A preparation method of a gellan gum-sialyllactose pH-responsive graft

Abstract

The application discloses a preparation method of a gellan gum-sialyllactose pH responsive graft, and belongs to the technical field of functional biological polymer production. The gellan gum-sialyllactose pH responsive graft is synthesized by an amidation reaction and a reductive amination reaction of raw materials gellan gum and sialyllactose, and is obtained by alcohol precipitation dialysis and freeze drying. The gellan gum-sialyllactose pH responsive graft is similar to a natural mucin structure and functions, and is a negatively charged sialylated glycan. The graft can resist digestion in the gastrointestinal tract, directly reaches the colon for intestinal microorganism utilization, has good antioxidant, anti-inflammatory and antiviral functions, is non-toxic to cells, reduces cell damage and inflammatory reactions of an LPS-induced inflammatory cell model, and has a wide application in the preparation of functional food and medicines.

Description

Technical Field

[0001] This invention relates to a method for preparing a gellan gum-sialic acid lactose pH-responsive graft, belonging to the field of functional biopolymer production technology. Background Technology

[0002] With social development and improved living standards, life expectancy has increased, leading to a significant aging trend in the population. Health issues among the elderly have become a core concern of this aging phenomenon. Epidemiological statistics show that Alzheimer's disease (AD) has become one of the most serious threats to the health of the elderly. The gut microbiota of the elderly is closely related to their metabolic diseases, immune diseases, gastrointestinal diseases, cardiovascular diseases, and even mental illnesses. As people age, organ function declines, and the gut microbiota changes accordingly. Furthermore, the gut microbiota is closely related to the physiological functions of the central nervous system. Currently, sialic acid polysaccharides have become a popular adjunctive treatment for the prevention and treatment of intestinal diseases.

[0003] Sialic acid is a family of α-ketoaldehydes with a nine-carbon pyranose structure, generally referring to the family of N- and O-substituted derivatives of neuraminic acid. Sialic acid is ubiquitous in biological systems. In humans, N-acetylneuraminic acid is the most common form of sialic acid in human glycoconjugates and is widely expressed throughout the body, including glycoproteins (typically mucins), glycolipids, and lactic oligosaccharides. Sialic acid plays a crucial role in neural development, synaptic transmission, cognition, and immune function, and can serve as a recognition site for some invading pathogens. In the brain, polysialic acid can regulate cellular stress responses by binding to β-amyloid (Aβ) to block Aβ aggregation or by binding to cell surface protein receptors, thereby reducing neuroinflammation and cytotoxicity. Furthermore, the gut microbiota has been widely recognized for its important role in host health; oral administration of sialic acid polysaccharides may alter the pathological state and microbial composition of the gut, modulate immune and antioxidant activity, and further regulate physiological health through the brain-gut axis.

[0004] Sialic acid can regulate the composition of the gut microbiota, shaping a healthy gut microbiota by promoting the growth of beneficial bacteria such as *Ackermania proteoglycans*, *Lactobacillus*, *Bifidobacterium*, and *Ruminococcus*, while reducing the relative abundance of potentially pathogenic bacteria such as *Helicobacter pylori*, *Salmonella*, *Shigella*, and *Klebsiella*. Simultaneously, sialic acid also promotes the release of beneficial metabolites such as short-chain fatty acids or lactose to further promote gut health. Furthermore, studies have shown that sialyl polysaccharides can promote the adhesion of beneficial bacteria to the mucus layer and bind to receptors of pathogenic bacteria, parasites, or viruses, facilitating the clearance of pathogens from the gut. Colonic bacteria express many glycosidases, including sialylases, which release free sialic acid from sialylated oligosaccharides, which then serve as substrates for bacterial energy metabolism. Therefore, the gut's sialic acid needs can be met by ingesting sialyl polysaccharides. The sialic acid excretion / metabolism ratio is strongly dependent on the retention time and properties of sialylated molecules in the gut.

[0005] Studies have shown that after free sialic acid is ingested by the body through the small intestine, a large portion of it is excreted in urine. However, bound sialic acid glycans, such as sialylated glycoproteins (mucin), are not absorbed and reach the colon directly. They remain in the intestine for a relatively long time and can be fully utilized by gut microbiota, promoting gut microbiota balance and protecting the intestinal barrier.

[0006] Sialic acid-terminated glycans are key receptors for many viruses. Viral attachment to sialic acid glycans / sialic acid glycoproteins involves extensive contact with one side of the sialic acid ring. This preference likely forms two key contact sites: one is the carboxyl group of sialic acid with a negatively charged group, and the other involves the nitrogen atom in the N-acetyl group. In recent years, research on constructing complexes of sialic acid oligosaccharides with polysaccharides has been gradually developing. For example, there are reports of ionic bonding of sialic acid oligosaccharides with chitosan to form complexes (CN200610011918.1, Sialic acid oligosaccharide-chitosan complex and its preparation method and application), or of compositions of sialic acid oligosaccharides formed through compounding (CN202180027387.8, Sialic acid composition for inhibiting and treating coronavirus infection; CN202210518150.6, A sialic acid composition and its application in alleviating inflammation). However, these sialic acid oligosaccharide complexes disrupt the integrity of sialic acid to some extent, reducing its inhibitory effect on viruses and pathogenic bacteria. Furthermore, the sialic acid oligosaccharide complexes formed by sialic acid oligosaccharide blends and electrostatic interactions are structurally unstable in their action on the intestines, reducing the utilization rate of sialic acid oligosaccharides. In contrast, natural mucins rich in sialic acid (such as bird's nest) have the ability to withstand digestive enzymes and can reach the colon without absorption, acting on the intestinal flora.

[0007] Therefore, developing a macromolecular sialylated glycan graft with pH-responsive properties and structure and function similar to natural mucins may be a novel strategy for inhibiting intestinal inflammation, oxidative stress, suppressing viruses, and promoting intestinal health. Summary of the Invention

[0008] To address the shortcomings and deficiencies of existing technologies, the present invention aims to overcome the problem of low bioavailability of orally administered sialic acid by providing a macromolecular sialylated polysaccharide graft with pH responsiveness and structure and function similar to natural mucin. This graft has low production cost, similar structure and physiological function to natural mucin, is responsive to different pH conditions in the gastrointestinal tract, has a high viral adsorption rate, and can directly reach the colon for utilization by intestinal flora, thereby improving and balancing the intestinal flora.

[0009] The preparation of the pH-responsive sialylated polysaccharide graft of the present invention selects sialyl lactose and gellan gum as the main raw materials. Gellan gum has excellent properties such as structural stability, heat resistance, acid and alkali resistance and thermal reversibility. This makes the obtained gellan gum-sialyl lactose graft similar to natural mucin in structure and physiological function. Furthermore, the negative charge and pH responsiveness enhance the practical application of this graft.

[0010] Gellan gum-sialyl lactose pH-responsive graft is a structurally stable, pH-responsive macromolecular sialylated polysaccharide. This property allows it to exhibit different swelling phenomena depending on the pH of the gastrointestinal tract, resisting gastrointestinal digestion and reaching directly to the colon. In the colon, it can be fully utilized by intestinal microorganisms, balancing the intestinal flora, improving antioxidant activity, and enhancing the body's immunity.

[0011] The first objective of this invention is to provide a gellan gum-sialic acid lactose pH-responsive graft, the chemical structure of which is shown below:

[0012] n = 100-120; the gellan gum-sialic acid lactose pH-responsive graft is synthesized from raw materials gellan gum and sialic acid lactose through amide reaction, reductive amination reaction, alcohol precipitation dialysis, and freeze drying.

[0013] A second objective of this invention is to provide a method for preparing the gellan gum-sialic acid lactose pH-responsive graft described above, the synthetic route of which is shown below:

[0014]

[0015] n = 100 - 120;

[0016] The preparation method includes the following steps:

[0017] (1) Dissolve the gellan gum shown in Formula 1 in water, mix well, let stand, place in a constant temperature water bath at 80-90℃ and stir for 1-3 hours, then adjust the pH to obtain a gellan gum solution.

[0018] (2) The gellan gum solution obtained in step (1) is reacted with a catalyst and the pH is controlled at 4 to 6. Then, ethylenediamine is added to continue the reaction and the pH is controlled at 7 to 8. After the reaction is completed, alcohol precipitation and dialysis are performed, the solution is concentrated by rotary evaporation under reduced pressure, and then freeze-dried to obtain the amidated gellan gum (GG-NH2) shown in Formula 2.

[0019] (3) Dissolve the sialic acid lactose shown in Formula 3 in water, control the pH at 6-7, add NaBH3CN and the amidated gellan gum obtained in step (2), and react. After the reaction is completed, perform alcohol precipitation and dialysis, concentrate by rotary evaporation under reduced pressure, freeze dry, and obtain the gellan gum-sialic acid lactose graft (GG-SL) shown in Formula 4.

[0020] In one embodiment, the gellan gum in step (1) has a molecular weight of 65,000-85,000 Da and a degree of polymerization of 100-120 DP.

[0021] In one embodiment, the concentration of the gellan gum solution in step (1) is 1 to 2 g / L.

[0022] In one embodiment, the gellan gum solution in step (1) refers to a clear, transparent, and flocculent solution in which gellan gum is completely dissolved.

[0023] In one embodiment, the pH value adjusted in step (1) is 4 to 6.

[0024] In one embodiment, the catalyst in step (2) is EDC·HCl and NHS, and the amidation reaction is carried out. The order of addition is to first add EDC·HCl and react at room temperature for 20-40 min, and then add NHS and react at room temperature for 20-40 min. The reaction molar ratio is n(GG):n(EDC·HCl):n(NHS)=1:1:1.

[0025] In one embodiment, the addition of ethylenediamine in step (2) and the continued reaction are carried out at room temperature for 24 to 36 hours.

[0026] In one embodiment, the amount of ethylenediamine added in step (2) is such that the molar ratio of gellan gum to ethylenediamine is n(GG):n(ethylenediamine) = 1:1.

[0027] In one embodiment, the alcohol precipitation dialysis in step (2) involves: precipitating the precipitate with 95% ethanol solution, redissolving the precipitate in water, and dialyzing it in deionized water using a 10000Da dialysis bag to remove unreacted small molecules such as ethylenediamine.

[0028] In one embodiment, step (2) involves vacuum rotary evaporation concentration at 0.02–0.08 MPa and 40–60 °C.

[0029] In one embodiment, step (2) involves freeze-drying at -20 to -40°C.

[0030] In one embodiment, the concentration of sialic acid lactose obtained by dissolving sialic acid lactose in water in step (3) is 10-30 g / L; preferably 20 g / L.

[0031] In one embodiment, the sialic acid lactose in step (3) is 3'-sialic acid lactose or 6'-sialic acid lactose.

[0032] In one embodiment, the molar ratio of sialyl lactose and amidated gellan gum in step (3) is n(SL):n(GG-NH2)=2:1, and the final concentration of NaBH3CN is 0.05mol / L.

[0033] In one embodiment, the reaction in step (3) is carried out at room temperature for 12 to 36 hours.

[0034] The third objective of this invention is to provide an application of the gellan gum-sialic acid lactose pH-responsive graft described above in the preparation of functional foods and pharmaceuticals.

[0035] The fourth objective of this invention is to provide the application of the gellan gum-sialic acid lactose pH-responsive graft described above in the preparation of functional foods or special medical foods for inhibiting intestinal inflammation and oxidative stress.

[0036] The beneficial effects of this invention are:

[0037] The gellan gum-sialic acid lactose pH-responsive graft prepared by this invention has the following advantages:

[0038] (1) In the preparation of the gellan gum-sialic acid-lactose pH-responsive graft in this invention, the sialic acid used is 3'-sialic acid-lactose or 6'-sialic acid-lactose. Compared with other sialic acid polysaccharides, sialic acid-lactose is relatively inexpensive and easy to obtain. The macromolecular low-acyl gellan gum has good water solubility, stable structure, heat resistance, acid and alkali resistance, excellent gelling properties, good biocompatibility and pH responsiveness. It has low swelling under low pH conditions and increased swelling under pH>7 conditions, which improves its pH responsiveness. Furthermore, gellan gum is a repeating polymer composed of four monosaccharide molecules with carboxyl groups. It can form a stable ester with EDC·HCl and NHS for subsequent reactions. It is a green, zero-length crosslinking agent. The synthesized gellan gum-sialic acid-lactose graft will not introduce too many intermediates.

[0039] (1) This graft has a stable pH-responsive macromolecular sialylated polysaccharide that can exhibit different swelling phenomena depending on the pH of the gastrointestinal tract. It can resist digestion in the gastrointestinal tract and directly reach the colon, where it can be fully utilized by intestinal microorganisms, balancing the intestinal flora, improving antioxidant activity, and enhancing the body's immunity. It overcomes the disadvantage that a large portion of the absorbed sialic acid is excreted in the urine after sialic acid lactose is ingested. This graft retains the physiological and structural characteristics of sialic acid, has good antioxidant, anti-inflammatory, and antiviral functions, is non-toxic to cells, and reduces cell damage and inflammatory response in LPS-induced inflammatory cell models. It has wide applications in the preparation of functional foods and pharmaceuticals. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the reaction route for preparing the gellan gum-sialic acid lactose graft in Example 1 of the present invention;

[0041] Figure 2 The infrared spectra of the gellan gum-sialic acid lactose grafts prepared in Examples 1 and 2 of this invention are shown below.

[0042] Figure 3 The above are the 1H NMR spectra of the gellan gum-sialic acid lactose grafts prepared in Examples 1 and 2 of this invention.

[0043] Figure 4 The swelling diagrams of the gellan gum-sialic acid lactose grafts prepared in Examples 1 and 2 of this invention under different pH conditions are shown.

[0044] Figure 5 The in vitro antioxidant properties of the gellan gum-sialic acid lactose grafts prepared in Examples 1 and 2 of this invention are shown in the figure.

[0045] Figure 6 The graph shows the cytotoxicity (MTT) results of the gellan gum-sialic acid lactose grafts prepared in Examples 1 and 2 of this invention.

[0046] Figure 7 The figure shows the experimental results of the gellan gum-sialic acid lactose grafts prepared in Examples 1 and 2 of this invention improving the inflammatory response in an inflammatory cell model.

[0047] Figure 8 The graph shows the hemagglutination inhibition results of the complexes prepared in Examples 1-2 and Comparative Example 1 of this invention, which are electrostatically bound to chitosan oligosaccharide-sialic acid lactose. Detailed Implementation

[0048] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0049] The sialic acid lactose involved in this invention was purchased from Chr. Hansen Biotechnology Co., Ltd.; the gellan gum was purchased from Spico Inc. (USA, food grade), with a molecular weight of 65,000-85,000 Da.

[0050] Example 1

[0051] A method for preparing a gellan gum-sialic acid lactose pH-responsive graft, the method comprising the following steps:

[0052] (1) Weigh 1g of gellan gum into a 1000ml flask, add 1000ml of deionized water, mix the solution, and let it stand at room temperature for 20min; place the gellan gum solution after standing in a constant temperature magnetic stirrer at 85℃ and stir for 1h to fully dissolve the gellan gum and present a clear, transparent and colorless solution. Then cool the solution naturally to room temperature and adjust the pH of the solution to 4-6 to obtain the gellan gum solution.

[0053] (2) Weigh 959 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and dissolve it in an appropriate amount of water. Add the gellan gum solution obtained in step (1) and react at room temperature for 30 min. Then add a solution containing 575 mg of N-hydroxysuccinimide (NHS) and react at room temperature for 30 min. Add 201 μL of ethylenediamine to the reaction solution and react at room temperature for 24 h. During the reaction, the pH is controlled between 7 and 8. After the reaction, perform a 3-fold alcohol precipitation with 95% ethanol solution. Take the precipitate, redissolve it in water, and dialyze it in deionized water using a 10000 Da dialysis bag to remove unreacted ethylenediamine and other small molecules. After dialysis, concentrate the gellan gum under reduced pressure at 0.06 MPa and 50 °C, and freeze dry it at -70 °C to obtain the amidated gellan gum product GG-NH2.

[0054] (3) Dissolve 2g of 3'-sialic acid lactose in 100mL of deionized water, control the pH at 6-7, add 315mg of NaBH3CN, and then add 1mg / mL of amidated gellan gum product GG-NH2, so that the molar ratio of sialic acid lactose to amidated gellan gum is n(SL):n(GG-NH2)=2:1. React at room temperature for 24h. After the reaction is completed, perform 3-fold alcohol precipitation with 95% ethanol solution, take the precipitate and redissolve it in water, dialyze it in deionized water with a 10000Da dialysis bag to remove unreacted sialic acid lactose and small molecules such as NaBH3CN. After dialysis, concentrate under reduced pressure at 0.06Mpa and 50℃, freeze dry at -40℃ to obtain gellan gum-sialic acid lactose graft (GG-SL).

[0055] The performance of the gellan gum-sialic acid lactose graft (GG-SL) prepared in this embodiment was characterized.

[0056] Figure 2 The infrared spectrum of the gellan gum-sialyl lactose graft is shown at 1610 cm⁻¹. -1 1151cm -1 1030cm -1 The characteristic absorption peak of sialic acid is enhanced at 3200 cm⁻¹. -1 The enhanced peak value is due to the introduction of NH bonds. This demonstrates the successful synthesis of the gellan gum-sialic acid lactose graft.

[0057] Figure 3 The 1H NMR spectrum of the gellan gum-sialic acid-lactose graft is shown. Characteristic peaks appear near δ 5.25, 2.33, and 2, respectively, corresponding to the characteristic peaks of -NH- in sialic acid, -CH2- in Sia, and MeCOO. This step indicates the successful synthesis of the sialic acid-gellan gum graft.

[0058] Example 2

[0059] A method for preparing a gellan gum-sialic acid lactose pH-responsive graft, the method comprising the following steps:

[0060] (1) Weigh 1g of gellan gum into a 1000ml flask, add 1000ml of deionized water, mix the solution, and let it stand at room temperature for 20min; place the gellan gum solution after standing in a constant temperature magnetic stirrer at 85℃ and stir for 1h to fully dissolve the gellan gum and present a clear, transparent and colorless solution. Then cool the solution naturally to room temperature and adjust the pH of the solution to 4-6 to obtain the gellan gum solution.

[0061] (2) Weigh 959 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and dissolve it in an appropriate amount of water. Add the gellan gum solution obtained in step (1) and react at room temperature for 30 min. Then add a solution containing 575 mg of N-hydroxysuccinimide (NHS) and react at room temperature for 30 min. Add 201 μL of ethylenediamine to the reaction solution and react at room temperature for 24 h. During the reaction, the pH is controlled between 7 and 8. After the reaction, perform a 3-fold alcohol precipitation with 95% ethanol solution. Take the precipitate, redissolve it in water, and dialyze it in deionized water using a 10000 Da dialysis bag to remove unreacted ethylenediamine and other small molecules. After dialysis, concentrate the gellan gum under reduced pressure at 0.06 MPa and 50 °C, and freeze dry it at -70 °C to obtain the amidated gellan gum product GG-NH2.

[0062] (3) Dissolve 2g of 6'-sialic acid lactose in 100mL of deionized water, control the pH at 6-7, add 315mg of NaBH3CN, and then add 1mg / mL of amidated gellan gum product GG-NH2, so that the molar ratio of sialic acid lactose to amidated gellan gum is n(SL):n(GG-NH2)=2:1. React at room temperature for 24h. After the reaction is completed, perform 3-fold alcohol precipitation with 95% ethanol solution, take the precipitate and redissolve it in water, dialyze it in deionized water with a 10000Da dialysis bag to remove unreacted sialic acid lactose and small molecules such as NaBH3CN. After dialysis, concentrate under reduced pressure at 0.06Mpa and 50℃, freeze dry at -40℃ to obtain gellan gum-sialic acid lactose graft (GG-SL).

[0063] Comparative Example 1

[0064] A method for preparing gellan gum-sialic acid lactose complexes via ion-binding includes the following steps:

[0065] (1) Weigh 1g of gellan gum into a 1000ml flask, add 1000ml of deionized water, mix the solution, and let it stand at room temperature for 20min; place the gellan gum solution after standing in a constant temperature magnetic stirrer at 85℃ and stir for 1h to fully dissolve the gellan gum and present a clear, transparent and colorless solution. Then cool the solution naturally to room temperature and adjust the pH of the solution to 4-6 to obtain the gellan gum solution.

[0066] (2) Weigh 281 mg EDC·HCl and dissolve it in an appropriate amount of water. Add the gellan gum solution obtained in step (1) and react at room temperature for 30 min. Then add a solution containing 169 mg NHS and react at room temperature for 30 min. Add 88 μL ethylenediamine to the reaction solution and react at room temperature for 24 h. During the reaction, the pH is controlled between 7 and 8. After the reaction, use 95% ethanol solution for 3-fold alcohol precipitation. Take the precipitate and redissolve it in water. Dialyze it in deionized water using a 10000 Da dialysis bag to remove unreacted ethylenediamine, sialic acid and other small molecules. After dialysis, concentrate it under reduced pressure at 0.06 M and 50 °C. Freeze-dry it at -40 °C to obtain the amidated gellan gum product GG-NH2.

[0067] (3) The amidated gellan gum product GG-NH2 was dissolved in deionized water at a concentration of 1 mg / ml and the pH was controlled in the range of pH 4 to 7. Then 3'-sialic acid lactose / 6'-sialic acid lactose was added so that the molar ratio of sialic acid lactose to amidated gellan gum was n(SL):n(GG-NH2) = 2:1. The reaction was carried out at room temperature for 24 h. After the reaction was completed, the product was precipitated three times with 95% ethanol solution. The precipitate was redissolved in water and dialyzed in deionized water using a 10000 Da dialysis bag to remove unreacted sialic acid lactose. After dialysis, the product was concentrated by rotary evaporation under reduced pressure at 0.06 MPa and 50 °C and then freeze-dried at -40 °C to obtain the gellan gum-sialic acid lactose complex (GG / SL).

[0068] Results Measurement

[0069] 1. The gellan gum-sialic acid lactose grafts prepared in Examples 1 and 2 were transferred to a dense nylon bag and immersed in PBS buffer solutions with pH values ​​of 2, 4, 6, 8, and 10 at 25°C for 24 hours. The samples were taken out and weighed at regular intervals. When the swelling reached equilibrium, the moisture in the nylon bag was wiped off with filter paper and the samples were weighed (W). The samples were dried in an oven at 30°C until constant weight (W0). The degree of swelling of the samples was calculated by gravimetric method according to the formula: degree of swelling S = (W - W0) / W0.

[0070] The results are as follows Figure 4 As shown, the gellan gum-sialyl lactose graft exhibits low swelling degree under low pH conditions. When pH > 6, the swelling degree of the gellan gum-sialyl lactose graft increases significantly with increasing pH, while the swelling degree remains relatively unchanged when pH > 8, indicating that the gellan gum-sialyl lactose graft exhibits pH responsiveness. After entering the digestive tract, it exhibits different swelling degrees depending on the environmental changes in the stomach (pH 1.5–3.5), small intestine (pH 7.6), and large intestine (pH 8.3), increasing the contact area of ​​the gellan gum-sialyl lactose graft in the colon and promoting its full utilization by the intestinal flora.

[0071] 2. Sialyl lactose, gellan gum, and the gellan gum-sialic acid lactose grafts prepared in Examples 1 and 2 were prepared into solutions with concentrations of 0, 1, 2, 3, 4, and 5 g / L, respectively. Vitamin C was prepared into solutions with concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, and 5 g / L.

[0072] (1) Take 2 mL of the above sample solutions of different concentrations and mix them with 2 mL of DPPH solution (0.2 mmol / L, dissolved in anhydrous ethanol) by vortexing. React in the dark for 30 min and then measure the absorbance at 517 nm. Vitamin C (Vc) is used as a positive control and deionized water is used as a blank control. The zeroing is set with deionized water. Calculate the DPPH free radical scavenging rate according to the following formula.

[0073]

[0074] (2) Take 1 mL of the above sample solutions of different concentrations, 1 mL of ferrous sulfate solution (9 mmol / L), and 1 mL of hydrogen peroxide solution (9 mmol / L) and vortex to mix. After reacting at 37℃ for 10 min, add 1 mL of salicylic acid solution (9 mmol / L, dissolved in anhydrous ethanol) and mix well. React at 37℃ for 30 min and measure the absorbance at a wavelength of 510 nm. Vc is a positive control, and deionized water is a blank control. Zero the instrument with deionized water. Calculate the hydroxyl radical scavenging rate according to the following formula.

[0075]

[0076] The results are as follows Figure 5 As shown, the DPPH radical scavenging rate and hydroxyl radical scavenging rate of the gellan gum-sialic acid lactose grafts prepared in Examples 1 and 2 are higher than those of gellan gum at the same concentration, but lower than those of sialic acid monomer and vitamin C, exhibiting better in vitro antioxidant properties.

[0077] 3. Cytotoxicity (MTT) assay of gellan gum-sialic acid lactose graft

[0078] RAW264.7 cells were digested with 0.25% trypsin, counted using a hemocytometer, and then the concentration was adjusted. Cells were seeded at 100 μL / well in 96-well plates with three replicates, resulting in a cell concentration of 1.0 × 10⁶ cells / well. 5 Cells / wells were cultured at 37°C and 5% CO2 for 24 hours. Then, 100 μL of gellan gum-sialyl lactose grafts (50, 100, 200, 400 and 800 μg / mL) prepared in Examples 1 and 2 were added to 96-well plates. After incubation for 24 hours, the culture medium was removed, and 10 μL of LTT solution was added. The cells were cultured at 37°C and 5% CO2 for 3 hours. After incubation, the culture medium in the wells was removed, and 200 μL of dimethyl sulfoxide was added. The cells were shaken at room temperature for 10 minutes. The absorbance at 490 nm was measured using a microplate reader.

[0079] The results are as follows Figure 6 As shown, within the range of 50–800 μg / mL, the activity of RAW264.7 cells increased with increasing concentration of gellan gum-sialic acid lactose graft, reaching its highest level at 800 μg / mL. This indicates that the gellan gum-sialic acid lactose graft has no cytotoxicity to RAW264.7 cells and exhibits good biocompatibility.

[0080] 4. The anti-inflammatory effect of gellan gum-sialic acid lactose grafts

[0081] LPS RAW 264.7 cells were induced with 1 μg / mL LPS for 24 h, resulting in a cell concentration of 2.0 × 10⁻⁶ cells / mL. 5 Cells / well were then added, and different concentrations of gellan gum-sialic acid lactose grafts prepared in Examples 1 and 2 (50, 100, 200, 400 and 800 μg / mL) were added and cultured for 24 h; the blank control group consisted of RAW264.7 cells without LPS treatment and without the addition of gellan gum-sialic acid lactose grafts; after incubation, the supernatant was collected by centrifugation, and the levels of IL-1β and IL-6 in the supernatant were detected by ELISA kit.

[0082] The results are as follows Figure 7 As shown, LPS-induced inflammatory cytokine levels significantly increased in LPS-treated RAW 264.7 cells. After incubation with gellan gum-sialyl lactose graft for 24 h, the levels of IL-1β and IL-6 decreased with increasing gellan gum-sialyl lactose graft concentration. This indicates that within the range of 50–800 μg / mL, gellan gum-sialyl lactose graft can inhibit the release of inflammatory cytokines induced by LPS in RAW 264.7 cells, and has the ability to improve the inflammatory response in inflammatory cell models.

[0083] 5. The inhibitory effect of gellan gum-sialic acid lactose grafts (GG-SL) prepared in Examples 1 and 2 and the gellan gum-sialic acid lactose complex (GG / SL) prepared in Comparative Example 1 on the hemagglutination of influenza virus hemagglutinin (HA).

[0084] Dilute A / FortMonmouth / 1 / 1947 (H1N1) with physiological saline at a ratio of 1:80. Add 25 μL of H1N1 diluent to each well of a 96-well microplate, and then add an equal volume of the test sample to each well to make the final sample concentrations 10, 20, 40, 80, and 160 μg / mL, respectively. The control group was the AH group. The samples were incubated at 4℃ for 30 min. After incubation, add 25 μL of chicken blood cells (1%) to each well, mix well, and let stand at room temperature for 30 min. Then observe the agglutination of red blood cells. The degree of agglutination was recorded as (+ to +++) from mild to severe, and no agglutination was recorded as (-).

[0085] The results are as follows Figure 8As shown, both the gellan gum-sialic acid lactose complex and the gellan gum-sialic acid lactose graft exhibit hemagglutination inhibition against H1N1 virus. However, the hemagglutination inhibition of the gellan gum-sialic acid lactose complex is significantly weaker than that of the gellan gum-sialic acid lactose graft. This is likely because the electrostatic binding between gellan gum and the sialic acid lactose complex disrupts the carboxyl structure of sialic acid, reducing its contact points with the virus and thus decreasing its adsorption capacity. In contrast, the gellan gum-sialic acid lactose graft retains the carboxyl binding sites of sialic acid, resulting in a higher adsorption effect on the virus.

[0086] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A gellan gum-sialic acid lactose pH-responsive graft, characterized in that, Its chemical structural formula is shown below: n=100-120; the preparation of the gellan gum-sialic acid lactose pH-responsive graft includes the following: (1) Dissolve gellan gum in water, mix well, let stand, place in a constant temperature water bath at 80~90℃ and stir for 1~3 hours, then adjust the pH to obtain gellan gum solution. (2) The gellan gum solution obtained in step (1) is reacted with a catalyst and the pH is controlled at 4-6; then ethylenediamine is added to continue the reaction and the pH is controlled at 7-8. After the reaction is completed, alcohol precipitation and dialysis are performed, the solution is concentrated by rotary evaporation under reduced pressure, and then freeze-dried to obtain amidated gellan gum GG-NH2; the catalyst is EDC·HCl and NHS. (3) Dissolve sialic acid lactose in water, control the pH at 6-7, add NaBH3CN and the amidated gellan gum obtained in step (2), and react. After the reaction is completed, perform alcohol precipitation and dialysis, concentrate by rotary evaporation under reduced pressure, freeze dry, and obtain gellan gum-sialic acid lactose graft.

2. The method for preparing the gellan gum-sialic acid lactose pH-responsive graft according to claim 1, characterized in that, The synthetic route of the gellan gum-sialic acid lactose pH-responsive graft is shown below: ，n=100-120； The preparation method includes the following steps: (1) Dissolve the gellan gum shown in Formula 1 in water, mix well, let stand, place in a constant temperature water bath at 80~90℃ and stir for 1~3h, then adjust the pH to obtain a gellan gum solution. (2) The gellan gum solution obtained in step (1) is reacted with a catalyst and the pH is controlled at 4-6; then ethylenediamine is added to continue the reaction and the pH is controlled at 7-8. After the reaction is completed, alcohol precipitation and dialysis are performed, the solution is concentrated by rotary evaporation under reduced pressure, and then freeze-dried to obtain the amidated gellan gum GG-NH2 shown in Formula 2. (3) Dissolve the sialic acid lactose shown in Formula 3 in water, control the pH at 6-7, add NaBH3CN and the amidated gellan gum obtained in step (2), and react. After the reaction is completed, perform alcohol precipitation and dialysis, concentrate under reduced pressure by rotary evaporation, freeze dry, and obtain the gellan gum-sialic acid lactose graft GG-SL shown in Formula 4.

3. The method according to claim 2, characterized in that, The gellan gum mentioned in step (1) has a molecular weight of 65,000-85,000 Da and a degree of polymerization of 100-120 DP.

4. The method according to claim 2, characterized in that, The catalysts in step (2) are EDC·HCl and NHS, which are added in the order of addition. EDC·HCl is added first and reacted at room temperature for 20~40 min, and then NHS is added and reacted at room temperature for 20~40 min. The molar ratio of the reaction is n(GG):n(EDC·HCl):n(NHS)=1:1:

1.

5. The method according to claim 2, characterized in that, The amount of ethylenediamine added in step (2) is based on the molar ratio of gellan gum to ethylenediamine as n(GG):n(ethylenediamine) = 1:

1.

6. The method according to claim 2, characterized in that, The concentration of sialic acid lactose obtained by dissolving sialic acid lactose in water in step (3) is 10~30 g / L.

7. The method according to claim 2, characterized in that, The sialic acid lactose mentioned in step (3) is 3'-sialic acid lactose or 6'-sialic acid lactose.

8. The method according to claim 2, characterized in that, In step (3), the molar ratio of sialyl lactose and amidated gellan gum is n(SL):n(GG-NH2)=2:1, and the final concentration of NaBH3CN is 0.05mol / L.

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