, weak effect of promoting rice germination, large process pollution and the like are solved in a coordinated manner by optimizing the preparation process and application parameters, and the germination rate of rice seeds and the seedling quality are significantly improved, and the operation is simple, green and safe.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a method for preparing seaweed enzymatic hydrolysate with high content of brown algae bi and trisaccharides and its application. Background Technology

[0002] Seaweed is one of the oldest organisms on Earth, classified into three main categories: brown algae, red algae, and green algae. Kelp and wakame are major economically important brown algae. The cell walls of brown algae are rich in alginate (polymannuronic acid, polyguluronic acid), and the resulting alginate oligosaccharides are natural plant biostimulants that can induce crop stress resistance, promote seed germination, and stimulate root growth, making them highly valuable in the field of green rice cultivation.

[0003] Existing methods for preparing seaweed enzymatic hydrolysates have significant drawbacks. Chemical degradation methods involve high-temperature cleavage using strong acids / bases, resulting in violent reactions, non-selective breaking of oligosaccharide glycosidic bonds, and a disordered degree of polymerization in the product, with extremely low proportions of disaccharides and trisaccharides. This generates large amounts of acidic and alkaline waste liquid, polluting the environment, and destroying the active groups of oligosaccharides, significantly reducing the growth-promoting effect and failing to meet the requirements for rice seed soaking. Physical degradation methods only break down cell walls, resulting in incomplete degradation of alginate macromolecules. The high proportion of macromolecular polysaccharides in the product makes it difficult for rice seeds to absorb quickly, leading to large variations in the degree of polymerization between batches, unstable quality, and high energy consumption and costs for large-scale processing. Enzymatic hydrolysis directly hydrolyzes seaweed powder, but the hydrogen bonds and dense crystalline structure within alginate molecules hinder enzyme contact with cleavage sites, resulting in low enzyme catalytic efficiency, long reaction cycles, high average degree of polymerization in the product, and insufficient total proportions of highly active disaccharides and trisaccharides. Furthermore, alginate lysins have poor thermal stability, leading to high enzyme consumption per unit product and high industrial production costs. Summary of the Invention

[0004] To address the aforementioned shortcomings, the present invention aims to provide a method for preparing seaweed enzymatic hydrolysate with high content of brown algae bi and trisaccharides and its application. It also provides an integrated green preparation process that utilizes choline chloride-lactic acid DES pretreatment coupled with the directional degradation of brown algae lyase and high enrichment of brown algae bi and trisaccharides, while establishing a complete application method suitable for rice seed soaking and seedling cultivation.

[0005] The technical solution adopted by this invention to achieve its technical objectives is as follows:

[0006] This invention provides a method for preparing seaweed enzymatic hydrolysate with high content of brown algae bi and trisaccharides, comprising:

[0007] (1) DES pretreatment: mix seaweed powder with choline chloride-lactic acid eutectic solvent at a solid-liquid ratio of 1:15~1:25 g / mL, stir and extract at 60~100℃ for 1~3 h, and collect the extract after solid-liquid separation;

[0008] (2) Alcohol precipitation separation: Add 3 to 5 times the volume of anhydrous ethanol to the extract obtained in step (1), let it stand at low temperature for alcohol precipitation, collect the precipitate by centrifugation, and wash the precipitate with anhydrous ethanol.

[0009] (3) Targeted enzymatic hydrolysis reaction: After reconstituted with buffer solution, alginate lyase was added and enzymatically hydrolyzed for 2-24 h at 25-55℃ and pH 6.0-10.0.

[0010] (4) Centrifugation purification: Centrifuge the enzymatic hydrolysate obtained in step (3) and retain the supernatant, which is the seaweed enzymatic hydrolysate with high content of brown algae bi and trisaccharides.

[0011] Preferably, in step (1), the molar ratio of choline chloride to lactic acid in the choline chloride-lactic acid eutectic solvent is 1:2, and the water content is 10~30%.

[0012] Preferably, in step (2), the precipitate is collected by allowing the mixture to stand at 4°C for 12 hours for alcohol precipitation, centrifuging at 10000 r / min for 20 minutes.

[0013] Preferably, in step (3), the final concentration of the alginate lyase is 40~70 U / mL; the buffer solution is a PBS buffer solution with pH 6.0~10.0.

[0014] Preferably, in step (1), the extraction temperature is 70~85℃; in step (3), the enzymatic hydrolysis conditions are 35~45℃, pH 8.5~9.0, and time 10~14 h.

[0015] Preferably, in step (4), the centrifugation speed is 6000~12000 rpm and the centrifugation time is 20~30 minutes.

[0016] The present invention also provides a seaweed enzymatic hydrolysate with high content of alginate and trisaccharides, which is obtained by the above preparation method. In the seaweed enzymatic hydrolysate, the active components are alginate and trisaccharides, which are 42-48 wt% and 35-40 wt% respectively.

[0017] This invention further provides the application of seaweed enzymatic hydrolysate in promoting rice growth, including:

[0018] To promote rice seed germination: Place the sterilized clean rice seeds in the seaweed enzymatic hydrolysate diluted 50 to 1000 times (more preferably 150 to 250 times) and soak for 24 to 48 hours to promote germination.

[0019] To promote rice seedling growth: Spray the leaves with the seaweed enzymatic hydrolysate diluted 150-250 times when the rice seedlings are in the two-leaf-one-heart stage.

[0020] Preferably, promoting rice seedling growth includes increasing seedling length, root length, and the number of lateral roots.

[0021] Preferably, the following method is used to obtain clean rice seeds after disinfection: Select rice seeds of uniform size and plumpness, wash them with clean water 2-3 times, soak them in a 1-10% sodium hypochlorite solution for 15-25 minutes for disinfection, and then wash them with clean water 2-5 times to remove any residual solution.

[0022] Preferably, the seeds are germinated by soaking in a constant temperature and light incubator at 27~29℃, with a light intensity of 3000 lx and a light / dark cycle of 14 h / 10 h.

[0023] The beneficial effects of this invention are as follows:

[0024] A green method for preparing seaweed enzymatic hydrolysate is adopted by combining a eutectic solvent with enzymatic hydrolysis, which has a synergistic effect: the hydrogen bond dissociation of DES disrupts the regular crystalline structure of alginate, exposing a large number of hidden enzyme cleavage sites; subsequent enzymatic hydrolysis can precisely break the intermediate glycosidic bonds of alginate, rather than random degradation, ultimately achieving a content of 42-48% fucoidan (DP2) and 35-40% fucoidan (DP3), with a total proportion ≥77% and an average degree of polymerization of only 2.4. Compared with the simple enzymatic hydrolysis process (average degree of polymerization 4.2 (Liu Ruizhi, 2009), the total proportion of fucoidan and DP3 is less than 60%), an unexpected enrichment effect of oligosaccharide components is produced; the acquisition of low-polymerization products makes the seaweed enzymatic hydrolysate prepared by this invention have higher plant growth-promoting and stress-resistance-inducing activities.

[0025] A customized solution for rice seed germination and seedling root development, including dilution concentration, soaking time, temperature and humidity, and foliar spraying, has been developed. This solution significantly promotes rice growth and can substantially improve germination rate, root length, and number of lateral roots. Attached Figure Description

[0026] Figure 1 Liquid chromatograms of DP2 and DP3 in Example 1 of this invention.

[0027] Figure 2 The growth of rice on the 4th day of germination during hydroponics in Example 9 of this invention.

[0028] Figure 3 The root growth of rice seedlings on day 6 during hydroponics in Example 9 of this invention.

[0029] Figure 4 Statistical results of root length, seedling length and number of lateral roots at the end of rice seedling cultivation in Example 10 of this invention. Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0031] In this invention, unless otherwise specified, all raw materials, equipment and instruments used in the preparation are commercially available products well known to those skilled in the art.

[0032] In the following examples, the alginate lyase used is AlgVS, derived from the genus *Bacillus* (…). Paenibacillus lautus A2), belonging to the PL7 family, has an enzyme activity of 750 U / mL, an optimal temperature of 40℃, and an optimal pH of 8.5. One unit of enzyme activity (1U) is defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute. This application does not specify the source or method of its production.

[0033] Example 1: Preparation of alginate and trisaccharides from kelp powder by pretreatment with eutectic solvent (DES)

[0034] (1) Preparation of eutectic solvent: choline chloride and lactic acid are mixed at a molar ratio of 1:2, the water content is adjusted to 10% (w / w), and stirred at 60°C for 30 minutes to form a uniform and transparent DES.

[0035] (2) Kelp powder pretreatment: Kelp powder and DES are mixed at a ratio of 1:20 (g / mL), stirred and extracted at 80℃ for 2 hours, and the extract is collected by filtration.

[0036] (3) Alcohol precipitation purification: Add 4 times the volume of anhydrous ethanol to the extract, let it stand at 4℃ for 12 hours for alcohol precipitation, centrifuge at 10000 r / min for 20 minutes, and collect the precipitate.

[0037] (4) Enzymatic hydrolysis: The precipitate was reconstituted with pH 9.0 PBS buffer solution, and crude alginate lyase AlgVS was added to a concentration of 50 U / mL. The mixture was then hydrolyzed for 12 hours at 40°C and pH 8.5.

[0038] (5) Centrifugation collection: After the enzymatic hydrolysis is completed, centrifuge at 8000 r / min for 30 min and collect the supernatant, which is the brown algae bi and trisaccharide hydrolysate.

[0039] (6) Detection methods: High performance liquid chromatography (HPLC) was used to detect the product components. The chromatographic column used was an OHpak SB-806M HQ 300 × 8.0 mm, 13 μm. The mobile phase was 0.1 M sodium chloride solution, the flow rate was 0.5 mL / min, a differential detector was used, the sample loading was 20 μL, and the column temperature was 30℃. The total sugar content was determined by the carbazole-sulfuric acid method, and the reducing sugar content was determined by the DNS method. The average degree of polymerization was calculated as total sugar concentration / reducing sugar concentration.

[0040] The enzymatic hydrolysate of brown algae prepared according to the above process was analyzed to have the following product composition: DP2=45.2%, DP3=37.6%, DP2+DP3=82.8%, and an average degree of polymerization of 2.4. Figure 1 As shown.

[0041] Example 2: Optimization of Eutectic Solvent Ratio

[0042] Following the method in Example 1, the following eutectic solvent ratios were used for the pretreatment of kelp powder:

[0043]

[0044] The remaining steps are the same as in Example 1, and the reconstituted solutions prepared by each treatment are subjected to subsequent enzymatic hydrolysis.

[0045]

[0046] Example 3: Optimization of water content in eutectic solvent

[0047] Following the method in Example 1, the following low eutectic solvent water contents were set for kelp powder pretreatment:

[0048]

[0049] The remaining steps are the same as in Example 1, and the reconstituted solutions prepared by each treatment are subjected to subsequent enzymatic hydrolysis.

[0050]

[0051] Example 4: Optimization of the ratio of kelp powder to eutectic solvent

[0052] Following the method of Example 1, with a fixed eutectic solvent ratio of choline chloride:lactic acid = 1:2 (molar ratio) and a water content of 10%, the following material-liquid ratios were set for kelp powder pretreatment:

[0053]

[0054] The remaining steps are the same as in Example 1.

[0055]

[0056] Example 5: Optimization of Enzymatic Hydrolysis Temperature

[0057] The kelp powder pretreatment solution (reconstituted to 100 mL) was prepared according to the method in Example 1. A crude alginate lyase AlgVS was added to bring the final enzyme activity concentration to 50 U / mL. The pH was adjusted to 8.5, and the enzymatic hydrolysis reaction was carried out at the following temperatures for 12 hours:

[0058]

[0059] The remaining steps are the same as in Example 1. After the enzymatic hydrolysis is completed, each treatment group is centrifuged at 8000 r / min for 30 min and the supernatant is collected.

[0060]

[0061] Example 6 Optimization of Enzymatic Hydrolysis Time

[0062] The kelp powder pretreatment solution (reconstituted to 100 mL) was prepared according to the method in Example 1. A crude alginate lyase AlgVS solution was added to bring the final enzyme activity concentration to 50 U / mL. The pH was adjusted to 8.5, and the solution was enzymatically hydrolyzed at 40°C for the following times:

[0063]

[0064] The remaining steps are the same as in Example 1. After the enzymatic hydrolysis is completed, each treatment group is centrifuged at 8000 r / min for 30 min and the supernatant is collected.

[0065]

[0066] Example 7 Enzymatic hydrolysis pH optimization

[0067] The kelp powder pretreatment solution (reconstituted to 100 mL) was prepared according to the method in Example 1. A crude alginate lyase AlgVS was added to bring the final enzyme activity concentration to 50 U / mL. The solution was then adjusted to the following pH values ​​at 40°C and enzymatically hydrolyzed for 12 hours:

[0068]

[0069] The remaining steps are the same as in Example 1. After the enzymatic hydrolysis is completed, each treatment group is centrifuged at 8000 r / min for 30 min and the supernatant is collected.

[0070]

[0071] Example 8 Enzyme Dosage Optimization

[0072] The kelp powder pretreatment solution (reconstituted to 100 mL) was prepared according to the method in Example 1. Alginate lyase AlgVS crude enzyme solution was added to the following final enzyme activity concentrations, and enzymatic hydrolysis was performed at 40°C and pH 8.5 for 12 hours:

[0073]

[0074] The remaining steps are the same as in Example 1. After the enzymatic hydrolysis is completed, each treatment group is centrifuged at 8000 r / min for 30 min and the supernatant is collected.

[0075]

[0076] Example 9: Application of treatment with brown algae bi and trisaccharide hydrolysate in rice germination

[0077] The supernatant of brown algae bisaccharide hydrolysis was prepared according to the method in Example 1.

[0078] The enzymatic hydrolysis supernatant was diluted to the following concentrations for use in rice germination experiments:

[0079]

[0080] Seed disinfection: Select plump, disease-free Nanjing 46 rice seeds, disinfect them with 10% sodium hypochlorite solution for 20 minutes, rinse them 5 times with sterile water, and air dry.

[0081] Seed soaking treatment: Disinfected rice seeds were placed in the respective treatment solutions and germinated in a 28℃ constant temperature and light incubator (light intensity 3000 lx, light / dark cycle 14 h / 10 h). Deionized water was used as a control (CK). The growth of rice on the 4th day of germination is shown in the figure. Figure 2 As shown, the root growth on day 6 is as follows: Figure 3 As shown.

[0082] Measurement indicators: Germination potential was measured on the 4th day after sowing, and germination rate was measured on the 10th day after sowing. (Germination potential: the percentage of rice seeds that germinated normally on the 4th day of germination; Germination rate: the percentage of rice seeds that ultimately germinated normally on the 10th day of germination.)

[0083]

[0084] Example 10: Application and Verification of Brown Algae Bis and Trisaccharide Hydrolysate in Promoting Rice Growth

[0085] The brown algae bi and trisaccharide hydrolysate was prepared according to the method in Example 1, diluted 200 times, and used for rice seedling cultivation.

[0086] (1) Seed disinfection: Select plump, disease-free Nanjing 46 rice seeds, disinfect them with 10% sodium hypochlorite solution for 20 minutes, rinse them with sterile water 5 times, and air dry them.

[0087] (2) Soaking and germination: Soak the disinfected seeds in deionized water for 48 hours and germinate them in a 28°C constant temperature dark incubator.

[0088] (3) Foliar spraying: Select rice seeds with uniform germination and transplant them into hydroponic boxes. Starting from the 10th day of hydroponics, spray the leaves with a 200-fold dilution of brown algae bi and trisaccharide hydrolysate (spray once every two days, just enough to keep the leaves moist). Grow in a constant temperature and light incubator at 28℃ (light intensity 3000 lx, light / dark cycle 14 h / 10 h). Deionized water was used as the control group.

[0089] (4) Evaluation of effects: Cultivation ended on day 17. Compared with the control group, the root length of the treatment group increased by 36.6%, the number of lateral roots increased by 68.5%, and the seedling length increased by 34.7% (e.g., ...). Figure 4 (As shown).

[0090] Comparative Example 1

[0091] No pretreatment was performed on the kelp powder. The kelp powder and pure water were mixed at a material-to-liquid ratio of 1:20 (g / mL), and crude alginate lyase AlgVS was added to a concentration of 50 U / mL. Enzymatic hydrolysis was carried out at 40℃ and pH 8.5 for 12 hours. After the enzymatic hydrolysis was completed, the mixture was centrifuged at 8000 r / min for 30 min, and the supernatant was collected to obtain the alginate bi / trisaccharide hydrolysate.

[0092] Comparative Example 2

[0093] Based on Example 1, the preparation of the eutectic solvent in step (1) is carried out using the following scheme, and the rest is the same as in Example 1.

[0094] Preparation of eutectic solvent: Choline chloride and glycerol are mixed at a molar ratio of 1:1, and the water content is adjusted to 10% (w / w). The mixture is stirred at 60°C for 30 minutes to form a homogeneous and transparent DES.

[0095] Comparative Example 3

[0096] Based on Example 1, the preparation of the eutectic solvent in step (1) is carried out using the following scheme, and the rest is the same as in Example 1.

[0097] Preparation of eutectic solvent: Choline chloride and glycerol are mixed at a molar ratio of 1:2, and the water content is adjusted to 10% (w / w). The mixture is stirred at 60°C for 30 minutes to form a homogeneous and transparent DES.

[0098] Comparative Example 4

[0099] Based on Example 1, the preparation of the eutectic solvent in step (1) is carried out using the following scheme, and the rest is the same as in Example 1.

[0100] Preparation of eutectic solvent: Choline chloride and glycerol are mixed at a molar ratio of 1:3, and the water content is adjusted to 10% (w / w). The mixture is stirred at 60°C for 30 minutes to form a homogeneous and transparent DES.

[0101] Comparative Example 5

[0102] Based on Example 1, the preparation of the eutectic solvent in step (1) is carried out using the following scheme, and the rest is the same as in Example 1.

[0103] Preparation of eutectic solvent: Choline chloride and urea are mixed at a molar ratio of 1:1, and the water content is adjusted to 10% (w / w). The mixture is stirred at 60°C for 30 minutes to form a uniform and transparent DES.

[0104] Comparative Example 6

[0105] Based on Example 1, the preparation of the eutectic solvent in step (1) is carried out using the following scheme, and the rest is the same as in Example 1.

[0106] Preparation of eutectic solvent: Choline chloride and urea are mixed at a molar ratio of 1:2, and the water content is adjusted to 10% (w / w). The mixture is stirred at 60°C for 30 minutes to form a uniform and transparent DES.

[0107] Comparative Example 7

[0108] Based on Example 1, the preparation of the eutectic solvent in step (1) is carried out using the following scheme, and the rest is the same as in Example 1.

[0109] Preparation of eutectic solvent: Choline chloride and urea are mixed at a molar ratio of 1:3, and the water content is adjusted to 10% (w / w). The mixture is stirred at 60°C for 30 minutes to form a uniform and transparent DES.

[0110] Table 1

[0111]

[0112] In Table 1, comparing Comparative Example 1 with Example 1, the DP2+DP3 content obtained in Example 1 was 82.8%, and the average degree of polymerization was 2.4, which was higher than that of Comparative Example 1, which had a DP2+DP3 content of 49.5% and an average degree of polymerization of 4.0. This indicates that the DES used in this method can selectively destroy the dense hydrogen bond crystal structure of alginate and directionally expose enzyme cleavage sites to achieve the directional enrichment of alginate and trisaccharides and the controllable reduction of the average degree of polymerization of the product, while also improving the germination-promoting activity.

[0113] In Comparative Examples 2-4, the choline chloride-glycerol system could not effectively disrupt the dense hydrogen bond crystal structure of alginate, and the products were mainly medium- to high molecular weight oligosaccharides, with limited germination-promoting activity.

[0114] In comparative examples 5-7, the choline chloride-urea system damaged the activity of alginate lyase, resulting in low enzymatic hydrolysis efficiency, high molecular weight polysaccharides as the main products, and low germination-promoting activity.

[0115] Compared with Comparative Examples 3 and 6, and the A2 treatment group in Example 2, the DP2+DP3 content obtained by the choline chloride-lactic acid system was 87.3%, and the average degree of polymerization was 2.6, which were higher than those of Comparative Examples 3 and 6. The germination potential was increased by 36.1%~54.2%, and the germination rate was increased by 36.0%~47.2%.

Claims

1. A method for preparing a seaweed enzymatic hydrolysate with high content of brown algae disaccharides and trisaccharides, comprising: (1) DES pretreatment: mix seaweed powder with choline chloride-lactic acid eutectic solvent at a solid-liquid ratio of 1:15~1:25 g / mL, stir and extract at 60~100℃ for 1~3 h, and collect the extract after solid-liquid separation; (2) Alcohol precipitation separation: Add 3 to 5 times the volume of anhydrous ethanol to the extract obtained in step (1), let it stand at low temperature for alcohol precipitation, collect the precipitate by centrifugation, and wash the precipitate with anhydrous ethanol. (3) Targeted enzymatic hydrolysis reaction: After reconstituted with buffer solution, alginate lyase was added and enzymatically hydrolyzed for 2-24 h at 25-55℃ and pH 6.0-10.

0. (4) Centrifugation purification: Centrifuge the enzymatic hydrolysate obtained in step (3) and retain the supernatant, which is the seaweed enzymatic hydrolysate with high content of brown algae bi and trisaccharides.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of choline chloride to lactic acid in the choline chloride-lactic acid eutectic solvent is 1:2, and the water content is 10~30%.

3. The preparation method according to claim 1, characterized in that, In step (2), the precipitate was allowed to stand at 4°C for 12 hours for alcohol precipitation, then centrifuged at 10,000 r / min for 20 minutes to collect the precipitate.

4. The preparation method according to claim 1, characterized in that, In step (3), the final concentration of the alginate lyase is 40-70 U / mL; the buffer solution is a PBS buffer solution with pH 6.0-10.

0.

5. The preparation method according to claim 1, characterized in that, In step (4), the centrifugation speed is 6000~12000 rpm and the centrifugation time is 20~30 minutes.

6. A seaweed enzymatic hydrolysate with high content of brown algae disaccharides and trisaccharides, characterized in that, The seaweed enzymatic hydrolysate is obtained by the preparation method according to any one of claims 1 to 5, wherein the active components are fucoidan 42-48 wt% and fucoidan trisaccharide 35-40 wt%.

7. The application of the seaweed enzymatic hydrolysate according to claim 6 in promoting rice growth, comprising: To promote rice seed germination: Place the sterilized clean rice seeds in the seaweed enzymatic hydrolysate diluted 50 to 1000 times and soak for 24 to 48 hours to promote germination. To promote rice seedling growth: Spray the leaves with the seaweed enzymatic hydrolysate diluted 150-250 times when the rice seedlings are in the two-leaf-one-heart stage.

8. The application according to claim 7, characterized in that, Promoting rice seedling growth includes increasing seedling length, root length, and the number of lateral roots.

9. The application according to claim 7, characterized in that, To obtain clean, disinfected rice seeds, use the following method: Select rice seeds that are uniform in size, round and plump, wash them 2-3 times with clean water, then soak them in a 1-10% sodium hypochlorite solution for 15-25 minutes for disinfection, and wash them 2-5 times with clean water to remove any residual solution.

10. The application according to claim 8, characterized in that, Seeds were soaked and germinated in a constant temperature and light incubator at 27~29℃ with a light intensity of 3000 lx and a light / dark cycle of 14 h / 10 h.

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