Ulva polysaccharide gel and preparation method thereof

Abstract

The invention discloses ulva polysaccharide gel and a preparation method thereof, relates to the technical field of ulva polysaccharide metal ion gels, and solves the problems that ulva polysaccharide gel is not reported and the application of ulva polysaccharide in gels is rare. The preparation method comprises the following steps: step 1, crushing dried ulva into powder by using a crusher; 2, soaking the ulva powder with 95% ethanol for 24 hours, and drying at 30 DEG C; 3, stirring the dehydrated powder in water at 95 DEG C for 4 hours, and centrifuging to obtain supernate A; 4, concentrating the supernate A to 40mL by using a rotary evaporator; 5, ethanol with the volume 3 times that of the crude polysaccharide is added, the crude polysaccharide is precipitated at the temperature of 4 DEG C for 24 h, the crude polysaccharide precipitate is separated, dissolved and centrifuged, supernatant is taken, and a polysaccharide solution A is obtained; according to the invention, a natural acidic polysaccharide gel is developed, the value of ulva lactuca is increased, and the polysaccharide gel can be applied to delivery of probiotics.

Description

Technical Field

[0001] The invention relates to the technical field of ulva polysaccharide metal ion gel, in particular to a ulva polysaccharide gel and a preparation method thereof. Background Art

[0002] Ulva is a large green algae with an annual production of nearly 1,800 tons. It is rich in nutrients such as polysaccharides, proteins, and inorganic minerals. Its main components include glucuronic acid, iduronic acid, rhamnose, and xylose. It is composed of β-d-glucuronic acid (1→4)-α-l-3-sulfate rhamnose and α-l-iduronic acid (1→4)-α-l-3-sulfate rhamnose in two main chain forms. Its unique chemical structure and sulfidation characteristics give it multiple biological functions such as antioxidant and immunomodulatory. Due to its significant biological activity, Ulva polysaccharides have broad application prospects as probiotic encapsulation agents.

[0003] There are no reports on Ulva polysaccharide gel, and its application in gel is rare. As a new type of wall material, Ulva polysaccharide not only gives full play to its good safety and biocompatibility from its natural source, but also plays its role as a prebiotic. The gel helps probiotics pass through the gastric acid environment and provides a new method for its application in protecting the delivery system. In this regard, we proposed a Ulva polysaccharide gel and a preparation method thereof. Summary of the invention

[0004] The purpose of the present invention is to provide a Ulva polysaccharide gel and a preparation method thereof, so as to solve the problem that there is no report on Ulva polysaccharide gel and the application of Ulva polysaccharide in gel mentioned in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a Ulva polysaccharide gel, comprising Ulva polysaccharide and trivalent iron ions.

[0006] Preferably, the content of trivalent iron ions is 5.73% to 5.95%.

[0007] A method for preparing ulva polysaccharide gel comprises the following steps:

[0008] Step 1: Use a crusher to grind the dried Ulva into powder;

[0009] Step 2: Soak the Ulva powder in 95% ethanol for 24 h and dry it at 30 °C;

[0010] Step 3: Stir the dehydrated powder in 95°C water for 4 hours and obtain supernatant A after centrifugation;

[0011] Step 4: Concentrate the supernatant A to 40 mL using a rotary evaporator;

[0012] Step 5: Add 3 times the volume of ethanol to precipitate crude polysaccharides at 4°C for 24 hours, separate and dissolve the crude precipitate, and centrifuge to obtain the supernatant to obtain polysaccharide solution A;

[0013] Step 6: Prepare Sevag reagent with n-butanol and chloroform in a volume ratio of 1:4, add a quarter volume of Sevag reagent to polysaccharide solution A, centrifuge and collect the aqueous phase, repeat this process 10 times to obtain polysaccharide solution B;

[0014] Step 7: Then put the polysaccharide solution B into a dialysis bag and dialyze it against deionized water for 24 hours;

[0015] Step 8: obtaining Ulva polysaccharide with a purity greater than 90%, dissolving it in deionized water to obtain a mixed solution;

[0016] Step 9: Stir the mixed solution of step 8 for 2 hours to dissolve, and heat it in a water bath at 60 degrees Celsius;

[0017] Step 10: dissolve ferric chloride in deionized water to a concentration of 0.05-0.2 M, add the Ulva polysaccharide solution to the ferric chloride solution, and age for half an hour;

[0018] Step 11: Take out the formed Ulva polysaccharide gel.

[0019] Preferably, the concentration range of the mixed solution is 10-15 mg / mL.

[0020] The ulva polysaccharide gel prepared by the preparation method is used for encapsulating and delivering probiotics.

[0021] A method for preparing probiotic gel microspheres containing ulva polysaccharide gel obtained by the preparation method, characterized in that it comprises the following steps:

[0022] S1: uniformly mixing the prepared Ulva polysaccharide solution and the probiotic L. gasseri solution;

[0023] S2: The mixed solution is dropped into the FeCl3 solution with a syringe, and the probiotic gel microspheres are formed after aging.

[0024] Preferably, the concentration of the probiotic L. gasseri solution is ×10 8 CFU / mL.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention develops a natural acidic polysaccharide gel, which increases the value of Ulva; the polysaccharide gel can be used for probiotic delivery and has practical value;

[0027] 2. The present invention integrates ULP+Fe3+ gel and probiotics into capsule microspheres through the droplet injection method. Compared with the unencapsulated strains, the survival rate of live bacteria in the gastrointestinal tract is significantly improved, thereby providing protection for the probiotics during the entire digestive process. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 forming an image for the gel of the present invention;

[0029] Figure 2 is a rheological analysis diagram of the Ulva polysaccharide gel of the present invention;

[0030] Figure 3 is a curve diagram showing the effect of iron ion concentration on the rheological properties of gel;

[0031] Figure 4 This is a curve diagram showing the effect of Ulva polysaccharide on the rheological properties of gel;

[0032] Figure 5 This is a schematic diagram of the infrared analysis results of Ulva polysaccharide gel;

[0033] Figure 6 To form images of gel microspheres under a microscope;

[0034] Figure 7 Schematic diagram of the characterization of probiotic gel microspheres for probiotic delivery and protection. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0036] The present invention provides five embodiments:

[0037] Embodiment 1:

[0038] The invention discloses an ulva polysaccharide gel, comprising ulva polysaccharide and trivalent iron ion, wherein the content of the trivalent iron ion is 5.73% to 5.95%.

[0039] A method for preparing ulva polysaccharide gel comprises the following steps:

[0040] Step 1: Use a crusher to grind the dried Ulva into powder;

[0041] Step 2: Soak the Ulva powder in 95% ethanol for 24 h and dry it at 30 °C;

[0042] Step 3: Stir the dehydrated powder in 95°C water for 4 hours and obtain supernatant A after centrifugation;

[0043] Step 4: Concentrate the supernatant A to 40 mL using a rotary evaporator;

[0044] Step 5: Add 3 times the volume of ethanol to precipitate the crude polysaccharide (ULP) at 4°C for 24 hours, separate and dissolve the crude precipitate, and centrifuge to obtain the supernatant to obtain a polysaccharide solution A;

[0045] Step 6: Prepare Sevag reagent with n-butanol and chloroform in a volume ratio of 1:4, add a quarter volume of Sevag reagent to polysaccharide solution A, centrifuge and collect the aqueous phase, repeat this process 10 times to obtain polysaccharide solution B;

[0046] Step 7: Then put the polysaccharide solution B into a dialysis bag and dialyze it against deionized water for 24 hours;

[0047] Step 8: obtaining Ulva polysaccharide with a purity greater than 90%, dissolving it in deionized water to obtain a mixed solution, wherein the concentration range of the mixed solution is 10-15 mg / mL;

[0048] Step 9: Stir the mixed solution of step 8 for 2 hours to dissolve, and heat it in a water bath at 60 degrees Celsius;

[0049] Step 10: dissolve ferric chloride in deionized water to a concentration of 0.05-0.2 M, add the Ulva polysaccharide solution to the ferric chloride solution, and age for half an hour;

[0050] Step 11: Take out the formed Ulva polysaccharide gel.

[0051] The ulva polysaccharide gel prepared by the above preparation method is used for encapsulating and delivering probiotics.

[0052] Image characterization of the formed gel, such as Figure 1 shown.

[0053] Embodiment 2:

[0054] The specific method for determining the rheological properties of the Ulva polysaccharide obtained by the above preparation method is as follows:

[0055] Rheological analyses were all performed by a TADHR-2 rheometer equipped with stainless steel parallel plates (25 mm in diameter). Strain sweep test The Ulva polysaccharide gel was fixed in shape and placed on a shear platform, and strain sweep measurements were performed in the range of 0.1% to 10% to determine the linear viscoelastic region (IVR) at 25°C with a frequency of 1 Hz. For the frequency sweep test, a constant strain of 0.1% (within the LVR region) was selected, the frequency range was 10 to 0.1 Hz, and the elastic modulus (storage modulus, G′) and viscous modulus (loss modulus, G″) were recorded. For the temperature sweep, the sample was sealed with silicone oil. A fixed strain of 0.1% and a constant frequency of 1 Hz were used to monitor the storage modulus (G′) and loss modulus (G″). Heating from 25°C to 85°C at a rate of 5°C / min, and then cooling from 85°C to 25°C at a rate of 5°C / min.

[0056] As the instruction manual Figure 2 As shown, Fe 3+ It can effectively promote the gel formation of Ulva polysaccharide. Stress relaxation experiments were carried out, which showed that after mixing the Ulva polysaccharide solution with the FeCl3 solution, a strong gel was formed, producing G'>G". The elastic modulus (G') and loss modulus (G") of the Ulva polysaccharide gel with a frequency of 0.1 to 10 Hz were evaluated.

[0057] As the instruction manual Figure 3 And the instruction manual Figure 4 As shown, both the elastic modulus (G′) and loss modulus (G″) of the Ulva polysaccharide gel increased at different Fe3+ ion concentrations. This indicates that more molecules were added to the Ulva polysaccharide gel network, leading to network integration. The increase in ULP concentration also showed the same trend, indicating that the gel strength is related to the concentration.

[0058] Embodiment three:

[0059] The infrared analysis results and formation mechanism of the Ulva polysaccharide gel prepared in Example 1 were evaluated as follows:

[0060] The samples were subjected to FTIR analysis (transmission mode) using a TENSORII spectrometer (Frontier, PerkinElmer, UK). The samples were mixed with KBr at a ratio of 1:100 (w / w) and pressed into tablets using a tablet press. The FTIR spectrum of the samples was 4000-400 cm -1 The wavenumber range is 4 cm, and the resolution is 4 cm. -1 .

[0061] Based on the above results, we can speculate that the formation mechanism of Ulva polysaccharide gel is that some hydrogen bonds in the ULP chains first interact to form weak viscoelastic entanglements, and then Fe 3+Ions cross-link with carboxyl and hydroxyl groups to form a dynamic network, as shown in the attached manual. Figure 5 As shown, it was demonstrated that the composite hydrogel was prepared without sacrificing the bioactive functional groups in the polysaccharide backbone structure.

[0062] Embodiment 4:

[0063] A method for preparing probiotic gel microspheres containing the ulva polysaccharide gel obtained by the above preparation method comprises the following steps:

[0064] S1: The prepared Ulva polysaccharide solution was mixed evenly with the probiotic L. gasseri solution. The concentration of the probiotic L. gasseri solution was ×10 8 CFU / mL;

[0065] S2: The mixed solution is dropped into the FeCl3 solution with a syringe, and the probiotic gel microspheres are formed after aging.

[0066] Microscope images of the formed gel were taken as shown in the attached manual. Figure 6 shown.

[0067] The probiotic delivery protection effect of the ulva polysaccharide gel microspheres prepared in Example 5 was evaluated. The specific method is as follows: In the in vitro anti-digestion simulation experiment of ulva polysaccharide gel microspheres, equal amounts of natural Lactobacillus gasseri and ulva polysaccharide encapsulated Lactobacillus gasseri were resuspended in 1 mL of simulated gastric fluid (SGF, pH = 2.0), simulated intestinal fluid (SIF, pH = 6.8) and bile salt (0.3 mg / mL). The samples were then gently shaken and further incubated at 37°C. At the predetermined time point, 50 μL of each sample was taken, washed with NaCl (0.15 M), and counted on a spot plate. Preparation of SIF, each 100 mL ddH2O contained 0.68 g KH2PO4 and 1 g trypsin; the pH was adjusted to 6.8 with NaOH, and filtered through a 0.22 μm membrane. Preparation of SGF, each 100mL ddH2O contains 0.2g NaCl and 0.32g pepsin; pH is adjusted to 2.0 using diluted HCl and 0.22μm membrane filtration.

[0068] As the instruction manual Figure 7 As shown, under the conditions of SGF, SIF and bile salt, the results showed significant differences between free cells and ULP-encapsulated L. gasseri (P<0.05). The number of live bacteria in the gel-encapsulated probiotic group was higher than that in the natural L. gasseri group, indicating that the use of ULP+Fe3+ gel-encapsulated probiotics has a protective effect. Further comparison showed that the protective effect of gel microspheres on probiotics is mainly reflected in SGF. The number of live bacteria can reach 30% (≥10 7CFU / mL), which is three times that of the natural probiotic group. Its basic principle is based on the ability of the gel formed by ULP to maintain its structural integrity in an acidic environment.

[0069] The encapsulation and protection effect of the Ulva polysaccharide gel of the present invention on probiotics was evaluated through in vitro digestion, and the ULP+Fe3+ gel was integrated with probiotics through the droplet injection method to prepare capsule microspheres. Compared with the strains that were not encapsulated, the survival rate of live bacteria in the gastrointestinal tract was significantly improved, thereby providing protection for the probiotics throughout the digestive process.

[0070] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A Ulva polysaccharide gel, characterized in that: Includes Ulva polysaccharide and trivalent iron ions.

2. The Ulva polysaccharide gel and the preparation method thereof according to claim 1, characterized in that: The content of trivalent iron ions is 5.73% to 5.95%.

3. A method for preparing the Ulva polysaccharide gel according to any one of claims 1 to 2, characterized in that: The steps include: Step 1: Use a crusher to grind the dried Ulva into powder; Step 2: Soak the Ulva powder in 95% ethanol for 24 h and dry it at 30 °C; Step 3: Stir the dehydrated powder in 95°C water for 4 hours and obtain supernatant A after centrifugation; Step 4: Concentrate the supernatant A to 40 mL using a rotary evaporator; Step 5: Add 3 times the volume of ethanol to precipitate crude polysaccharides at 4°C for 24 hours, separate and dissolve the crude precipitate, and centrifuge to obtain the supernatant to obtain polysaccharide solution A; Step 6: Prepare Sevag reagent with n-butanol and chloroform in a volume ratio of 1:4, add a quarter volume of Sevag reagent to polysaccharide solution A, centrifuge and collect the aqueous phase, repeat this process 10 times to obtain polysaccharide solution B; Step 7: Then put the polysaccharide solution B into a dialysis bag and dialyze it against deionized water for 24 hours; Step 8: obtaining Ulva polysaccharide with a purity greater than 90%, dissolving it in deionized water to obtain a mixed solution; Step 9: Stir the mixed solution of step 8 for 2 hours to dissolve, and heat it in a water bath at 60 degrees Celsius; Step 10: dissolve ferric chloride in deionized water to a concentration of 0.05-0.2 M, add the Ulva polysaccharide solution to the ferric chloride solution, and age for half an hour; Step 11: Take out the formed Ulva polysaccharide gel.

4. The method for preparing the Ulva polysaccharide gel according to claim 3, characterized in that: The concentration range of the mixed solution is 10-15 mg / mL.

5. The ulva polysaccharide gel prepared by the preparation method according to any one of claims 3 to 4 is used for encapsulating and delivering probiotics.

6. A method for preparing probiotic gel microspheres comprising the ulva polysaccharide gel obtained by the preparation method according to any one of claims 3 to 4, characterized in that: The steps include: S1: uniformly mixing the prepared Ulva polysaccharide solution and the probiotic L. gasseri solution; S2: The mixed solution is dropped into the FeCl3 solution with a syringe, and the probiotic gel microspheres are formed after aging.

7. The Ulva polysaccharide gel and the preparation method thereof according to claim 6, characterized in that: The concentration of the probiotic L. gasseri solution is ×10 8 CFU / mL.

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