CN112167268A - Method for producing agricultural seaweed extract by physical and biological combination method and seaweed extract - Google Patents
Method for producing agricultural seaweed extract by physical and biological combination method and seaweed extract Download PDFInfo
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Abstract
The invention discloses a method for producing an agricultural seaweed extracting solution by a physical and biological combination method and the seaweed extracting solution. The invention comprises the following steps: 1) pretreatment: pulverizing dry Sargassum to 40-80 mesh to obtain Sargassum powder; 2) wall breaking: mixing the keep-alive agent with the seaweed powder, and physically breaking the wall, wherein the weight ratio of the seaweed powder to the keep-alive agent is 1:5-10 to obtain a mixture; 3) enzymolysis: adding alginate lyase into the mixture, wherein the enzymolysis temperature is 35-55 ℃, and the enzymolysis time is 4-8 h; 4) inactivation: inactivating at 90-100 deg.C for 5-10 min; 5) filtering, and packaging to obtain Sargassum extract. The seaweed extract has high extraction rate and low molecular weight of active ingredients, can be absorbed by plants, keeps the activity of plant growth regulators, can be stored for a long time, and has the effects of promoting plant growth, regulating soil ecological environment, improving fertilizer utilization rate, resisting biotic stress and abiotic stress and the like.
Description
Technical Field
The invention belongs to the technical field of plant extracts, and particularly relates to a method for producing an agricultural seaweed extract by a physical and biological combination method and the seaweed extract.
Background
The ocean covers about 71% of the surface area of the earth, and the land occupation ball occupies 97% of the total water, which is the largest resource treasure house on the earth. As the largest biological population in the ocean, seaweed contains a large amount of highly active biomass components including polysaccharides, proteins, mannitol, betaine, iodine, polyphenols, unsaturated fatty acids, sterol compounds, terpenoids, mineral elements, polyamines, plant growth regulators, vitamins, pigments and the like, has excellent biological activity, is widely used in various fields such as biomedicine, functional food, animal feed, cosmetics and the like, and has important application value in ecological fertilizers.
At present, the extraction method of the active ingredients of the seaweed mainly comprises a physical method, a chemical method and a biological method. The physical method comprises breaking cell wall by physical method (such as ultrasonic wave, microwave, freeze thawing, etc.), and extracting effective components in cells with cold water, wherein the physical method ensures that the active components are not destroyed, but has low extraction rate, and the active components such as polysaccharide have large molecular weight and are not easy to be absorbed by plants; the chemical method is that under the condition of certain temperature and pressure, the cell wall of the seaweed is dissolved and broken by acid, alkali and the like, and simultaneously, active ingredients with large molecular weight are degraded into effective ingredients with low molecular weight which can be absorbed by plants, but the method is easy to destroy the active ingredients of plant growth regulators; the biological method is to degrade the seaweed by enzyme generated by microbial degradation or microbial fermentation, although the biological method is fully extracted, the alginic acid in the seaweed can not be degraded by conventional cellulase, protease, saccharomycetes and the like, and the production process has strict control requirements and high cost. In addition, the seaweed extract is rich in nutrient components and is extremely easy to decay and deteriorate, especially under the high-temperature condition; moreover, plant growth regulator substances in the seaweed extract are gradually lost in the long-term storage process, for example, gibberellin GA3 is rearranged or isomerized in a water solution at 25 ℃ under the relatively mild condition, the activity of gibberellin GA3 is reduced more and more obviously along with the prolonging of the storage time, and the activity of the seaweed extract is lost, so that the using effect of the seaweed extract is influenced.
Disclosure of Invention
The invention aims to provide a method for producing an agricultural seaweed extract by a physical and biological combination method and the seaweed extract, and aims to solve the problems that the seaweed extract prepared by the prior art is extremely easy to decay and deteriorate, and the active ingredients can not be stored for a long time due to short storage time.
In order to solve the technical problems, the method is mainly realized by the following technical scheme:
in one aspect, the invention relates to a method for producing an agricultural seaweed extract by a physical and biological combination method, which comprises the following steps: 1) pretreatment: pulverizing dry Sargassum to 40-80 mesh to obtain Sargassum powder; 2) wall breaking: taking a keep-alive agent, uniformly mixing the seaweed powder obtained in the step 1) with the keep-alive agent, and carrying out physical wall breaking to obtain a mixture, wherein the weight ratio of the seaweed powder to the keep-alive agent is 1: 5-10; 3) enzymolysis: taking alginate lyase, adding 2-8 per mill of alginate lyase into the mixture obtained in the step 2) for enzymolysis, wherein the enzymolysis temperature is 35-55 ℃, and the enzymolysis time is 4-8 hours, so as to obtain an alga enzymolysis liquid; 4) inactivation: inactivating the seaweed enzymolysis liquid obtained in the step 3) at 90-100 ℃ for 5-10 min; 5) filtering, and packaging to obtain Sargassum extract.
In the production method of the seaweed extract, the keep-alive agent is added at the initial stage of extraction of the seaweed active ingredients, so that the reduction of the activity of substances such as a plant growth regulator is avoided, meanwhile, the seaweed is subjected to efficient wall breaking by adopting a physical method, and then, alginic acid lyase is adopted for enzymolysis, so that alginic acid with high molecular weight is degraded into low molecular weight alginate oligosaccharides which can be absorbed by plants; the production method of the invention has high extraction rate and low molecular weight of active ingredients, can be absorbed by plants, and can maintain the activity of plant growth regulator substances; the seaweed extract prepared by the method can be stored for a long time, and has the effects of promoting plant growth, regulating soil ecological environment, improving fertilizer utilization rate, resisting biotic stress and abiotic stress and the like. The addition amount of the alginate lyase is measured by volume, namely mL, and 2-8 per thousand of the alginate lyase is added into 1000g of the mixture, namely 2-8mL of the alginate lyase is added into the mixture.
In a preferred embodiment, in step 2), the keep-alive agent is any one of a phosphate buffer solution having a pH value of 5.8 to 7.0 and a concentration of 0.2mol/L, a citrate buffer solution having a pH value of 4.4 to 6.6, and a borate buffer solution having a pH value of 7.4 to 8.0. The keep-alive agent can be phosphate buffer solution, citrate buffer solution or borate buffer solution, is easy to prepare, can resist the influence of adding a small amount of acid or alkali outside, can maintain the pH value basically unchanged, has small influence of temperature on the pH value, and has small change of the pH value after dilution, for example, the change of the pH value after 10 times of dilution is less than 0.1; therefore, the activity of the active ingredients of the seaweed can be kept basically unchanged during the reaction process, and simultaneously, the activity of the enzyme can be kept basically unchanged during the reaction process.
As a preferred embodiment, the alginate lyase comprises the following components: 1, 4-beta-D-mannuronic acid lyase, the enzyme activity is 20000-30000U/mL; 1, 4-alpha-L-guluronic acid lyase with the enzyme activity of 20000-30000U/mL; polymannuronic acid and polyguluronic acid lyase, the enzyme activity is 25000-35000U/mL. In the alginate lyase, 1, 4-beta-D-mannuronic acid lyase has specificity to polymannuronic acid (PolyM), 1, 4-alpha-L-guluronic acid lyase has specificity to polyguluronic acid (PolyG), and polymannuronic acid and polyguluronic acid lyase have specificity to both PolyM and PolyG, namely the alginate lyase has substrate specificity to PolyM, PolyG and PolyMG.
As a preferred embodiment, the enzymatic activity of the alginate lyase is 20000-35000U/mL. The alginate lyase can be a complex enzyme generated by a complex strain which is screened and separated from seaweed compost and has alginate degradation capability, can cleave 4-O-glycosyl bonds of alginic acid through beta elimination reaction, simultaneously forms double bonds between C-4 and C-5, generates 4-deoxy-L-erythro-hex-4-enepyranosyluronate at the non-reducing end of the generated oligosaccharide, and has a strong absorption peak at 230-240 nm; the speed of catalyzing the degradation of the macromolecular alginic acid into the micromolecular alginic acid is high, the dosage is small, and the method is economical and applicable.
In a preferred embodiment, the dried seaweed is air-dried seaweed, and the moisture content of the seaweed is not more than 10%. The air-dried seaweed is preferably selected, the water content of the seaweed cannot be too high, otherwise, seaweed powder is not easy to form; generally, the pretreatment process of the present invention is carried out by pulverizing the dried seaweed using a pulverizer.
In a preferred embodiment, the seaweed is any one or more of Ascophyllum nodosum, Macrocystis japonica, Laminaria japonica, Sargassum fulvum, Undaria pinnatifida, Cyrtymenia Sparsa, Surgassum thunbergii, Sargassum pallidum, Fucus vesiculosus, Coccinum, Gymnema sylvestre, and Hordeum japonicum. The seaweed has various varieties, can be selected according to actual conditions, has high yield and is convenient to purchase; moreover, the seaweeds are brown seaweeds, belong to large individual multicellular seaweeds, are fast in growth and high in yield, can generate 3.3-11.3 kilograms of biomass in dry weight per square meter of sea surface every year, are low in production cost, and are important raw materials for producing alginic acid.
As a preferred embodiment, the physical wall breaking method in step 2) is any one or more of a high-speed shearing method, a high-pressure homogenization method, an ultrasonic method and a sanding method. The invention can select various methods to carry out physical wall breaking, and the method has simple operation and is easy to realize industrialization. After the physical wall breaking is carried out by the methods, the method has short extraction time and high extraction efficiency, keeps the natural structure of the bioactive substances in the raw materials to the maximum extent, avoids the problems of denaturation, loss, activity reduction and the like caused by heat effect and the like, has simple process operation, and is suitable for large-scale and modern production.
As a preferred embodiment, the high speed shearing method has a shearing rotation speed of 10000-; the homogenization pressure of the high-pressure homogenization method is 60-170MPa, and the homogenization time is 15-30 min; the ultrasonic power of the ultrasonic method is 200-600W, and the ultrasonic time is 10-30 min; the grinding time of the sand grinding method is 60-90 min. The invention can control the physical wall breaking parameters of high-speed shearing method, high-pressure homogenizing method, ultrasonic method and sand grinding method, and has easy control and convenient operation.
In a preferred embodiment, in the step 5), the inactivated enzymolysis solution is filtered by a plate and frame filter press, and then is subjected to ultrafiltration by using a ceramic membrane with the particle size of 100-200 nm. The inactivated enzymolysis liquid is firstly subjected to rough filtration and then to ultrafiltration, so that impurities are effectively filtered, and the effective components of the obtained seaweed extract are fully ensured; the filtering method has the advantages of simple equipment, convenient operation, no phase change, no damage to active ingredients, low energy consumption, high separation efficiency, continuous production and no pollution, and can be used for filtering at normal temperature.
In another aspect, the seaweed extract is prepared by the method for producing the agricultural seaweed extract according to any one of the above physical and biological combination methods. The seaweed extract has low molecular weight of active ingredients, can be absorbed by plants, keeps the activity of plant growth regulators, can be stored for a long time, and has the effects of promoting plant growth, regulating soil ecological environment, improving fertilizer utilization rate, resisting biotic stress and abiotic stress and the like.
Compared with the prior art, the invention has the beneficial effects that: in the production method of the seaweed extract, the keep-alive agent is added at the initial stage of extraction of seaweed active ingredients, so that the reduction of the activity of substances such as a plant growth regulator is avoided, meanwhile, the seaweed is subjected to efficient wall breaking by adopting a physical method, and then, alginic acid lyase is adopted for enzymolysis, so that alginic acid with high molecular weight is degraded into low molecular weight alginate oligosaccharides which can be absorbed by plants; the production method of the invention has high extraction rate and low molecular weight of active ingredients, can be absorbed by plants, and can maintain the activity of plant growth regulator substances; the seaweed extract prepared by the method can be stored for a long time, and has the effects of promoting plant growth, regulating soil ecological environment, improving fertilizer utilization rate, resisting biotic stress and abiotic stress and the like.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to a method for producing an agricultural seaweed extracting solution by a physical and biological combined method, which comprises the following steps:
1) pretreatment: pulverizing dry Sargassum to 40-80 mesh to obtain Sargassum powder;
2) wall breaking: taking a keep-alive agent, uniformly mixing the seaweed powder obtained in the step 1) with the keep-alive agent, and carrying out physical wall breaking to obtain a mixture, wherein the weight ratio of the seaweed powder to the keep-alive agent is 1: 5-10;
3) enzymolysis: taking alginate lyase, adding 2-8 per mill of alginate lyase into the mixture obtained in the step 2) for enzymolysis, wherein the enzymolysis temperature is 35-55 ℃, and the enzymolysis time is 4-8 hours, so as to obtain an alga enzymolysis liquid;
4) inactivation: inactivating the seaweed enzymolysis liquid obtained in the step 3) at 90-100 ℃ for 5-10 min;
5) filtering, and packaging to obtain Sargassum extract.
Preferably, in the step 2), the keep-alive agent is any one of a phosphate buffer solution with a pH value of 5.8-7.0 and a concentration of 0.2mol/L, a citrate buffer solution with a pH value of 4.4-6.6, and a borate buffer solution with a pH value of 7.4-8.0.
Further, the alginate lyase comprises the following components: 1, 4-beta-D-mannuronic acid lyase, the enzyme activity is 20000-30000U/mL; 1, 4-alpha-L-guluronic acid lyase with the enzyme activity of 20000-30000U/mL; polymannuronic acid and polyguluronic acid lyase, the enzyme activity is 25000-35000U/mL.
Specifically, the enzyme activity of the alginate lyase is 20000-35000U/mL.
More preferably, the dried seaweed is air-dried seaweed, and the moisture content of the seaweed is not more than 10%.
Furthermore, the seaweed is one or more of Ascophyllum nodosum, Macrocystis, Laminaria japonica, Sargassum, Laminaria japonica, Hizikia fusiforme, Sargassum thunbergii, Sargassum pallidum, Fucus vesiculosus, Botrytis cinerea, Gymnema sylvestre, and Hordeum japonicum.
More specifically, the physical wall breaking method in the step 2) is any one or more of a high-speed shearing method, a high-pressure homogenizing method, an ultrasonic method and a sanding method.
Preferably, the shearing rotating speed of the high-speed shearing method is 10000-; the homogenization pressure of the high-pressure homogenization method is 60-170MPa, and the homogenization time is 15-30 min; the ultrasonic power of the ultrasonic method is 200-600W, and the ultrasonic time is 10-30 min; the grinding time of the sand grinding method is 60-90 min.
Furthermore, in the step 5), the filtration is to filter the inactivated enzymolysis solution by using a plate and frame filter press, and then to perform ultrafiltration by using a ceramic membrane with the particle size of 100-.
The seaweed extract is prepared by the method for producing the agricultural seaweed extract by the physical and biological combination method.
Example one
The invention relates to a method for producing an agricultural seaweed extracting solution by a physical and biological combined method, which comprises the following steps:
1) pretreatment: taking dry giant kelp, and crushing the giant kelp into 40 meshes to obtain giant kelp powder;
2) wall breaking: taking a keep-alive agent, sequentially adding 10Kg of the kelp powder obtained in the step 1) and 50Kg of the keep-alive agent into a reaction kettle, opening and stirring, uniformly mixing, carrying out high-pressure homogenization treatment for 20min at the homogenization pressure of 80MPa, and carrying out physical wall breaking to obtain a mixture;
the keep-alive agent is phosphate buffer solution with pH value of 6.6 and concentration of 0.2mol/L, and the preparation method comprises the following steps:
a)0.2mol/L NaH2PO4preparation of the solution: weighing NaH2PO4·2H2O31.2 g or NaH2PO4·H2Adding 27.6g of O, dissolving in water, and fixing the volume to 1000 mL;
b)0.2mol/L Na2HPO4preparation of the solution: weighing Na2HPO4·12H2O71.632 g or Na2HPO4·7H2O53.6 g or Na2HPO4·2H235.6g of O, adding water to dissolve, and fixing the volume to 1000 mL;
c) taking 0.2mol/L NaH obtained in the step a)2PO462.5mL and 0.2mol/L Na from step b)2HPO437.5mL, and uniformly mixing to obtain a phosphate buffer solution;
3) enzymolysis: adding 320mL of alginate lyase into the mixture obtained in the step 2) for enzymolysis, wherein the enzymolysis temperature is 55 ℃, and the enzymolysis time is 4 hours, so as to obtain seaweed enzymolysis liquid;
4) inactivation: inactivating the seaweed enzymolysis liquid obtained in the step 3), wherein the inactivation temperature is 90 ℃, and the inactivation time is 10 min;
5) filtering, namely filtering the inactivated enzymolysis liquid in the step 4) by using a plate-and-frame filter press, then performing ultrafiltration by using a 100nm ceramic membrane, and subpackaging to obtain the seaweed extract.
Example two
The invention relates to a method for producing an agricultural seaweed extracting solution by a physical and biological combined method, which comprises the following steps:
1) pretreatment: taking dry phyllocladium nodosum, and crushing the dry phyllocladium nodosum to 60 meshes by using a crusher to obtain phyllocladium nodosum powder;
2) wall breaking: taking a keep-alive agent, sequentially adding 10Kg of the Ascophyllum nodosum powder obtained in the step 1) and 60Kg of the keep-alive agent into a reaction kettle, opening and stirring, uniformly mixing, performing ultrasonic treatment for 20min at the ultrasonic power of 400W, and performing physical wall breaking to obtain a mixture;
the keep-alive agent is borate buffer solution with the pH value of 7.4, and the preparation method comprises the following steps:
a) preparing 0.05mol/L borax solution: accurately weighing 19.07g of borax, dissolving the borax by using distilled water, and fixing the volume to 1000 mL;
b) preparation of 0.2mol/L boric acid: accurately weighing 12.37g of boric acid, dissolving the boric acid by using distilled water, and fixing the volume to 1000 mL;
c) uniformly mixing 10.0mL of 0.05mol/L borax solution obtained in the step a) and 90.0mL of 0.2mol/L boric acid obtained in the step b) to obtain a borate buffer solution;
3) enzymolysis: adding 560mL of alginate lyase into the mixture obtained in the step 2) for enzymolysis, wherein the enzymolysis temperature is 45 ℃ and the enzymolysis time is 6h, so as to obtain an alga enzymolysis liquid;
4) inactivation: inactivating the seaweed enzymolysis liquid obtained in the step 3), wherein the inactivation temperature is 90 ℃, and the inactivation time is 10 min;
5) filtering, namely filtering the inactivated enzymolysis liquid in the step 4) by using a plate-and-frame filter press, then performing ultrafiltration by using a 100nm ceramic membrane, and subpackaging to obtain the seaweed extract.
EXAMPLE III
The invention relates to a method for producing an agricultural seaweed extracting solution by a physical and biological combined method, which comprises the following steps:
1) pretreatment: taking dry kelp, and crushing the kelp into 80 meshes by using a crusher to obtain kelp powder;
2) wall breaking: taking a keep-alive agent, sequentially adding 10Kg of kelp powder obtained in the step 1) and 70Kg of the keep-alive agent into a reaction kettle, opening and stirring, uniformly mixing, shearing for 10min at the shearing rotating speed of 13000r/min, and carrying out physical wall breaking to obtain a mixture;
the keep-alive agent is a citrate buffer solution with the pH value of 4.8, and the preparation method comprises the following steps:
a)0.2mol/L Na2HPO4the preparation of (1): weighing Na2HPO4·12H2O71.632 g or Na2HPO4·7H2O53.6 g or Na2HPO4·2H235.6g of O, adding water to dissolve, and fixing the volume to 1000 mL;
b) preparation of 0.1mol/L citric acid solution: weighing C6H8O7·H2O21.014 g, adding water to dissolve, and fixing the volume to 1000 mL;
c) taking 0.2mol/L of Na obtained in the step a)2HPO49.86mL and 10.14mL of the 0.1mol/L citric acid solution obtained in the step b) are mixed uniformly to obtain a citrate buffer solution;
3) enzymolysis: adding 160mL of alginate lyase which is a complex enzyme generated by a complex strain with alginic acid degradation capability screened and separated from seaweed compost into the mixture obtained in the step 2) for enzymolysis at 40 ℃ for 4 hours to obtain seaweed enzymatic hydrolysate;
4) inactivation: inactivating the seaweed enzymolysis liquid obtained in the step 3), wherein the inactivation temperature is 90 ℃, and the inactivation time is 10 min;
5) filtering, namely filtering the inactivated enzymolysis liquid in the step 4) by using a plate-and-frame filter press, then performing ultrafiltration by using a 200nm ceramic membrane, and subpackaging to obtain the seaweed extract.
Example four
The invention relates to a method for producing an agricultural seaweed extracting solution by a physical and biological combined method, which comprises the following steps:
1) pretreatment: pulverizing sargassum to 80 mesh with a pulverizer to obtain sargassum powder;
2) wall breaking: taking a keep-alive agent, sequentially adding 10Kg of sargassum powder obtained in the step 1) and 100Kg of the keep-alive agent into a reaction kettle, opening and stirring, uniformly mixing, and physically breaking the walls by a sand grinding method for 80min to obtain a mixture;
the keep-alive agent is a citrate buffer solution with the pH value of 4.4, and the preparation method comprises the following steps:
a)0.2mol/L Na2HPO4the preparation of (1): weighing Na2HPO4·12H2O71.632 g or Na2HPO4·7H2O53.6 g or Na2HPO4·2H235.6g of O, adding water to dissolve, and fixing the volume to 1000 mL;
b) preparation of 0.1mol/L citric acid solution: weighing C6H8O7·H2O21.014 g, adding water to dissolve, and fixing the volume to 1000 mL;
c) taking 0.2mol/L of Na obtained in the step a)2HPO48.82mL and 11.18mL of the 0.1mol/L citric acid solution obtained in the step b) are mixed uniformly to obtain a citrate buffer solution;
3) enzymolysis: adding 400mL of alginate lyase into the mixture obtained in the step 2) for enzymolysis, wherein the enzymolysis temperature is 35 ℃, and the enzymolysis time is 8 hours, so as to obtain an alga enzymolysis liquid;
4) inactivation: inactivating the seaweed enzymolysis liquid obtained in the step 3) at the temperature of 100 ℃ for 5 min;
5) filtering, namely filtering the inactivated enzymolysis liquid in the step 4) by using a plate-and-frame filter press, then performing ultrafiltration by using a 200nm ceramic membrane, and subpackaging to obtain the seaweed extract.
Experiment one
TABLE 1 results of storage property test of different seaweed extracts
TABLE 2 results of storage property test of different seaweed extracts
Respectively carrying out storage performance test experiments on four seaweed extracting solutions obtained in the first to fourth embodiments of the invention, an imported seaweed extracting solution (a comparison sample I) and a domestic seaweed extracting solution (a comparison sample II), detecting the content of alginic acid by using a m-hydroxybiphenyl method specified in NY/T3174-2017, detecting the content of seaweed polysaccharide by using a phenol-sulfuric acid method, detecting the contents of mannitol, auxin, cytokinin, gibberellin and brassinolide by using a liquid chromatography-mass spectrometry method, and detecting the content of polyamine by using a high performance liquid chromatography; storing the Sargassum extractive solution in the open air for 6 months (2018, 9, 1 day) after the preparation (2018, 3, 1 day), and periodically detecting the above indexes, and respectively recording the detection results of 1 month, 3 months and 6 months in tables 1 and 2.
As can be seen from tables 1 and 2, the contents of alginic acid, algal polysaccharides and mannitol in four seaweed extracts obtained in the first to fourth examples of the present invention did not change significantly during 6 months of storage, indicating that the seaweed extracts prepared in the first to fourth examples of the present invention did not spoil; furthermore, the contents of auxin, cytokinin, gibberellin, brassinolide and polyamine in the seaweed extract solutions prepared in the first to fourth embodiments of the present invention were not significantly changed during 6 months of storage, which indicates that the auxin, cytokinin, gibberellin, brassinolide and polyamine in the seaweed extract solutions prepared in the first to fourth embodiments of the present invention were not lost during storage, and the activities of various physiologically active substances were maintained for a long period of time, thereby promoting the growth and development of plants. However, during the storage process of 6 months, the contents of alginic acid, algal polysaccharides and mannitol of certain imported seaweed solution, namely the control sample I and the seaweed solution produced in certain country, namely the control sample II, all have obvious reduction trends, wherein the reduction range of the mannitol content of the control sample I is the largest, and the reduction range of the alginic acid content of the control sample II is the largest; the result shows that the seaweed extract of the control sample I and the seaweed extract of the control sample II are both rotten to a certain degree; in addition, during the storage process of 6 months, the contents of auxin, cytokinin, gibberellin, brassinolide and polyamine also appear in certain imported seaweed liquid, namely a control sample I and seaweed liquid produced in certain country, namely a control sample II, and particularly, the reduction range of the brassinolide of the control sample I is the largest, and the reduction range of the gibberellin of the control sample II is the largest; this indicates that auxin, cytokinin, gibberellin, brassinolide and polyamine in the seaweed extracts of the control sample I and the control sample II are all obviously lost in the storage process, and the activities of various physiologically active substances cannot be maintained for a long time.
TABLE 3 extraction yield results
In addition, the extraction rate was calculated from the alginic acid content of the seaweed extract and the alginic acid content of the seaweed powder itself used, and the alginic acid content of the four seaweed extract obtained in the first to fourth examples of the present invention and the alginic acid content of the seaweed powder itself used and the extraction rate are shown in table 3.
As can be seen from table 3, the extraction rate of alginic acid obtained in example one of the present invention is 94.78%, the extraction rate of alginic acid obtained in example two is 95.16%, the extraction rate of alginic acid obtained in example three is 97.15%, and the extraction rate of alginic acid obtained in example four is 97.12%, which is much higher than the extraction rate of alginic acid obtained by a common physical method (30% to 50%), so that the present invention greatly improves the extraction rate of alginic acid, and reduces resource consumption and production cost.
Experiment two
The four seaweed extracts obtained in the first to fourth embodiments of the present invention, an imported seaweed extract (control one) and a domestic seaweed extract (control two) were subjected to a corn rooting test, a rape seedling growth test and a tomato flowering and fruiting test, respectively.
Corn rooting experiment
The method is carried out in seedling raising pots in a laboratory of Qingdao agricultural university, wherein 20 pots are used for each pot, and the number of pots is 18;
the experiment was set up with 6 treatments: respectively diluting four parts of seaweed extract, imported seaweed extract (a comparison sample I) and domestic seaweed extract (a comparison sample II) obtained in the first to fourth embodiments of the invention by 300 times, soaking seeds for 10 hours by using diluent, taking out after 10 hours, airing the seeds, and transplanting the seeds into a seedling pot filled with a seedling substrate to grow for 10 days; the root length, surface area, fresh weight and dry weight of the root system after the corn growth were measured and are shown in table 4.
TABLE 4 corn growth assay results
| Seaweed extract | Root length (cm) | Surface area (cm)2) | Fresh weight (g) | Dry weight (g) |
| Example one | 28.46 | 108.17 | 2.87 | 0.23 |
| Example two | 27.98 | 106.33 | 2.69 | 0.21 |
| EXAMPLE III | 27.69 | 105.90 | 2.83 | 0.22 |
| Example four | 28.81 | 106.50 | 2.75 | 0.22 |
| Comparison sample one | 21.89 | 93.37 | 1.75 | 0.14 |
| Control 2 | 22.02 | 93.03 | 1.80 | 0.15 |
As can be seen from Table 4, the root length of the corn seedlings treated by the seaweed extract prepared in the first to fourth examples of the present invention is 27-29cm, the root length of the corn seedlings treated by the first and second control samples is about 22cm, and the root length of the corn seedlings treated by the seaweed extract prepared in the first to fourth examples of the present invention is significantly longer than the root length of the corn seedlings treated by the first and second control samples. The root surface area of the corn seedling treated by the seaweed extract prepared in the first to fourth embodiments of the invention is 105-109cm2The surface areas of the root systems of the corn seedlings treated by the first control sample and the second control sample are both 93cm2The root surface area of the corn seedlings treated by the seaweed extracting solution prepared in the first to fourth embodiments of the invention is obviously larger than that of the corn seedlings treated by the first and second control samples. The fresh weight of the corn seedlings treated by the seaweed extract prepared in the first to fourth embodiments of the invention is 2.7-2.9g, the fresh weight of the corn seedlings treated by the first and second control samples is only about 1.8g, and the fresh weight of the corn seedlings treated by the seaweed extract prepared in the first to fourth embodiments of the invention is obviously greater than that of the corn seedlings treated by the first and second control samples. The dry weight of the corn seedlings treated by the seaweed extract prepared in the first to fourth embodiments of the invention is 0.21-0.23g, the dry weight of the corn seedlings treated by the first and second control samples is only about 0.15g, and the dry weight of the corn seedlings treated by the seaweed extract prepared in the first to fourth embodiments of the invention is also obviously greater than that of the corn seedlings treated by the first and second control samples. This shows that the seaweed extract prepared in the first to fourth embodiments of the present invention has a significant effect of promoting corn seedling rooting and root growth compared to the first and second control samples, and is beneficial to the corn seedling to absorb nutrients.
Second, rape seedling growth experiment
The method is carried out in a test field of agricultural science research institute in Qingdao city, and the area of a test cell is 60m2。
The experiment was set up with 6 treatments: when the rape seedlings grow to 3 leaves and 1 heart, the seaweed extract prepared in the first to fourth examples of the invention, an imported seaweed solution (a control sample I) and a domestic seaweed solution (a control sample II) are respectively diluted by 300 times, and root irrigation is carried out, and the seaweed extract, the imported seaweed solution and the domestic seaweed solution are applied once every 15 days and are used for 3 times in total. The rape seedling growth results, including plant height, leaf area, chlorophyll value and fresh weight, were recorded and the test results are included in table 5.
TABLE 5 rape growth assay results
| Seaweed extract | Plant height (cm) | Leaf area (cm)2) | Chlorophyll value | Fresh weight (g) |
| Example one | 24.35 | 257.21 | 53.37 | 90.56 |
| Example two | 24.12 | 249.92 | 52.99 | 88.65 |
| EXAMPLE III | 23.88 | 247.78 | 52.31 | 88.14 |
| Example four | 23.70 | 255.39 | 53.12 | 89.73 |
| Comparison sample one | 20.97 | 209.18 | 45.87 | 71.89 |
| Control 2 | 20.67 | 207.93 | 45.96 | 72.23 |
As can be seen from Table 5, the plant heights of the rape seedlings treated by the seaweed extract solutions prepared in the first to fourth examples of the present invention were about 24cm, the plant heights of the rape seedlings treated by the first and second control samples were only about 21cm, and the plant heights of the rape seedlings treated by the seaweed extract solutions prepared in the first to fourth examples of the present invention were significantly greater than the plant heights of the rape seedlings treated by the first and second control samples. The total area of the leaves of the rape seedlings treated by the seaweed extract prepared in the first to fourth embodiments of the invention, namely the leaf area, is 250cm2On the left and right sides, the total area of the leaves of the rape seedlings treated by the control sample I and the control sample II, namely the area of the leaves, is only 208cm2In the embodiments I to IV, the total leaf area of the rape seedlings treated by the seaweed extract prepared in the embodiments I to IV is obviously larger than the total leaf area of the rape seedlings treated by the control sample I and the control sample IIThe area is the leaf area. The chlorophyll value of the rape seedlings treated by the seaweed extract prepared in the first to fourth embodiments of the invention is about 53, the chlorophyll value of the rape seedlings treated by the first and second control samples is only about 46, and the chlorophyll value of the rape seedlings treated by the seaweed extract prepared in the first to fourth embodiments of the invention is obviously greater than that of the rape seedlings treated by the first and second control samples. The fresh weight of the rape seedlings treated by the seaweed extract prepared in the first to fourth embodiments of the invention is about 89g, the fresh weight of the rape seedlings treated by the first and second control samples is only about 72g, and the fresh weight of the rape seedlings treated by the seaweed extract prepared in the first to fourth embodiments of the invention is obviously greater than that of the rape seedlings treated by the first and second control samples. This shows that the seaweed extract prepared in the first to fourth examples of the present invention has a significant effect of promoting the growth of rape seedlings as compared with the control sample one and the control sample two.
Third, tomato blossom and fruit test
The test is carried out in the test field of agricultural science research institute in Qingdao city, and the area of the test cell is 120m2。
The experiment was set up with 6 treatments: the seaweed extract, an imported seaweed solution (control one) and a seaweed solution produced in a country (control two) prepared in the first to fourth examples of the present invention were diluted 750 times, respectively, subjected to leaf spraying treatment before flowering, during fruit setting and during fruit expansion, and the number of flowers, number of fruits, fruit setting rate and single fruit weight during tomato growth were recorded, respectively, and the soluble solid content and the vitamin C content at fresh weight in tomato fruits were measured, and the test results are shown in table 6.
TABLE 6 tomato growth assay results
As can be seen from Table 6, the number of flowers in the flowering period of the tomatoes is basically 32, and after the seaweed extract prepared in the first to fourth examples of the present invention, the imported seaweed solution (control one) and the seaweed solution produced in a country (control two) are subjected to leaf spray treatment before flowering, at the fruit setting period and at the fruit expanding period, the number of fruits of the tomatoes treated with the seaweed extract prepared in the first to fourth examples of the present invention is about 24, the number of fruits of the tomatoes treated with the control one and the control two is only 21, and the number of fruits of the tomatoes treated with the seaweed extract prepared in the first to fourth examples of the present invention is greater than the number of fruits of the tomatoes treated with the control one and the control two. The tomato treated by the seaweed extract prepared in the first to fourth embodiments of the present invention has a fruit setting rate of 74%, the tomato treated by the first and second control samples has a fruit setting rate of only 67%, and the tomato treated by the seaweed extract prepared in the first to fourth embodiments of the present invention has a fruit setting rate significantly higher than that of the tomato treated by the first and second control samples. The weight of the single fruit of the tomato treated by the seaweed extract prepared in the first to fourth examples of the present invention is about 258g, the weight of the single fruit of the tomato treated by the first and second control samples is only about 220g, and the weight of the single fruit of the tomato treated by the seaweed extract prepared in the first to fourth examples of the present invention is greater than that of the tomato treated by the first and second control samples. The content of soluble solids in the tomato fruits treated by the seaweed extract prepared in the first to fourth embodiments of the present invention is about 4.50%, the content of soluble solids in the tomato fruits treated by the first and second control samples is only about 4.00%, and the content of soluble solids in the tomato fruits treated by the seaweed extract prepared in the first to fourth embodiments of the present invention is greater than the content of soluble solids in the tomato fruits treated by the first and second control samples. The content of the vitamin C in the tomato fruits treated by the seaweed extract prepared in the first to fourth embodiments of the invention is more than 155mg/Kg in fresh weight, the content of the vitamin C in the tomato fruits treated by the first and second control samples is only about 118mg/Kg in fresh weight, and the content of the vitamin C in the tomato fruits treated by the seaweed extract prepared in the first to fourth embodiments of the invention is obviously more than the content of the vitamin C in the tomato fruits treated by the first and second control samples in fresh weight. The results show that the seaweed extract prepared in the first to fourth embodiments of the present invention can effectively increase the fruit setting rate of tomatoes, promote fruit expansion, increase fruit yield, and significantly increase the content of soluble solids and vitamin C in the tomato fruits and significantly improve the tomato quality compared with the control sample one and the control sample two.
Therefore, compared with the prior art, the invention has the beneficial effects that: in the production method of the seaweed extract, the keep-alive agent is added at the initial stage of extraction of seaweed active ingredients, so that the reduction of the activity of substances such as a plant growth regulator is avoided, meanwhile, the seaweed is subjected to efficient wall breaking by adopting a physical method, and then, alginic acid lyase is adopted for enzymolysis, so that alginic acid with high molecular weight is degraded into low molecular weight alginate oligosaccharides which can be absorbed by plants; the production method of the invention has high extraction rate and low molecular weight of active ingredients, can be absorbed by plants, and can maintain the activity of plant growth regulator substances; the seaweed extract prepared by the method can be stored for a long time, and has the effects of promoting plant growth, regulating soil ecological environment, improving fertilizer utilization rate, resisting biotic stress and abiotic stress and the like.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A method for producing an agricultural seaweed extracting solution by a physical and biological combined method is characterized by comprising the following steps: the method comprises the following steps:
1) pretreatment: pulverizing dry Sargassum to 40-80 mesh to obtain Sargassum powder;
2) wall breaking: taking a keep-alive agent, uniformly mixing the seaweed powder obtained in the step 1) with the keep-alive agent, and carrying out physical wall breaking to obtain a mixture, wherein the weight ratio of the seaweed powder to the keep-alive agent is 1: 5-10;
3) enzymolysis: taking alginate lyase, adding 2-8 per mill of alginate lyase into the mixture obtained in the step 2) for enzymolysis, wherein the enzymolysis temperature is 35-55 ℃, and the enzymolysis time is 4-8 hours, so as to obtain an alga enzymolysis liquid;
4) inactivation: inactivating the seaweed enzymolysis liquid obtained in the step 3) at 90-100 ℃ for 5-10 min;
5) filtering, and packaging to obtain Sargassum extract.
2. The method for producing the agricultural seaweed extract in combination with physical and biological methods as claimed in claim 1, wherein:
in the step 2), the keep-alive agent is any one of a phosphate buffer solution with a pH value of 5.8-7.0 and a concentration of 0.2mol/L, a citrate buffer solution with a pH value of 4.4-6.6, and a borate buffer solution with a pH value of 7.4-8.0.
3. The method for producing the agricultural seaweed extract in combination with physical and biological methods as claimed in claim 1, wherein:
the alginate lyase comprises the following components: 1, 4-beta-D-mannuronic acid lyase, the enzyme activity is 20000-30000U/mL; 1, 4-alpha-L-guluronic acid lyase with the enzyme activity of 20000-30000U/mL; polymannuronic acid and polyguluronic acid lyase, the enzyme activity is 25000-35000U/mL.
4. The method for producing the agricultural seaweed extract in combination with physical and biological methods as claimed in claim 3, wherein:
the enzyme activity of the alginate lyase is 20000-35000U/mL.
5. The method for producing the agricultural seaweed extract in combination with physical and biological methods as claimed in claim 1, wherein:
the dried seaweed is air-dried seaweed, and the water content of the seaweed is not more than 10%.
6. The method for producing the agricultural seaweed extract in combination with physical and biological methods as claimed in claim 5, wherein:
the seaweed is one or more of Ascophyllum nodosum, Macrocystis japonica, herba Zosterae Marinae, Sargassum, thallus laminariae, Cyrtymenia Sparsa, Sargassum thunbergii, Sargassum pallidum, sea weed, sea oak, bull algae, goose palm, and Hordeum vulgare.
7. The method for producing an agricultural seaweed extract by the physical and biological combination method according to any one of claims 1 to 6, wherein:
the physical wall breaking method in the step 2) is any one or more of a high-speed shearing method, a high-pressure homogenizing method, an ultrasonic method and a sand milling method.
8. The method for producing the agricultural seaweed extract in combination with physical and biological methods as claimed in claim 7, wherein:
the shearing rotating speed of the high-speed shearing method is 10000-; the homogenization pressure of the high-pressure homogenization method is 60-170MPa, and the homogenization time is 15-30 min; the ultrasonic power of the ultrasonic method is 200-600W, and the ultrasonic time is 10-30 min; the grinding time of the sand grinding method is 60-90 min.
9. The method for producing the agricultural seaweed extract in combination with physical and biological methods as claimed in claim 1, wherein:
in the step 5), the filtration is to filter the inactivated enzymolysis liquid by a plate-and-frame filter press, and then to carry out ultrafiltration by a ceramic membrane of 100-200 nm.
10. A seaweed extract is characterized in that:
the seaweed extract is prepared by the method for producing the agricultural seaweed extract by the physical and biological combination method according to any one of claims 1 to 9.
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