Preparation method of high-activity chondrus crispus extract, functional additive and toothpaste

Abstract

The invention belongs to the technical field of oral care products, and relates to a preparation method of a high-activity chondrus crispus extract, a functional additive and toothpaste. According to the method, during raw material pretreatment, an anti-oxidation protection solution containing sodium sulfite and cysteine is used, the high-activity extract is prepared through the steps of enzymolysis, centrifugation, ultrafiltration, reverse osmosis and the like, and the problems that in a traditional water-alcohol extraction technology preparation method, the extraction amount and purity of active ingredients are limited and the like can be solved. The extract is excellent in antioxidant, antibacterial and anti-inflammatory activity, a functional additive prepared from xylitol, hydroxyapatite and other components is added into the toothpaste, the residual area ratio of dental plaque can be reduced to 10%-15%, gingival inflammation can be effectively improved, and the gingival bleeding index of animals is reduced by 50%-60%. Meanwhile, the toothpaste is good in stability, the viscosity change under different temperature and humidity conditions is within + / -10%, and the particle size distribution is stable. Therefore, the problem that traditional toothpaste has limitation in the aspect of preventing and improving oral diseases is effectively solved.

Description

Technical Field

[0001] The invention belongs to the technical field of oral care products, and particularly relates to a preparation method of a high-activity Chondrus crispus extract, a functional additive and a toothpaste. Background Art

[0002] As a must-have for daily oral cleaning, the efficacy and quality of toothpaste have always been the focus of research and development. Traditional toothpaste mainly focuses on the cleaning function, removing plaque and stains through abrasives, but has limitations in preventing and improving oral diseases. As people pay more attention to oral health, the demand for toothpaste with special functions is growing.

[0003] In recent years, many oral care industry studies have been devoted to exploring the innovative application of natural ingredients in toothpaste. Chondrus crispus extract has become a research hotspot due to its unique biological activity. In a study with patent application number 202210335111.2, Guangzhou Luojie Biotechnology Co., Ltd. found that a mixed extract composed of Leonurus japonicus, Angelica sinensis, Chuanxiong, peach blossom, Centella asiatica, chamomile and Chondrus crispus not only has the ability to scavenge DPPH free radicals and ABTS free radicals, but also effectively inhibits tyrosinase activity and melanin production, and has a soothing and modifying effect. Chondrus crispus extract, as a seaweed extract, has anti-inflammatory and antibacterial effects. Because it has a certain inhibitory effect on certain leukocyte proteases, it has anti-inflammatory activity, anti-inflammatory and antibacterial effects, can relieve the symptoms of gingival swelling and pain, has a strong inhibitory effect on Staphylococcus aureus, Helicobacter pylori and its influenza virus, and can keep teeth healthy for a long time. Therefore, adding Chondrus crispus extract to toothpaste can not only improve the cleaning effect, but also effectively prevent and improve oral inflammation and improve the overall health of the oral cavity.

[0004] At present, the traditional preparation methods of Chondrus crispus extract are mainly divided into two types: water and alcohol extraction technology and microfluidic extraction technology. For example, the Chondrus crispus extraction process proposed in Application No. 202210454047.X combines alcohol extraction with water extraction, including washing and chopping Chondrus crispus and then stirring and filtering with ethanol aqueous solution at constant temperature to obtain the first extract and filter residue, and the filter residue is treated by freezing, ultrasound, secondary ultrasound, cooking, filter pressing, etc., and then the extracts of each time are mixed and stirred at constant temperature, distilled under reduced pressure, and freeze-dried to obtain the extract. However, the pertinence and effectiveness in cell activity regulation and impurity removal are insufficient, resulting in limited extraction amount and purity of active ingredients, and ultimately showing low activity. In addition, the microfluidic extraction technology is mainly used in Application No. 202210335111.2, the principle of which is to use high-speed fluid to impact plant raw materials to break cells and release active ingredients. However, due to high-speed impact, some active ingredients may cause a certain degree of mechanical damage, affecting their activity. Therefore, in the subsequent processing process, although purification and concentration were carried out, the activity of the final extract was relatively low due to the relatively low purity of the active ingredients extracted in the early stage and the possible presence of more impurities.

[0005] However, in the existing toothpaste market, although there are some products with added plant extracts, the physical and chemical properties of Chondrus crispus extract are not good, such as uneven particle size distribution and poor water solubility, which makes it difficult to disperse evenly in the toothpaste formula, affecting the overall quality and use effect of the toothpaste. Therefore, it is urgent to develop a method for preparing a highly active Chondrus crispus extract and successfully apply it to toothpaste to improve the efficacy of toothpaste in preventing oral diseases and maintaining the balance of oral microecology, which has become a key direction for the current research and development of oral care products. Summary of the invention

[0006] The background technology discloses that the existing extraction and application methods of Chondrus crispus extract have many defects. In the present invention, the use of antioxidant protection solution (containing sodium sulfite and cysteine) in the raw material pretreatment stage effectively prevents the oxidation and browning of the raw materials in the initial stage of treatment, maintains the color of the raw materials and the integrity of the active ingredients, and lays a good foundation for subsequent extraction. In the entire extraction process, each step is closely linked, and impurities, unreacted enzymes and small molecules that may affect the activity in the raw materials are orderly removed through operations such as enzymatic hydrolysis, centrifugation, ultrafiltration, and reverse osmosis, so that the extract has high purity and rich active ingredient content. Based on this, a preparation method, functional additive and toothpaste of a highly active Chondrus crispus extract are proposed.

[0007] To achieve the above object, the present invention adopts the following technical scheme: a method for preparing a highly active Chondrus crispus extract, which mainly comprises the following steps: S1. Raw material pretreatment: First, fresh Chondrus crispus is placed in a stainless steel container with an antioxidant protection solution and soaked for 20-30 minutes; then, the Chondrus crispus is taken out and rinsed with deionized water for 4-6 times to remove the residual antioxidant protection solution; then, the Chondrus crispus is transferred to a vacuum drying oven and vacuum dried at a temperature of 35-45° C. until the water content is reduced to less than 8%; finally, the dried Chondrus crispus is crushed into a particle size of 50-70 meshes using a universal crusher and stored for later use; S2 enzyme-assisted extraction: add the crushed Chondrus crispus powder into a reactor of a buffer solution, and add 1%-3% of a composite enzyme preparation at a mass volume ratio of 1:15-1:20 to the buffer solution, and perform enzymolysis at 45-55°C for 3-5 hours; after the enzymolysis, heat to 80-90°C and maintain for 10-20 minutes to inactivate the enzyme, then transfer the reaction solution to a high-speed centrifuge, centrifuge at 5000-7000 rpm for 15-25 minutes, and collect the supernatant; S3 Aqueous Two-Phase Extraction: Add an aqueous two-phase extractant to the supernatant, stir evenly with a stirring paddle in a plastic extraction tank, let stand to separate, and collect the upper phase rich in active ingredients; then add an equal volume of deionized water to the upper phase, repeat the extraction 1-2 times, and combine all the upper phases; S4 ultrafiltration and reverse osmosis concentration: The combined upper phase is first ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 8-12kDa to remove small molecular impurities and unreacted enzymes, and then the ultrafiltered liquid is transferred to a reverse osmosis concentration device and concentrated to 1 / 5-1 / 3 of the original volume at a pressure of 2-4MPa; S5 Freeze drying and pulverization: The above concentrate is transferred to a freeze dryer, pre-frozen at -45 to -35°C for 3-5 hours, and then freeze-dried at a vacuum degree of 0.08-0.12Pa for 24-36 hours to obtain a dry extract, which is then pulverized using a jet mill to a particle size of 10-30 μm to obtain a highly active Chondrus crispus extract.

[0008] Furthermore, in the S1 raw material pretreatment, the antioxidant protection liquid contains 0.2%-0.4% sodium sulfite and 0.1%-0.3% cysteine ​​in a mass volume ratio. The synergistic effect of sodium sulfite and cysteine ​​can effectively prevent the oxidation and browning of the raw materials in the initial stage of treatment, and maintain the color of the raw materials and the integrity of the active ingredients.

[0009] Furthermore, in the S2 enzyme-assisted extraction, the complex enzyme preparation is composed of mannanase, xylanase and alginate lyase in a ratio of 3:2:1, which is used for the corresponding polysaccharide components in the cell wall and intracellular structure of Chondrus crispus, synergistically destroying the cell structure and promoting the release of active ingredients; the buffer solution is a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer with a pH of 5.5-6.5.

[0010] Furthermore, in the S3 aqueous two-phase extraction, the aqueous two-phase extractant contains 15%-25% by mass of polyethylene glycol and 10%-20% by mass of ammonium sulfate.

[0011] A functional additive comprises the highly active Chondrus crispus extract obtained by the above preparation method and the following auxiliary ingredients: 5%-10% by mass of xylitol, 3%-6% by mass of hydroxyapatite, 2%-5% by mass of lactobacillus fermentation product, and 0.5%-1.5% by mass of bromelain, wherein the mass fraction of the highly active Chondrus crispus extract is 20%-50%.

[0012] A toothpaste, characterized in that it contains the above-mentioned functional additives and toothpaste basic ingredients, wherein the toothpaste basic ingredients include, by weight, 40-50 parts of water, 12-18 parts of sorbitol, 15-20 parts of hydrated silica, 4-6 parts of polyethylene glycol-400, 3-5 parts of sodium lauryl sulfate, 1-2 parts of edible flavor, 0.8-1.2 parts of cellulose gum, 0.2-0.5 parts of titanium dioxide, and 3-5 parts of glucose, wherein the mass percentage of the functional additives in the toothpaste is 8%-15%.

[0013] Furthermore, the particle size of the hydrated silica is 8-15 μm, and its spherical structure has a low friction coefficient with the tooth surface during brushing, which can effectively remove dental plaque and stains, reduce the wear on tooth enamel, and synergize with functional additives to enhance the cleaning and protection effects of teeth.

[0014] Furthermore, the purity of the sodium lauryl sulfate is not less than 90%. As a surfactant, it can produce rich and dense foam, effectively remove oral foreign matter, and has little irritation to the gums and oral mucosa, and maintains oral hygiene together with other ingredients.

[0015] Furthermore, the viscosity of the cellulose gum is 800-1200 mPa·s, which can make the toothpaste form a uniform and stable paste, which is well dispersed and adhered in the oral cavity, ensuring that the functional additives and other ingredients are evenly distributed in the oral cavity and fully exert their effects.

[0016] Furthermore, the toothpaste also contains 0.5%-1% by mass of butylene glycol and 0.3%-0.6% by mass of dipotassium glycyrrhizinate as auxiliary additives. Butylene glycol is used for moisturizing, and dipotassium glycyrrhizinate has anti-inflammatory and soothing effects, which further enhances the toothpaste's ability to protect the oral mucosa and relieve inflammation.

[0017] Compared with the prior art, the present invention has the following advantages: 1. The present invention breaks through the traditional limitations in the extract preparation process, and uses an antioxidant protective liquid in the pretreatment stage to effectively prevent oxidation and browning of the raw materials and maintain the stability of the active ingredients. Subsequently, impurities are removed through precise steps such as enzymolysis, centrifugation, ultrafiltration, and reverse osmosis, greatly improving the purity of the extract and the content of active ingredients. The extract of the present invention has been verified by experiments to be far superior to products prepared by traditional processes in terms of antioxidant, antibacterial, and anti-inflammatory activities. It can efficiently remove free radicals, inhibit the growth of harmful bacteria, and reduce inflammatory reactions, providing strong protection for oral health.

[0018] 2. The present invention applies the highly active Chondrus crispus extract to toothpaste, and utilizes the synergistic effect of the extract with other ingredients in the toothpaste, especially with bromelain, to effectively decompose food residues and bacterial biofilms, deeply clean the gaps and surfaces of teeth, and significantly reduce dental plaque residues. Comparative experiments show that the toothpaste containing the extract of the present invention has excellent effects in cleaning dental plaque, can make teeth cleaner, and reduce the generation of bad breath in the mouth.

[0019] 3. In terms of oral care, in addition to cleaning, the toothpaste of the present invention can inhibit the acid production of oral bacteria with xylitol, reduce the risk of tooth decay; hydroxyapatite can supplement tooth minerals and enhance the acid resistance of teeth; lactobacillus fermentation products can regulate oral microecology and maintain the balance of bacterial flora; and the highly active Chondrus crispus extract can relieve gingival inflammation and reduce gingival bleeding, redness and swelling due to its anti-inflammatory and antibacterial properties.

[0020] 4. In the toothpaste formula design of the present invention, basic ingredients such as hydrated silica, sodium lauryl sulfate, and cellulose gum are carefully selected and proportioned to ensure that the viscosity of the toothpaste changes very little under different temperature and humidity conditions, and the particle size distribution of the functional additives is stable. Therefore, the toothpaste has reliable performance during storage and use, and will not have problems such as stratification and thinning, ensuring that consumers can always get a stable use experience. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a comparison chart of the activities of the highly active Chondrus crispus extract prepared by the embodiment of the present invention and the control group; Figure 2 It is a dose-effect curve diagram of the antioxidant activity of the high-activity Chondrus crispus extract of the present invention and the control group; Figure 3 This is a comparison chart of the antibacterial activity of the highly active Chondrus crispus extract of the present invention and the control group; Figure 4 The dose-effect curve of the anti-inflammatory activity of the highly active Chondrus crispus extract of the present invention and the control group; Figure 5 This is a SEM comparison of the cleaning ability and improvement of gingival inflammation of the toothpaste of the present invention and the control toothpaste; Figure 6A comparison chart of viscosity stability between the toothpaste of the present invention and the control toothpaste under different temperature-humidity conditions; Figure 7 The present invention is a schematic diagram of the packaging information, ingredients and instructions for use of a toothpaste containing a highly active Chondrus crispus extract. DETAILED DESCRIPTION

[0022] In order to further illustrate the technical solution of the present invention, we will combine the attached Figures 1 to 7 , the present invention is further illustrated by 12 embodiments.

[0023] 1. Example and Control Group Setup In order to study the biological activity of Chondrus crispus extract more comprehensively and deeply, the number of examples was increased and the setting of the control group was adjusted accordingly during the research and development of this project. By carefully comparing the differences between different examples and the control group, we can more accurately explore the specific effects of various conditions and factors on the activity of the extract. Example 1

[0024] This example is set as a basic preparation process and serves as a reference standard for subsequent experiments. The specific steps are as follows: S1 Raw material pretreatment: Take fresh Chondrus crispus and place it in a stainless steel container containing an antioxidant protection liquid containing 0.3% sodium sulfite and 0.2% cysteine ​​in a mass volume ratio and soak it for 25 minutes. After taking it out, rinse it with deionized water 5 times, transfer it to a 40℃ vacuum drying oven and dry it to a moisture content of 7%, and then use a universal grinder to grind it to a particle size of 60 mesh for later use.

[0025] S2 enzyme-assisted extraction: The crushed Chondrus crispus powder was added into a pH 6.0 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution reactor containing 2% composite enzyme preparation by mass fraction at a mass volume ratio of 1:18, and enzymatic hydrolysis was carried out at 48°C for 4 hours, and the temperature was raised to 85°C and maintained for 15 minutes to inactivate the enzyme. The supernatant was collected by centrifugation at 5500 rpm for 20 minutes. In this embodiment, the composite enzyme preparation is preferably composed of mannanase, xylanase and alginate lyase in a ratio of 3:2:1).

[0026] S3 two-phase aqueous extraction: add a two-phase aqueous extractant containing 20% ​​polyethylene glycol and 10% ammonium sulfate by mass to the supernatant, stir evenly in a plastic extraction tank and let stand to separate the layers, collect the upper phase, add an equal volume of deionized water and repeat the extraction once, then combine the upper phases.

[0027] S4 ultrafiltration and reverse osmosis concentration: The combined upper phases were ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and then concentrated to 1 / 4 of the original volume by reverse osmosis at a pressure of 3 MPa.

[0028] S5 freeze drying and pulverization: the concentrate was pre-frozen at -40°C for 4 hours, freeze-dried at 0.1 Pa vacuum for 30 hours, and pulverized by a jet mill to a particle size of 20 μm to obtain a highly active Chondrus crispus extract. Example 2

[0029] In this example, the effect of the ratio of the antioxidant protection liquid components on the activity of the extract was studied. The specific preparation steps are as follows: S1 raw material pretreatment: fresh Chondrus crispus was placed in an antioxidant protection liquid containing 0.2% sodium sulfite and 0.1% cysteine ​​in a mass volume ratio for 25 minutes, and the centrifugal speed in S2 was increased to 7000 rpm. The subsequent treatment was the same as the remaining steps of S1 in Example 1. S2-S5 are the same as Example 1. Example 3

[0030] In this example, the effect of changing the mass-volume ratio of enzymatic hydrolysis on the activity of the extract was studied. The specific preparation steps are as follows: S2 enzyme-assisted extraction: the crushed Chondrus crispus powder was added to a pH 6.0 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution reactor containing 2% composite enzyme preparation by mass volume ratio of 1:15, and the subsequent treatment was the same as the remaining steps of S2 in Example 1. S1, S3-S5 are the same as Example 1. Example 4

[0031] In this example, the effect of changing the mass fraction of the complex enzyme preparation on the activity of the extract was studied. The specific preparation steps are as follows: S2 enzyme-assisted extraction: the crushed Chondrus crispus powder was added to a pH 6.0 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution reactor containing 1% of the complex enzyme preparation at a mass volume ratio of 1:18, and the subsequent treatment was the same as the remaining steps of S2 in Example 1. S1, S3-S5 are the same as Example 1. Example 5

[0032] In this example, the effect of changing the enzymatic hydrolysis temperature on the activity of the extract was studied. The specific preparation steps are as follows: S2 enzyme-assisted extraction: the crushed Chondrus crispus powder was added to a pH 6.0 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution reactor containing 2% composite enzyme preparation by mass fraction at a mass volume ratio of 1:18, and enzymatic hydrolysis was performed at 42°C for 4 hours. The subsequent treatment was the same as the remaining steps of S2 in Example 1. S1, S3-S5 are the same as Example 1. Example 6

[0033] In this example, the effect of enzymatic hydrolysis time on the activity of the extract was studied. The specific preparation steps are as follows: S2 enzyme-assisted extraction: The crushed Chondrus crispus powder was added to a pH 6.0 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution reactor containing 2% of a composite enzyme preparation (mannanase, xylanase and alginate lyase in a ratio of 3:2:1) at a mass volume ratio of 1:18, and enzymatic hydrolysis was performed at 48°C for 3 hours. The subsequent treatment was the same as the remaining steps of S2 in Example 1. S1, S3-S5 are the same as Example 1. Example 7

[0034] In this example, the influence of the mass fraction of polyethylene glycol as a two-phase extractant on the activity of the extract was studied. The specific preparation steps are as follows: S3 two-phase extraction: a two-phase extractant containing 15% polyethylene glycol and 20% ammonium sulfate by mass fraction was added to the supernatant, and the subsequent treatment was the same as the remaining steps of S3 in Example 1. S1, S2, S4-S5 were the same as in Example 1. Example 8

[0035] In this example, the mass fraction of ammonium sulfate in the two-phase aqueous extraction agent was changed to study its effect on the activity of the extract. The specific preparation steps are as follows: S3 two-phase aqueous extraction: a two-phase aqueous extraction agent containing 20% ​​polyethylene glycol and 15% ammonium sulfate by mass fraction was added to the supernatant, and the subsequent treatment was the same as the remaining steps of S3 in Example 1. S1, S2, S4-S5 are the same as Example 1. Example 9

[0036] In this example, the effect of changing the molecular weight cut-off of the ultrafiltration membrane on the activity of the extract was studied. The specific preparation steps are as follows: S4 ultrafiltration and reverse osmosis concentration: The upper phases were combined and ultrafiltered through an ultrafiltration membrane with a molecular weight cut-off of 8 kDa, and the subsequent treatment was the same as the remaining steps of S4 in Example 1. S1-S3 and S5 were the same as in Example 1. Example 10

[0037] In this example, the effect of reverse osmosis concentration pressure on the activity of the extract was studied. The specific preparation steps are as follows: S4 ultrafiltration and reverse osmosis concentration: The upper phases were combined and ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and then reverse osmosis concentrated to 1 / 4 of the original volume at a pressure of 2 MPa. S1-S3 and S5 are the same as in Example 1. Embodiment 11

[0038] In this example, the effect of changing the freeze drying pre-freezing temperature on the activity of the extract was studied. The specific preparation steps are as follows: S5 freeze drying and pulverization: the concentrate was pre-frozen at -30°C for 4 hours, and the subsequent treatment was the same as the remaining steps of S5 in Example 1. S1-S4 were the same as in Example 1. Example 12

[0039] In this example, the effect of freeze drying time on the activity of the extract was studied. The specific preparation steps are as follows: S5 freeze drying and pulverization: the concentrate was pre-frozen at -40°C for 4 hours, freeze dried at 0.1 Pa vacuum for 20 hours, and the subsequent treatment was the same as the remaining steps of S5 in Example 1. S1-S4 were the same as in Example 1.

[0040] Control group 1 The specific steps of the traditional water extraction and alcohol extraction process are as follows: Referring to the process of patent application number 202210454047.X, the wrinkled chondrion is washed and chopped, and a 60% ethanol aqueous solution is used for constant temperature stirring and filtering at 55°C to obtain a first extract and a residue. The residue is frozen at -12°C and crushed, and 70% ethanol is added for ultrasonic treatment for 0.8 hours, and then distilled water containing sodium hydroxide (pH8) (ethanol volume 250%) is added for a second ultrasonic treatment for 3 hours. After filtration, the second residue and distilled water are mixed in a ratio of 1:8 and steamed at 85°C for 2.5 hours, and the temperature is naturally reduced and filtered to obtain a third extract. The three extracts are mixed and stirred at 85°C for 3 hours, and the extract is distilled under reduced pressure and freeze-dried.

[0041] Control group 2 The specific steps of the non-enzyme-assisted extraction process are as follows: S1 raw material pretreatment is the same as in Example 1. S2 direct extraction: the crushed Chondrus crispus powder is added to a pH 6.0 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution reactor at a mass volume ratio of 1:18, treated at 48°C for 4 hours, heated to 85°C for 15 minutes, centrifuged at 5500 rpm for 20 minutes, and the supernatant is collected. The subsequent S3-S5 steps are the same as in Example 1.

[0042] 2. Study on the activity of Chondrus crispus extract As attached Figure 1 As shown, Example 1 of the present invention achieves the optimal level in antioxidant, antibacterial and anti-inflammatory activities by optimizing the ratio of antioxidant protection solution, enzymatic hydrolysis conditions and aqueous two-phase extraction parameters. By adjusting single variables (such as enzyme concentration, temperature, freeze-drying time, etc.) in Examples 2-12, the activity index fluctuates within the range of 66.2%-84.1%, indicating that the present invention has a certain fault tolerance. Control group 1 suffers from serious loss of active ingredients due to the lack of antioxidant protection and enzymatic hydrolysis steps; Control group 2 suffers from insufficient release of active ingredients due to the failure to destroy the cell wall structure, and all indicators of both groups are significantly lower than those of the embodiment group. The experimental results verify the significant advantages of the process of the present invention in improving the activity of Chondrus crispus extract.

[0043] In order to further verify the activity of the Chondrus crispus extract, we took Example 1 and two control groups as examples and conducted multiple experiments to study the activity of the extract one by one.

[0044] As attached Figures 2 to 4As shown, the specific experimental scheme and results are as follows: the antioxidant experiment uses the DPPH method to set a concentration gradient of 0.1-1.0 mg / mL, and it is found that the DPPH free radical scavenging rate of Example 1 is significantly enhanced with increasing concentration, and is better than the control group 1 and the control group 2 at each concentration, and is close to the effect of vitamin C, reaching about 85% at 1.0 mg / mL; the antibacterial experiment uses the filter paper diffusion method, and the results show that the diameter of the inhibition zone of Example 1 against Escherichia coli and Staphylococcus aureus is greater than that of the control group, such as the inhibition zone of Staphylococcus aureus is about 20 mm; the anti-inflammatory experiment uses a cell model to set a concentration gradient of 10-50 μg / mL, which confirms that the NO inhibition rate of Example 1 increases with increasing concentration, reaching about 70% at 50 μg / mL, which is significantly better than the control group, and is close to the inhibition effect of dexamethasone.

[0045] It should be noted that the above experiments selected part of the research and development process as the experimental data support of the present invention to ensure that the results are scientific and rigorous. Through comparative analysis, the specific effects of different processes on the activity of the extracts are revealed, providing an empirical basis for optimizing the process.

[0046] It can be seen that the Chondrus crispus extract obtained according to the preparation process of Example 1 of the present invention exhibits good activity in anti-oxidation, antibacterial and anti-inflammatory aspects, and its process optimization effect is significantly better than that of the control group.

[0047] After many experiments and process optimization, we successfully extracted highly active Chondrus crispus extract. During the research and development process, after a lot of research, we applied highly active Chondrus crispus extract to toothpaste formula and designed a series of experiments to verify its effect. In response to this, we developed a toothpaste with high cleaning efficiency, anti-inflammatory and antibacterial properties and good stability.

[0048] 3. Overall formula design of toothpaste As attached Figure 7As shown, the toothpaste basic ingredients developed by the present invention include water, sorbitol, hydrated silica, polyethylene glycol-400, sodium lauryl sulfate, etc. Water is the solvent of toothpaste, sorbitol provides moisturizing effect, hydrated silica is the main abrasive, polyethylene glycol-400 helps to dissolve and disperse other ingredients, and sodium lauryl sulfate is used as a surfactant to produce rich foam and enhance the cleaning effect. Hydrated silica screened hydrated silica with a particle size of 8-15μm. Sodium lauryl sulfate uses sodium lauryl sulfate with a purity of not less than 90% to ensure its high efficiency as a surfactant and reduce the side effects that may be caused by impurities. The viscosity of cellulose gum is controlled at 800-1200mPa・s, which makes the toothpaste have a uniform and stable texture. Butylene glycol and dipotassium glycyrrhizinate are used as auxiliary additives. The role of butanediol is to moisturize and prevent the toothpaste from drying out during storage and use. Dipotassium glycyrrhizinate has anti-inflammatory and soothing effects, which can reduce the irritation that may be caused to the oral mucosa during brushing. The proportion of highly active Chondrus crispus extract in the functional additives is set at 20%-50%, which is the core ingredient of toothpaste to exert anti-inflammatory and antibacterial effects. Xylitol accounts for 5%-10% of the functional additives. It can not only add sweetness to toothpaste and replace traditional artificial sweeteners, but also has the important function of preventing caries. Xylitol can inhibit the utilization of sucrose by oral bacteria and reduce the production of acidic substances, thereby reducing the risk of caries. Hydroxyapatite accounts for 3%-6%. Its composition is highly similar to the main inorganic components of teeth, replenishing the minerals lost by teeth in daily use, enhancing the acid resistance of teeth, and preventing tooth sensitivity and caries. Lactobacillus fermentation products account for 2%-5%. The oral cavity is a complex microbial ecosystem, and the excessive growth of harmful bacteria can cause a variety of oral problems. Lactobacillus fermentation products can regulate the balance of oral microecology, inhibit the growth of harmful bacteria, promote the growth of beneficial bacteria, and create a good microbial environment for oral health. Bromelain accounts for 0.5%-1.5%. It has the ability to decompose proteins and can help decompose food debris and bacterial biofilm during oral cleaning, making the tooth surface cleaner and reducing the formation of dental plaque.

[0049] Of course, in order to further improve the performance and quality of toothpaste, the toothpaste can also add the following trace ingredients: sodium methylparaben, butylene glycol, forsythia fruit extract, licorice root extract, 1,2-hexanediol, p-hydroxyacetophenone, caprylhydroxamic acid, ethylhexylglycerin, phenoxyethanol, magnesium nitrate, methylchloroisothiazolinone, magnesium chloride, methylisothiazolinone. The total proportion of the above trace ingredients in toothpaste is strictly controlled within 0.1%, which is a conventional added ingredient in the toothpaste production industry. Therefore, the addition of trace ingredients does not fall within the scope of protection of the present invention.

[0050] In order to further study the effect of toothpaste containing highly active Chondrus crispus extract in cleaning dental plaque and improving gingival inflammation. In this research experiment, the example toothpaste selected the basic ingredients and functional additives of the present invention, wherein the toothpaste contained highly active Chondrus crispus extract; the control group selected toothpaste purchased on the market, which did not contain Chondrus crispus extract. At the same time, by simulating different environmental conditions, the stability of the example toothpaste and the control group toothpaste was verified to ensure the stable performance of the toothpaste during storage and use.

[0051] As attached Figure 5 As shown, in order to fully verify the efficacy of toothpaste containing highly active Chondrus crispus extract, we conducted the following cleaning ability and gingival inflammation improvement experiments: In the cleaning ability experiment, artificial dental plaque was prepared on sterile enamel slices using standard oral bacterial strains and simulated saliva culture medium, and the two toothpastes were used for treatment and rinsing after grouping. Electron microscopy observation and image analysis showed that the residual plaque area of ​​the embodiment toothpaste (Figure a) was significantly lower than that of the control group (Figure b) due to the synergistic effect of highly active Chondrus crispus extract and bromelain and other ingredients; in the gingival inflammation improvement experiment, after gingival inflammation was induced in experimental animals, the corresponding toothpaste was used to brush teeth and the indicators were observed. After the end, gingival tissue section analysis found that the embodiment toothpaste (Figure c) reduced the animal gingival bleeding index by 50%-60% with anti-inflammatory ingredients such as highly active Chondrus crispus extract, which was better than the 20%-30% of the control group (Figure d), and the gingival redness and swelling were lighter, and the tissue inflammatory cell infiltration was less.

[0052] As attached Figure 6 As shown, we put the toothpastes of the embodiment and the control group into toothpaste tubes of the same specifications, placed them in a stability test box with different temperature (4°C, 25°C, 40°C) and humidity (30%, 60%, 90%) combinations for 3-6 months, and used a rheometer to measure the viscosity and a particle size analyzer to measure the particle size distribution of the functional additives every month to test the stability of the toothpaste of the present invention. It can be seen that due to the interaction of ingredients and reasonable formulation, the viscosity of the toothpaste of the embodiment changes within ±10% under different conditions and the particle size distribution is stable, which is better than the obvious viscosity change or widening of the particle size distribution that may occur in the control group.

[0053] In summary, the highly active Chondrus crispus extract prepared by the present invention and the toothpaste containing its functional additives perform well in stability, cleaning power and anti-inflammatory effects, verifying its potential to significantly improve oral health and providing strong support for innovation in the toothpaste industry.

[0054] The above shows and describes the main features and advantages of the present invention. For those skilled in the art, it is obvious that the specific implementation of the present invention is not limited to the details of the above exemplary embodiments, and the creative ideas and design ideas of the present invention can be realized in other specific forms without departing from the spirit or basic features of the present invention, which should be equivalent to the protection scope disclosed in the technical solution of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention.

[0055] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing a highly active Chondrus crispus extract, characterized in that: The following steps are involved: S1. Raw material pretreatment: First, fresh Chondrus crispus is placed in a stainless steel container with an antioxidant protection solution and soaked for 20-30 minutes; then, the Chondrus crispus is taken out and rinsed with deionized water for 4-6 times to remove the residual antioxidant protection solution; then, the Chondrus crispus is transferred to a vacuum drying oven and vacuum dried at a temperature of 35-45° C. until the water content is reduced to less than 8%; finally, the dried Chondrus crispus is crushed into a particle size of 50-70 meshes using a universal crusher and stored for later use; S2 enzyme-assisted extraction: add the crushed Chondrus crispus powder into a reactor of a buffer solution, and add 1%-3% of a composite enzyme preparation at a mass volume ratio of 1:15-1:20 to the buffer solution, and perform enzymolysis at 45-55°C for 3-5 hours; after the enzymolysis, heat to 80-90°C and maintain for 10-20 minutes to inactivate the enzyme, then transfer the reaction solution to a high-speed centrifuge, centrifuge at 5000-7000 rpm for 15-25 minutes, and collect the supernatant; S3 Aqueous Two-Phase Extraction: Add an aqueous two-phase extractant to the supernatant, stir evenly with a stirring paddle in a plastic extraction tank, let stand to separate the layers, and collect the upper phase rich in active ingredients; Then add an equal volume of deionized water to the upper phase, repeat the extraction 1-2 times, and combine all the upper phases; S4 ultrafiltration and reverse osmosis concentration: The combined upper phase is first ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 8-12kDa to remove small molecular impurities and unreacted enzymes, and then the ultrafiltered liquid is transferred to a reverse osmosis concentration device and concentrated to 1 / 5-1 / 3 of the original volume at a pressure of 2-4MPa; S5 Freeze drying and pulverization: The above concentrate is transferred to a freeze dryer, pre-frozen at -45 to -35°C for 3-5 hours, and then freeze-dried at a vacuum degree of 0.08-0.12Pa for 24-36 hours to obtain a dry extract, which is then pulverized using a jet mill to a particle size of 10-30 μm to obtain a highly active Chondrus crispus extract.

2. The method for preparing the highly active Chondrus crispus extract according to claim 1, characterized in that: In the S1 raw material pretreatment, the antioxidant protection solution contains 0.2%-0.4% sodium sulfite and 0.1%-0.3% cysteine ​​in a mass volume ratio. The synergistic effect of sodium sulfite and cysteine ​​can effectively prevent the oxidation and browning of the raw materials in the initial stage of treatment, and maintain the color of the raw materials and the integrity of the active ingredients.

3. The method for preparing the highly active Chondrus crispus extract according to claim 1, characterized in that: In the S2 enzyme-assisted extraction, the composite enzyme preparation is composed of mannanase, xylanase and alginate lyase in a ratio of 3:2:1, which is used for the corresponding polysaccharide components in the cell wall and intracellular of Chondrus crispus to synergistically destroy the cell structure and promote the release of active ingredients; the buffer solution is a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer with a pH of 5.5-6.

5.

4. The method for preparing the highly active Chondrus crispus extract according to claim 1, characterized in that: In the S3 aqueous two-phase extraction, the aqueous two-phase extractant contains 15%-25% by mass of polyethylene glycol and 10%-20% by mass of ammonium sulfate.

5. A functional additive, characterized in that: The invention comprises the highly active Chondrus crispus extract prepared according to claim 1, and the following auxiliary ingredients: 5%-10% by mass of xylitol, 3%-6% by mass of hydroxyapatite, 2%-5% by mass of lactobacillus fermentation product, 0.5%-1.5% by mass of bromelain, and 20%-50% by mass of the highly active Chondrus crispus extract.

6. A toothpaste comprising a toothpaste base component, which comprises, by weight, 40-50 parts of water, 12-18 parts of sorbitol, 15-20 parts of hydrated silica, 4-6 parts of polyethylene glycol-400, 3-5 parts of sodium lauryl sulfate, 1-2 parts of edible flavor, 0.8-1.2 parts of cellulose gum, 0.2-0.5 parts of titanium dioxide, and 3-5 parts of glucose, characterized in that: It also contains the functional additive according to claim 5, whose mass percentage in the toothpaste is 8%-15%.

7. The toothpaste according to claim 6, characterized in that The particle size of the hydrated silica is 8-15 μm, and its spherical structure has a low friction coefficient with the tooth surface during brushing, which can effectively remove dental plaque and stains and reduce the wear on tooth enamel, and synergize with functional additives to enhance the cleaning and protection effects of teeth.

8. The toothpaste according to claim 6, characterized in that The purity of the sodium lauryl sulfate is not less than 90%. As a surfactant, it can produce rich and dense foam, effectively remove oral foreign matter, and has little irritation to the gums and oral mucosa, and maintains oral hygiene together with other ingredients.

9. The toothpaste according to claim 6, characterized in that The viscosity of the cellulose gum is 800-1200 mPa·s, which can make the toothpaste form a uniform and stable paste, which is well dispersed and adhered in the oral cavity, ensuring that the functional additives and other ingredients are evenly distributed in the oral cavity and fully exert their effects.

10. The toothpaste according to claim 6, characterized in that The toothpaste also contains 0.5%-1% by mass of butylene glycol and 0.3%-0.6% by mass of dipotassium glycyrrhizinate as auxiliary additives. Butylene glycol is used for moisturizing, and dipotassium glycyrrhizinate has anti-inflammatory and soothing effects, which further enhances the toothpaste's ability to protect the oral mucosa and relieve inflammation.

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