Preparation methods and applications of κ-carrageenan oligosaccharides

Abstract

This invention discloses a method for preparing κ-carrageenan oligosaccharides and their applications. The preparation method includes the following steps: (1) preparing a mixed solution of κ-carrageenan, H2O2, and ascorbic acid; (2) placing the mixed solution in a reaction environment at a temperature of 40℃-60℃ and a pressure of 20MPa-60MPa for 5min-30min to obtain a κ-carrageenan degradation solution; (3) separating and retaining the κ-carrageenan degradation solution to obtain κ-carrageenan oligosaccharides with a molecular weight of 1000Da-3000Da. The method for preparing κ-carrageenan oligosaccharides provided by this invention uses the H2O2-ascorbic acid system as the main component to oxidize and degrade κ-carrageenan under low temperature and high pressure. It has many advantages such as simple process, high yield, short time consumption, and environmental friendliness, and can be industrialized. The antibacterial composition with the above-mentioned κ-carrageenan oligosaccharides as the active ingredient has good antibacterial activity.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide derivative preparation technology, specifically relating to a method for preparing κ-carrageenan oligosaccharide and its application. Background Technology

[0002] Carrageenan, also known as carrageenan, is a linear sulfated polysaccharide extracted from the cell walls of marine red algae (such as *Euphorbia*). It is one of the three major marine algal colloids (carrageenan, agar, and alginate). Carrageenan is composed of alternating 3-β-D-galactose (G-units) and 4-linked α-D-galactopyran (D-units) or 4-linked 3,6-endoether-α-D-galactopyran (DA-units) units, exhibiting high molecular weight (100-1000 kDa) and high dispersibility. Based on the presence of DA- units and the number and position of sulfate groups in the disaccharide units, carrageenan can be roughly divided into kappa(κ)-, iota(ι)-, lambda(λ)-, mu(μ)-, nu(ν)-, gamma(γ)-, alpha(α)-, delta(δ), theta(θ)-, and beta(β)- carrageenan. Among them, the three most common commercial carrageenan are κ-, ι-, and λ- carrageenan, with 1, 2, and 3 sulfate groups in each repeating disaccharide unit, respectively.

[0003] Carrageenan, due to its unique physicochemical properties, has broad application prospects in the food, chemical, and biopharmaceutical fields. However, its high molecular weight and high viscosity result in low permeability and limited physiological activity, restricting its widespread application. Studies have shown that low molecular weight carrageenan oligosaccharides, due to their lower molecular weight, have better solubility and bioavailability, exhibiting better biological activities than carrageenan, including antioxidant, hypoglycemic, hypolipidemic, and anti-inflammatory activities.

[0004] Currently, the main methods for degrading carrageenan include chemical degradation, physical degradation, and enzymatic degradation. Physical degradation of carrageenan is pollution-free and does not damage its skeletal structure, but the product yield is low, and it is generally used as an auxiliary method. Enzymatic degradation of carrageenan is gentle and highly specific, but most carrageenan-degrading enzymes are still in the laboratory stage, with low enzyme activity, making industrial production difficult. Among chemical degradation methods, acid degradation is a mature process but is highly destructive and easily causes environmental pollution. Oxidative degradation, also a type of chemical degradation, has attracted much attention due to its highly active and environmentally friendly degradation products, but current oxidative degradation methods have low degradation efficiency and are time-consuming. Therefore, developing a simple and high-yield carrageenan degradation process is of great significance for the production of carrageenan oligosaccharides. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects of the prior art and provide a method for preparing κ-carrageenan oligosaccharides.

[0006] Another object of the present invention is to provide the application of the κ-carrageenan oligosaccharide obtained by the above preparation method.

[0007] The technical solution of the present invention is as follows:

[0008] The preparation method of κ-carrageenan oligosaccharides includes the following steps:

[0009] (1) Prepare a mixed solution of κ-carrageenan, H2O2 and ascorbic acid;

[0010] (2) Place the above mixed solution in a reaction environment with a temperature of 40℃-60℃ and a pressure of 20MPa-60MPa for 5min-30min to obtain κ-carrageenan degradation solution;

[0011] (3) The above κ-carrageenan degradation solution was separated and retained to obtain κ-carrageenan oligosaccharides with a molecular weight of 1000Da-3000Da.

[0012] In a preferred embodiment of the present invention, the concentration of κ-carrageenan solution in the mixed solution is 1.5wt%-2.5wt%, the concentration of H2O2 in the mixed solution is 10mM-50mM, and the concentration of ascorbic acid is 5mM-50mM.

[0013] More preferably, the concentration ratio of H2O2 to ascorbic acid in the mixed solution is 10:1-10.

[0014] In a preferred embodiment of the present invention, step (3) is performed as follows: centrifuge the κ-carrageenan degradation solution, collect the supernatant, then perform nanofiltration on the supernatant, collect the retentate, and obtain κ-carrageenan oligosaccharide.

[0015] More preferably, the centrifugation speed is 5000 r / min-8000 r / min, and the centrifugation time is 10 min-20 min.

[0016] More preferably, the nanofiltration temperature is 40°C, the pressure is 0.2MPa-1.0MPa, and the molecular weight cutoff is 100Da-300Da.

[0017] In a preferred embodiment of the present invention, step (4) is further included: κ-carrageenan oligosaccharide is subjected to vacuum concentration and freeze drying operations in sequence to obtain solid κ-carrageenan oligosaccharide.

[0018] An antibacterial composition, the active ingredient of which is κ-carrageenan oligosaccharide prepared by the above method.

[0019] In a preferred embodiment of the present invention, the bacteria are Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas schrenckii, or Shigella flexneri.

[0020] Application of κ-carrageenan oligosaccharides prepared by the above method in the preparation of antibacterial compositions.

[0021] The beneficial effects of this invention are:

[0022] 1. The method for preparing κ-carrageenan oligosaccharides provided by the present invention uses the H2O2-ascorbic acid system as the main component to oxidize and degrade κ-carrageenan under low temperature and high pressure. It has many advantages such as simple process, high yield, short time and environmental friendliness, and can be industrialized.

[0023] 2. The κ-carrageenan oligosaccharide prepared by the method of the present invention exhibits good antibacterial activity against Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Pseudomonas hydrophila, Pseudomonas stutzeri, and Shigella flexneri, and can be used as an antibacterial composition, feed additive, food additive, or daily chemical additive. Attached Figure Description

[0024] Figure 1 This is a high-performance gel permeation chromatogram of κ-carrageenan oligosaccharide from Example 1 of the present invention.

[0025] Figure 2 This is a scanning electron microscope image of κ-carrageenan.

[0026] Figure 3 This is a scanning electron microscope image of the κ-carrageenan oligosaccharide from Example 1 of the present invention.

[0027] Figure 4 Infrared spectra of κ-carrageenan and κ-carrageenan oligosaccharides of Examples 1-5 of this invention. Detailed Implementation

[0028] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0029] Comparative Example 1

[0030] The preparation method of κ-carrageenan oligosaccharides includes the following steps:

[0031] (1) Prepare a mixed solution of κ-carrageenan, H2O2 and ascorbic acid.

[0032] The solvent for this mixed solution is water, the concentration of κ-carrageenan is 2 wt%, the concentration of H2O2 is 10 mM, and the concentration of ascorbic acid is 10 mM.

[0033] (2) The mixed solution was placed in a reaction environment at 50°C and normal pressure for 3 hours to obtain κ-carrageenan degradation solution.

[0034] (3) Centrifuge the κ-carrageenan degradation solution, collect the supernatant, then perform nanofiltration on the supernatant, collect the retentate, and obtain the κ-carrageenan oligosaccharide.

[0035] The centrifugation speed was 5000 r / min, the centrifugation time was 20 min, and the centrifugation operation could remove undegraded κ-carrageenan with a large molecular weight. The nanofiltration temperature was 40℃, the nanofiltration pressure was 0.5 MPa, and the molecular weight cutoff was 100 Da-300 Da.

[0036] (4) The κ-carrageenan oligosaccharide was subjected to vacuum concentration and freeze-drying operations in sequence to obtain solid κ-carrageenan oligosaccharide. The vacuum concentration temperature was 60°C.

[0037] (5) Weigh the items and calculate the yield. The formula for calculating the yield is as follows:

[0038] Yield = A / B × 100%

[0039] Where A represents 10g of κ-carrageenan oligosaccharide and B represents the mass (g) of κ-carrageenan.

[0040] Calculations show that the yield of κ-carrageenan oligosaccharides in this embodiment is 56.23%, and the production of κ-carrageenan oligosaccharides is 5.62g.

[0041] Comparative Example 2

[0042] According to publicly available literature (Li Xinjiang, Zhao Shanzhen, He Qiyu, Lu Haiyan, Liu Yao, Hou Hu, Zhao Xue. Study on high temperature and high pressure degradation process and degradation mechanism of κ-carrageenan [J]. Journal of Food Safety and Quality Testing, 2022, 13(13): 4242-4249)

[0043] The preparation method of κ-carrageenan oligosaccharides includes the following steps:

[0044] (1) Prepare a κ-carrageenan solution with a concentration of 2wt%. The solvent of the mixed solution is water, and the pH is adjusted to 4.0 with acetic acid.

[0045] (2) Place it in a high-temperature and high-pressure sterilizer and degrade it at 120°C for 60 minutes to obtain a κ-carrageenan degradation solution.

[0046] (3) Centrifuge the κ-carrageenan degradation solution, collect the supernatant, then perform nanofiltration on the supernatant, collect the retentate, and obtain the κ-carrageenan oligosaccharide.

[0047] The centrifugation speed was 5000 r / min, the centrifugation time was 20 min, and the centrifugation operation could remove undegraded κ-carrageenan with a large molecular weight. The nanofiltration temperature was 40℃, the nanofiltration pressure was 0.5 MPa, and the molecular weight cutoff was 100 Da-300 Da.

[0048] (4) The κ-carrageenan oligosaccharide was subjected to vacuum concentration and freeze-drying operations in sequence to obtain solid κ-carrageenan oligosaccharide. The vacuum concentration temperature was 60°C.

[0049] (5) Weigh the items and calculate the yield. The formula for calculating the yield is as follows:

[0050] Yield = A / B × 100%

[0051] Where A represents 10g of κ-carrageenan oligosaccharide and B represents the mass (g) of κ-carrageenan.

[0052] Calculations showed that the yield of κ-carrageenan oligosaccharides in this comparative example was 73.45%, and the production of κ-carrageenan oligosaccharides was 7.34 g.

[0053] Example 1

[0054] The preparation method of κ-carrageenan oligosaccharides includes the following steps:

[0055] (1) Prepare a mixed solution of κ-carrageenan, H2O2 and ascorbic acid.

[0056] The solvent for this mixed solution is water, the concentration of κ-carrageenan is 2 wt%, the concentration of H2O2 is 10 mM, and the concentration of ascorbic acid is 10 mM.

[0057] (2) The mixed solution was placed in a reaction environment at a temperature of 50℃ and a pressure of 40MPa for 30 minutes to obtain a κ-carrageenan degradation solution.

[0058] (3) Centrifuge the κ-carrageenan degradation solution, collect the supernatant, then perform nanofiltration on the supernatant, collect the retentate, and obtain the κ-carrageenan oligosaccharide.

[0059] The centrifugation speed was 5000 r / min and the centrifugation time was 20 min. Centrifugation can remove undegraded κ-carrageenan with a large molecular weight. The nanofiltration temperature was 40℃ and the nanofiltration pressure was 0.5 MPa. The molecular weight cutoff was 100 Da-300 Da. The molecular weight of the obtained κ-carrageenan oligosaccharide was measured to be 1000 Da-3000 Da.

[0060] (4) The κ-carrageenan oligosaccharide was subjected to vacuum concentration and freeze-drying operations in sequence to obtain solid κ-carrageenan oligosaccharide. The vacuum concentration temperature was 60°C.

[0061] (5) Weigh the items and calculate the yield. The formula for calculating the yield is as follows:

[0062] Yield = A / B × 100%

[0063] Where A represents 10g of κ-carrageenan oligosaccharide and B represents the mass (g) of κ-carrageenan.

[0064] Calculations show that the yield of κ-carrageenan oligosaccharides in this embodiment is 88.12%, and the production of κ-carrageenan oligosaccharides is 8.81g.

[0065] The physicochemical properties of the κ-carrageenan oligosaccharides prepared in Example 1, Comparative Example 1, and Comparative Example 2 were determined using the following methods:

[0066] (1) Determination of total sugar content in κ-carrageenan oligosaccharides

[0067] In this invention, the total sugar content of κ-carrageenan oligosaccharides is determined using the phenol-sulfuric acid method, and the steps are as follows:

[0068] Construction of the standard curve: Measure 0.00, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, and 1.00 mL of 0.20 mg / mL D-(+)-galactose solution into glass test tubes, respectively, and add distilled water to bring the volume to 1.00 mL. Add 1.00 mL of 5.0% (w / v) phenol solution and 5.00 mL of concentrated sulfuric acid to each test tube, mix well, let stand for 20 min, and measure the absorbance of the mixture in each test tube at 490 nm to construct the standard curve.

[0069] Determination of total sugar content of κ-carrageenan oligosaccharides: Dissolve 0.0020 g of κ-carrageenan oligosaccharides in distilled water and bring the volume to 10.00 mL. Take 1.00 mL of the sample solution into a test tube, then add 1.00 mL of 5.0% (w / v) phenol solution and 5.00 mL of concentrated sulfuric acid, mix well, let stand for 20 min, and measure the absorbance of the mixture in the test tube at a wavelength of 490 nm.

[0070] (2) Determination of sulfate content in κ-carrageenan oligosaccharides

[0071] The sulfate content in κ-carrageenan oligosaccharides was determined using the gelatin-barium chloride method, as follows:

[0072] Plotting the standard curve: Accurately weigh 0.1088 g of K2SO4 dried to constant weight at 105℃, dissolve it in 1.00 mol / L HCl solution, and dilute to 100.00 mL in a volumetric flask. Shake well to obtain the sulfate standard stock solution (0.60 mg / mL). Take 0.00, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, and 0.20 mL of sulfate standard stock solution into test tubes, respectively, and add 1.00 mol / L HCl solution to each tube to a final volume of 0.20 mL. Use 0.20 mL of HCl solution as a blank control. Add 3.80 mL of 3.0% (w / v) trichloroacetic acid and 1.00 mL of 0.5% (w / v) gelatin-1.0% (w / v) barium chloride solution, shake well, and let stand at room temperature for 15 min. Measure the absorbance A1 at 360 nm. Repeat the above operation using 1.00 mL of 0.5% gelatin solution instead of barium chloride-gelatin solution, and measure the absorbance A2. 2- Plot a standard curve with concentration on the x-axis and absorbance difference (A1-A2) on the y-axis.

[0073] Determination of sulfate content in κ-carrageenan oligosaccharides: Accurately weigh 0.0100 g of κ-carrageenan oligosaccharides, dissolve in 5.00 mL of 1.00 mol / L HCl solution, boil in a water bath at 100℃ for 5 h, take 0.20 mL of sample solution, and determine the absorbance of the κ-carrageenan oligosaccharide solution according to the above method. Calculate the sulfate content in the sample according to the sulfate content standard curve.

[0074] (3) Determination of 3,6-lacto-D-galactose content in κ-carrageenan oligosaccharides

[0075] The operation steps are as follows:

[0076] Preparation of resorcinol reagent: (1) Weigh 0.3750g of resorcinol, add an appropriate amount of distilled water to dissolve it, and make up to 250.00mL; (2) Prepare 50.00mL of 0.04% (w / v) 1,1-acetal solution. (3) Take 9.00mL of resorcinol solution, 1.00mL of 1,1-acetal solution and 100mL of 12.00mol / L concentrated hydrochloric acid and mix them evenly.

[0077] Construction of the standard curve: Prepare 50.00 mL of a 1.40 μmol / mL fructose solution and store it for later use. Take 0.00, 0.20, 0.40, 0.60, 0.80, and 1.00 mL of the fructose solution into stoppered glass test tubes, respectively, and bring the volume to 1 mL with distilled water. Place the test tubes in an ice-water bath for 5 min. Then, add 5.00 mL of resorcinol reagent to each test tube, shake well, and react at 80℃ for 15 min. After the reaction, remove the tubes and place them in ice water for 2.0 min. Measure the absorbance of the solution in each test tube at 554 nm. Plot the standard curve.

[0078] Determination of 3,6-lacto-D-galactose content in κ-carrageenan oligosaccharides: Dissolve 0.0050 g of κ-carrageenan oligosaccharides in a beaker with an appropriate amount of distilled water, and bring the volume to 25.00 mL. Place 1.00 mL of the κ-carrageenan oligosaccharide solution in a glass test tube and determine the 3,6-lacto-D-galactose content in the κ-carrageenan oligosaccharides using the method described above.

[0079] Table 1 shows the yield, total sugar content, sulfate content, and 3,6-lacto-D-galactose content of the κ-carrageenan oligosaccharides prepared in Example 1, Comparative Example 1, and Comparative Example 2 of this invention:

[0080] Table 1

[0081]

[0082] As shown in Table 1, the total sugar content of the κ-carrageenan oligosaccharide prepared in Example 1 is comparable to that of Comparative Examples 1 and 2, but the sulfate content and yield are higher than those of Comparative Examples 1 and 2, and the 3,6-endoether-D-galactose content is lower than that of Comparative Examples 1 and 2. This indicates that the preparation method of the present invention reduces the damage to sulfate and effectively prevents the formation of 3,6-endoether-D-galactose in the κ-carrageenan oligosaccharide. Based on the prior art, it is inferred that the κ-carrageenan oligosaccharide prepared by the present invention may have higher bioactivity in subsequent applications.

[0083] (4) The molecular weight of κ-carrageenan oligosaccharides was determined by high performance gel permeation chromatography.

[0084] Accurately weigh 25.0 mg of κ-carrageenan oligosaccharide, dissolve it in ultrapure water, and dilute to 5 mL. Filter the solution through a 0.45 μm microporous membrane and perform HPGPC detection. The chromatographic conditions were as follows: HPGPC system: high performance liquid chromatography; column: gel chromatography; detector: differential refractive index detector; mobile phase: ultrapure water; flow rate: 0.4 mL / min; column temperature: 40 °C; injection volume: 20 μL. Simultaneously, prepare 5.0 mg / mL standard solutions of dextran with different molecular weights (5 kDa, 25 kDa, 50 kDa, 150 kDa, and 270 kDa, respectively), filter through a 0.45 μm microporous membrane, and perform HPGPC detection to plot a standard curve. Based on this, the molecular weight of κ-carrageenan oligosaccharide was calculated, and the results are shown in [Figure not provided]. Figure 1 .

[0085] (5) The microstructure of κ-carrageenan and κ-carrageenan oligosaccharides was observed by scanning electron microscopy.

[0086] The samples were sprayed onto the sample holder with conductive adhesive, and the surface morphology of κ-carrageenan and κ-carrageenan oligosaccharides was observed in a vacuum sample chamber. The results are shown in [Figure number missing]. Figure 2 and Figure 3 .

[0087] (6) Antibacterial activity test was performed on the κ-carrageenan oligosaccharide prepared in Example 1.

[0088] The strains involved include: Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Pseudomonas hydrophila, Pseudomonas stutzeri, and Shigella flexneri. All strains were purchased from the China General Microbiological Culture Collection Center.

[0089] The testing method is as follows:

[0090] ① The preserved Staphylococcus aureus, Listeria, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas schrenckii, and Shigella freundii were spread on nutrient broth plates and incubated upside down at the suitable growth temperature of each bacterium for 18-24 hours.

[0091] ② Pick colonies from each plate and inoculate them onto the corresponding culture slant, then continue culturing for 18-24 hours. Wash the bacteria off the slant with sodium phosphate buffer (10mM, pH=7.4), adjust the bacterial suspension concentration using a 10-fold dilution method, so that the final bacterial concentration is 5×10⁻⁶. 5 CFU / mL.

[0092] ③ Dissolve κ-carrageenan oligosaccharide in sterile MiliiQ water, filter through a 0.22μM filter membrane, and place on ice for later use.

[0093] ④ In 96-well cell culture plates, blank control group, negative control group and test experimental group were set up for each test bacteria, and three replicates were set up for each group. Each group was serially diluted to a κ-carrageenan oligosaccharide concentration of 1.25 mg / mL. The initial composition of each group is shown below:

[0094] a. Blank control group: 100 μL of 80 mg / mL κ-carrageenan oligosaccharide sample and 100 μL of culture medium;

[0095] negative control group: 100 μL of sterile MilliQ water and 100 μL of bacterial suspension;

[0096] c. Test group: 100 μL of 80 mg / mL κ-carrageenan oligosaccharide sample and 100 μL of bacterial suspension.

[0097] ⑤ Place the 96-well cell culture plate in a 28℃ incubator and incubate for 1-2 days. Determine the minimum inhibitory concentration (MIC) of each bacterium. After thoroughly mixing the experimental groups by pipetting, take an appropriate amount of bacterial suspension and drop it onto the corresponding solid culture medium plate, spread it evenly, and incubate upside down at a suitable temperature for 1-2 days. Observe the minimum bactericidal concentration (MBC) results. The results are shown in Table 2.

[0098] Table 2. Analysis of the antibacterial activity of κ-carrageenan oligosaccharides

[0099]

[0100] The κ-carrageenan oligosaccharide prepared by the extraction method of this invention has broad-spectrum antibacterial activity. Because it is derived from natural seaweed polysaccharides, it has good safety and can be used as an antibacterial agent in feed additives, as well as in the preparation of food, cosmetics or disinfectants. It can also be prepared into antibacterial compositions, etc., and has good application prospects.

[0101] Example 2

[0102] The preparation method of κ-carrageenan oligosaccharides includes the following steps:

[0103] (1) Prepare a mixed solution of κ-carrageenan, H2O2 and ascorbic acid.

[0104] The solvent for this mixed solution is water, the concentration of κ-carrageenan is 2 wt%, the concentration of H2O2 is 20 mM, and the concentration of ascorbic acid is 20 mM.

[0105] (2) Place the mixed solution in a reaction environment at a temperature of 50℃ and a pressure of 40MPa and react for 30min to obtain κ-carrageenan degradation solution.

[0106] (3) Centrifuge the κ-carrageenan degradation solution, collect the supernatant, then perform nanofiltration on the supernatant, collect the retentate, and obtain the κ-carrageenan oligosaccharide.

[0107] The centrifugation speed was 5000 r / min and the centrifugation time was 20 min. Centrifugation can remove undegraded κ-carrageenan with a large molecular weight. The nanofiltration temperature was 40℃ and the nanofiltration pressure was 0.5 MPa. The molecular weight cutoff was 100 Da-300 Da. The molecular weight of the obtained κ-carrageenan oligosaccharide was measured to be 1000 Da-3000 Da.

[0108] (4) The κ-carrageenan oligosaccharide was subjected to vacuum concentration and freeze-drying operations in sequence to obtain solid κ-carrageenan oligosaccharide. The vacuum concentration temperature was 60°C.

[0109] (5) Weigh the items and calculate the yield. The formula for calculating the yield is as follows:

[0110] Yield = A / B × 100%

[0111] Where A represents 10g of κ-carrageenan oligosaccharide and B represents the mass (g) of κ-carrageenan.

[0112] Calculations show that the yield of κ-carrageenan oligosaccharides in this embodiment is 90.63%, and the production of κ-carrageenan oligosaccharides is 9.06g.

[0113] Example 3

[0114] The preparation method of κ-carrageenan oligosaccharides includes the following steps:

[0115] (1) Prepare a mixed solution of κ-carrageenan, H2O2 and ascorbic acid.

[0116] The solvent for this mixed solution is water, the concentration of κ-carrageenan is 2 wt%, the concentration of H2O2 is 10 mM, and the concentration of ascorbic acid is 10 mM.

[0117] (2) Place the mixed solution in a reaction environment at a temperature of 60℃ and a pressure of 60MPa and react for 10min to obtain κ-carrageenan degradation solution.

[0118] (3) Centrifuge the κ-carrageenan degradation solution, collect the supernatant, then perform nanofiltration on the supernatant, collect the retentate, and obtain the κ-carrageenan oligosaccharide.

[0119] The centrifugation speed was 5000 r / min and the centrifugation time was 20 min. Centrifugation can remove undegraded κ-carrageenan with a large molecular weight. The nanofiltration temperature was 40℃ and the nanofiltration pressure was 0.5 MPa. The molecular weight cutoff was 100 Da-300 Da. The molecular weight of the obtained κ-carrageenan oligosaccharide was measured to be 1000 Da-3000 Da.

[0120] (4) The κ-carrageenan oligosaccharide was subjected to vacuum concentration and freeze-drying operations in sequence to obtain solid κ-carrageenan oligosaccharide. The vacuum concentration temperature was 60°C.

[0121] (5) Weigh the items and calculate the yield. The formula for calculating the yield is as follows:

[0122] Yield = A / B × 100%

[0123] Where A represents 10g of κ-carrageenan oligosaccharide and B represents the mass (g) of κ-carrageenan.

[0124] Calculations show that the yield of κ-carrageenan oligosaccharides in this embodiment is 86.91%, and the production volume of κ-carrageenan oligosaccharides is 8.69g.

[0125] Example 4

[0126] The preparation method of κ-carrageenan oligosaccharides includes the following steps:

[0127] (1) Prepare a mixed solution of κ-carrageenan, H2O2 and ascorbic acid.

[0128] The solvent for this mixed solution is water, the concentration of κ-carrageenan is 2 wt%, the concentration of H2O2 is 30 mM, and the concentration of ascorbic acid is 30 mM.

[0129] (2) The mixed solution was placed in a reaction environment at a temperature of 40℃ and a pressure of 20MPa for 20 minutes to obtain a κ-carrageenan degradation solution.

[0130] (3) Centrifuge the κ-carrageenan degradation solution, collect the supernatant, then perform nanofiltration on the supernatant, collect the retentate, and obtain the κ-carrageenan oligosaccharide.

[0131] The centrifugation speed was 5000 r / min and the centrifugation time was 20 min. Centrifugation can remove undegraded κ-carrageenan with a large molecular weight. The nanofiltration temperature was 40℃ and the nanofiltration pressure was 0.5 MPa. The molecular weight cutoff was 100 Da-300 Da. The molecular weight of the obtained κ-carrageenan oligosaccharide was measured to be 1000 Da-3000 Da.

[0132] (4) The κ-carrageenan oligosaccharide was subjected to vacuum concentration and freeze-drying operations in sequence to obtain solid κ-carrageenan oligosaccharide. The vacuum concentration temperature was 60°C.

[0133] (5) Weigh the items and calculate the yield. The formula for calculating the yield is as follows:

[0134] Yield = A / B × 100%

[0135] Where A represents 10g of κ-carrageenan oligosaccharide and B represents the mass (g) of κ-carrageenan.

[0136] Calculations show that the yield of κ-carrageenan oligosaccharides in this embodiment is 84.43%, and the production of κ-carrageenan oligosaccharides is 8.43g.

[0137] Example 5

[0138] The preparation method of κ-carrageenan oligosaccharides includes the following steps:

[0139] (1) Prepare a mixed solution of κ-carrageenan, H2O2 and ascorbic acid.

[0140] The solvent for this mixed solution is water, the concentration of κ-carrageenan is 2 wt%, the concentration of H2O2 is 20 mM, and the concentration of ascorbic acid is 10 mM.

[0141] (2) Place the mixed solution in a reaction environment at a temperature of 50℃ and a pressure of 40MPa and react for 30min to obtain κ-carrageenan degradation solution.

[0142] (3) Centrifuge the κ-carrageenan degradation solution, collect the supernatant, then perform nanofiltration on the supernatant, collect the retentate, and obtain the κ-carrageenan oligosaccharide.

[0143] The centrifugation speed was 5000 r / min and the centrifugation time was 20 min. Centrifugation can remove undegraded κ-carrageenan with a large molecular weight. The nanofiltration temperature was 40℃ and the nanofiltration pressure was 0.5 MPa. The molecular weight cutoff was 100 Da-300 Da. The molecular weight of the obtained κ-carrageenan oligosaccharide was measured to be 1000 Da-3000 Da.

[0144] (4) The κ-carrageenan oligosaccharide was subjected to vacuum concentration and freeze-drying operations in sequence to obtain solid κ-carrageenan oligosaccharide. The vacuum concentration temperature was 60°C.

[0145] (5) Weigh the items and calculate the yield. The formula for calculating the yield is as follows: 2

[0146] Yield = A / B × 100%

[0147] Where A represents 10g of κ-carrageenan oligosaccharide and B represents the mass (g) of κ-carrageenan.

[0148] Calculations show that the yield of κ-carrageenan oligosaccharides in this embodiment is 85.14%, and the production of κ-carrageenan oligosaccharides is 8.51g.

[0149] Example 6

[0150] The preparation method of κ-carrageenan oligosaccharides includes the following steps:

[0151] (1) Add H2O2 solution and ascorbic acid solution to κ-carrageenan solution to obtain a mixed solution.

[0152] The solvent for this mixed solution is water, the concentration of κ-carrageenan solution is 2 wt%, the concentration of H2O2 solution is 50 mM, and the concentration of ascorbic acid solution is 5 mM.

[0153] (2) The mixed solution was placed in a reaction environment at a temperature of 50℃ and a pressure of 30MPa for 30 min to obtain a κ-carrageenan degradation solution.

[0154] (3) Centrifuge the κ-carrageenan degradation solution, collect the supernatant, then perform nanofiltration on the supernatant, collect the retentate, and obtain the κ-carrageenan oligosaccharide.

[0155] The centrifugation speed was 5000 r / min and the centrifugation time was 20 min. Centrifugation can remove undegraded κ-carrageenan with a large molecular weight. The nanofiltration temperature was 40℃ and the nanofiltration pressure was 0.5 MPa. The molecular weight cutoff was 100 Da-300 Da. The molecular weight of the obtained κ-carrageenan oligosaccharide was measured to be 1000 Da-3000 Da.

[0156] (4) The κ-carrageenan oligosaccharide was subjected to vacuum concentration and freeze-drying operations in sequence to obtain solid κ-carrageenan oligosaccharide. The vacuum concentration temperature was 60°C.

[0157] (5) Weigh the items and calculate the yield. The formula for calculating the yield is as follows:

[0158] Yield = A / B × 100%

[0159] Wherein, A represents 10g of κ-carrageenan oligosaccharide, and B represents the mass (g) of κ-carrageenan. Calculations show that the yield of κ-carrageenan oligosaccharide in this example is 64.22%, and the production volume of κ-carrageenan oligosaccharide is 6.42g.

[0160] Although the yield of Example 6 was slightly lower than that of Comparative Example 2, Example 6 was still considered superior to Comparative Example 2 overall because the reaction time of Example 6 was halved, which improved the reaction efficiency, and the reaction temperature was significantly reduced, which prevented the caramelization reaction of κ-carrageenan oligosaccharides.

[0161] Example 7

[0162] The preparation method of κ-carrageenan oligosaccharides includes the following steps:

[0163] (1) Add H2O2 solution and ascorbic acid solution to κ-carrageenan solution to obtain a mixed solution.

[0164] The solvent for this mixed solution is water, the concentration of κ-carrageenan solution is 2 wt%, the concentration of H2O2 solution is 50 mM, and the concentration of ascorbic acid solution is 50 mM.

[0165] (2) The mixed solution was placed in a reaction environment at a temperature of 40℃ and a pressure of 20MPa for 30 minutes to obtain a κ-carrageenan degradation solution.

[0166] (3) Centrifuge the κ-carrageenan degradation solution, collect the supernatant, then perform nanofiltration on the supernatant, collect the retentate, and obtain the κ-carrageenan oligosaccharide.

[0167] The centrifugation speed was 5000 r / min and the centrifugation time was 20 min. Centrifugation can remove undegraded κ-carrageenan with a large molecular weight. The nanofiltration temperature was 40℃ and the nanofiltration pressure was 0.5 MPa. The molecular weight cutoff was 100 Da-300 Da. The molecular weight of the obtained κ-carrageenan oligosaccharide was measured to be 1000 Da-3000 Da.

[0168] (4) The κ-carrageenan oligosaccharide was subjected to vacuum concentration and freeze-drying operations in sequence to obtain solid κ-carrageenan oligosaccharide. The vacuum concentration temperature was 60°C.

[0169] (5) Weigh the items and calculate the yield. The formula for calculating the yield is as follows:

[0170] Yield = A / B × 100%

[0171] Where A represents 10g of κ-carrageenan oligosaccharide and B represents the mass (g) of κ-carrageenan.

[0172] Calculations show that the yield of κ-carrageenan oligosaccharides in this embodiment is 89.53%, and the production volume of κ-carrageenan oligosaccharides is 8.95g.

[0173] Example 8

[0174] The infrared spectra of raw κ-carrageenan and the κ-carrageenan oligosaccharides prepared in Examples 1-5 were detected using Fourier transform infrared spectroscopy. Before testing, the dried samples were mixed uniformly with dried KBr and pressed into sheets for testing. The scanning range was set to 400 cm⁻¹. -1 -4000cm -1 The resolution is 4cm. -1 The results are shown Figure 4 .

[0175] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for preparing κ-carrageenan oligosaccharides, characterized in that, Includes the following steps: (1) Prepare a mixed solution of κ-carrageenan, H2O2 and ascorbic acid; the concentration of κ-carrageenan solution in the mixed solution is 1.5wt%-2.5wt%, the concentration of H2O2 is 10mM-50 mM, the concentration of ascorbic acid is 10mM-50 mM, and the concentration ratio of H2O2 to ascorbic acid is 1:1; (2) Place the above mixed solution in a reaction environment with a temperature of 40℃-60℃ and a pressure of 20MPa-60MPa, and react for 5min-30min to obtain κ-carrageenan degradation solution; (3) Centrifuge the above κ-carrageenan degradation solution, collect the supernatant, and then perform nanofiltration on the supernatant to collect the retentate to obtain the κ-carrageenan oligosaccharide; the centrifugation speed is 5000r / min-8000r / min, and the time is 10min-20min; the nanofiltration temperature is 40℃, the pressure is 0.2MPa-1.0MPa, and the molecular weight cutoff is 100Da-300Da.

2. The preparation method according to claim 1, characterized in that, The process also includes step (4): subjecting the κ-carrageenan oligosaccharide to vacuum concentration and freeze-drying operations in sequence to obtain the solid κ-carrageenan oligosaccharide.

3. An antibacterial composition, characterized in that, Its active ingredient is κ-carrageenan oligosaccharide prepared by the preparation method described in claim 1 or 2.

4. The antibacterial composition according to claim 3, characterized in that, The bacteria mentioned are Staphylococcus aureus, Listeria, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas schrenckii, or Shigella flexneri.

5. The use of the κ-carrageenan oligosaccharide prepared by the method according to claim 1 or 2 in the preparation of antibacterial compositions.

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