WO2020143353A1 - Method for preparing k-potassium carrageenan having low molecular weight by means of enzymolysis, and application - Google Patents

Abstract

A method for preparing k-potassium carrageenan having a low molecular weight by means of enzymolysis, comprising the following steps: (1) preparing a k-carrageenan aqueous solution having a mass concentration of 0.5-1.5% from k-carrageenan powder in a 40-50°C water bath kettle, stirring uniformly, adding k-carrageenanase, and stirring for enzymolysis for 6-8 h; (2) subjecting an enzymatic hydrolysate to alcohol precipitation to obtain k-carrageenan oligosaccharide having a low molecular weight; and (3) preparing a solution having a mass concentration of 4-10% from the obtained k-carrageenan oligosaccharide, mixing the solution with a potassium chloride aqueous solution having a mass concentration of 0.1-2%, and subjecting the mixed solution to alcohol washing and salt removing to obtain k-potassium carrageenan having a low molecular weight. The method has simple steps and mild conditions; the molecular weight of prepared k-potassium carrageenan having a low molecular weight is stabilized within a small range; and the potassium ion binding rate is high. K-potassium carrageenan can be added into food as a food additive to obtain healthcare products, and can also be prepared into medicines as drugs for preventing or treating hypertension.

Description

Method and application for preparing low molecular weight κ-carrageenan potassium by enzymatic hydrolysis Technical field

The invention relates to the technical field of preparation of low molecular weight κ-carrageenan potassium, and more particularly to a method and application of enzymatic hydrolysis for preparing low molecular weight κ-carrageenan potassium.

Background technique

As the original producer of organic matter in the ocean, marine algae have special carbohydrates and minerals indispensable for human normal physiological metabolism. Seaweed polysaccharides refer to various high-molecular-weight carbohydrates with high viscosity and coagulation ability. With the vigorous development of marine drugs in recent years, the research of seaweed polysaccharides has been paid more and more attention. As an extract of red algae, one of the three major algae in the ocean, κ-carrageenan has attracted the attention of contemporary biology, biochemistry, and medicine circles due to its unique physical and chemical properties and a wide range of uses. It has become a research hotspot.

κ-Carrageenan is a linear sulfated polysaccharide formed by alternately connecting 1,3-β-D-galactopyranose and 1,4-α-D-galactopyranose as the basic skeleton, and mainly exists in the red algae class In the cell walls of the genus Kirinia, carrageen, genus Xanthium and genus L. κ-Carrageenan is used in the food industry and can be used as coagulant, adhesive, stabilizer, emulsifier, suspending agent, thickener, etc. It is widely used in dairy products, beverages, milk pudding, coffee, jelly, canned food, etc. Industrially. In addition, κ-carrageenan also has very important biological activities, such as subcutaneous injection of κ-carrageenan can promote the growth of connective tissue and collagen, increase bone absorption of calcium; κ-carrageenan has a curative effect on rheumatoid arthritis After subcutaneous injection or intravenous injection of polysaccharide complex of κ-carrageenan, it can prevent granulation in the synovium and make the synovial cells proliferate significantly, which plays a therapeutic role; κ-carrageenan also shows in pathological research Can prevent gastric ulcers and duodenal ulcers, because acid can degrade κ-carrageenan to adjust the sulfur content in gastric mucus, prevent ulcers, and can combine with mucin on the mucosal layer to form a more resistant Membrane structure, protects the gastrointestinal mucosa; κ-carrageenan also has important physiological activities in antiviral, anticoagulant and immunity.

However, at present, there are few studies on low molecular weight κ-carrageenan potassium for lowering blood pressure, nor is there any research on the preparation process of κ-carrageenan potassium in any molecular weight range.

Therefore, how to provide a method for preparing a low-molecular-weight κ-carrageenan potassium with a simple step, mild conditions, and capable of enzymatic hydrolysis with a hypotensive effect is an urgent problem to be solved by those skilled in the art.

Summary of the invention

In view of this, the present invention provides a method for preparing low molecular weight κ-carrageenan potassium with antihypertensive effect by enzymolysis, using refined κ-carrageenan powder from red algae as raw material, enzymatic hydrolysis and alcohol precipitation by specific enzymes After that, low molecular weight κ-carrageenan oligosaccharides with molecular weights of 1-4 kDa are obtained. After potassium salt mixing treatment, low molecular weight κ-carrageenan potassium is obtained. The method of the invention has simple steps and mild conditions, the molecular weight of the prepared low molecular weight κ-carrageenan potassium is stable in a small range, and the potassium ion content is high.

In order to achieve the above objectives, the present invention adopts the following technical solutions:

An enzymatic method for preparing low molecular weight κ-carrageenan potassium includes the following steps:

(1) In a water bath at 40-50°C, mix κ-carrageenan powder into a 0.5-1.5% mass concentration of κ-carrageenan aqueous solution, stir evenly, add κ-carrageenan and stir for 6-8h ;

(2) The enzymolysis solution undergoes alcohol precipitation to obtain low molecular weight κ-carrageenan oligosaccharides;

(3) The obtained κ-carrageenan oligosaccharide is prepared into a solution with a mass concentration of 4-10%, and mixed with a potassium chloride aqueous solution with a mass concentration of 0.1-2%; the mixed solution is washed with alcohol to obtain a low molecular weight Κ-carrageenan potassium.

Preferably, in step (1), the molecular weight of kappa-carrageenan is 100-700 kDa, and the potassium ion content is 6.0-6.5%; the source of kappa-carrageenan is natural red algae such as Kirinia, Amaryllidaceae, and Carrageenan.

The enzymolysis substrate has a large molecular weight and a low potassium ion content. Under specific enzymatic hydrolysis conditions, κ-carrageenase is used for enzymatic hydrolysis to prepare a low molecular weight κ-carrageenan oligosaccharide with stable molecular weight and high biological activity.

Preferably, the volume fraction of the initial addition of kappa-carrageenase in step (1) is 0.5-3%.

The form of enzymolysis is one-time addition of enzyme, and no enzyme solution is added during the enzymatic hydrolysis.

Further preferably, 1% kappa-carrageenase is added, and the original pH is enzymatically hydrolyzed at 40°C for 8 hours.

The added amount of κ-carrageenase, enzymolysis time and enzymolysis temperature all ensure the stability of enzymatic hydrolysis products.

Preferably, the gene encoding κ-carrageenase is shown in SEQ ID NO:1.

Further preferably, the preparation method of κ-carrageenase is as follows:

1) Design the upstream primer F1: 5’-CGGGGTACCATGACAAAACTAAAGTTTAACGGC-3’, SEQ ID NO: 2;

And the downstream primer R1: 5’-ATAAGAATGCGGCCGCTTAAGCCGAAGTTCCGGGCG-3’, SEQ ID NO: 3;

After PCR amplification, the PCR product shown in SEQ ID NO: 1 was purified with a purification kit. After electrophoresis imaging, the target band and the empty vector pPIC9K were subjected to AvrII and NotI double digestion, respectively, and the target gene and vector were connected overnight. The ligation product was transformed into DH-5α competent cells, positive clones were cultured and plasmids were extracted, introduced into Pichia pastoris using an electroporator, cultured, and recombinant Pichia pastoris selected.

2) Inoculate the selected recombinant Pichia pastoris into YPD medium and incubate at 26-32°C for 20-24h; inoculate 0.5-2% inoculation amount in BMGY fermentation broth medium for 36-48h, add methanol, 20 Incubate at -24°C for 20-24h. Repeat adding methanol 3-4 times to continue culturing. After the cultivation, centrifuge to obtain the supernatant, namely κ-carrageenase.

Preferably, the amount of methanol added is 1-2%.

Further preferably, the amount of methanol added is 1%.

A specific amount of methanol can induce the expression of κ-carrageenase.

Preferably, the molecular weight of the low molecular weight kappa-carrageenan oligosaccharide in step (2) is 1-4 kDa; the low molecular weight kappa-carrageenan oligosaccharide includes kappa-carrageenan behenose and kappa-carrageenan twenty-four sugar, Both accounted for more than 40%.

Preferably, the alcohol precipitation method in step (2) is as follows:

1) Centrifuge the enzymolysis solution, take the supernatant, add 95% ethanol, make the alcohol meter read 45-55%, and let it stand refrigerated for 1-2h;

2) Take the supernatant, concentrate it 5-6 times, add 95% ethanol to make the alcohol meter read 75-78%, and let it stand refrigerated for 1-2h;

3) Take the precipitate, dissolve it in water, rotary evaporate, and freeze-dry to obtain low molecular weight κ-carrageenan oligosaccharide.

Preferably, the low molecular weight κ-carrageenan potassium in step (3) has a molecular weight of 1-4 kDa.

Preferably, the potassium ion content of the low molecular weight κ-carrageenan in step (3) is 8.2-8.8%.

Preferably, the specific steps in step (3) are as follows:

1) Prepare a κ-carrageenan potassium oligosaccharide solution with a mass concentration of 4-10%, mix it with an equal volume of a mass concentration of 0.1-2% potassium chloride, spin-concentrate the mixed solution twice, add 95% ethanol, and make the alcohol meter read Between 75-78%, let stand for 1-2h, centrifuge, take the precipitate;

2) The precipitation is reconstituted with water to the volume of the mixed solution after rotary evaporation, and 95% ethanol is added again to make the alcohol meter reading between 75-78%, it is allowed to stand for 1-2h, centrifuged, and the precipitate is taken;

3) Precipitation, reconstitution, and rotary evaporation to remove ethanol, and freeze drying to obtain low molecular weight κ-carrageenan potassium.

Further preferably, the mass concentration of κ-carrageenan potassium oligosaccharide solution is 4-8%, and the mass concentration of potassium chloride is 0.1-1%.

The application of the low molecular weight κ-carrageenan potassium prepared by the above method for preparing low molecular weight κ-carrageenan potassium in the preparation of drugs for preventing or treating hypertension.

According to the above technical solution, compared with the prior art, the present disclosure provides a method for preparing low molecular weight κ-carrageenan potassium by enzymolysis. After dissolving the macromolecular κ-carrageenan powder in water, κ- Carrageenase is hydrolyzed and mixed with potassium chloride solution to obtain low molecular weight κ-carrageenan potassium with high purity and high potassium ion content. It can be used as a food additive, as well as medicine and functional food.

BRIEF DESCRIPTION

The drawings needed to be used in the embodiments will be briefly described below. Obviously, the drawings in the following description are only embodiments of the present invention. For those of ordinary skill in the art, the premise of not paying creative labor Below, other drawings can also be obtained from the provided drawings.

FIG. 1 is a graph of the salt peak of the low-molecular-weight κ-carrageenan potassium in an alcohol wash in Example 1;

2 is a graph showing the salt peak of the low-molecular-weight κ-carrageenan potassium secondary alcohol wash in Example 1;

Figure 3 is a macromolecular κ-carrageenan GPC diagram;

4 is a GPC chart of low molecular weight κ-carrageenan oligosaccharide in Example 1;

5 is a GPC chart of low molecular weight κ-carrageenan potassium in Example 1;

6 is a graph showing the salt peak of the low molecular weight κ-carrageenan potassium in one alcohol wash in Example 2;

7 is a graph showing the salt peak of the low molecular weight κ-carrageenan potassium secondary alcohol wash in Example 2;

8 is a GPC chart of low molecular weight κ-carrageenan oligosaccharide in Example 2;

9 is a GPC chart of low molecular weight κ-carrageenan potassium in Example 2;

10 is a GPC chart of κ-carrageenan oligosaccharide in Comparative Example 1;

FIG. 11 is a GPC chart of κ-carrageenan potassium in Comparative Example 1. FIG.

detailed description

The technical solutions in the embodiments of the present invention will be described clearly and completely below with reference to the drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

Example 1

1. Preparation of κ-carrageenase

Based on the entire gene sequence of κ-carrageenase derived from Zobellia sp.ZM-2, design the upstream primer F1: 5'-CGGGGTACCATGACAAAACTAAAGTTTAACGGC-3', SEQ ID ID NO: 2; and the downstream primer R1: 5'-ATAAGAATGCGGCCGCTTAAGCCGAAGTTCCGGGCG-3 ', SEQ ID NO: 3;

The amplification process uses genomic DNA as a template and high-fidelity pfu polymerase (Takara) to amplify the target gene. The amplification reaction system (50 μL) is shown in Table 1.

Table 1 Amplification reaction system

The PCR amplification procedure is as follows: pre-denaturation 98℃, 2min; denaturation 94℃, 30s; annealing 56℃, 30s; extension 72℃, 1min; final extension 72℃, 10min; cycle number 30 times.

After PCR amplification, the PCR product purification kit was used for purification. After electrophoresis imaging, the target band and empty vector pPIC9K were subjected to AvrII and NotI double digestion, and the target gene was ligated with the vector overnight, and the ligation product was transformed into DH-5α competent state. Cells, positive clones were cultured and plasmids were extracted, introduced into Pichia pastoris using an electroporator, cultured, and recombinant Pichia pastoris selected.

Select the successfully constructed recombinant strain, inoculate into YPD medium, and incubate at 30℃ for 24h; inoculate with 1% inoculation in BMGY fermentation broth medium for 38h, add 1% volume of methanol, incubate at 22℃ for 24h, add 1% The methanol was incubated at 22°C for 24h, 1% methanol was added, 22°C was incubated for 24h, and 1% methanol was added. After incubation at 22°C for 24h, the supernatant was centrifuged to obtain κ-carrageenase crude enzyme solution.

2. Enzymatic hydrolysis

Aqueous solution of κ-carrageenan with a mass concentration of 1% is prepared with a molecular weight of κ-carrageenan powder, which is dissolved by stirring under a 40°C water bath; the crude enzyme solution is centrifuged at 10000r/min for 10 minutes at high speed to take the supernatant, and added to the volume of 1% by volume In the κ-carrageenan aqueous solution, continue to enzymatically hydrolyze at 40℃ for 8h.

3. Alcohol precipitation

Centrifuge the enzymolysis solution, take the supernatant, spin-concentrate and double it, add 95% ethanol to keep the alcohol meter reading at 50%, and let it stand at 4°C for 1.5h; centrifuge to take the supernatant, concentrate 6 times, add 95% ethanol, Keep the reading of the alcohol meter at 75%, and let stand at 4°C for 1 h; take the precipitate by centrifugation, re-dissolve it in 400 ml of distilled water, rotate and evaporate at 60°C, concentrate as much as possible, and freeze-dry to obtain low molecular weight κ-carrageenan oligosaccharides.

4. Alcohol eluting salt

The obtained low molecular weight κ-carrageenan oligosaccharide was made into 6% aqueous solution, mixed with equal volume of 0.1% potassium chloride solution, the mixture was concentrated by rotary evaporation, and 95% ethanol was added to keep the alcohol meter reading at 75% , Leave at 4℃ for 2h, centrifuge to take the precipitate, reconstitute to the volume after rotary evaporation and concentration, take part to measure the concentration of free salt ions using Sephadex G-10 desalting column, the result is shown in Figure 1; add 95% ethanol again to make alcohol Keep the reading of the meter at 75%, let stand at 4℃ for 2h, take the precipitate by centrifugation, reconstitute enough distilled water, spin to evaporate at 60℃, concentrate as much as possible, freeze-drying is the low molecular weight κ-carrageenan potassium with high potassium ion, partly used Sephadex G-10 desalting column measures the salt concentration. The results are shown in Figure 2. The salt peak (ie free salt ion concentration) is negligible.

The molecular weights of undigested κ-carrageenan, low molecular weight κ-carrageenan oligosaccharides and low molecular weight κ-carrageenan potassium obtained by high performance liquid chromatography combined with TSKgel G4000PW _XL column were measured. 3. As shown in Figures 4 and 5, the weight average molecular weights are 700 kDa, 3.5 kDa, and 3.5 kDa, respectively.

Flame atomic absorption spectrometry determined that the potassium content in κ-carrageenan was 6.0%, and the potassium content in low molecular weight κ-carrageenan was 8.2%.

Example 2

1. Preparation of κ-carrageenase

Recombinant strains were inoculated into YPD medium in the same way, and cultured at 30°C for 24h; inoculated with 1% inoculation amount in BMGY fermentation broth cultured for 40h, added 1.2% by volume of methanol, cultured at 22°C for 24h, and added 1.2% of methanol Incubate at 22°C for 24h, add 1.2% methanol, 22°C for 24h, add 1.2% methanol, and incubate at 22°C for 24h. After centrifugation, the supernatant is κ-carrageenase crude enzyme solution.

2. Enzymatic hydrolysis

Prepare 1% κ-carrageenan aqueous solution (same as in Example 1), stir and dissolve in a 40°C water bath; centrifuge the crude enzyme solution at 10000r/min for 10min at high speed to take the supernatant, add κ-carrageenan at a volume ratio of 2% In the solution, the enzymolysis was continued for 7 hours under the condition of 40°C water bath.

3. Alcohol precipitation

The enzyme solution was centrifuged, the supernatant was taken, concentrated by rotary evaporation, and 95% ethanol was added to keep the alcohol meter reading at 50%, and left at 4°C for 2h; the supernatant was taken by centrifugation, concentrated 5 times, and 95% ethanol was added to make Keep the reading of the alcohol meter at 75%, and let stand at 4°C for 1 h; take the precipitate by centrifugation, re-dissolve it in water, rotate and evaporate at 60°C, concentrate as much as possible, and freeze-dry to obtain low molecular weight κ-carrageenan oligosaccharides.

4. Alcohol eluting salt

The obtained κ-carrageenan oligosaccharide was formulated into 8% aqueous solution, mixed with 0.5% potassium chloride solution in equal volume, the mixture was concentrated by rotary evaporation, and 95% ethanol was added to keep the alcohol meter reading at 75%. 4 Stand at ℃ for 1h, centrifuge to take the precipitate, reconstitute to the volume after rotary evaporation and concentration, take part to measure the concentration of free salt ions using Sephadex G-10 desalting column, the result is shown in Figure 6; add 95% ethanol again, make the alcohol meter read Keep at 75%, let stand at 4°C for 1h, centrifuge to take the precipitate, reconstitute enough distilled water, spin to evaporate at 60°C, concentrate as much as possible, freeze-drying is the low molecular weight κ-carrageenan potassium with high potassium ion, and partly uses Sephadex G The -10 desalting column measures the salt concentration. The results are shown in Figure 7. The salt peak (ie, free salt ion concentration) is negligible.

The molecular weights of low molecular weight κ-carrageenan oligosaccharide and low molecular weight κ-carrageenan potassium obtained in this example were measured by high performance liquid chromatography combined with PL Aquagel-OH 30 column. The results are shown in Figure 8 and Figure 9, respectively. The average molecular weight is 3.8 kDa.

Flame atomic absorption spectrometry determined that the potassium content in κ-carrageenan was 6.5%, and the potassium content in low molecular weight κ-carrageenan was 8.5%.

Comparative Example 1

1. Preparation of κ-carrageenase

Recombinant strain in the same way, inoculated into YPD medium and cultivated at 30°C for 24h; inoculated with 1% inoculation amount in BMGY fermentation broth cultured for 38h, added 1% volume of methanol, cultured at 22°C for 24h, added 1% methanol Incubate at 22°C for 24h, add 1% methanol, incubate at 24°C for 24h, add 1% methanol, and incubate at 22°C for 24h. After centrifugation, the supernatant is κ-carrageenase crude enzyme solution.

2. Enzymatic hydrolysis

A kappa-carrageenan aqueous solution with a mass concentration of 1% was prepared with kappa-carrageenan powder, and stirred and dissolved in a 40°C water bath; the crude enzyme solution was centrifuged at 10,000r/min for 10 minutes at high speed to take the supernatant, and added to the kappa- 1% by volume. In the carrageenan aqueous solution, continue to maintain the enzymolysis at 40 ℃ water bath conditions for 8h.

3. Alcohol precipitation

The enzymolysis solution was centrifuged, the supernatant was taken, concentrated by rotary evaporation, and 95% ethanol was added to keep the reading of the alcohol meter at 50%, and it was allowed to stand at 4°C for 2 hours; the precipitate was taken by centrifugation, reconstituted in sufficient distilled water, and rotated at 60°C Evaporate, concentrate as much as possible, and freeze-dry to obtain κ-carrageenan oligosaccharides.

4. Alcohol eluting salt

The obtained κ-carrageenan oligosaccharide was formulated into a 6% aqueous solution, mixed with an equal volume of 0.1% potassium chloride solution, the mixture was concentrated by rotary evaporation, and 95% ethanol was added to keep the alcohol meter reading at 50%. 4 Stand at ℃ for 2h, take the precipitate by centrifugation, reconstitute to the volume after rotary evaporation and concentration, add 95% ethanol again to keep the alcohol meter reading at 50%, stand at 4°C for 2h, take the precipitate by centrifugation, reconstitute enough distilled water, 60 Rotate steam at ℃, concentrate as much as possible, freeze-drying is κ-carrageenan potassium.

The molecular weights of κ-carrageenan oligosaccharides and κ-carrageenan potassium obtained in Comparative Example 1 were determined by high performance liquid chromatography combined with PL Aquagel-OH 30 column. The results are shown in Figure 10 and Figure 11, respectively. It can be seen that the molecular weight of κ-carrageenan oligosaccharides obtained by only one-fold alcohol precipitation is not uniform and messy. Compared with Example 1, about 20% of polysaccharides with a molecular weight of 500 kDa or more are present. Due to the complex composition, it is not suitable for studying the activity of specific molecular weight κ-carrageenan oligosaccharides.

Example 3 Hypotensive effect of low molecular weight κ-carrageenan on spontaneously hypertensive rats

9-week-old SPF male spontaneously hypertensive rats (SHR) were selected and randomly grouped according to blood pressure and body weight. Ten rats in the blank group were given intragastric distilled water; six rats in the experimental group were intragastrically administered Example 1. Low molecular weight κ-carrageenan Potassium, gavage dose is 600mg/kgbw/day; 6 captopril group, captopril gavage dose is 10mg/kg bw/day; systolic blood pressure (SBP) and heart rate are measured once a week, weighed 2 times (for calculation of intragastric volume).

The effect of low molecular weight κ-carrageenan potassium on the SBP of SHR is shown in Table 2. It can be seen that after the third week of intragastric administration, the systolic blood pressure of the experimental group has been significantly reduced. The statistical analysis results show that it is similar to the blank group. There is a significant difference (P = 0.034), indicating that low molecular weight κ-carrageenan potassium has a hypotensive effect from the third week, and the statistical analysis results of the fourth week show that the antihypertensive effect remains stable (P = 0.027) , No signs of rebound. It shows that low molecular weight potassium κ-carrageenan has a sustained and stable antihypertensive effect on SHR and can be applied in the field of biomedicine.

Table 2 The effect of low molecular weight κ-carrageenan potassium on SHR SBP

Note: * means P<0.05 compared with blank control; ** means P<0.01 compared with blank control.

The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown in this document, but should conform to the widest scope consistent with the principles and novel features disclosed in this document.

Claims (10)

1. A method for preparing low molecular weight κ-carrageenan potassium by enzymolysis is characterized in that it includes the following steps:

   (1) In a water bath at 40-50°C, kappa-carrageenan powder is formulated into a 0.5-1.5% mass concentration of kappa-carrageenan aqueous solution, stir evenly, add kappa-carrageenase and stir for 6-8h ;

   (2) The enzymolysis solution undergoes alcohol precipitation to obtain low molecular weight κ-carrageenan oligosaccharides;

   (3) The obtained κ-carrageenan oligosaccharide is prepared into a solution with a mass concentration of 4-10%, and mixed with a potassium chloride aqueous solution with a mass concentration of 0.1-2%; the mixed solution is washed with alcohol to obtain a low molecular weight Κ-carrageenan potassium.
2. The method for preparing low molecular weight κ-carrageenan potassium according to claim 1, wherein the molecular weight of κ-carrageenan in step (1) is 100-700kDa, and the potassium ion content is 6.0- 6.5%; The source of κ-carrageenan is natural red algae.
3. The method for preparing low molecular weight κ-carrageenan potassium by enzymatic hydrolysis according to claim 1, wherein the volume fraction of the initial addition of κ-carrageenase in step (1) is 0.5-3%.
4. The method for preparing low molecular weight κ-carrageenan potassium according to claim 1, wherein the gene encoding the κ-carrageenase is shown in SEQ ID NO:1.
5. The method for preparing potassium of low molecular weight κ-carrageenan by enzymolysis according to claim 1, characterized in that the molecular weight of the low molecular weight κ-carrageenan oligosaccharide in the step (2) is 1-4kDa; low molecular weight κ-carrageenan oligosaccharides include κ-carrageenan behenose and κ-carrageenan twenty-four sugar, both of which account for more than 40%.
6. The method for preparing low molecular weight κ-carrageenan potassium by enzymolysis according to claim 1, characterized in that the alcohol precipitation method in the step (2) is as follows:

   1) Centrifuge the enzymolysis solution, take the supernatant, add 95% ethanol, make the alcohol meter read 45-55%, and let it stand in the refrigerator;

   2) Take the supernatant, concentrate it 5-6 times, add 95% ethanol, make the alcohol meter read 75-78%, and store it in a refrigerator;

   3) Take the precipitate, dissolve it in water, rotary evaporate, and freeze-dry to obtain low molecular weight κ-carrageenan oligosaccharide.
7. The method for preparing low molecular weight κ-carrageenan potassium according to claim 1, wherein the molecular weight of the low molecular weight κ-carrageenan potassium in step (3) is 1-4 kDa.
8. The method for preparing low molecular weight κ-carrageenan potassium by enzymatic hydrolysis according to claim 1, characterized in that the potassium ion content of the low molecular weight κ-carrageenan potassium in step (3) is 8.2-8.8%.
9. A method for preparing low molecular weight κ-carrageenan potassium by enzymatic hydrolysis according to claim 1, wherein the specific steps in the step (3) are as follows:

   1) Prepare a κ-carrageenan potassium oligosaccharide solution with a mass concentration of 4-10%, mix it with an equal volume of a mass concentration of 0.1-2% potassium chloride, spin-concentrate the mixed solution twice, add 95% ethanol, and make the alcohol meter read Between 75-78%, let stand, centrifuge, take the precipitate;

   2) The precipitation is reconstituted with water to the volume of the mixed solution after rotary evaporation, alcohol precipitation again, keeping the alcohol meter reading between 75-78%, standing, centrifugation, and taking the precipitate;

   3) Precipitation and reconstitution rotary evaporation to remove ethanol, freeze-drying to obtain low molecular weight κ-carrageenan potassium.
10. The application of the method for preparing low molecular weight κ-carrageenan potassium by enzymatic hydrolysis according to any one of claims 1-9 in the preparation of a drug for preventing or treating hypertension.

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