A kind of oligomeric agarose sol and its preparation method and application

Abstract

The present invention belongs to the field of human body care technology, and discloses an oligomeric agarose sol, a preparation method, and an application thereof. The oligomeric agarose sol provided by the present invention is in the form of an opaque semi-fluid colloid or a solidified colloid at room temperature, and the degree of polymerization n is 2 to 10,000. The oligomeric agarose sol with such a special form and a specific degree of polymerization enables the polymer to have excellent moisturizing ability and the ability to promote the healing of micro-wounds. It can be used as a moisturizing ingredient in cosmetics, and has an excellent moisturizing effect regardless of whether it is in a high humidity environment or a dry environment, and has a significant effect on improving the expression of filaggrin in human keratinocytes; in the field of medical dressings, it can be used as a skin dressing to promote the healing of micro-wounds. Therefore, the oligomeric agarose sol provided by the present invention has a high application value as a bioactive substance.

Description

Technical Field

[0001] The invention belongs to the technical field of human body care, and particularly relates to an oligomeric agarose sol, a preparation method and an application thereof. Background Art

[0002] Agar, also known as agar gel, agar-agar, agar-agar, and jelly powder, is primarily found in the cell walls of red algae such as Agar-agar and Gelidium agar. It is a large hydrophilic polysaccharide. Its structure is composed of linear chain molecules consisting of alternating (1,3)-O-β-D-galactose and (1,4)-O-3,6-ether-α-L-galactose.

[0003] In recent years, agar has been primarily used in the food industry and as a microbial culture medium. However, due to the high molecular weight of agar polysaccharides, its application is relatively limited. To expand the high-value utilization of marine red algae polysaccharides, methods are often used to modify these polysaccharides. Researchers have employed various methods to hydrolyze agar to obtain relatively low-molecular-weight ultra-oligosaccharides or agar-oligosaccharides. Acid hydrolysis and enzymatic hydrolysis are the most commonly used methods.

[0004] Acid hydrolysis is a traditional method for degrading agar. Acids act on the α-(1,3) glycosidic bonds of agar, producing ultra-oligosaccharides or agar oligosaccharides with β-D-galactose as the non-reducing end and 3,6-endo-α-L-galactose as the reducing end. Acid hydrolysis is a harsh reaction method that places high demands on production equipment. The resulting product has poor uniformity and presents challenges in product analysis and recovery. Furthermore, the acids used in the production process can pollute the environment.

[0005] Enzymatic hydrolysis involves the degradation of agar using agarase. Agarase is an important class of algal polysaccharide-degrading enzymes that catalyzes the cleavage of glycosidic bonds in agar. Agarases can be divided into α-agarases and β-agarases based on how they cleave glycosidic bonds. β-agarases are the most commonly studied agarases. β-Agarase hydrolyzes the β-(1,4) glycosidic bonds of agarose, producing ultra-oligosaccharides or agar-oligosaccharides with β-D-galactose as the reducing end and 3,6-endo-α-L-galactose as the non-reducing end.

[0006] Agar polysaccharide is a neutral polysaccharide with a large number of hydroxyl groups within its structural units, which can bind to water molecules and exhibit a certain degree of hygroscopicity. In the existing technology, most agar polysaccharides are degraded into ultra-oligosaccharides (generally with a degree of polymerization of 10 to 30) or agar oligosaccharides (generally with a degree of polymerization of 4 to 10) with relatively low molecular weight by enzymatic or acidic hydrolysis. As the glycosidic bonds break, a large number of free hydroxyl groups are further released, which are conducive to binding with water molecules and enhancing its water absorption performance. However, at the same time, the network structure of the polysaccharide molecules is destroyed, forming a loose short-chain structure, which weakens its water retention. Summary of the Invention

[0007] The present invention aims to overcome the technical problem of weak water retention in ultra-oligosaccharides or agar oligosaccharides obtained by degrading agar using existing acid hydrolysis and enzymatic hydrolysis methods. The present invention provides a special oligomeric agarose sol in an opaque semi-fluid colloid or solidified colloid at room temperature, as well as a preparation method and application thereof. The oligomeric agarose sol has a network structure similar to that of agar polysaccharide, easily forming a water-retaining film on the skin surface. Furthermore, the oligomeric agarose sol contains small-molecule agar oligosaccharides, exhibiting good hygroscopicity and being absorbable by organisms. The oligomeric agarose sol not only has excellent water retention and moisture absorption capacity but also promotes the healing of minor wounds, thus playing an important role in the fields of cosmetics and medical dressings.

[0008] Specifically, the first aspect of the present invention provides an oligomeric agarose sol, wherein the oligomeric agarose sol has a structure as shown in formula (I) and is in an opaque semi-fluid colloid form or a solidified colloid form at room temperature.

[0009]

[0010] Among them, n is 2 to 10000, n is 2 to 1000 accounts for 1 to 5%, n is 1001 to 4000 accounts for 20 to 50%, n is 4001 to 7000 accounts for 20 to 50%, n is 7001 to 10000 accounts for 4 to 5%, and the weight average molecular weight Mw of the oligomeric agarose sol is 5×10 5 ~6×10 5 g / mol.

[0011] A second aspect of the present invention provides a method for preparing an oligomeric agarose sol, wherein the method comprises the following steps:

[0012] (1) Decolorization: Use bleaching agent to decolorize agar powder;

[0013] (2) Rinse: After decolorization, filter to remove the bleach and rinse the agar powder with pure water;

[0014] (3) Dissolving: Add pure water to the rinsed agar powder and heat to completely dissolve it to obtain an agar solution;

[0015] (4) Enzymolysis: After the agar solution in step (3) is cooled, agarase is added thereto and an enzymolysis reaction is carried out to obtain an enzymolysis reaction liquid; the agarase is β-agarase, and its amino acid sequence and nucleotide sequence are SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the enzymolysis conditions include a concentration of 20 U / L to 200 U / L agarase, an enzymolysis temperature of 40 to 55° C., and an enzymolysis time of 10 min to 120 min;

[0016] (5) Inactivating the enzyme: heating the enzymatic hydrolysis reaction solution in step (4) until the agarase is inactivated to terminate the reaction and cooling to room temperature to obtain an enzyme-inactivated enzymatic hydrolysis reaction solution;

[0017] (6) Dispersion: The enzyme-inactivated enzymatic hydrolysis reaction solution in step (5) is dispersed to form a uniform opaque semi-fluid colloid or solidified colloid.

[0018] In a preferred embodiment, in step (1), the decolorization conditions include a bleaching agent of 0.05% to 0.2% sodium hypochlorite solution, and a decolorization time of 10 to 20 minutes.

[0019] In a preferred embodiment, in step (2), the rinsing conditions include filtering with a nylon mesh, rinsing for 3 to 5 times, and each rinsing time for 5 to 10 minutes.

[0020] In a preferred embodiment, in step (3), the concentration of the agar solution is 0.5% to 5%, the dissolution temperature is 110° C. to 120° C., and the dissolution time is 10 min to 20 min.

[0021] In a preferred embodiment, in step (5), the enzyme inactivation conditions include a heating temperature of 80 to 100° C. and a heating time of 5 to 10 minutes.

[0022] In a preferred embodiment, in step (6), the dispersion method is a physical dispersion method.

[0023] In a preferred embodiment, the dispersion method is a homogenization method and / or an ultrasonic dispersion method.

[0024] The third aspect of the present invention provides an oligomeric agarose sol prepared by the above-mentioned preparation method.

[0025] A fourth aspect of the present invention provides a use of the above-mentioned oligomeric agarose sol or the oligomeric agarose sol prepared by the above-mentioned preparation method in the field of human body care.

[0026] In a preferred embodiment, the application scenarios include applications in the fields of cosmetics and medical dressings.

[0027] After in-depth research, the inventors of the present invention found that the amino acid sequence and nucleotide sequence are SEQ ID NO: 1 and SEQ ID NO:2 β-agarase is used as a catalyst, and the enzymolysis temperature is controlled at 40-55°C, and the enzymolysis time is controlled at 10-120 minutes. The enzymolysis product obtained is an oligomeric agarose sol, which is different from ultra-oligosaccharides or agar oligosaccharides, and has an opaque semi-fluid colloid form or a solidified colloid form. Its degree of polymerization n is 2-10000, of which n is 2-1000 accounts for 1-5%, n is 1001-4000 accounts for 20-50%, n is 4001-7000 accounts for 20-50%, and n is 7001-10000 accounts for 4-5%. The oligomeric agarose sol has a network structure similar to that of agar polysaccharide and is very easy to form a water-retaining film on the skin surface. At the same time, it contains small molecule agar oligosaccharides, has good hygroscopicity, and can be absorbed and utilized by organisms. The oligomeric agarose sol, with its unique morphology and specific degree of polymerization, possesses excellent water-retention and moisture-absorbing properties, and promotes the healing of microwounds. It can be used as a moisturizing ingredient in cosmetics, exhibiting excellent moisturizing effects in both high-humidity and dry environments. It also significantly enhances the expression of filaggrin in human keratinocytes. In the field of medical dressings, it can be used as a skin dressing to promote the healing of microwounds. Therefore, the oligomeric agarose sol provided by the present invention has high application value as a bioactive substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The moisturizing effects of the 0.1% oligo-agarose sol of Preparation Example 1 and the 0.1% agar oligosaccharide, 10% glycerol and 0.1% sodium hyaluronate of Comparative Preparation Example 1 after 12 hours of experiment at humidity of 40% and 80% respectively;

[0029] Figure 2 (A) is the results of filaggrin staining in human keratinocytes under a fluorescence microscope for (B) 0.02% sodium hyaluronate, (C) 0.02% agar oligosaccharide of Comparative Example 1, (D) 0.1% agar oligosaccharide of Comparative Example 1, (E) 0.02% oligo agarose sol of Preparation Example 1, and (F) 0.1% oligo agarose sol of Preparation Example 1;

[0030] Figure 3This is a graphic illustration of the expression levels of filaggrin in the blank control group, 0.02% sodium hyaluronate, 0.02% agar oligosaccharide of Comparative Preparation Example 1, 0.1% agar oligosaccharide of Comparative Preparation Example 1, 0.02% oligomeric agarose sol of Preparation Example 1, and 0.1% oligomeric agarose sol of Preparation Example 1. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below through examples.

[0032] The first aspect of the present invention provides an oligomeric agarose sol, wherein the oligomeric agarose sol has a structure as shown in formula (I) and is in an opaque semi-fluid colloid form or a solidified colloid form at room temperature.

[0033]

[0034] Wherein, n is preferably 2 to 10,000, specifically, n can be 2, 10, 100, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000. Wherein, n is 2 to 1000 and the proportion can be 1 to 5%, for example, 1%, 2%, 3%, 4%, 5%. Wherein, n is 1001 to 4000 and the proportion can be 20 to 50%, for example, 20%, 30%, 40%, 50%. Wherein, n is 4001 to 7000 and the proportion can be 20 to 50%, for example, 20%, 30%, 40%, 50%. Wherein, n is 7001 to 10,000 and the proportion can be 4 to 5%, for example, 4%, 4.2%, 4.5%, 4.8%, 5%. The weight average molecular weight Mw of the oligomeric agarose sol is preferably 5×10 5 ~6×10 5 g / mol, specifically, Mw can be 5×10 5 g / mol, 5.5×10 5 g / mol, 6×10 5 Specifically, in the present invention, the weight average molecular weight of the oligo-agarose sol is determined by gel permeation chromatography, and the test conditions are as follows.

[0035] The test instrument is a gel chromatography instrument (model ELEOS System, manufacturer Wyatt); the basic configuration is a Waters 515 pump, a laser detector (LS), and a differential refractive index detector (DRI); the chromatographic column is a Shodex Ohpak series SB-806 series 803; the mobile phase is an aqueous solution containing 0.02% sodium azide; the flow rate is 1 mL / min; the column temperature is 40°C; and the injection volume is 500 μL.

[0036] A second aspect of the present invention provides a method for preparing an oligomeric agarose sol, wherein the method comprises the following steps:

[0037] (1) Decolorization: Use bleaching agent to decolorize agar powder;

[0038] (2) Rinse: After decolorization, filter to remove the bleach and rinse the agar powder with pure water;

[0039] (3) Dissolving: Add pure water to the rinsed agar powder and heat to completely dissolve it to obtain an agar solution;

[0040] (4) Enzymolysis: After the agar solution in step (3) is cooled, agarase is added thereto and an enzymolysis reaction is carried out to obtain an enzymolysis reaction liquid; the agarase is β-agarase, and its amino acid sequence and nucleotide sequence are SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the enzymolysis conditions include a concentration of 20 U / L to 200 U / L agarase, an enzymolysis temperature of 40 to 55° C., and an enzymolysis time of 10 min to 120 min;

[0041] (5) Inactivating the enzyme: heating the enzymatic hydrolysis reaction solution in step (4) until the agarase is inactivated to terminate the reaction and cooling to room temperature to obtain an enzyme-inactivated enzymatic hydrolysis reaction solution;

[0042] (6) Dispersion: The enzyme-inactivated enzymatic hydrolysis reaction solution in step (5) is dispersed to form a uniform opaque semi-fluid colloid or solidified colloid.

[0043] In the present invention, the amino acid sequence of the β-agarase is SEQ ID NO: 1.

[0044] SEQ ID NO: 1:

[0045] MRRKFITIHATSDDDGQTKELILRDDLIRKADVYVGREFFGMIKTVYNVSKQAAWRGVRGAWTSLQDVKRYIATEHLHPFWPIVFPGGTATGEYMANFLAKFFEIPAFVEVMNEPLYDLVDFARTSQTKLFEFHSTIAAQVDYKTWKMKVIGYCTSFPNHEAQPFERWEERDKLFIDLAGEGTDFFTIHLYDFPCADKQMYRWQSNSEAVFDIMEQYSYPVRGEVKPFMISEYGAPDHALGHYKLPERDWLFLIAFNGLLMSFLERTDNICYAMPFAMLKSEWGYQSWATWWGYKTTWRLTEFSKTYQLWSDLVVNAYAEGKKAYVILNNLEEETVDVKLNSSVSGLQSVRTKSLKTDGGFAVLDEQTSDKLPESVTLGKEATLVLQLNYTQDVQISNAIRTQSYYANKYLQNFPINGIKTGKGSAILRLSIGRPHHTVAFPKVIINGTEVETPFQISGDDQKRDGFFGSLHIPVNNSQLRSSNTVTLMYPDGGHVSSVVLSVN

[0046] In the present invention, the nucleotide sequence of the β-agarase is SEQ ID NO:2.

[0047] SEQ ID NO:2:

[0048]

[0049] The nucleotide sequence (SEQ ID NO: 2) provided by the present invention can generally be obtained by polymerase chain reaction (PCR) amplification, recombination, or synthetic methods. For example, those skilled in the art can easily obtain a template and primers based on the nucleotide sequence and amplify the relevant sequence using PCR.

[0050] Acid hydrolysis is a traditional method for degrading agar. Acids act on the α-(1,3) glycosidic bonds of agar, producing ultra-oligosaccharides or agar oligosaccharides with β-D-galactose as the non-reducing end and 3,6-endo-α-L-galactose as the reducing end. Acid hydrolysis is a harsh reaction method that places high demands on production equipment. The resulting product has poor uniformity and presents challenges in product analysis and recovery. Furthermore, the acids used in the production process can pollute the environment.

[0051] Enzymatic hydrolysis involves the degradation of agar using agarase. Agarase is an important class of seaweed polysaccharide-degrading enzymes that catalyzes the cleavage of glycosidic bonds in agar. Agarases can be divided into α-agarases and β-agarases based on how they cleave glycosidic bonds. β-agarases are the most commonly studied agarases. β-Agarase hydrolyzes the β-(1,4) glycosidic bonds of agarose, producing ultraoligosaccharides or agar-oligosaccharides with β-D-galactose as the reducing end and 3,6-endo-α-L-galactose as the non-reducing end. Compared to traditional acid hydrolysis, enzymatic hydrolysis offers advantages such as mild degradation conditions, high specificity, and environmental friendliness. The products of enzymatic hydrolysis of agar using existing agarases are generally ultraoligosaccharides or agar-oligosaccharides, which have poor moisture retention. The present invention uses the above-mentioned β-agarase to perform random cutting inside the polysaccharide molecules. By strictly controlling the enzymatic reaction conditions, a special oligomeric agarose sol in the form of an opaque semi-fluid colloid or a solidified colloid at room temperature is produced. The oligomeric agarose sol has a network structure similar to that of agar polysaccharide and easily forms a water-retaining film on the skin surface. At the same time, it contains small-molecule agar oligosaccharides, has good hygroscopicity, and can be absorbed and utilized by organisms, so that it plays an important application value in the fields of cosmetics and medical dressings.

[0052] The agar powder used in the present invention is a commercially available product purchased from Fujian Huanhai Biotechnology Co., Ltd. In the present invention, preferably, in step (1), the decolorization conditions include a bleaching agent that can be sodium hypochlorite with a concentration of 0.05% to 0.2%, such as 0.05%, 0.1%, 0.15%, and 0.2%. The decolorization time can be 10 to 20 minutes, such as 10 minutes, 15 minutes, and 20 minutes. Preferably, in step (2), the rinsing conditions include filtering with a nylon mesh, and the number of rinsing times can be 3 to 5 times, such as 3 times, 4 times, and 5 times. The rinsing time for each time can be 5 to 10 minutes, such as 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, and 10 minutes. Preferably, in step (3), the concentration of the agar solution can be 0.5% to 5%, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, and 5%. The dissolution temperature can be 110°C to 120°C, for example, 110°C, 115°C, and 120°C. The dissolution time can be 10min to 20min, for example, 10min, 15min, and 20min. Preferably, in step (4), the enzymatic hydrolysis conditions include agarase at a concentration of 20U / L to 200U / L, for example, 20U / L, 50U / L, 100U / L, 150U / L, and 200U / L. The enzymatic hydrolysis temperature can be 40 to 55°C, for example, 40°C, 45°C, 50°C, and 55°C. The enzymatic hydrolysis time can be 10min to 120min, for example, 10min, 30min, 50min, 80min, 100min, and 120min. Preferably, in step (5), there is no particular limitation on the enzyme inactivation conditions, as long as they can inactivate the enzyme. Preferably, a heating method is selected, and the heating temperature can be 80-100°C, for example, 80°C, 90°C, 100°C. The heating time can be 5-10min, for example, 5min, 6min, 7min, 8min, 9min, 10min. Preferably, in step (6), there is no particular limitation on the dispersion of the obtained enzyme-inactivated enzymatic hydrolysis reaction liquid, as long as it can form a uniform opaque semi-fluid colloid or solidified colloid. Preferably, a physical dispersion method is selected to disperse it, and specific examples of the physical dispersion method include but are not limited to: homogenization and / or ultrasonic dispersion method. The homogenization method is to disperse it by homogenizing it for 10min at room temperature using a homogenizer. The ultrasonic dispersion method is to disperse it by ultrasonicating it for 30min at room temperature using an ultrasonic machine.

[0053] The third aspect of the present invention provides an oligomeric agarose sol prepared by the above-mentioned preparation method. The oligomeric agarose sol prepared by the above-mentioned method has a structure as shown in formula (II) and is in an opaque semi-fluid colloid form or a solidified colloid form at room temperature.

[0054]

[0055] Wherein, n is preferably 2 to 10,000, specifically, n can be 2, 10, 100, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000. Wherein, n is 2 to 1000 and the proportion can be 1 to 5%, for example, 1%, 2%, 3%, 4%, 5%. Wherein, n is 1001 to 4000 and the proportion can be 20 to 50%, for example, 20%, 30%, 40%, 50%. Wherein, n is 4001 to 7000 and the proportion can be 20 to 50%, for example, 20%, 30%, 40%, 50%. Wherein, n is 7001 to 10,000 and the proportion can be 4 to 5%, for example, 4%, 4.2%, 4.5%, 4.8%, 5%. The weight average molecular weight Mw of the oligomeric agarose sol is preferably 5×10 5 ~6×10 5 g / mol, specifically, Mw can be 5×10 5 g / mol, 5.5×10 5 g / mol, 6×10 5 Specifically, in the present invention, the weight average molecular weight of the oligo-agarose sol is determined by gel permeation chromatography, and the test conditions are as follows.

[0056] The test instrument is a gel chromatography instrument (model ELEOS System, manufacturer Wyatt); the basic configuration is a Waters 515 pump, a laser detector (LS), and a differential refractive index detector (DRI); the chromatographic column is a Shodex Ohpak series SB-806 series 803; the mobile phase is an aqueous solution containing 0.02% sodium azide; the flow rate is 1 mL / min; the column temperature is 40°C; and the injection volume is 500 μL.

[0057] A fourth aspect of the present invention provides an application of the oligomeric agarose sol or the oligomeric agarose sol prepared by the above-mentioned preparation method in the field of human body care. Preferably, the application scenarios include applications in the fields of cosmetics and medical dressings.

[0058] Preparation Example 1: Preparation of semi-fluid oligomeric agarose sol by enzymatic hydrolysis

[0059] Weigh 10g of agar powder, add a sodium hypochlorite solution with a concentration of 0.1% thereto, and stir to decolorize for 15 minutes. Then filter the decolorized agar powder with a 250-mesh nylon mesh and rinse it repeatedly with pure water 5 times to remove residual sodium hypochlorite. Add 1L of pure water to the rinsed agar powder, place it in a high-temperature wet heat sterilizer, and keep it warm at 110°C for 10 minutes to completely dissolve the agar powder to obtain an agar solution. Place the agar solution in a 45°C shaking table for shaking and cooling. When the temperature of the agar solution drops to 45°C, add 20U of agarase (final concentration in the system is 20U / L) thereto, and enzymolyze for 20 minutes to obtain an enzymolysis reaction solution. Immediately boil at 100°C to terminate the reaction and cool to room temperature to obtain an enzyme-inactivated enzymolysis reaction solution. The obtained enzyme-inactivated enzymatic hydrolysis reaction solution was homogenized and dispersed using a homogenizer to form a uniform semi-fluid oligomeric agarose sol, which was recorded as P-1. The weight average molecular weight Mw was measured to be 5.017×10 5 g / mol.

[0060] Preparation Example 2: Preparation of solidified oligomeric agarose sol by enzymatic hydrolysis

[0061] Weigh 50g of agar powder, add a sodium hypochlorite solution with a concentration of 0.1% thereto, and stir to decolorize for 20min. Then filter with a 250-mesh nylon mesh, filter the decolorized agar powder, and rinse repeatedly with pure water 5 times to remove residual sodium hypochlorite. Add 1L of pure water to the rinsed agar powder, place it in a high-temperature wet heat sterilizer, and keep it warm at 110°C for 20min to completely dissolve the agar powder to obtain an agar solution. Place the agar solution in a 45°C shaking table and shake to cool it down. When the temperature of the agar solution drops to 45°C, add 100U of agarase (final concentration in the system is 100U / L) thereto, and enzymolysis for 60min to obtain an enzymolysis reaction solution. Immediately thereafter, boil at 100°C to terminate the reaction and cool to room temperature to obtain an enzyme-inactivated enzymolysis reaction solution. The obtained enzyme-inactivated enzymatic hydrolysis reaction solution was homogenized and dispersed using a homogenizer to form a uniform solidified oligomeric agarose sol, which was recorded as P-2. The weight average molecular weight Mw was measured to be 5.982×10 5 It should be noted that in actual production applications, the solidified oligo-agarose sol is convenient for storage and transportation. When finally used, the solidified sol needs to be diluted into a semi-fluid sol.

[0062] Comparative Preparation Example 1 Preparation of Agar Oligosaccharide by Enzymatic Hydrolysis

[0063] Weigh 10g of agar powder, add 1L of pure water thereto, place in a high-temperature moist heat sterilizer, keep warm at 110°C for 10min to completely dissolve the agar powder to obtain an agar solution. The agar solution is placed in a 50°C shaker for shaking and cooling. When the temperature of the agar solution drops to 50°C, 200U of agarase (the final concentration in the system is 200U / L, its amino acid sequence and nucleotide sequence are shown in CN114214302A) is added thereto, and enzymolysis is carried out for 4h to obtain an enzymatic hydrolysis reaction solution. Immediately thereafter, boil at 100°C to terminate the reaction and cool to room temperature. The reaction solution is centrifuged at low temperature and high speed, and the supernatant is taken to obtain an enzymatic hydrolysis agar oligosaccharide solution. TLC analysis test shows that the obtained product is a mixture of neoagarotetrose and neoagarotehexose.

[0064] Test Example 1: In vitro moisturizing performance test of oligo-agarose sol under different humidity environments

[0065] In the skin care cosmetics on the market, the commonly used moisturizing functional raw materials are glycerin and sodium hyaluronate. The amount of glycerin added in skin care products is generally 5-10%, and the amount of sodium hyaluronate added is generally 0.02-0.1%. 1g of 0.1% oligomeric agarose sol of Preparation Example 1, 1g of 0.1% agar oligosaccharide after enzymatic hydrolysis in Comparative Preparation Example 1, 1g of 10% glycerin (purchased from Sinopharm Chemical Reagent Co., Ltd.) and 1g of 0.1% sodium hyaluronate (purchased from Huaxi Freda Biotechnology Co., Ltd.) were used as raw materials (all of the above raw materials are aqueous solutions). All of the above raw materials were applied on a glass plate with medical breathable tape. The moisturizing properties of each raw material were tested under relative humidity of 40% and 80%. The experimental time was 12h. Accurate weighing was performed and the moisturizing rate was calculated. The experimental results are as follows. Figure 1 shown.

[0066] Moisture retention rate (%) = W1 / W0×100%

[0067] Where W0 is the water content weight measured before placement, in g; W1 is the water content weight measured after placement for 12 hours, in g.

[0068] from Figure 1 It can be seen that in an environment with relatively high relative humidity (80% RH), the moisturizing effect of the oligomeric agarose sol is comparable to that of agar oligosaccharide, glycerol, and sodium hyaluronate; in a dry environment (40% RH), the moisturizing ability of the oligomeric agarose sol is significantly better than that of agar oligosaccharide, glycerol, and sodium hyaluronate. Thus, the oligomeric agarose sol provided by the present invention has an excellent moisturizing effect, good moisturizing properties, and can be used as a moisturizing ingredient in cosmetics, whether in a high humidity environment or a dry environment.

[0069] Test Example 2: Moisturizing performance test of oligomeric agarose sol at the cellular level

[0070] Using human keratinocytes as a model, and filaggrin (FLG) expression as a detection indicator, the moisturizing ability of oligo-agarose sol was evaluated. FLG is primarily present in the stratum corneum of the skin. Protease hydrolysis of FLG produces free amino acids and derivatives, which, as important components of the natural moisturizing factor (NMF), play a crucial role in maintaining stratum corneum moisture. A blank control group and experimental groups were set up according to Table 1. The specific experimental steps for this test example are as follows:

[0071] (1) Cell seeding: 2×10 4 Human keratinocytes were seeded into a 24-well plate containing a coverslip at a cell seeding density of 100 cells / well and incubated overnight at 37°C in a 5% CO2 incubator.

[0072] (2) Liquid preparation: Set the test substances and experimental groups according to Table 1 and prepare the test samples. A is a pure water blank control group; B is 0.02% sodium hyaluronate; C is 0.02% agar oligosaccharide of Comparative Preparation Example 1; D is 0.1% agar oligosaccharide of Comparative Preparation Example 1; E is 0.02% oligomeric agarose sol of Preparation Example 1; and F is 0.1% oligomeric agarose sol of Preparation Example 1.

[0073] Table 1 Immunofluorescence detection group design

[0074]

[0075] (3) Drug administration: According to the experimental design in Table 1, when the cell plating rate in the 24-well plate reaches 30% to 40%, the cells are divided into groups for drug administration, with three replicate wells per group. The cells are cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0076] (4) Sample collection: After the incubation, discard the culture medium in the 24-well plate, wash the slides three times with PBS buffer solution, fix the cells with 4% paraformaldehyde at room temperature for 30 minutes, and store in a refrigerator at 4°C.

[0077] (5) Cell immunofluorescence detection: The cells were permeabilized and blocked with goat serum blocking solution at 37°C for 30 min; the cells were incubated with primary antibody working solution at 4°C overnight; and the cells were incubated with secondary antibody working solution (Goat Anti-Rabbit IgG antibody at a dilution of 1:200, Goat Anti-Mouse IgG FITC antibody at a dilution of 1:200) at 37°C for 2 h.

[0078] (6) Contrast staining: Hochest staining solution is used to stain the cell nuclei (the stained nuclear area can be excited by ultraviolet light to emit blue fluorescence).

[0079] (7) Photographing: Take photos under an upright fluorescence microscope. In the same field of view, use the blue light channel to excite the green fluorescence of the target protein and the ultraviolet light channel to excite the blue fluorescence of the cell nucleus. Take photos separately. Combine the two images to form a merge image.

[0080] (8) Data analysis: Image-pro Plus (IPP) software was used to analyze the green fluorescence intensity in the photos of cells emitting green fluorescence under blue light, and the fluorescence intensity per unit area was calculated. The experimental results were statistically analyzed using the T-test method.

[0081] (9) Experimental results: The expression results of filaggrin in human keratinocytes are as follows: Figure 2 The mean fluorescence intensity results are shown in Figure 3 shown.

[0082] from Figure 2 and Figure 3 It can be seen that compared with the blank control group, sodium hyaluronate, agar oligosaccharide of comparative preparation example 1 and oligomeric agarose sol of preparation example 1 all have a certain promoting effect on the expression of filaggrin in human keratinocytes at a concentration of 0.02% (P*<0.05). When the concentration of the oligomeric agarose sol of preparation example 1 is 0.1%, it has a very significant promoting effect on the expression of filaggrin in human keratinocytes (P**<0.01). The promoting effect of sodium hyaluronate at the same concentration on the expression of filaggrin in human keratinocytes is significantly lower than that of the oligomeric agarose sol at the same concentration, and the promoting effect of the agar oligosaccharide of comparative preparation example 1 at the same concentration on the expression of filaggrin in human keratinocytes is lower than that of the oligomeric agarose sol at the same concentration. Therefore, the oligomeric agarose sol provided by the present invention has a significant promoting effect on the expression of filaggrin in human keratinocytes and can be used as a moisturizing ingredient in the field of cosmetics.

[0083] Test Example 3: Oligomeric agarose sol promotes microwound healing

[0084] (1) Test samples: Rats were selected as experimental animals. The hair on the test area on the back of the rats was removed, and a wound about 10 mm long and 1.5 mm deep was made on each rat as the test sample.

[0085] (2) Experimental process: The experiment was divided into two groups, a control group and a treatment group, with 10 test samples in each group. The dressings of the control group and the treatment group were respectively applied to the wound surface of the test samples, and the wound healing of the test samples was observed during the 10-day observation period. Control group: a single-layer gauze cloth pre-soaked in pure water was used as a dressing; Treatment group 1: a single-layer gauze cloth pre-soaked in 0.1% sodium hyaluronate was used as a dressing; Treatment group 2: a single-layer gauze cloth pre-soaked in 0.1% agar oligosaccharide prepared in Comparative Preparation Example 1 was used as a dressing; Treatment group 3: a single-layer gauze cloth pre-soaked in 0.1% oligomeric agarose sol prepared in Preparation Example 1 was used as a dressing. It should be noted that the soaking time and soaking method of the above experiments were the same.

[0086] (3) Test results: The test results are listed in Table 2. As can be seen from the test results in Table 2, the wound healing of treatment group 3 was 2 to 4 days earlier than that of the control group and treatment groups 1 to 2. This shows that the oligomeric agarose sol provided by the present invention has the ability to promote the healing of micro-wounds and can be used as a skin dressing in the field of medical dressings.

[0087] Table 2 Wound healing of test samples

[0088]

[0089] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A method for preparing oligomeric agarose sol, characterized in that: The method comprises the following steps: (1) Decolorization: Use bleaching agent to decolorize agar powder; (2) Rinse: After decolorization, filter to remove the bleach and rinse the agar powder with pure water; (3) Dissolving: Add pure water to the rinsed agar powder and heat to completely dissolve it to obtain an agar solution; (4) Enzymolysis: After the agar solution in step (3) is cooled, agarase is added thereto and an enzymolysis reaction is carried out to obtain an enzymolysis reaction liquid; the agarase is β-agarase, and its amino acid sequence and nucleotide sequence are SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the enzymolysis conditions include a concentration of 20 U / L to 200 U / L agarase, an enzymolysis temperature of 40 to 55° C., and an enzymolysis time of 10 to 120 min; (5) Inactivating the enzyme: heating the enzymatic hydrolysis reaction solution in step (4) until the agarase is inactivated to terminate the reaction and cooling to room temperature to obtain an enzyme-inactivated enzymatic hydrolysis reaction solution; (6) Dispersion: The enzyme-inactivated enzymatic hydrolysis reaction solution in step (5) is dispersed to form a uniform opaque semi-fluid colloid or solidified colloid.

2. The method for preparing oligomeric agarose sol according to claim 1, wherein In step (1), the decolorization conditions include a bleaching agent of 0.05% to 0.2% sodium hypochlorite solution and a decolorization time of 10 to 20 minutes.

3. The method for preparing oligomeric agarose sol according to claim 1, wherein In step (2), the rinsing conditions include filtering with a nylon mesh, rinsing 3 to 5 times, and each rinsing time is 5 to 10 minutes.

4. The method for preparing oligomeric agarose sol according to claim 1, wherein In step (3), the concentration of the agar solution is 0.5% to 5%, the dissolution temperature is 110° C. to 120° C., and the dissolution time is 10 min to 20 min.

5. The method for preparing oligomeric agarose sol according to claim 1, wherein In step (5), the enzyme inactivation conditions include a heating temperature of 80 to 100° C. and a heating time of 5 to 10 minutes.

6. The method for preparing oligomeric agarose sol according to claim 1, wherein In step (6), the dispersion method is a physical dispersion method.

7. The method for preparing oligomeric agarose sol according to claim 6, wherein: The dispersion method is a homogenization method and / or an ultrasonic dispersion method.

8. The oligomeric agarose sol prepared by the preparation method according to any one of claims 1 to 7.

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