Mannan with firming and wrinkle-removing effects, and preparation method and application thereof

Abstract

The present application relates to a kind of mannans with firming and wrinkle-removing efficacy and its preparation method and application, the preparation method includes the following steps: (1) konjac powder and water are mixed, first enzymolysis and second enzymolysis are carried out in turn, after enzyme hydrolysate is separated by solid-liquid, take supernatant, obtain crude extract of mannans;(2) after the crude extract of mannans is sterilized, bacteriostatic agent is added, and supernatant is taken after standing;(3) the supernatant is sequentially subjected to ultrafiltration, first adsorption of activated carbon, cation exchange resin purification and second adsorption of activated carbon, to obtain mannans.The preparation method of the mannans provided by the present application contains multiple enzymolysis and purification procedures, the product obtained has high extraction rate, low molecular weight and high purity, effectively improves the solubility and transdermal absorption effect of mannans, and also has moisturizing and firming and wrinkle-removing performance, and has good application prospect in the field of cosmetics.

Description

Technical Field

[0001] This invention relates to the field of cosmetic raw material technology, and in particular to a mannan with firming and wrinkle-reducing effects, its preparation method and application. Background Technology

[0002] Mannans are highly branched polymers of mannose, widely found in various life forms. Mannans possess the unique physical properties of being colorless, non-toxic, and odorless, giving them broad potential in scientific research and practical applications. The sources of mannans are abundant, and based on their origin, they can be categorized as follows: plant-derived konjac glucomannan, galactomannan, aloe-derived mannan; mannans and their sulfated oligosaccharide derivatives derived from brewer's yeast; α-mannopeptides derived from human alpha-hemolytic streptococci; and mannans derived from various marine bacteria and fungi.

[0003] The properties of mannan vary depending on its source. Konjac mannan, a soluble plant fiber with a molecular weight of approximately 1.1 million, possesses water-holding, thickening, rheological, and film-forming properties, and can be widely used in food, biomedicine, environmental protection, textile industry, and cosmetics.

[0004] CN106317239A discloses a method for preparing konjac mannan, the preparation process of which is as follows: (1) material preparation; (2) initial treatment; (3) enzymatic hydrolysis; (4) drying; (5) mixing; (6) impurity removal; (7) water extraction; (8) column chromatography; (9) primary alcohol precipitation; (10) secondary alcohol precipitation; (11) tertiary alcohol precipitation.

[0005] CN108342430A discloses a method for preparing konjac glucomannan oligosaccharide, comprising the following steps: (1) mixing ethanol solution with konjac powder thoroughly and stirring evenly to make the konjac powder fully swollen and form a suspension; (2) adding α-amylase to the suspension and performing ultrasonic treatment; (3) filtering with gauze and taking the sieve residue; (4) adding water to swell and obtaining konjac glucomannan extract; (5) adding cellulase to the konjac glucomannan extract and water bath to obtain an enzymatic hydrolysate; (6) adding ethanol solution to the enzymatic hydrolysate, centrifuging and filtering to obtain a precipitate; (7) drying, grinding and packaging the obtained precipitate.

[0006] Existing technologies for preparing konjac mannan involve the use of organic solvents and alcohol precipitation, resulting in lower purity and content. Therefore, this paper proposes a method for preparing mannan with firming and wrinkle-reducing effects, improving its purity and content while reducing its molecular weight, thereby enhancing transdermal absorption and solubility. This method offers promising application prospects for mannan with excellent moisturizing and firming effects. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a mannan with firming and wrinkle-reducing effects, its preparation method, and its applications. The mannan preparation method provided by this invention includes multiple enzymatic hydrolysis and purification steps, resulting in a product with high extraction rate, low molecular weight, and high purity. This effectively improves the solubility and transdermal absorption of mannan, while also possessing moisturizing, firming, and wrinkle-reducing properties, showing promising application prospects in the cosmetics field.

[0008] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing mannan, the method comprising the following steps: (1) Mix konjac powder and water, and perform first and second enzymatic hydrolysis in sequence. After solid-liquid separation, take the supernatant to obtain crude mannan extract. (2) After sterilizing the crude mannan extract, add an antibacterial agent, let it stand and take the supernatant; (3) The supernatant was subjected to ultrafiltration, first adsorption with activated carbon, purification with cation exchange resin and second adsorption with activated carbon in sequence to obtain mannan.

[0009] In step (1) of this invention, a two-step enzymatic hydrolysis process is adopted to gradually achieve deep and effective enzymatic hydrolysis of konjac flour. This not only improves the extraction efficiency of effective components, but also reduces the molecular weight of mannan, thereby improving the transdermal absorption effect and solubility of mannan.

[0010] In step (3) of this invention, a four-stage purification process is adopted, consisting of ultrafiltration, first adsorption with activated carbon, purification with cation exchange resin, and second adsorption with activated carbon. Ultrafiltration removes high molecular weight impurities, first adsorption with activated carbon removes low molecular weight impurities, cation exchange resin separates, purifies, and concentrates ions, and second adsorption with activated carbon removes floating flocculent matter. The multiple purification processes are progressive and interconnected, which together improve the purity and extraction rate of the product.

[0011] The preparation method provided by this invention does not introduce any organic solvents throughout the process, and the resulting product is safe and non-irritating, and can be directly applied to cosmetics. Furthermore, due to the product's low molecular weight and suitable viscosity, it also has excellent moisturizing and firming and wrinkle-reducing properties, showing promising application prospects.

[0012] Preferably, in step (1), the enzyme used in the first enzymatic hydrolysis includes any one or a combination of at least two of β-glucanase, cellulase or pectinase.

[0013] Preferably, the enzymes used in the first enzymatic hydrolysis include a combination of β-glucanase, cellulase and pectinase.

[0014] In this invention, β-glucanase can effectively decompose β-glucan in the cell wall of konjac and reduce the viscosity of the extract; cellulase hydrolyzes or degrades the components of the cell wall and impurities such as mucilage, destroying the plant cell wall and exposing and dissolving the effective components in the solvent, increasing the solubility of the effective components, thereby extracting the effective components from the cells; pectinase can accelerate decomposition, improve efficiency, accelerate juice filtration, and promote clarification.

[0015] This invention improves enzymatic hydrolysis efficiency by rationally combining enzymes, with β-glucanase, cellulase and pectinase working together synergistically.

[0016] Preferably, the mass ratio of β-glucanase, cellulase and pectinase is 1:(0.5-1.5):(0.5-1.5).

[0017] The values ​​in the above (0.5-1.5) can be, for example, 0.6, 0.8, 1, 1.2, 1.4, etc.

[0018] In this invention, when the amount of compound enzyme is within the above-mentioned ratio range, it has a better enzymatic hydrolysis effect.

[0019] Preferably, in step (1), the enzyme used in the second enzymatic hydrolysis includes lysozyme.

[0020] In this invention, lysozyme is used to perform deep enzymatic hydrolysis of the system, which has the effects of hydrolyzing pathogenic bacteria and reducing polysaccharide viscosity. The second enzymatic hydrolysis and the first enzymatic hydrolysis work together to improve the enzymatic hydrolysis efficiency and product quality.

[0021] Preferably, in step (1), the mass ratio of konjac powder to water is 1:(0.8-1.6), for example, it can be 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.4, 1:1.5, etc.

[0022] Preferably, in step (1), the amount of enzyme used in the first enzymatic hydrolysis is 1.5-2.5% of the substrate mass, for example, it can be 1.6%, 1.8%, 2%, 2.2%, 2.4%, etc.

[0023] In this invention, the substrate is a solid-liquid mixture of the system.

[0024] Preferably, in step (1), the temperature of the first enzymatic hydrolysis is 40-65℃ (e.g., 45℃, 50℃, 55℃, 60℃, etc.), and the time is 40-56 h (e.g., 42 h, 44 h, 46 h, 48 h, 50 h, 52 h, 54 h, etc.).

[0025] Preferably, in step (1), the pH adjustment operation is further included after the first enzymatic hydrolysis.

[0026] Preferably, the pH is adjusted to a system pH of 5-6, for example, 5.2, 5.4, 5.5, 5.6, 5.8, etc.

[0027] Preferably, the pH adjustment is performed using a buffer solution of disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0028] Preferably, the concentration of the buffer solution is 0.1-0.2 mol / L, for example, it can be 0.12 mol / L, 0.14 mol / L, 0.15 mol / L, 0.16 mol / L, 0.18 mol / L, etc.

[0029] Preferably, the pH of the buffer solution is 6-7, for example, it can be 6.2, 6.4, 6.5, 6.6, 6.8, etc.

[0030] Preferably, in step (1), the amount of enzyme used in the second enzymatic hydrolysis is 0.2-0.3% of the substrate mass, for example, it can be 0.22%, 0.24%, 0.25%, 0.26%, 0.28%, etc.

[0031] Preferably, in step (1), the temperature of the second enzymatic hydrolysis is 35-50℃ (e.g., 36℃, 38℃, 40℃, 42℃, 44℃, 46℃, 48℃, etc.), and the time is 1.5-2.5 h (e.g., 1.6 h, 1.8 h, 2 h, 2.2 h, 2.4 h, etc.).

[0032] Preferably, in step (1), the solid-liquid separation includes sequential filtration and centrifugation operations.

[0033] Preferably, the centrifugation speed is 7000-8000 rpm (e.g., 7200 rpm, 7400 rpm, 7500 rpm, 7600 rpm, 7800 rpm, etc.), and the centrifugation time is 5-8 min (e.g., 5.5 min, 6 min, 6.5 min, 7 min, 7.5 min, etc.).

[0034] Preferably, in step (2), the sterilization temperature is 70-90℃ (e.g., 72℃, 75℃, 78℃, 80℃, 82℃, 85℃, 88℃, etc.), and the sterilization time is 25-35 min (e.g., 26 min, 28 min, 30 min, 32 min, 34 min, etc.).

[0035] Preferably, in step (2), the amount of the antibacterial agent is 2.5-3.5% of the crude mannan extract, for example, it can be 2.6%, 2.8%, 3%, 3.2%, 3.4%, etc.

[0036] Preferably, in step (2), the antibacterial agent includes 1,2-hexanediol.

[0037] Preferably, in step (2), the settling time is 20-28 hours, for example, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, etc.

[0038] Preferably, in step (3), the ultrafiltration membrane used includes a polyethersulfone membrane.

[0039] Preferably, in step (3), the molecular weight cutoff of the ultrafiltration is 8000-12000, for example, it can be 8500, 9000, 9500, 10000, 10500, 11000, 11500, etc.

[0040] Preferably, in step (3), the particle size of the activated carbon in the first adsorption is 4.5-6 mm, for example, it can be 4.6 mm, 4.8 mm, 5 mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, etc.

[0041] Preferably, in step (3), the adsorption time of the first adsorption of activated carbon is 30-40 min, for example, it can be 32 min, 34 min, 35 min, 36 min, 38 min, etc.

[0042] Preferably, in step (3), in the first adsorption of activated carbon, based on a system of 100 mL after ultrafiltration, the amount of activated carbon used is 0.5-1 g, for example, 0.6 g, 0.7 g, 0.8 g, 0.9 g, etc.

[0043] Preferably, in step (3), the structure of the cation exchange resin includes a sulfonic acid-modified styrene-divinylbenzene copolymer.

[0044] Preferably, the eluent used for purifying the cation exchange resin includes water.

[0045] Preferably, in step (3), the activated carbon in the second adsorption process has a particle size of 1.5-2.5 mm, such as 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, etc.

[0046] Preferably, in step (3), the adsorption time in the second adsorption of activated carbon is 30-50 min, for example, it can be 32 min, 35 min, 38 min, 40 min, 42 min, 45 min, 48 min, etc.

[0047] Preferably, in step (3), in the second adsorption of activated carbon, the amount of activated carbon used is 2-3 g, based on a system purified by cation exchange resin of 100 mL. For example, it can be 2.2 g, 2.4 g, 2.5 g, 2.6 g, 2.8 g, etc.

[0048] Preferably, the preparation method includes the following steps: (1) Mix konjac powder and water at a mass ratio of 1:(0.8-1.6), add 1.5-2.5% of the substrate mass of the compound enzyme, and enzymatically hydrolyze at 40-65℃ for 40-56 h to complete the first enzymatic hydrolysis; adjust the pH to 5-6 using a 0.1-0.2 mol / L, pH 6-7 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, add 0.2-0.3% of the substrate mass of lysozyme, and enzymatically hydrolyze at 35-50℃ for 1.5-2.5 h to complete the second enzymatic hydrolysis; then filter, centrifuge the filtrate at 7000-8000 rpm for 5-8 min, and take the supernatant to obtain the crude mannan extract; The complex enzyme comprises a combination of β-glucanase, cellulase, and pectinase in a mass ratio of 1:(0.5-1.5):(0.5-1.5). (2) After sterilizing the crude mannan extract at 70-90℃ for 25-35 min, add 2.5-3.5% of the crude mannan extract as an antibacterial agent, let stand for 20-28 h and take the supernatant. (3) The supernatant was ultrafiltered using a filter membrane with a molecular weight cutoff of 8000-12000; the retentate was treated with activated carbon with a particle size of 4.5-6 mm for 30-40 min to complete the first adsorption by activated carbon; then purified by cation exchange resin using a styrene-divinylbenzene copolymer with a structure including sulfonic acid group modification; then treated with activated carbon with a particle size of 1.5-2.5 mm for 30-50 min to complete the second adsorption by activated carbon, and mannan was obtained. In the first adsorption of activated carbon, the amount of activated carbon used is 0.5-1g, based on a system volume of 100 mL after ultrafiltration. In the second adsorption of activated carbon, the amount of activated carbon used is 2-3g, based on a system volume of 100 mL after purification by cation exchange resin.

[0049] Secondly, the present invention provides a mannan, which is prepared by the mannan preparation method described in the first aspect.

[0050] Thirdly, the present invention provides the application of mannan according to the second aspect in the preparation of cosmetics.

[0051] Preferably, the cosmetic includes facial mask essence, facial mask, lotion, cream or toner.

[0052] Compared with the prior art, the present invention has at least the following beneficial effects: The mannan preparation method provided by this invention includes multiple enzymatic hydrolysis and purification steps, resulting in a product with high extraction rate, low molecular weight, and high purity, effectively improving the solubility and transdermal absorption of mannan. The preparation method provided by this invention does not introduce any organic solvents throughout the process, making it safe and non-irritating, and can be directly applied to cosmetics. The resulting product has suitable viscosity and good film-forming properties, is well miscible with amphoteric surfactants, and can improve the dry and wet combing properties of shampoos and conditioners. It also possesses moisturizing, firming, and wrinkle-reducing properties. Furthermore, it has significant effects in treating increased dandruff, dermatitis, and keratosis caused by dry skin. Detailed Implementation

[0053] To facilitate understanding of the present invention, the following embodiments are provided. Those skilled in the art should understand that these embodiments are merely illustrative and should not be construed as limiting the scope of the invention.

[0054] The materials used in the following embodiments are from the following sources: The β-glucanase model is BGS, and the brand is Xiasheng FDG-2219. The cellulase model is CEL-01, and the brand is Xiasheng. Pectinase activity: 30,000-300,000 u / g, brand: Xindeli Biotechnology; The brand of lysozyme is Xiasheng FDG-2269; Alkaline protease, brand name: Dongheng Huadao; Activated carbon 1, particle size 6 mm, model BH0012, brand Boheng; Activated carbon 2, particle size 2 mm, model BH0012, brand Boheng.

[0055] Example 1 This embodiment provides a method for preparing mannan, the preparation method comprising the following steps: (1) Konjac powder and water were mixed at a mass ratio of 1:1.04. 2% of the total mass of konjac powder and water was added to the mixture, and the mixture was hydrolyzed at 50℃ for 48 h to complete the first hydrolysis. The pH was adjusted to 5.5 using a 0.15 mol / L, pH 6.7 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution. 0.25% of the substrate mass of lysozyme was added, and the mixture was hydrolyzed at 40℃ for 2 h to complete the second hydrolysis. The mixture was then filtered, and the filtrate was centrifuged at 7500 rpm for 6.5 min. The supernatant was collected to obtain crude mannan extract. The complex enzyme comprises a combination of β-glucanase, cellulase, and pectinase in a mass ratio of 1:1:1. (2) After sterilizing the crude mannan extract at 80℃ for 30 min, add 3% of the crude mannan extract in 1,2-hexanediol and let it stand for 24 h to collect the supernatant. (3) The supernatant was ultrafiltered using a PES membrane with a molecular weight cutoff of 10,000; the retentate was treated with 0.7% (w / v) activated carbon 1 for 35 min to complete the first adsorption of activated carbon; then purified with cation exchange resin HPR1200Na and eluted with deionized water; finally treated with 2.5% (w / v) activated carbon 2 for 40 min to complete the second adsorption of activated carbon and obtain mannan.

[0056] Example 2 This embodiment provides a method for preparing mannan, the preparation method comprising the following steps: (1) Konjac powder and water were mixed at a mass ratio of 1:0.8. A compound enzyme of 1.5% of the total mass of konjac powder and water was added and enzymatically hydrolyzed at 40℃ for 56 h to complete the first enzymatic hydrolysis. The pH was adjusted to 6 using a 0.1 mol / L, pH 7 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution. Lysozyme of 0.2% of the substrate mass was added and enzymatically hydrolyzed at 35℃ for 2.5 h to complete the second enzymatic hydrolysis. The mixture was then filtered, and the filtrate was centrifuged at 7000 rpm for 8 min. The supernatant was collected to obtain crude mannan extract. The complex enzyme comprises a combination of β-glucanase, cellulase, and pectinase in a mass ratio of 1:0.5:1.5. (2) After sterilizing the crude mannan extract at 70℃ for 35 min, add 2.5% of the crude mannan extract in 1,2-hexanediol, let stand for 20 h and take the supernatant. (3) The supernatant was ultrafiltered using a PES membrane with a molecular weight cutoff of 8000; the retentate was treated with 0.5% (w / v) activated carbon 1 for 40 min to complete the first adsorption of activated carbon; then purified with cation exchange resin HPR1200Na and eluted with deionized water; finally treated with 2% (w / v) activated carbon 2 for 30 min to complete the second adsorption of activated carbon and obtain mannan.

[0057] Example 3 This embodiment provides a method for preparing mannan, the preparation method comprising the following steps: (1) Konjac powder and water were mixed in a mass ratio of 1:1.6. A compound enzyme of 2.5% of the total mass of konjac powder and water was added and enzymatically hydrolyzed at 65℃ for 40 h to complete the first enzymatic hydrolysis. The pH was adjusted to 5 using a 0.2 mol / L, pH 6, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution. Lysozyme of 0.3% of the substrate mass was added and enzymatically hydrolyzed at 50℃ for 1.5 h to complete the second enzymatic hydrolysis. The mixture was then filtered, and the filtrate was centrifuged at 8000 rpm for 5 min. The supernatant was collected to obtain crude mannan extract. The complex enzyme comprises a combination of β-glucanase, cellulase, and pectinase in a mass ratio of 1:1.5:0.5. (2) After sterilizing the crude mannan extract at 90℃ for 25 min, add 3.5% of the crude mannan extract in 1,2-hexanediol and let stand for 28 h to collect the supernatant. (3) The supernatant was ultrafiltered using a PES membrane with a molecular weight cutoff of 12000; the retentate was treated with 1% (w / v) activated carbon 1 for 30 min to complete the first adsorption of activated carbon; then purified with cation exchange resin HPR1200Na and eluted with deionized water; finally treated with 3% (w / v) activated carbon 2 for 50 min to complete the second adsorption of activated carbon and obtain mannan.

[0058] Example 4 This embodiment provides a method for preparing mannan, which differs from Example 1 only in that, in step (1), the first enzymatic hydrolysis uses a combination of β-glucanase and cellulase in a mass ratio of 1:1.

[0059] Example 5 This embodiment provides a method for preparing mannan, which differs from Example 1 only in that, in step (1), the first enzymatic hydrolysis uses a combination of β-glucanase and pectinase in a mass ratio of 1:1.

[0060] Example 6 This embodiment provides a method for preparing mannan, which differs from Example 1 only in that, in step (1), the first enzymatic hydrolysis uses a combination of cellulase and pectinase in a mass ratio of 1:1.

[0061] Example 7 This embodiment provides a method for preparing mannan, which differs from Example 1 only in that, in step (1), the enzyme used for the second enzymatic hydrolysis is an alkaline protease.

[0062] Comparative Example 1 This comparative example provides a method for preparing mannan, which differs from Example 1 only in that the first enzymatic hydrolysis is not performed in step (1), and the other steps are the same as in Example 1.

[0063] Comparative Example 2 This comparative example provides a method for preparing mannan, which differs from Example 1 only in that the second enzymatic hydrolysis is not performed in step (1), and the other steps are the same as in Example 1.

[0064] Comparative Example 3 This comparative example provides a method for preparing mannan, which differs from Example 1 only in that ultrafiltration is not performed in step (3), while the other steps are the same as in Example 1.

[0065] Comparative Example 4 This comparative example provides a method for preparing mannan, which differs from Example 1 only in that activated carbon adsorption is not performed in step (3), and the other steps are the same as in Example 1.

[0066] Comparative Example 5 This comparative example provides a method for preparing mannan, which differs from Example 1 only in that cation exchange resin purification is not performed in step (3), while other steps are the same as in Example 1.

[0067] Comparative Example 6 This comparative example provides a method for preparing mannan, which differs from Example 1 only in that activated carbon adsorption is not performed in step (3), and the other steps are the same as in Example 1.

[0068] Test Example 1 Extraction rate and purity test The extraction rates and purity results of the preparation methods provided in Examples 1-7 and Comparative Examples 1-6 are shown in Table 1.

[0069] Table 1 The test results show that: (1) As can be seen from Examples 1 to 7, the present invention adopts a multi-stage enzymatic hydrolysis and multi-stage purification process, with each step closely linked and progressively advancing, thereby improving extraction efficiency and product purity.

[0070] (2) By comparing Examples 1 and Examples 4-7, it can be seen that the first enzymatic hydrolysis of the present invention rationally combines β-glucanase, cellulase and pectinase, and the three enzymes have a synergistic effect; at the same time, the second enzymatic hydrolysis uses a specific lysozyme for deep enzymatic hydrolysis, which together improves the extraction rate and purity of the product.

[0071] (3) By comparing Example 1 and Comparative Examples 1-2, it can be seen that the present invention improves the quality of the product through a two-step enzymatic hydrolysis process; by comparing Example 1 and Comparative Examples 3-6, it can be seen that the present invention uses a multi-stage purification process of ultrafiltration, activated carbon first adsorption, cation exchange resin purification and activated carbon second adsorption in sequence, with each step progressing in turn, which together improves the extraction efficiency and product purity.

[0072] Test Example 2 Viscosity and molecular weight testing Sample preparation: Prepare 28 g of 5% (w / w, on a dry basis) sample sol with distilled water and place it in the dedicated measuring cup of the Rapid Viscosity Analyzer (RVA).

[0073] Test method: (1) Place the treated sample in the special measuring cup of the rapid viscosity analyzer (RVA) to obtain the viscosity of the cosmetic. (2) Calculate the molecular weight using gel permeation chromatography (GPC). Instrument: Waters 1525 liquid chromatograph, chromatographic column: PL aquagel-OH MIXED 8 μm, 300×7.5 mm; detector: differential refractive index detector (RID). The injection volume was 50 μL, the mobile phase was 0.05 M anhydrous Na2SO4 aqueous solution, the flow rate was 1.0 mL / min, the column temperature was 30℃, the column pressure was 3.3 MPa, the running time was 20 min, and the weight-average molecular weight of the sample was measured.

[0074] The products obtained by the preparation methods provided in Examples 1-7 and Comparative Examples 1-6 were tested, and the results are shown in Table 2.

[0075] Table 2 The test results show that: (1) As can be seen from Examples 1 to 7, the present invention uses a multi-stage enzymatic hydrolysis and multi-stage purification process to obtain products with a small weight-average molecular weight, suitable viscosity and good film-forming properties.

[0076] (2) By comparing Examples 1 and Examples 4-7, it can be seen that the first enzymatic hydrolysis of the present invention rationally combines β-glucanase, cellulase and pectinase, and the three enzymes have a synergistic effect; at the same time, the second enzymatic hydrolysis uses a specific lysozyme for deep enzymatic hydrolysis, and the resulting product has a suitable viscosity and a small weight-average molecular weight.

[0077] (3) By comparing Example 1 and Comparative Examples 1-2, it can be seen that the present invention improves the quality of the product through a two-step enzymatic hydrolysis process; by comparing Example 1 and Comparative Examples 3-6, it can be seen that the present invention uses a multi-stage purification process of ultrafiltration, activated carbon first adsorption, cation exchange resin purification and activated carbon second adsorption in sequence, with each step progressing in turn, which together improves the quality of the product.

[0078] Test Example 3 elastase inhibition rate test Test method: (1) Add 0.2 mL of sample solution of the same concentration to the sample tube and the sample background, add 1.0 mL of pH 8.8 borate buffer to the sample background, add 0.2 mL of pH 8.8 borate buffer to the enzyme reaction tube, and add 1.0 mL of pH 8.8 borate buffer to the solvent background.

[0079] (2) Add 10 mg of lichen red-elastin and 1.0 mL of elastase to the sample tube and enzyme reaction tube, respectively. Mix the mixture thoroughly and place it in a constant temperature water bath shaker for 20 min.

[0080] (3) Add 2 mL of pH 6.0 phosphate buffer to each sample tube, sample background, enzyme reaction tube and solvent background, and then use an equal volume of pH 8.8 borate buffer and pH 6.0 phosphate buffer to make up to 5 mL; centrifuge for 5 min and take 3 mL of the supernatant.

[0081] (4) Transfer each reaction solution into a 1 cm cuvette and measure the absorbance at 590 nm.

[0082] The mannan stock solution obtained by the preparation method provided in Example 1 was tested, and the results are shown in Table 3.

[0083] Table 3 The test results show that the mannan stock solution prepared by the method provided by the present invention has an elastase inhibition rate of 60.08%. Compared with the blank group, the elastase inhibition rate of the sample group is higher and the difference is significant (P<0.05). This proves that the mannan prepared by the method provided by the present invention has an inhibitory effect on elastase under the experimental conditions and has a certain firming effect.

[0084] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for preparing mannan with firming and wrinkle-reducing effects, characterized in that, The preparation method includes the following steps: (1) Mix konjac powder and water, and perform first and second enzymatic hydrolysis in sequence. After solid-liquid separation, take the supernatant to obtain crude mannan extract. The enzymes used in the first enzymatic hydrolysis include a combination of β-glucanase, cellulase, and pectinase; the mass ratio of β-glucanase, cellulase, and pectinase is 1:(0.5-1.5):(0.5-1.5). The enzyme used in the second enzymatic hydrolysis includes lysozyme; In the first enzymatic hydrolysis, the amount of enzyme used is 1.5-2.5% of the substrate weight; in the second enzymatic hydrolysis, the amount of enzyme used is 0.2-0.3% of the substrate weight. (2) After sterilizing the crude mannan extract, add an antibacterial agent, let it stand and take the supernatant; (3) The supernatant was subjected to ultrafiltration, first adsorption with activated carbon, purification with cation exchange resin and second adsorption with activated carbon in sequence to obtain mannan.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of konjac powder to water is 1:(0.8-1.6).

3. The preparation method according to claim 1, characterized in that, In step (1), the temperature of the first enzymatic hydrolysis is 40-65℃ and the time is 40-56 h.

4. The preparation method according to claim 1, characterized in that, In step (1), the first enzymatic hydrolysis is followed by a pH adjustment operation.

5. The preparation method according to claim 4, characterized in that, The pH is adjusted to a level of 5-6.

6. The preparation method according to claim 1, characterized in that, In step (1), the temperature of the second enzymatic hydrolysis is 35-50℃ and the time is 1.5-2.5 h.

7. The preparation method according to claim 1, characterized in that, In step (2), the sterilization temperature is 70-90℃ and the sterilization time is 25-35 min.

8. The preparation method according to claim 1, characterized in that, In step (2), the amount of the antibacterial agent used is 2.5-3.5% of the crude mannan extract.

9. The preparation method according to claim 1, characterized in that, In step (2), the antibacterial agent includes 1,2-hexanediol.

10. The preparation method according to claim 1, characterized in that, In step (2), the settling time is 20-28 hours.

11. The preparation method according to claim 1, characterized in that, In step (3), the ultrafiltration membrane used includes a polyethersulfone membrane.

12. The preparation method according to claim 1, characterized in that, In step (3), the molecular weight cutoff of the ultrafiltration is 8000-12000.

13. The preparation method according to claim 1, characterized in that, In step (3), the particle size of the activated carbon in the first adsorption is 4.5-6 mm.

14. The preparation method according to claim 1, characterized in that, In step (3), the adsorption time in the first adsorption of activated carbon is 30-40 min.

15. The preparation method according to claim 1, characterized in that, In step (3), in the first adsorption of activated carbon, the amount of activated carbon used is 0.5-1 g, based on a system volume of 100 mL after ultrafiltration.

16. The preparation method according to claim 1, characterized in that, In step (3), the structure of the cation exchange resin includes a sulfonic acid-modified styrene-divinylbenzene copolymer.

17. The preparation method according to claim 1, characterized in that, The elution solution used for cation exchange resin purification includes water.

18. The preparation method according to claim 1, characterized in that, In step (3), the activated carbon in the second adsorption process has a particle size of 1.5-2.5 mm.

19. The preparation method according to claim 1, characterized in that, In step (3), the adsorption time in the second adsorption of activated carbon is 30-50 min.

20. The preparation method according to claim 1, characterized in that, In step (3), in the second adsorption of activated carbon, the amount of activated carbon used is 2-3 g, based on a system purified by cation exchange resin of 100 mL.

21. The preparation method according to claim 1, characterized in that, The preparation method includes the following steps: (1) Mix konjac powder and water at a mass ratio of 1:(0.8-1.6), add 1.5-2.5% of the substrate mass of the compound enzyme, and enzymatically hydrolyze at 40-65℃ for 40-56 h to complete the first enzymatic hydrolysis; adjust the pH to 5-6, add 0.2-0.3% of the substrate mass of lysozyme, and enzymatically hydrolyze at 35-50℃ for 1.5-2.5 h to complete the second enzymatic hydrolysis; then filter, centrifuge the filtrate at 7000-8000rpm for 5-8 min, and take the supernatant to obtain crude mannan extract; The complex enzyme comprises a combination of β-glucanase, cellulase, and pectinase in a mass ratio of 1:(0.5-1.5):(0.5-1.5). (2) After sterilizing the crude mannan extract at 70-90℃ for 25-35 min, add 2.5-3.5% of the crude mannan extract as an antibacterial agent, let stand for 20-28 h and take the supernatant. (3) The supernatant was ultrafiltered using a filter membrane with a molecular weight cutoff of 8000-12000; the retentate was treated with activated carbon with a particle size of 4.5-6 mm for 30-40 min to complete the first adsorption of activated carbon; then purified with cation exchange resin using a styrene-divinylbenzene copolymer with a structure including sulfonic acid group modification; then treated with activated carbon with a particle size of 1.5-2.5 mm for 30-50 min to complete the second adsorption of activated carbon, and mannan was obtained. In the first adsorption of activated carbon, the amount of activated carbon used is 0.5-1 g, based on a system volume of 100 mL after ultrafiltration. In the second adsorption of activated carbon, the amount of activated carbon used is 2-3 g, based on a system volume of 100 mL after purification by cation exchange resin.

22. A mannan with firming and wrinkle-reducing effects, characterized in that, The mannan is prepared by the method for preparing mannan with firming and wrinkle-reducing effects as described in any one of claims 1-21.

23. The application of the mannan with firming and wrinkle-reducing effects according to claim 22 in the preparation of cosmetics.

24. The application according to claim 23, characterized in that, The cosmetics include face masks, lotions, creams, or toners.

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