CN114989984B - Method for preparing seaweed extract by enzymolysis - Google Patents
Method for preparing seaweed extract by enzymolysis Download PDFInfo
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- CN114989984B CN114989984B CN202210878770.0A CN202210878770A CN114989984B CN 114989984 B CN114989984 B CN 114989984B CN 202210878770 A CN202210878770 A CN 202210878770A CN 114989984 B CN114989984 B CN 114989984B
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Abstract
The invention relates to the technical field of seaweed fertilizer preparation, in particular to a method for preparing seaweed extract by an enzymolysis method. The method specifically comprises the following steps: (1) Mixing and soaking seaweed with the mixed acid solution to obtain a material 1; (2) Carrying out ultrasonic treatment on the material 1 obtained in the step (1), and regulating the pH value to 6.8-7.6 to obtain a material 2; (3) And (3) mixing the material 2 obtained in the step (2) with enzyme liquid for enzymolysis, and inactivating enzyme. The invention firstly carries out preliminary destruction on seaweed cells by acid solution, then further destroys the seaweed cell walls by utilizing ultrasonic action, and then carries out enzymolysis by using complex enzyme, thus leading the seaweed enzymolysis to be complete and improving the content of active ingredients in seaweed extract.
Description
Technical Field
The invention relates to the technical field of seaweed fertilizer preparation, in particular to a method for preparing seaweed extract by an enzymolysis method.
Background
The traditional chemical fertilizer has single fertilizer efficiency and serious pollution, and can lead to the destruction of soil after long-term use. The seaweed fertilizer belongs to natural seaweed extract, is harmless to human and livestock, has no pollution to the environment, is listed as a special fertilizer for producing organic foods abroad, and is a natural, efficient and novel organic fertilizer.
The seaweed contains seaweed polysaccharide, phenols, multipolymer, mannitol, betaine, alginic acid, plant growth regulating substances, nitrogen, phosphorus, potassium, iron, boron, molybdenum, iodine and other elements, is not chemically synthesized, can greatly reduce the pollution to the environment after being used, and the agricultural products planted by the seaweed can reach the standard of green and nuisance free. Therefore, the seaweed fertilizer completely accords with the development trend of modern agriculture, has very wide market space and gradually replaces chemical fertilizers.
The method for extracting the seaweed extract comprises a physical method, a chemical method and a biological enzymolysis method, and a plurality of technologies for extracting the seaweed by using enzyme combination exist at present, such as patent number CN106834358A and the like, but the seaweed also contains a plurality of polysaccharides which are not subjected to enzymolysis due to the specificity of the enzyme, so that the problem of incomplete enzymolysis exists.
Therefore, how to provide a method for preparing seaweed extract by an enzymolysis method so as to solve the technical problem of incomplete hydrolysis of seaweed by an enzymatic method in the prior art is a problem which needs to be solved by a person skilled in the art.
Disclosure of Invention
The invention aims to provide a method for preparing seaweed extract by an enzymolysis method.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for preparing seaweed extract by an enzymolysis method, which comprises the following steps:
(1) Mixing and soaking seaweed with the mixed acid solution to obtain a material 1;
(2) Carrying out ultrasonic treatment on the material 1 obtained in the step (1), and regulating the pH value to 6.8-7.6 to obtain a material 2;
(3) And (3) mixing the material 2 obtained in the step (2) with enzyme liquid for enzymolysis, and inactivating enzyme.
Preferably, the power of the ultrasonic wave in the step (2) is 120-160W.
Preferably, the time of the ultrasonic treatment in the step (2) is 20-40 min.
Preferably, the mixed acid solution in the step (1) comprises hydrochloric acid, acetic acid and citric acid; the final concentration of hydrochloric acid in the mixed acid solution is 0.5-1.5 mol/L, the final concentration of acetic acid is 0.1-0.3 mol/L, and the final concentration of citric acid is 0.1-0.2 mol/L.
Preferably, the mass ratio of the seaweed to the mixed acid solution in the step (1) is 1:10 to 16.
Preferably, the soaking time in the step (1) is 3-5 hours.
Preferably, the temperature of the enzymolysis in the step (3) is 28-32 ℃.
Preferably, the enzymolysis time in the step (3) is 3-5 hours.
Preferably, in the step (3), the mass ratio of the material 3 to the enzyme solution is 60-80: 1.5 to 2.5.
Preferably, the enzyme solution of step (3) comprises alginic acid lyase, glucoamylase, cellulase and xylanase.
Preferably, in the step (3), the mass ratio of the alginic acid lyase to the glucoamylase to the cellulase to the xylanase is 22-32: 22-32: 18 to 22: 12-16.
Preferably, the temperature of the inactivation in the step (3) is 75-85 ℃.
Preferably, the time of the inactivation in the step (3) is 2-4 hours.
Compared with the prior art, the invention has the following beneficial effects:
1. the acid is added in the step (1) to primarily degrade the kelp, and the simple acid solution can influence the activity of the effective components dissolved after wall breaking.
According to the invention, the seaweed cells are subjected to wall breaking in an acid treatment and ultrasonic treatment mode, the seaweed cells are firstly subjected to preliminary damage by the acid solution, then the seaweed cell walls are further damaged by utilizing the ultrasonic action, and if the ultrasonic power is too high or too long, the loss of active substances can be caused, and if the ultrasonic power is too low or too short, the wall breaking is incomplete, so that the extraction rate is affected.
2. The traditional method for extracting seaweed extract only adds alginic acid lyase and cellulase, which can only act on 1-4-O-glycosidic bond, double bond is formed between C4 and C5 on six-membered ring, 4-O-glycosidic bond is eliminated, alginic acid is degraded, but seaweed has 1, 6-glycosidic bond, so the traditional enzymolysis method has the problem of incomplete glycosidic bond enzymolysis. The glucoamylase and the glucoamylase can simultaneously carry out enzymolysis on the 1, 6-glycosidic bond and the 1, 4-glycosidic bond. Thus, the alginic acid lyase and the glucoamylase can play a role in synergistic enzymolysis. The cellulase and xylanase have the function of synergistically decomposing cellulose.
Detailed Description
The invention provides a method for preparing seaweed extract by an enzymolysis method, which comprises the following steps:
(1) Mixing and soaking seaweed with the mixed acid solution to obtain a material 1;
(2) Carrying out ultrasonic treatment on the material 1 obtained in the step (1), and regulating the pH value to 6.8-7.6 to obtain a material 2;
(3) And (3) mixing the material 3 obtained in the step (2) with enzyme liquid for enzymolysis, and inactivating enzyme.
In the present invention, the step (2) is preferably to adjust the pH to 7.0 to 7.4; further preferably 7.2.
In the invention, the power of the ultrasonic wave in the step (2) is 120-160W; preferably 130 to 150W; further preferably 140W.
In the invention, the ultrasonic time in the step (2) is 20-40 min; preferably 24-36 min; further preferably 28 to 32 minutes; more preferably 30min.
In the invention, the mixed acid solution in the step (1) comprises hydrochloric acid, acetic acid and citric acid; the final concentration of hydrochloric acid in the mixed acid solution is 0.5-1.5 mol/L, the final concentration of acetic acid is 0.1-0.3 mol/L, and the final concentration of citric acid is 0.1-0.2 mol/L; preferably, the final concentration of hydrochloric acid is 0.7-1.3 mol/L, the final concentration of acetic acid is 0.2mol/L, and the final concentration of citric acid is 0.15mol/L; further preferably, the final concentration of hydrochloric acid is 0.9 to 1.1mol/L, the final concentration of acetic acid is 0.2mol/L, and the final concentration of citric acid is 0.15mol/L; more preferably, the final concentration of hydrochloric acid is 1mol/L, the final concentration of acetic acid is 0.2mol/L, and the final concentration of citric acid is 0.15mol/L
In the invention, the mass ratio of the seaweed to the mixed acid solution in the step (1) is 1:10 to 16; preferably 1: 11-15; further preferably 1: 12-14; more preferably 1:13.
in the invention, the soaking time in the step (1) is 3-5 hours; preferably 4h.
In the invention, the enzymolysis temperature in the step (3) is 28-32 ℃; preferably 29 to 31 ℃; further preferably 30 ℃.
In the invention, the enzymolysis time in the step (3) is 3-5 h; preferably 4h.
In the invention, the mass ratio of the material 2 to the enzyme solution in the step (3) is 60-80: 1.5 to 2.5; preferably 64 to 76:1.7 to 2.3; more preferably 68 to 72:1.9 to 2.1; more preferably 70:2.
in the present invention, the enzyme solution of step (3) includes alginic acid lyase, glucoamylase, cellulase and xylanase.
In the invention, the mass ratio of the alginic acid lyase to the glucoamylase to the cellulase to the xylanase in the step (3) is 22-32: 22-32: 18 to 22: 12-16; preferably 24 to 30: 24-30: 19 to 21: 13-15; more preferably 25 to 29:25 to 29:20:14; more preferably 27:27:20:14.
in the invention, the inactivating temperature in the step (3) is 75-85 ℃; preferably 77-83 ℃; further preferably 79 to 81 ℃; more preferably 80 ℃.
In the invention, the inactivation time in the step (3) is 2-4 h; preferably 3h.
In the invention, the seaweed is one of kelp, gulfweed, undaria pinnatifida, sargassum fusiforme, sargassum thunbergii, sargassum pallidum and goose palmate; preferably kelp.
The alginic acid lyase, glucoamylase, cellulase and xylanase used in the present invention were purchased from Hebei run Biotechnology Co.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The alginic acid lyase, glucoamylase, cellulase and xylanase used in the following examples were purchased from Hebei run Biotech Co.
Example 1
The invention provides a method for preparing seaweed extract by an enzymolysis method, which comprises the following steps:
(1) Mixing undaria pinnatifida and a mixed acid solution according to the mass ratio of 1:10, mixing and soaking for 3 hours to obtain a material 1;
(2) Carrying out ultrasonic treatment on the material 1 obtained in the step (1) at 120W for 20min, and regulating the pH value to 6.8 to obtain a material 2;
(3) And (3) mixing the material 2 obtained in the step (2) with enzyme solution according to the mass ratio of 60:1.5 mixing the materials and the enzymolysis solution at 28 ℃ for 3 hours, then heating the enzymolysis solution to 75 ℃, and stabilizing the temperature for 2 hours to inactivate enzymes;
the final concentration of hydrochloric acid in the mixed acid solution is 0.5mol/L, the final concentration of acetic acid is 0.1mol/L and the final concentration of citric acid is 0.1mol/L;
the enzyme solution comprises alginic acid lyase, glucoamylase, cellulase and xylanase; the mass ratio is 22:22:18:12.
(4) Cooling the enzymolysis liquid obtained in the step (3) to normal temperature, and centrifuging at 2000rpm for 15min to separate and remove residues.
Example 2
The invention provides a method for preparing seaweed extract by an enzymolysis method, which comprises the following steps:
(1) The method comprises the steps of mixing the amanita and the mixed acid solution according to the mass ratio of 1:16, mixing and soaking for 5 hours to obtain a material 1;
(2) Carrying out ultrasonic treatment on the material 1 obtained in the step (1) at 160W for 40min, and regulating the pH value to 7.6 to obtain a material 2;
(3) And (3) mixing the material 2 obtained in the step (2) with enzyme solution according to the mass ratio of 80:2.5 mixing the materials, carrying out enzymolysis for 5 hours at 32 ℃, then heating the enzymolysis liquid to 85 ℃, and stabilizing the temperature for 4 hours to inactivate enzymes;
the mixed acid solution has a final concentration of hydrochloric acid of 1.5mol/L, acetic acid of 0.3mol/L and citric acid of 0.2mol/L;
the enzyme solution comprises alginic acid lyase, glucoamylase, cellulase and xylanase; the mass ratio is 32:32:22:16.
(4) Cooling the enzymolysis liquid obtained in the step (3) to normal temperature, and then centrifuging at 3000rpm for 25min to separate and remove residues.
Example 3
The invention provides a method for preparing seaweed extract by an enzymolysis method, which comprises the following steps:
(1) The kelp and the mixed acid solution are mixed according to the mass ratio of 1:13, mixing and soaking for 4 hours to obtain a material 1;
(2) Carrying out ultrasonic treatment on the material 1 obtained in the step (1) at 140W for 30min, and regulating the pH value to 7.2 to obtain a material 2;
(3) And (3) mixing the material 2 obtained in the step (2) with enzyme solution according to the mass ratio of 70:2, mixing the materials and the enzymolysis solution at 30 ℃ for enzymolysis for 4 hours, heating the enzymolysis solution to 80 ℃, and stabilizing the temperature for 3 hours to inactivate enzymes;
the mixed acid solution has a final concentration of 1mol/L hydrochloric acid solution, a final concentration of 0.2mol/L acetic acid solution and a final concentration of 0.15mol/L citric acid solution;
the enzyme solution comprises alginic acid lyase, glucoamylase, cellulase and xylanase; the mass ratio is 27:27:20:14.
(4) Cooling the enzymolysis liquid obtained in the step (3) to normal temperature, and centrifuging at 2500rpm for 20min to separate and remove residues.
Experimental example 1
Comparative experiments, the procedure of example 3 was used as the experimental group.
Control group 1: the procedure of example 3 was followed with the following modifications: replacing the mixed acid solution with water of equal mass;
control group 2: the procedure of example 3 was followed with the following modifications: replacing the acetic acid solution and the citric acid solution in the mixed acid solution with water with equal mass;
control group 3: the procedure of example 3 was followed with the following modifications: replacing acetic acid solution in the mixed acid solution with water with equal mass;
control group 4: the procedure of example 3 was followed with the following modifications: replacing the citric acid solution in the mixed acid solution with water with equal mass;
control group 5: the procedure of example 3 was followed with the following modifications: no sonication was performed;
control group 6: the procedure of example 3 was followed with the following modifications: replacing the glucoamylase with equal mass of water;
control group 7: the procedure of example 3 was followed with the following modifications: replacing cellulase and xylanase with water with equal mass;
control group 8: the procedure of example 3 was followed with the following modifications: replacing alginic acid lyase with water of equal mass;
blank control group: the conventional seaweed extract preparation method (CN 101580852A, using its enzymatic hydrolysis, supernatant after centrifugation) was used.
Two parallel experiments are set up for each test group, the content of the effective components in the seaweed extract obtained by each test group is measured, and the average value is taken, and the results are shown in Table 1.
Table 1 content of active ingredient in each test group
As is clear from the contents shown in Table 1, the experimental groups contained the most of the active ingredients and contained rich phytohormone ingredients, which were significantly higher than those of the other experimental groups and the conventional methods.
From the data of the control groups 1 to 5, it is apparent that if the seaweed is not treated with the mixed acid solution first or only with a part of the acid solution, the extraction effect is affected, because the cell wall structure is stable, the simple ultrasonic wall breaking effect is poor, and the optimal wall breaking effect can be achieved only by the acid treatment and the ultrasonic treatment.
From the data of control groups 6-8, it can be seen that the use of glucoamylase has a promoting effect on the enzymatic hydrolysis, and the addition of cellulase and xylanase also affects the enzymatic hydrolysis. If the alginic acid lyase is not used, the complete enzymolysis function cannot be realized by the pure glucoamylase, the pure cellulase and the complete enzymolysis function cannot be realized by only using the mixed enzyme solution provided by the invention.
Experimental example 2
Comparative experiment, tomato seed germination experiment:
the seaweed extract obtained from each test group was diluted 20 times and then the tomato seeds were soaked with the dilution to seed ratio being 5 ml/min. Soaking at normal temperature for 3h, culturing in a constant temperature incubator at 25deg.C, supplementing distilled water for 1 time every day, testing for 15 days, collecting the soaked seeds as clear water groups, setting two groups of parallel experiments for each test group, calculating germination rate and germination vigor of tomato seeds, taking average value, and measuring the results shown in Table 2.
Table 2 germination test data for each test group
As can be seen from Table 2, the seaweed extract prepared by the method has obvious promotion effect on the germination of tomato seeds after dilution, and the data of a control group is obviously lower than that of an experimental group. The comparison of the data of the blank control group shows that the fertilizer effect of the seaweed extract prepared by the method is obviously superior to that of the seaweed extract prepared by the traditional method group, and the superiority of the method can be proved.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. A method for preparing seaweed extract by an enzymolysis method, which is characterized by comprising the following steps:
(1) Mixing and soaking seaweed with the mixed acid solution to obtain a material 1;
(2) Carrying out ultrasonic treatment on the material 1 obtained in the step (1), and regulating the pH value to 6.8-7.6 to obtain a material 2;
(3) Mixing the material 2 obtained in the step (2) with enzyme liquid for enzymolysis, and inactivating enzyme;
the power of the ultrasonic wave in the step (2) is 120-160W, and the time of the ultrasonic wave is 20-40 min;
the acid in the mixed acid solution in the step (1) consists of hydrochloric acid, acetic acid and citric acid; the final concentration of hydrochloric acid in the mixed acid solution is 0.5-1.5 mol/L, the final concentration of acetic acid is 0.1-0.3 mol/L, and the final concentration of citric acid is 0.1-0.2 mol/L;
the enzyme solution in the step (3) comprises alginic acid lyase, glucoamylase, cellulase and xylanase.
2. The method for preparing seaweed extract by the enzymolysis method according to claim 1, wherein the mass ratio of the seaweed to the mixed acid solution in the step (1) is 1:10 to 16.
3. The method for preparing seaweed extract of claim 1, wherein the soaking time in the step (1) is 3 to 5 hours.
4. The method for preparing seaweed extract by using the enzymolysis method according to claim 1, wherein the temperature of the enzymolysis in the step (3) is 28-32 ℃, and the time of the enzymolysis is 3-5 h.
5. The method for preparing seaweed extract by using the enzymolysis method as claimed in claim 1 or 4, wherein the mass ratio of the material 2 to the enzyme solution in the step (3) is 60-80: 1.5 to 2.5.
6. The method for preparing seaweed extract by using the enzymolysis method according to claim 1, wherein the mass ratio of the alginic acid lyase to the glucoamylase to the cellulase to the xylanase in the step (3) is 22-32: 22-32: 18 to 22: 12-16.
7. The method for preparing seaweed extract of claim 1, wherein the inactivation temperature in the step (3) is 75-85 ℃ and the time is 2-4 h.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2011012036A (en) * | 2009-07-06 | 2011-01-20 | Konan Kako Kk | Method for producing cellulose decomposed product from seaweed residue |
| CN113880612A (en) * | 2021-10-25 | 2022-01-04 | 中国农业大学烟台研究院 | Method for preparing seaweed biological organic liquid fertilizer, seaweed biological organic liquid fertilizer and application |
| CN114181009A (en) * | 2021-12-27 | 2022-03-15 | 广西源藻生物科技有限公司 | Method for preparing bio-organic fertilizer by degrading seaweed with complex enzyme |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011012036A (en) * | 2009-07-06 | 2011-01-20 | Konan Kako Kk | Method for producing cellulose decomposed product from seaweed residue |
| CN113880612A (en) * | 2021-10-25 | 2022-01-04 | 中国农业大学烟台研究院 | Method for preparing seaweed biological organic liquid fertilizer, seaweed biological organic liquid fertilizer and application |
| CN114181009A (en) * | 2021-12-27 | 2022-03-15 | 广西源藻生物科技有限公司 | Method for preparing bio-organic fertilizer by degrading seaweed with complex enzyme |
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