Honeysuckle flower polysaccharide with oral health care efficacy and preparation and application thereof

Abstract

The present application relates to the field of functional food and microbial technology, and particularly relates to a honeysuckle polysaccharide with oral health and tooth care effects, and preparation and application thereof.The preparation method comprises the following steps: S1 surface modification: L-arginine and honeysuckle polysaccharide are respectively dissolved in water, and the pH value is adjusted, and then mixed and stirred; S2 modification liquid purification: the modification liquid is dialyzed by using a dialysis bag, and the unmodified L-arginine is removed; S3 drying: the solution is dried.The honeysuckle polysaccharide with oral health and tooth care effects is modified by using a simple hydrogen bond effect L-arginine, and the method is simple, green and easy to operate; the polysaccharide has enhanced growth inhibition function of Streptococcus mutans, and can directly intervene in the formation process of dental caries; the polysaccharide has antibacterial and anti-inflammatory effects, and the functions are synergistic, can effectively maintain the stability of the oral environment, and can be widely applied to food with cariogenic effects as a high-quality raw material to reduce the cariogenicity of food.

Description

Technical Field

[0001] This invention relates to the field of functional foods and microbial technology, specifically to a honeysuckle polysaccharide with oral health and dental care effects, its preparation and application. Background Technology

[0002] Oral health is an important indicator of overall health and quality of life, with the health of teeth and gums being particularly crucial. Tooth decay is primarily caused by pathogenic bacteria such as Streptococcus mutans, which metabolize acid and erode the hard tissues of the teeth. Gingivitis and periodontitis arise from plaque buildup leading to inflammation of the gum tissue. Currently, routine oral care often relies on products containing chemical antibacterial agents (such as fluoride and triclosan), which can cause dysbiosis or mucosal irritation with long-term use. In the food sector, the risk of tooth decay is often reduced by adding artificial sweeteners (such as xylitol) or minerals (such as calcium and phosphorus). However, artificial sweeteners are unsuitable for people with sensitive stomachs, and minerals have limited effect on strengthening the enamel of erupted teeth; existing strategies have significant limitations.

[0003] Honeysuckle, a traditional medicinal and edible resource, is rich in polysaccharides, chlorogenic acid, and other active ingredients, possessing broad-spectrum antibacterial, anti-inflammatory, and antioxidant properties. However, the functions of natural honeysuckle polysaccharide extracts are relatively limited, often only exhibiting anti-inflammatory effects, with unclear inhibitory effects on caries-causing bacteria, making it difficult to achieve a synergistic effect of both combating caries-causing bacteria and reducing gingival inflammation. For example, existing patent (CN113520936A) only uses honeysuckle extract as a toothpaste thickener, without addressing its effect on caries-causing bacteria. Furthermore, existing polysaccharide modification methods are complex and not environmentally friendly, hindering their large-scale integration and application in ordinary foods, health products, or daily oral care products.

[0004] Therefore, there is an urgent need for a honeysuckle polysaccharide prepared by a simple and green process that can effectively inhibit cariogenic bacteria and relieve gingival inflammation, thus providing a comprehensive natural raw material solution for dental and gingival health. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of the prior art by providing a honeysuckle polysaccharide with oral health and dental care benefits, its preparation method, and its application. The honeysuckle polysaccharide prepared by this method has both antibacterial and anti-inflammatory effects. It can not only effectively exert the anti-inflammatory effect of honeysuckle polysaccharide, but also has a significant antibacterial effect on Streptococcus mutans, a caries-causing bacterium, thus maintaining the stability of the oral microenvironment and reducing the probability of tooth decay.

[0006] The objective of this invention is achieved through the following technical solution: a method for preparing honeysuckle polysaccharide with oral health and dental care effects, comprising the following steps: S1 Surface Modification: L-arginine and honeysuckle polysaccharide were dissolved separately in water, and the pH values ​​of the L-arginine solution and honeysuckle polysaccharide solution were adjusted separately. Then, the L-arginine solution and honeysuckle polysaccharide solution were mixed and stirred to obtain the modified solution. S2 modification solution purification: Dialyze the modification solution obtained in step S1 using a dialysis bag to remove the unmodified L-arginine and obtain the purified honeysuckle polysaccharide modification solution in the bag; S3 Drying: The solution obtained in step S2 is dried to obtain the target honeysuckle polysaccharide.

[0007] Furthermore, in step S1, the honeysuckle polysaccharide that is surface-modified with L-arginine is a low molecular weight honeysuckle polysaccharide, wherein the molecular weight of the low molecular weight honeysuckle polysaccharide is 1000-5000 Da.

[0008] Furthermore, in step S1, the preparation method of honeysuckle polysaccharide with L-arginine surface modification includes the following steps: A1 Polysaccharide Extraction: Honeysuckle was soaked in water and boiled with hot water to obtain a crude extract of honeysuckle polysaccharides; A2 Polysaccharide Purification: The crude polysaccharide extract was centrifuged to obtain the supernatant, which was then filtered through a membrane and purified by dialysis. The solution in the dialysis bag was then freeze-dried to obtain honeysuckle polysaccharide.

[0009] The method for preparing honeysuckle polysaccharide with oral health and dental care effects obtained in this invention employs water extraction and charge effect, which is simple, green and easy to operate.

[0010] Furthermore, in step A1, the mass ratio of honeysuckle to water is 1:(20-30), the cold water soaking time is 20-30 minutes, and the hot water boiling is required until the volume of the crude extract is 1 / 6-1 / 2 of the initial added water volume.

[0011] Furthermore, in step A2, the centrifugation conditions are a relative centrifugal force of 4000-5000 g and a time of 3-6 minutes.

[0012] Furthermore, in step A2, the supernatant is subjected to two membrane filtrations. The membrane used for the first filtration has a pore size of 0.44 μm, and the membrane used for the second filtration has a pore size of 0.22 μm. Both filtrations are performed using vacuum filtration.

[0013] Furthermore, in step A2, the dialysis purification is performed using a dialysis bag with a molecular cutoff of 1000 Da, with a dialysis time of more than 24 hours and the water being changed at least 4 times every 24 hours.

[0014] Furthermore, in step S1, the mass ratio of L-arginine to low molecular weight honeysuckle polysaccharide is 1:(2-4).

[0015] Furthermore, in step S1, the pH of the L-arginine solution and the low molecular weight honeysuckle polysaccharide solution is adjusted to 5.0-7.5; the pH is adjusted using Tris-citric acid buffer; the stirring time is 2-4 hours.

[0016] Furthermore, in step S2, the molecular rejection capacity of the dialysis bag is 1000 Da, the dialysis time is more than 24 hours, and the water is changed at least 4 times every 24 hours.

[0017] Furthermore, in step S3, the solution obtained in step S2 is freeze-dried to obtain the target honeysuckle polysaccharide.

[0018] Another objective of this invention is to provide a honeysuckle polysaccharide with oral health and dental care effects, wherein the polysaccharide is prepared by the above-mentioned method for preparing honeysuckle polysaccharide with oral health and dental care effects.

[0019] Another objective of this invention is to provide the application of the honeysuckle polysaccharide with oral health and dental care effects in foods with cariogenic effects, such as candies.

[0020] The beneficial effects of this invention are as follows: The method for preparing honeysuckle polysaccharide with oral health and dental care effects obtained in this invention utilizes charge effect, and the method is simple, green, and easy to operate.

[0021] The honeysuckle polysaccharide obtained by this invention, which has oral health and dental care effects, has enhanced inhibition of Streptococcus mutans growth and can directly intervene in the formation process of tooth decay.

[0022] The honeysuckle polysaccharide obtained by this invention, which has oral health and dental care effects, is highly water-soluble and also has antibacterial and anti-inflammatory effects. Its functions are synergistic, which can effectively maintain the stability of the oral environment. It can be widely used as a high-quality raw material in foods with cariogenic effects (such as candy) to reduce the cariogenicity of the food, meet the market demand for diversified and high-efficiency health products, and has broad application prospects. Attached Figure Description

[0023] Figure 1 Electron micrographs of L-arginine, honeysuckle polysaccharide with oral health and dental care effects prepared in Example 1, and honeysuckle polysaccharide prepared in Comparative Example 1 dissolved in water.

[0024] Figure 2 Scanning electron microscope images of honeysuckle polysaccharide with oral health and dental care effects prepared in Example 1 and honeysuckle polysaccharide prepared in Comparative Example 1.

[0025] Figure 3The images shown are electron micrographs and scanning electron microscope images of the honeysuckle polysaccharide with oral health and dental care effects prepared in Example 2.

[0026] Figure 4 Fourier transform infrared spectra of L-arginine, honeysuckle polysaccharide with oral health and dental care effects prepared in Example 1, and honeysuckle polysaccharide prepared in Comparative Example 1.

[0027] Figure 5 Electron photographs of L-arginine, honeysuckle polysaccharides with oral health and dental care effects prepared in Examples 2 and 3, and honeysuckle polysaccharides prepared in Comparative Examples 1-3.

[0028] Figure 6 The ZETA potential diagrams of honeysuckle polysaccharide with oral health and tooth protection effects prepared in Example 2 and honeysuckle polysaccharides prepared in Comparative Examples 1-3 are shown.

[0029] Figure 7 The study compared the antibacterial effects of honeysuckle polysaccharide with oral health and dental care effects prepared in Example 2 and honeysuckle polysaccharide and L-arginine prepared in Comparative Example 1 against Streptococcus mutans.

[0030] Figure 8 The image shows the anti-inflammatory effects of honeysuckle polysaccharide with oral health and dental care effects prepared in Example 2 and honeysuckle polysaccharide and L-arginine prepared in Comparative Example 1. Detailed Implementation

[0031] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments. The content mentioned in the embodiments is not intended to limit the present invention.

[0032] In some embodiments of the present invention, a method for preparing honeysuckle polysaccharide with oral health and dental care effects includes the following steps: S1 Surface Modification: L-arginine and honeysuckle polysaccharide were dissolved separately in water, and the pH values ​​of the L-arginine solution and honeysuckle polysaccharide solution were adjusted separately. Then, the L-arginine solution and honeysuckle polysaccharide solution were mixed and stirred to obtain the modified solution. S2 modification solution purification: Dialyze the modification solution obtained in step S1 using a dialysis bag to remove the unmodified L-arginine and obtain the purified honeysuckle polysaccharide modification solution in the bag; S3 Freeze-drying: The solution obtained in step S2 is freeze-dried to obtain the target honeysuckle polysaccharide.

[0033] The honeysuckle polysaccharide with oral health and tooth protection effects provided by this invention is extracted from honeysuckle using a simple water immersion method, and then L-arginine is modified using the charge effect. The method is simple, green and easy to operate. The polysaccharide has an enhanced inhibitory function against the growth of Streptococcus mutans and can directly intervene in the formation process of tooth decay. The polysaccharide also has antibacterial and anti-inflammatory effects, with synergistic functions, which can effectively maintain the stability of the oral environment.

[0034] In some embodiments of the present invention, in step S1, the honeysuckle polysaccharide that is surface-modified with L-arginine is a low molecular weight honeysuckle polysaccharide, wherein the molecular weight of the low molecular weight honeysuckle polysaccharide is 1000-5000 Da.

[0035] In some embodiments of the present invention, the method for preparing honeysuckle polysaccharide with L-arginine surface modification in step S1 includes the following steps: A1 Polysaccharide Extraction: Honeysuckle was soaked in water and boiled with hot water to obtain a crude extract of honeysuckle polysaccharides; A2 Polysaccharide Purification: The crude polysaccharide extract was centrifuged to obtain the supernatant, which was then filtered through a membrane and purified by dialysis. The solution in the dialysis bag was then freeze-dried to obtain low molecular weight honeysuckle polysaccharide.

[0036] In some embodiments of the present invention, in step A1, the mass ratio of honeysuckle to water is 1:(20-30), the cold water soaking time is 20-30 minutes, and the hot water boiling is required until the volume of the crude extract is 1 / 6-1 / 2 of the initial added water volume.

[0037] In some embodiments of the present invention, in step A2, the centrifugation conditions are a relative centrifugal force of 4000-5000 g and a time of 3-6 minutes.

[0038] In some embodiments of the present invention, in step A2, the supernatant is subjected to two membrane filtrations. The membrane used for the first filtration has a pore size of 0.44 μm, and the membrane used for the second filtration has a pore size of 0.22 μm. Both filtrations are performed using vacuum filtration.

[0039] In some embodiments of the present invention, in step A2, the dialysis purification is performed using a dialysis bag with a molecular cutoff of 1000 Da, with a dialysis time of more than 24 hours and the water being changed at least 4 times every 24 hours.

[0040] In some embodiments of the present invention, in step S1, the mass ratio of L-arginine to low molecular weight honeysuckle polysaccharide is 1:(2-4), that is, during surface modification, the mixed mass ratio of L-arginine to low molecular weight honeysuckle polysaccharide is 1:(2-4); the pH value of the L-arginine solution and the low molecular weight honeysuckle polysaccharide solution is adjusted to 5.0-7.5; the pH value is adjusted using Tris-citric acid buffer; the stirring time is 2-4 hours; after the two solutions are mixed, the concentration of L-arginine in the mixed solution is 2-6 mg / mL, and the concentration of honeysuckle polysaccharide in the mixed solution is 4-24 mg / mL. The pH value can be adjusted to 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5, etc.

[0041] In some embodiments of the present invention, in step S2, the molecular rejection capacity of the dialysis bag is 1000 Da, the dialysis time is more than 24 hours, and the water is changed at least 4 times every 24 hours.

[0042] In this embodiment, a method for preparing honeysuckle polysaccharide with oral health and dental care effects includes the following steps: S1 Surface Modification: L-arginine and low molecular weight honeysuckle polysaccharide were dissolved separately in pure water at a mass ratio of 1:2, i.e., the mixing mass ratio of L-arginine and low molecular weight honeysuckle polysaccharide during surface modification was 1:2; the pH of the L-arginine solution and the low molecular weight honeysuckle polysaccharide solution was adjusted to 6 using Tris-citric acid buffer, and the two solutions were mixed and stirred for 4 hours to obtain the modified solution; after mixing the two solutions, the concentration of L-arginine in the mixed solution was 4 mg / mL, and the concentration of honeysuckle polysaccharide in the mixed solution was 8 mg / mL; S2 modification solution purification: Dialyze the modification solution obtained in step S1 again using a 1000Da dialysis bag for 24 hours to remove the unmodified L-arginine. During this period, the pure water was changed 4 times. Finally, the purified honeysuckle polysaccharide modification solution in the bag was obtained. S3 Freeze-drying: Freeze-dry the solution obtained in step S2 to obtain the target honeysuckle polysaccharide.

[0043] Furthermore, in step S1, the preparation method of honeysuckle polysaccharide with L-arginine surface modification includes the following steps: A1 Polysaccharide Extraction: Dried honeysuckle and pure water were soaked in cold water at a mass ratio of 1:30 for 30 minutes, and then heated. When the volume of the crude extract was about 1 / 3 of the initial volume of pure water, heating was stopped and the extract was cooled to room temperature to obtain the crude extract of honeysuckle polysaccharides. A2 Polysaccharide Purification: Centrifuge 5000g of crude honeysuckle polysaccharide extract for 3 minutes, collect the supernatant, first use a 0.44μm filter membrane for vacuum filtration to obtain the filtrate, then use a 0.22μm filter membrane for a second vacuum filtration to obtain the filtrate, and then use a 1000Da dialysis bag for dialysis purification for 24 hours, changing the pure water 4 times during the period, and finally freeze-dry the solution in the dialysis bag to obtain low molecular weight honeysuckle polysaccharide; the molecular weight of low molecular weight honeysuckle polysaccharide is 1000-5000Da.

[0044] Example 2 In this embodiment, a method for preparing honeysuckle polysaccharide with oral health and dental care effects includes the following steps: S1 Surface Modification: L-arginine and low molecular weight honeysuckle polysaccharide were separately dissolved in pure water at a mass ratio of 1:2. The pH of the L-arginine solution and the low molecular weight honeysuckle polysaccharide solution was adjusted to 6 using Tris-citric acid buffer. The two solutions were mixed and stirred for 3 hours to obtain the modified solution. After mixing the two solutions, the concentration of L-arginine in the mixed solution was 4 mg / mL, and the concentration of honeysuckle polysaccharide in the mixed solution was 8 mg / mL. The molecular weight of the low molecular weight honeysuckle polysaccharide was 1000-5000 Da. S2 modification solution purification: Dialyze the modification solution obtained in step S1 again using a 1000Da dialysis bag for 24 hours to remove the unmodified L-arginine. During this period, the pure water was changed 4 times. Finally, the purified honeysuckle polysaccharide modification solution in the bag was obtained. S3 Freeze-drying: Freeze-dry the solution obtained in step S2 to obtain the target honeysuckle polysaccharide.

[0045] Furthermore, in step S1, the preparation method of honeysuckle polysaccharide with L-arginine surface modification includes the following steps: A1 Polysaccharide Extraction: Dried honeysuckle and pure water were soaked in cold water at a mass ratio of 1:20 for 30 minutes, and then heated. When the volume of the crude extract was about 1 / 3 of the initial volume of pure water, heating was stopped, and the extract was cooled to room temperature to obtain a crude extract of honeysuckle polysaccharides. A2 Polysaccharide Purification: Centrifuge 4000g of crude honeysuckle polysaccharide extract for 6 minutes, collect the supernatant, first use a 0.44μm filter membrane for vacuum filtration to obtain the filtrate, then use a 0.22μm filter membrane for a second vacuum filtration to obtain the filtrate, and then use a 1000Da dialysis bag for dialysis purification for 24 hours, changing the pure water 4 times during the period, and finally freeze-dry the solution in the dialysis bag to obtain low molecular weight honeysuckle polysaccharide; the molecular weight of low molecular weight honeysuckle polysaccharide is 1000-5000Da.

[0046] Example 3 In this embodiment, a method for preparing honeysuckle polysaccharide with oral health and dental care effects includes the following steps: S1 Surface Modification: L-arginine and low molecular weight honeysuckle polysaccharide were separately dissolved in pure water at a mass ratio of 1:4. The pH of the L-arginine solution and the low molecular weight honeysuckle polysaccharide solution was adjusted to 6 using Tris-citric acid buffer. The two solutions were mixed and stirred for 4 hours to obtain the modified solution. After mixing the two solutions, the concentration of L-arginine in the mixed solution was 4 mg / mL, and the concentration of honeysuckle polysaccharide in the mixed solution was 16 mg / mL. The molecular weight of the low molecular weight honeysuckle polysaccharide was 1000-5000 Da. S2 modification solution purification: Dialyze the modification solution obtained in step S1 again using a 1000Da dialysis bag for 24 hours to remove the unmodified L-arginine. During this period, the pure water was changed 4 times. Finally, the purified honeysuckle polysaccharide modification solution in the bag was obtained. S3 Freeze-drying: Freeze-dry the solution obtained in step S2 to obtain the target honeysuckle polysaccharide.

[0047] Furthermore, in step S1, the preparation method of honeysuckle polysaccharide with L-arginine surface modification includes the following steps: A1 Polysaccharide Extraction: Dried honeysuckle and pure water were soaked in cold water at a mass ratio of 1:30 for 30 minutes, and then heated. When the volume of the crude extract was about 1 / 3 of the initial volume of pure water, heating was stopped, and the extract was cooled to room temperature to obtain a crude extract of honeysuckle polysaccharides. A2 Polysaccharide Purification: Centrifuge 5000g of crude honeysuckle polysaccharide extract for 3 minutes, collect the supernatant, first use a 0.44μm filter membrane for vacuum filtration to obtain the filtrate, then use a 0.22μm filter membrane for a second vacuum filtration to obtain the filtrate, and then use a 1000Da dialysis bag for dialysis purification for 24 hours, changing the pure water 4 times during the period, and finally freeze-dry the solution in the dialysis bag to obtain low molecular weight honeysuckle polysaccharide; the molecular weight of low molecular weight honeysuckle polysaccharide is 1000-5000Da.

[0048] Comparative Example 1 The preparation method of the target honeysuckle polysaccharide in this comparative example includes the following steps: A1 Polysaccharide Extraction: Dried honeysuckle and pure water were soaked in cold water at a mass ratio of 1:30 for 30 minutes, and then heated. When the volume of the crude extract was about 1 / 3 of the initial volume of pure water, heating was stopped and the extract was cooled to room temperature to obtain the crude extract of honeysuckle polysaccharides. A2 Polysaccharide Purification: The crude polysaccharide extract was centrifuged at 4000g for 6 minutes, and the supernatant was collected. The supernatant was first vacuum filtered through a 0.44μm filter membrane to obtain the filtrate, and then vacuum filtered again through a 0.22μm filter membrane to obtain the filtrate. Subsequently, the filtrate was purified by dialysis using a 1000Da dialysis bag for 24 hours, during which the pure water was changed 4 times. Finally, the solution in the dialysis bag was freeze-dried to obtain low molecular weight honeysuckle polysaccharide; the molecular weight of low molecular weight honeysuckle polysaccharide is 1000-5000Da.

[0049] Comparative Example 2 The preparation method of the target honeysuckle polysaccharide in this comparative example includes the following steps: S1 Surface Modification: L-arginine and low molecular weight honeysuckle polysaccharide were separately dissolved in pure water at a mass ratio of 1:2. The pH of the L-arginine solution and the low molecular weight honeysuckle polysaccharide solution was adjusted to 4.8 using Tris-citric acid buffer. The mixture was stirred for 3 hours to obtain the modified solution. After mixing the two solutions, the concentration of L-arginine in the mixed solution was 4 mg / mL, and the concentration of honeysuckle polysaccharide in the mixed solution was 8 mg / mL. S2 modification solution purification: Dialyze the modification solution obtained in step S1 again using a 1000Da dialysis bag for 24 hours to remove the unmodified L-arginine. During this period, the pure water was changed 4 times. Finally, the purified honeysuckle polysaccharide modification solution in the bag was obtained. S3 Freeze-drying: Freeze-dry the solution obtained in step S2 to obtain the target honeysuckle polysaccharide.

[0050] Furthermore, in step S1, the preparation method of the low molecular weight honeysuckle polysaccharide includes the following steps: A1 Polysaccharide Extraction: Dried honeysuckle and pure water were soaked in cold water at a mass ratio of 1:30 for 30 minutes, and then heated. When the volume of the crude extract was about 1 / 3 of the initial volume of pure water, heating was stopped and the extract was cooled to room temperature to obtain the crude extract of honeysuckle polysaccharides. A2 Polysaccharide Purification: Centrifuge 4000g of crude honeysuckle polysaccharide extract for 6 minutes, collect the supernatant, first use a 0.44μm filter membrane for vacuum filtration to obtain the filtrate, then use a 0.22μm filter membrane for a second vacuum filtration to obtain the filtrate, and then use a 1000Da dialysis bag for dialysis purification for 24 hours, changing the pure water 4 times during the period, and finally freeze-dry the solution in the dialysis bag to obtain low molecular weight honeysuckle polysaccharide; the molecular weight of low molecular weight honeysuckle polysaccharide is 1000-5000Da.

[0051] Comparative Example 3 The preparation method of the target honeysuckle polysaccharide in this comparative example includes the following steps: S1 Surface Modification: L-arginine and low molecular weight honeysuckle polysaccharide were dissolved separately in pure water at a mass ratio of 1:2. The pH of the L-arginine solution and the low molecular weight honeysuckle polysaccharide solution was adjusted to 8 using Tris-citric acid buffer. The mixture was stirred for 3 hours to obtain the modified solution. After mixing the two solutions, the concentration of L-arginine in the mixed solution was 4 mg / mL, and the concentration of honeysuckle polysaccharide in the mixed solution was 8 mg / mL. S2 modification solution purification: Dialyze the modification solution obtained in step S1 again using a 1000Da dialysis bag for 24 hours to remove the unmodified L-arginine. During this period, the pure water was changed 4 times. Finally, the purified honeysuckle polysaccharide modification solution in the bag was obtained. S3 Freeze-drying: Freeze-dry the solution obtained in step S2 to obtain the target honeysuckle polysaccharide.

[0052] Furthermore, in step S1, the preparation method of the low molecular weight honeysuckle polysaccharide includes the following steps: A1 Polysaccharide Extraction: Dried honeysuckle and pure water were soaked in cold water at a mass ratio of 1:30 for 30 minutes, and then heated. When the volume of the crude extract was about 1 / 3 of the initial volume of pure water, heating was stopped, and the extract was cooled to room temperature to obtain a crude extract of honeysuckle polysaccharides. A2 Polysaccharide Purification: Centrifuge 4000g of crude honeysuckle polysaccharide extract for 6 minutes, collect the supernatant, first use a 0.44μm filter membrane for vacuum filtration to obtain the filtrate, then use a 0.22μm filter membrane for a second vacuum filtration to obtain the filtrate, and then use a 1000Da dialysis bag for dialysis purification for 24 hours, changing the pure water 4 times during the period, and finally freeze-dry the solution in the dialysis bag to obtain low molecular weight honeysuckle polysaccharide; the molecular weight of low molecular weight honeysuckle polysaccharide is 1000-5000Da.

[0053] Test results and analysis: Figure 1 These are photographs of equal concentrations of L-arginine, honeysuckle polysaccharide prepared in Example 1, and honeysuckle polysaccharide prepared in Comparative Example 1 dissolved in 10 mL of pure water. It can be seen that the pure L-arginine aqueous solution is colorless and transparent, while the honeysuckle polysaccharide aqueous solution prepared in Comparative Example 1 without L-arginine modification is transparent and pale yellow. After L-arginine modification, the honeysuckle polysaccharide aqueous solution of Example 1 is transparent and green, indicating that L-arginine was successfully modified onto the honeysuckle polysaccharide, causing a conformational change in the honeysuckle polysaccharide of Comparative Example 1.

[0054] Figure 2 The images are scanning electron microscope (SEM) images of Comparative Example 1 and Example 1. Figure 3 The images shown are electron micrographs and scanning electron microscope images of the honeysuckle polysaccharide prepared in Example 2. In Comparative Example 1, due to the weak hydrogen bonding between pure polysaccharide molecules, the molecular chains are difficult to form regular aggregates, resulting in an irregular fragmented structure. In Examples 1 and 2, L-arginine and honeysuckle polysaccharide undergo electrostatic cross-linking, inducing cross-linking to form regular aggregates, thus exhibiting rod-shaped and spherical structures. This is a typical result of molecular assembly under weak interactions, demonstrating the successful preparation of honeysuckle polysaccharides with oral health and dental care effects in Examples 1 and 2.

[0055] Figure 4In Example 1, compared to Comparative Example 1, the honeysuckle polysaccharide with oral health and dental care effects prepared in Example 1, due to the effect of L-arginine, is attributed to the carboxyl group (-COO). - The asymmetric stretching vibration absorption peak of the ) redshifted (from 1743 cm⁻¹). -1 1630cm -1 Displaced to 1738cm -1 1613cm -1 And 1738cm -1 The weakening of the carboxyl peak intensity at 1440 cm⁻¹ is attributed to the superposition of in-plane vibrations of the hydroxyl group (-OH) and the bending vibrations of the sugar ring CH group. -1 Redshifted to 1417cm - The absorption peak at 1271 cm⁻¹ is attributed to the CO stretching vibration of the hydroxyl group (-OH) + the CO stretching vibration of the sugar ring. -1 Redshifted to 1247cm -1 This is due to the interaction between the carboxyl and hydroxyl groups of the polysaccharide and the amino group (-NH3) of L-arginine. + ), guanidino group (-NH-C(=NH2) + A new hydrogen bond was formed between NH2 and L-arginine, while the CH on the sugar ring was subjected to electrostatic interaction, resulting in a slight change in the local spatial conformation and alteration of the CH bending vibration environment. The above peak shift is one of the direct evidences of the effective binding of honeysuckle polysaccharide with L-arginine in Example 1, further demonstrating the successful preparation of honeysuckle polysaccharide with oral health and dental care effects in Example 1.

[0056] Figure 5 The images show photographs of L-arginine in Examples 2-3 and Comparative Examples 1-3. It is clearly visible that the color of Examples 2-3 has fundamentally changed compared to Comparative Example 1, appearing as a green, cotton-like solid. Conversely, the morphology of Comparative Examples 2-3 is similar to that of Comparative Example 1, indicating that L-arginine cannot be successfully modified onto pure honeysuckle polysaccharide when the pH value is too low (e.g., 4.8) or too high (e.g., 8). In conjunction with the other embodiments of the present invention, it is difficult to successfully modify L-arginine onto pure honeysuckle polysaccharide when the pH value is less than 5 or greater than 7.5. Therefore, the preferred pH value for the L-arginine solution and the low molecular weight honeysuckle polysaccharide solution of the present invention is 5-7.5.

[0057] Figure 6 In comparison with Comparative Example 1, the ZETA potential of Comparative Examples 2-3 did not change significantly; conversely, the ZETA potential of Example 2 was significantly different from that of Comparative Example 1, and the absolute value was greater than 30 mV, indicating that the stability of the honeysuckle obtained in Example 2 was significantly improved. This further proves that L-arginine cannot be successfully modified onto pure honeysuckle polysaccharide when the pH value is too low, such as 4.8, or too high, such as 8.

[0058] Figure 7 In comparison with L-arginine and the honeysuckle polysaccharide prepared in Comparative Example 1, the growth rate of Streptococcus mutans after treatment with the honeysuckle polysaccharide prepared in Example 2, which has oral health and dental care effects, was only 14.11%. This indicates that modifying pure honeysuckle polysaccharide with L-arginine can significantly enhance the antibacterial properties of both against Streptococcus mutans, a caries-causing bacterium, and directly intervene in the formation process of caries, achieving a 1+1>2 effect and thus having dental care effects.

[0059] The method for determining the growth rate of Streptococcus mutans includes the following steps: 1. Preparation of bacterial culture: Use brain heart extract broth (BHI) to culture Streptococcus mutans in an anaerobic tank at 37°C. When the OD600 absorbance of the culture medium is greater than 1, dilute the bacterial culture 1000 times for subsequent experiments.

[0060] 2. Groups: The blank control group was pure BHI medium; the L-arginine group was BHI medium containing 5 mg / mL L-arginine; Comparative Example 1 was BHI medium containing 10 mg / mL honeysuckle polysaccharide; Example 2 was BHI medium containing 10 mg / mL honeysuckle polysaccharide with oral health and dental care effects. 3. Treatment method: Add 10 μL of diluted bacterial solution to 1 mL of different group culture medium, and then incubate in an anaerobic jar at 37℃ for 24 hours. Measure the absorbance at OD600 to calculate the growth rate of Streptococcus mutans.

[0061] Figure 8 This figure shows the anti-inflammatory effects of honeysuckle polysaccharide and L-arginine prepared in Comparative Example 1, and honeysuckle polysaccharide with oral health and dental care effects prepared in Example 2, in a RAW264.7 cell inflammation model. As shown in the figure, after induction with lipopolysaccharide (LPS), the concentration of the representative pro-inflammatory factor—tumor necrosis factor α—significantly increased. Treatment with honeysuckle polysaccharide and L-arginine prepared in Comparative Example 1 failed to significantly reduce the concentration of the pro-inflammatory factor, showing no anti-inflammatory effect. However, treatment with an equal concentration of honeysuckle polysaccharide with oral health and dental care effects prepared in Example 2 significantly decreased the concentration of tumor necrosis factor α, indicating that the honeysuckle polysaccharide with oral health and dental care effects prepared in Example 2 has an anti-inflammatory effect, helping to maintain oral microenvironment homeostasis and protect gingival health.

[0062] The method for investigating the anti-inflammatory effect of honeysuckle polysaccharide in a RAW264.7 cell inflammation model includes the following steps: RAW264.7 macrophages were selected, and after adhesion and differentiation, they were treated with 0.1 μg / mL lipopolysaccharide (LPS) for 24 h to induce pro-inflammatory differentiation and form an inflammatory cell model.

[0063] Control group: Normal cells not treated with LPS Model group: cultured in medium containing LPS for 24 h; Comparative Example 1: Cultured in a medium containing 10 mg / mL honeysuckle polysaccharide and LPS for 24 h; L-arginine: Cultured in a medium containing 5 mg / mL L-arginine and LPS for 24 h; Example 2: Cultured in a medium containing 10 mg / mL honeysuckle polysaccharide, which has oral health and dental care effects, and LPS for 24 h; After treatment, the concentration of tumor necrosis factor α was detected using an ELISA kit to evaluate the anti-inflammatory effect.

[0064] In summary, the honeysuckle polysaccharide with oral health and dental care effects obtained in this invention is obtained by simple water extraction of honeysuckle polysaccharide followed by modification of L-arginine using a simple hydrogen bonding effect. The method is simple, green, and easy to operate. This polysaccharide has enhanced inhibition of Streptococcus mutans growth and can directly intervene in the formation process of tooth decay. This polysaccharide also has antibacterial and anti-inflammatory effects, with synergistic functions, which can effectively maintain the stability of the oral environment. It can be widely used as a high-quality raw material in foods with cariogenic effects to reduce the cariogenicity of food.

[0065] The specific embodiments described above are further illustrations of the technical solution and beneficial effects of the present invention, and are not intended to limit the implementation methods. For those skilled in the art, any obvious substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A method for preparing honeysuckle polysaccharide with oral health and dental care effects, characterized in that: Includes the following steps: S1 Surface Modification: L-arginine and honeysuckle polysaccharide were dissolved separately in water, and the pH values ​​of the L-arginine solution and honeysuckle polysaccharide solution were adjusted separately. Then, the L-arginine solution and honeysuckle polysaccharide solution were mixed and stirred to obtain the modified solution. S2 modification solution purification: Dialyze the modification solution obtained in step S1 using a dialysis bag to remove the unmodified L-arginine and obtain the purified honeysuckle polysaccharide modification solution in the bag; S3 Drying: The solution obtained in step S2 is dried to obtain the target honeysuckle polysaccharide.

2. The method for preparing honeysuckle polysaccharide with oral health and dental care effects according to claim 1, characterized in that: In step S1, the honeysuckle polysaccharide that is surface-modified with L-arginine is a low molecular weight honeysuckle polysaccharide, wherein the molecular weight of the low molecular weight honeysuckle polysaccharide is 1000-5000 Da.

3. The method for preparing honeysuckle polysaccharide with oral health and dental care effects according to claim 1, characterized in that: In step S1, the preparation method of honeysuckle polysaccharide with L-arginine surface modification includes the following steps: A1 Polysaccharide Extraction: Honeysuckle was soaked in water and boiled with hot water to obtain a crude extract of honeysuckle polysaccharides; A2 Polysaccharide Purification: The crude polysaccharide extract was centrifuged to obtain the supernatant, which was then filtered through a membrane and purified by dialysis. The solution in the dialysis bag was then freeze-dried to obtain honeysuckle polysaccharide.

4. The method for preparing honeysuckle polysaccharide with oral health and dental care effects according to claim 3, characterized in that: In step A1, the mass ratio of honeysuckle to water is 1:(20-30), and the soaking time in cold water is 20-30 minutes; when boiling in hot water, the volume of the crude extract should be 1 / 6-1 / 2 of the initial volume of water added.

5. The method for preparing honeysuckle polysaccharide with oral health and dental care effects according to claim 3, characterized in that: In step A2, the supernatant is subjected to two membrane filtrations. The first filtration uses a membrane with a pore size of 0.44 μm, and the second filtration uses a membrane with a pore size of 0.22 μm. Both filtrations are performed using vacuum filtration. The dialysis purification is carried out using a dialysis bag with a molecular weight cutoff of 1000 Da, with a dialysis time of more than 24 hours and the water is changed at least 4 times every 24 hours.

6. The method for preparing honeysuckle polysaccharide with oral health and dental care effects according to claim 1, characterized in that: In step S1, the mass ratio of L-arginine to honeysuckle polysaccharide is 1:(2-4).

7. The method for preparing honeysuckle polysaccharide with oral health and dental care effects according to claim 1, characterized in that: In step S1, the pH of the L-arginine solution and the honeysuckle polysaccharide solution is adjusted to 5.0-7.

5.

8. The method for preparing honeysuckle polysaccharide with oral health and dental care effects according to claim 1, characterized in that: In step S2, the molecular rejection capacity of the dialysis bag is 1000 Da, the dialysis time is more than 24 hours, and the water is changed at least 4 times every 24 hours.

9. A honeysuckle polysaccharide with oral health and dental care benefits, characterized in that: The polysaccharide is prepared by the method for preparing honeysuckle polysaccharide with oral health and dental care effects as described in any one of claims 1-8.

10. The application of the honeysuckle polysaccharide with oral health and dental care effects as described in claim 9 in foods with cariogenic effects.

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