Preparation method of saccharide isomeride with strong moisturizing and soothing effects as well as product and application of saccharide isomeride

Abstract

The invention relates to a preparation method of a saccharide isomeride with strong moisturizing and relieving effects as well as a product and application of the saccharide isomeride. The preparation method comprises the following steps: mixing corn starch with water to prepare starch milk, and performing acidolysis, neutralization, composite enzymolysis, saccharification and cooling crystallization to prepare D-glucose crystals; mixing the D-glucose crystal with water and glucose isomerase for reaction, and performing enzyme deactivation and concentration to obtain a saccharide isomeride; wherein the enzyme in the composite enzymatic hydrolysis comprises a combination of alpha-amylase, pullulanase and beta-amylase. The specific enzyme combination is adopted to decompose and extract the saccharide isomeride component in the corn starch, a potential synergistic relationship exists among the three enzymes, the extraction effect can be remarkably improved after the three enzymes are combined, and the prepared saccharide isomeride product has excellent moisturizing and soothing effects.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a method for preparing a sugar isomer with strong moisturizing and soothing effects, as well as its products and applications. Background Technology

[0002] Saccharide isomers are natural moisturizers extracted from natural plant saccharide complexes. Similar to the carbohydrate complexes (NMFs) in the stratum corneum of human skin, they possess excellent skin-friendly properties. Saccharide isomers achieve their moisturizing effect by assisting water in binding to keratin in the stratum corneum. Because they can bind tightly to keratin like a magnet, they are also known as "water-locking magnets."

[0003] The saccharide isomers, derived from edible corn sugar (plant-based D-glucose), can rapidly enhance skin hydration and moisture retention, thus improving dryness and flaking. Furthermore, testing has shown that these saccharide isomers can effectively stimulate and improve the expression of key genes in the skin barrier, specifically by stimulating silk protein and hyaluronic acid synthase-3. + It enhances the expression of genes related to lutein and acid sphingomyelinase, thereby increasing NMF and hyaluronic acid levels and improving skin hydration. It also stimulates ceramide synthesis and strengthens the stratum corneum by stimulating gene expression of lutein and acid sphingomyelinase. This saccharide isomer is a good hydrating agent, soothing agent, and emollient. It is an isomerized D-glucan manufactured using biochemical technology, possessing a compositional structure similar to the human stratum corneum. When applied to the skin, it binds to ε-amino acid functional groups, forming a strong bond like a magnet, thus maintaining long-lasting skin hydration.

[0004] Currently, carbohydrate isomers have certain applications in pharmaceuticals, food, and daily chemical products. However, due to the inconsistent moisturizing properties, the efficacy of carbohydrate isomers is somewhat affected, limiting their application in products. Modification of carbohydrate isomers has improved their water solubility, absorption, and therapeutic effects. Existing extraction techniques for improving the chemical properties of carbohydrate isomers mainly include acid hydrolysis and enzymatic hydrolysis. Therefore, developing an extraction and preparation process that can effectively increase the extraction rate of carbohydrate isomers, reduce their molecular weight, and improve product stability and uniformity has significant application value. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing a carbohydrate isomer with strong moisturizing and soothing effects, as well as its products and applications.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a saccharide isomer with strong moisturizing and soothing effects, the preparation method comprising the following steps:

[0008] Corn starch was mixed with water to prepare starch milk, which was then subjected to acid hydrolysis, neutralization, compound enzymatic hydrolysis, saccharification and cooling crystallization to prepare D-glucose crystals. The D-glucose crystals were then reacted with water and glucose isomerase, and the enzyme was inactivated, purified by ion exchange resin and concentrated to prepare carbohydrate isomers.

[0009] The enzymes in the complex enzymatic hydrolysis include a combination of α-amylase, pullulanase, and β-amylase.

[0010] This invention employs a process of bio-enzymatic hydrolysis for the extraction, saccharification, and isomerization of carbohydrate isomers. This process preserves the natural components of corn starch while maximizing its moisturizing properties. The addition of three bio-enzymes—α-amylase, pullulanase, and β-amylase—provides a synergistic effect, further enhancing the extraction efficiency of related carbohydrates and other components from corn starch. This increases the content of active components in the final product. Furthermore, the subsequent saccharification and isomerization processes further reduce the molecular weight of the carbohydrate isomers in the final product, making them easier for the skin to absorb. This results in a product with strong moisturizing and soothing effects.

[0011] The preparation method of corn starch includes the following steps:

[0012] Clean and soak the corn kernels to remove impurities. Use a pulping machine to pulp the corn kernels soaked at 50-60℃ for 12-24 hours to separate the fiber and protein from the corn pulp to obtain starch milk. Let the starch milk stand to precipitate, and repeatedly wash the precipitated starch with deionized water to remove residual protein and impurities. Dry to obtain corn starch.

[0013] Preferably, the mass ratio of α-amylase, pullulanase and β-amylase is (8-15):(3-6):(7-12);

[0014] Among them, "8-15" can be, for example, 8, 9, 10, 11, 12, 13, 14, 15, etc.;

[0015] The "3-6" mentioned above can be, for example, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.

[0016] The "7-12" mentioned above can be, for example, 7, 7.5, 8, 9, 10, 10.5, 11, 12, etc.

[0017] Preferably, the acid hydrolysis temperature is 90-110℃, such as 90℃, 92℃, 95℃, 98℃, 100℃, 102℃, 105℃, 108℃, 110℃, etc.; the time is 1-2h, such as 1h, 1.2h, 1.4h, 1.5h, 1.6h, 1.8h, 2h, etc.; and the pH is 1.5-2, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc.

[0018] Preferably, the neutralization involves adjusting the pH of the reaction system to 5-7, such as 5, 5.2, 5.5, 5.8, 6, 6.2, 6.5, 7, etc.

[0019] Preferably, the temperature of the compound enzymatic hydrolysis is 50-70℃, such as 50℃, 52℃, 55℃, 58℃, 60℃, 62℃, 65℃, 68℃, 70℃, etc.; and the time is 1-3h, such as 1h, 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h, 2.8h, 3h, etc.

[0020] Preferably, the compound enzymatic hydrolysis specifically includes: adding α-amylase and pullulanase at a temperature of 60-70℃ and reacting for 0.5-2 hours, adjusting the temperature to 50-60℃, and reacting for another 0.5-1 hour to obtain dextrin.

[0021] Preferably, the amount of the compound enzyme added is 0.04%-0.1% of the mass of corn starch, such as 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, etc.

[0022] Preferably, the corn starch in the starch milk is 25%-45% by mass, for example, 25%, 28%, 30%, 32%, 35%, 38%, 40%, 45%, etc.

[0023] Preferably, the amount of saccharifying enzyme added during saccharification is 0.6%-1.2% of the mass of corn starch, for example, it can be 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, etc.

[0024] Preferably, the saccharification temperature is 60-65℃, such as 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, etc.; and the time is 12-24h, such as 12h, 14h, 16h, 18h, 20h, 22h, 24h, etc.

[0025] Preferably, before cooling and crystallization, activated carbon adsorption for impurity removal and filtration are performed.

[0026] Preferably, the cooling crystallization temperature is 20-30℃, for example, it can be 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc.

[0027] Preferably, the cooled crystals are separated by centrifugation to obtain D-glucose crystals.

[0028] Preferably, the centrifugation speed is 3000-5000 rpm, such as 3000 rpm, 3200 rpm, 3500 rpm, 3700 rpm, 4000 rpm, 4250 rpm, 4500 rpm, 4750 rpm, 5000 rpm, etc.; the time is 8-16 min, such as 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, etc.

[0029] Preferably, the D-glucose crystals are mixed with water to obtain a D-glucose solution with a mass percentage of 10%-30% (e.g., 10%, 12%, 15%, 18%, 19%, 20%, 25%, 28%, 30%, etc.).

[0030] Preferably, the amount of glucose isomerase added is 0.5%-1% of the dry weight of D-glucose crystals, for example, it can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc.

[0031] Preferably, the mixed reaction system also contains Mg. 2+ ;Mg 2+ The concentration in the system is 1.5-2mM, such as 1.5mM, 1.6mM, 1.7mM, 1.8mM, 1.9mM, 2mM, etc.

[0032] Preferably, the temperature of the mixing reaction is 55-65℃, such as 55℃, 58℃, 60℃, 61℃, 62℃, 64℃, 65℃, etc.; the pH is 7-7.5, such as 7, 7.1, 7.2, 7.3, 7.4, 7.5, etc.; and the time is 2-4h, such as 2h, 2.2h, 2.5h, 2.8h, 3h, 3.2h, 3.4h, 3.6h, 3.8h, 4h, etc.

[0033] Preferably, the enzyme inactivation temperature is 85-100℃, for example, 85℃, 86℃, 87℃, 88℃, 90℃, 92℃, 95℃, 98℃, 100℃, etc.; and the time is 5-10min, for example, 5min, 6min, 7min, 8min, 9min, 10min, etc.

[0034] Preferably, the enzyme is further purified using an ion exchange resin after inactivation.

[0035] Preferably, the ion exchange resin includes type D301 and / or type 732, and more preferably type D301 and type 732.

[0036] Among them, the D301 type anion exchange resin is a styrene-divinylbenzene copolymer, which can effectively remove organic acid residues in the system, and the 732 type cation exchange resin is a 001×7 styrene-divinylbenzene copolymer, which can effectively remove metal ion residues in the system. The two work together to further remove impurities from the sugar isomer solution and improve its purity.

[0037] Preferably, the concentration is achieved by membrane separation concentration; the concentration temperature is 20-40℃, for example, 20℃, 22℃, 25℃, 28℃, 30℃, 32℃, 35℃, 38℃, 40℃, etc.

[0038] In a second aspect, the present invention provides a carbohydrate isomer prepared according to the preparation method described in the first aspect.

[0039] Thirdly, the present invention provides the application of the carbohydrate isomer described in the second aspect in the preparation of cosmetics.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] This invention employs a process of bio-enzymatic hydrolysis for the extraction, saccharification, and isomerization of carbohydrate isomers. This process preserves the natural components of corn starch while maximizing its moisturizing properties. The addition of three bio-enzymes—α-amylase, pullulanase, and β-amylase—provides a synergistic effect, further enhancing the extraction efficiency of related carbohydrates and other components from corn starch. This increases the content of active components in the final product. Furthermore, the subsequent saccharification and isomerization processes further reduce the molecular weight of the carbohydrate isomers in the final product, making them easier for the skin to absorb. This results in a product with strong moisturizing and soothing effects. Detailed Implementation

[0042] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0043] The enzyme information mentioned below is as follows: α-amylase (Yuanye, S10004), pullulanase (Yuanye, S10193), β-amylase (Yuanye, S10006), glucoamylase (Yuanye, S10017), and glucose isomerase (Maclean, G916744).

[0044] Preparation Example

[0045] This preparation example provides a corn starch, the preparation method of which is as follows:

[0046] Clean the corn kernels thoroughly to remove impurities, then soak them in 55°C warm water for 18 hours to soften. Use a pulping machine to pulp the soaked corn kernels into a paste, and use a sieve to separate the fiber and protein in the corn paste to obtain starch milk. Let it stand, collect the starch particles that have settled to the bottom, and repeatedly wash the precipitated starch with deionized water to remove residual protein and impurities. Dry the starch to obtain corn starch.

[0047] Example 1

[0048] This embodiment provides a carbohydrate isomer, and the specific steps are as follows:

[0049] (1) Corn starch was mixed with water to prepare a 35% starch slurry. Dilute hydrochloric acid was added for acid hydrolysis, and the pH was adjusted to 1.8. The reaction was carried out at 100℃ for 1.5 h, and sodium hydroxide was added to adjust the pH to 6. The starch slurry was cooled to 65℃, and α-amylase and pullulanase were added and reacted for 1 h. The temperature was adjusted to 55℃, and β-amylase was added and the reaction was continued for 0.8 h. The mass ratio of α-amylase, pullulanase and β-amylase was 10:5:8, and the total amount of the three enzymes added was 0.081% of the mass of corn starch.

[0050] (2) After adjusting the temperature of the starch milk system to 62℃, add 0.8% of corn starch mass of saccharifying enzyme and saccharify for 18h. Use filter cloth to remove insoluble impurities in the system, add 2% of corn starch mass of activated carbon to adsorb organic impurities, filter with a 5μm microfiltration membrane to remove activated carbon, concentrate, cool (25℃) crystallize, centrifuge (4000rpm, 12min) to separate and dry to obtain D-glucose crystals.

[0051] (3) Prepare a 20% D-glucose solution by mixing D-glucose crystals with water, adjust the pH of the reaction system to 7.2, and add 0.75% of the dry weight of D-glucose crystals of glucose isomerase and Mg. 2+ Mg 2+ The concentration in the system was 1.8 mM, and the reaction was carried out at a constant temperature of 60℃ for 3 h to inactivate the enzyme (88℃, 8 min). The metal ions and organic acids in the solution were removed by 732 type cation exchange resin and D301 type anion exchange resin, respectively. The eluent was collected and separated and concentrated using a polyethersulfone membrane at 30℃. After drying, the sugar isomers were obtained.

[0052] Example 2

[0053] This embodiment provides a carbohydrate isomer, and the specific steps are as follows:

[0054] (1) Mix corn starch with water to prepare a 40% starch slurry, add dilute hydrochloric acid for acid hydrolysis, adjust the pH to 1.5, react at 110℃ for 1 h, and add sodium hydroxide to adjust the pH to 5. Cool the starch slurry to 68℃, add α-amylase and pullulanase and react for 0.8 h, adjust the temperature to 58℃, add β-amylase and continue the reaction for 0.8 h. The mass ratio of α-amylase, pullulanase and β-amylase is 15:3:12, and the total amount of the three enzymes added is 0.1% of the mass of corn starch.

[0055] (2) After adjusting the temperature of the starch milk system to 64℃, add 1% of corn starch mass of saccharifying enzyme for saccharification reaction for 14h. Use filter cloth to remove insoluble impurities in the system, add 2% of corn starch mass of activated carbon to adsorb organic impurities, use 5μm microfiltration membrane to filter and remove activated carbon, concentrate, cool (28℃) crystallize, centrifuge (5000rpm, 8min) to separate and dry to obtain D-glucose crystals.

[0056] (3) Prepare a 10% D-glucose solution by mixing D-glucose crystals with water, adjust the pH of the reaction system to 7.5, and add glucose isomerase at 1% of the dry weight of D-glucose crystals and Mg. 2+ Mg 2+ The concentration in the system was 2 mM, and the reaction was carried out at a constant temperature of 58℃ for 3.5 h to inactivate the enzyme (88℃, 8 min). The metal ions and organic acids in the solution were removed by 732 type cation exchange resin and D301 type anion exchange resin, respectively. The eluent was collected and separated and concentrated using a polyethersulfone membrane at 35℃. After drying, the sugar isomers were obtained.

[0057] Example 3

[0058] This embodiment provides a carbohydrate isomer, and the specific steps are as follows:

[0059] (1) Mix corn starch with water to prepare a 30% starch slurry, add dilute hydrochloric acid for acid hydrolysis, adjust the pH to 2, react at 90℃ for 2 hours, and add sodium hydroxide to adjust the pH to 7. Cool the starch slurry to 60℃, add α-amylase and pullulanase and react for 2 hours, adjust the temperature to 50℃, add β-amylase and continue the reaction for 1 hour. The mass ratio of α-amylase, pullulanase and β-amylase is 8:6:7, and the total amount of the three enzymes added is 0.05% of the mass of corn starch.

[0060] (2) After adjusting the temperature of the starch milk system to 60℃, add 0.6% of corn starch mass of saccharifying enzyme for saccharification reaction for 24h. Use filter cloth to remove insoluble impurities in the system, add 2% of corn starch mass of activated carbon to adsorb organic impurities, use 5μm microfiltration membrane to filter and remove activated carbon, concentrate, cool (20℃) crystallize, centrifuge (3000rpm, 16min) to separate and dry to obtain D-glucose crystals.

[0061] (3) Prepare a 30% D-glucose solution by mixing D-glucose crystals with water, adjust the pH of the reaction system to 7, and add 0.5% of the dry weight of D-glucose crystals of glucose isomerase and Mg. 2+ Mg 2+ The concentration in the system was 1.5 mM, and the reaction was carried out at a constant temperature of 65℃ for 2 h to inactivate the enzyme (88℃, 8 min). The metal ions and organic acids in the solution were removed by 732 type cation exchange resin and D301 type anion exchange resin, respectively. The eluent was collected and separated and concentrated using a polyethersulfone membrane at 22℃. After drying, the sugar isomers were obtained.

[0062] Example 4

[0063] This embodiment provides a carbohydrate isomer using the starch milk from Example 1. The only difference between this isomer and Example 1 is the enzymatic hydrolysis process in step (1). Specifically, the starch milk is cooled to 60°C, and α-amylase, pullulanase, and β-amylase are added and reacted for 1.8 hours. The mass ratio of α-amylase, pullulanase, and β-amylase is 10:5:8, and the total amount of the three enzymes added is 0.081% of the mass of corn starch. The remaining steps and related parameters are the same as in Example 1.

[0064] Example 5

[0065] This embodiment provides a carbohydrate isomer, using the enzyme-inactivated solution system from Example 1. The only difference between this embodiment and Example 1 is that the ion exchange resin purification step is omitted. Instead, the enzyme-inactivated solution system is directly separated and concentrated using a polyethersulfone membrane at 22°C, and then dried to obtain the carbohydrate isomer.

[0066] Example 6

[0067] This embodiment provides a carbohydrate isomer using the starch milk from Example 1. The only difference between this embodiment and Example 1 is that the total amount of the three enzymes, α-amylase, pullulanase, and β-amylase, added is 0.01% of the mass of corn starch. All other parameters and process flow are the same as in Example 1.

[0068] Example 7

[0069] This embodiment provides a carbohydrate isomer, using the starch milk of Example 1. The only difference between this embodiment and Example 1 is that the total amount of the three enzymes, α-amylase, pullulanase and β-amylase, added is 1% of the mass of corn starch. All other parameters and process flow are the same as in Example 1.

[0070] Comparative Example 1

[0071] This comparative example provides a carbohydrate isomer using the starch milk of Example 1. The only difference between this isomer and Example 1 is that α-amylase is not added in step (1), and the reduced amount is proportionally allocated to pullulanase and β-amylase. The remaining steps and related parameters are the same as in Example 1.

[0072] Comparative Example 2

[0073] This comparative example provides a carbohydrate isomer using the starch milk of Example 1. The only difference between this isomer and Example 1 is that β-amylase is not added in step (1), and the reduced amount is proportionally allocated to pullulanase and α-amylase. The remaining steps and related parameters are the same as in Example 1.

[0074] Comparative Example 3

[0075] This comparative example provides a carbohydrate isomer using the starch milk of Example 1. The only difference between this isomer and Example 1 is that pullulanase is not added in step (1), and the reduced amount is proportionally allocated to α-amylase and β-amylase. The remaining steps and related parameters are the same as in Example 1.

[0076] Test Example 1

[0077] Moisturizing efficacy test

[0078] (1) Test principle: Based on the different forces that different moisturizers exert on water molecules, their ability to absorb and retain water also varies slightly. The moisturizing rate of a moisturizer can be tested by laboratory method (weighing method) to characterize the moisturizing effect of the ingredient.

[0079] (2) Instruments and materials:

[0080] Instrument: Constant temperature and humidity chamber;

[0081] Test materials: carbohydrate isomers (test substance), water (blank control), 10% glycerol solution (standard reference).

[0082] (3) Test environment: Temperature 20±1℃, humidity 45%±2RH%.

[0083] (4) Test method: Weigh the sample and place it in a constant temperature and humidity chamber. Weigh the sample at 2, 8 and 20 h, record the data and calculate the moisture retention rate. Select a reagent with known moisturizing ability as a standard reference.

[0084] (5) Calculation of test results: According to the experimental design, the values ​​of Mt (mass of test substance), Yt (mass of blank control), and Ht (mass of standard reference) were measured at each time period. The relative moisturizing rate of each group of test substances at each time period was calculated according to the following moisturizing rate calculation formula.

[0085] Relative moisturizing rate % = [P] 样 -P 空 ] / [P 标 -P 空 ]×100%;

[0086] In the formula, the moisturizing rate P 样 = (Mt / M0)×100%; Moisture retention rate P 空 = (Yt / Y0)×100%; Moisturizing rate P 标 = (Ht / H0) × 100%.

[0087] The experimental results are shown in Table 1.

[0088] Table 1

[0089]

[0090]

[0091] According to the data in the table:

[0092] (1) As can be seen from Examples 1 to 3, the sugar isomer products obtained by the preparation method provided by the present invention have excellent moisturizing effects.

[0093] (2) When comparing Example 1 and Example 4, it can be seen that adding α-amylase and pullulanase first for enzymatic hydrolysis and then adding β-amylase after cooling can effectively improve the efficiency of starch decomposition and effectively gelatinize it into dextrin components. However, when all three enzymes are added at the same time in Example 4, the enzymatic hydrolysis effect is worse than that in Example 1, and the moisturizing effect of the prepared sugar isomers is not as good as that in Example 1.

[0094] (3) When comparing Example 1 and Example 5, it can be seen that when ion exchange resin is used to further purify the sugar isomers, the resulting product has better purity and better moisturizing effect.

[0095] (4) When comparing Example 1 with Examples 6-7, it can be seen that when the amount of the compound enzyme used is within a certain range, it has a better decomposition effect. When the total amount of the three enzymes used in Examples 6-7 is not within a certain range, the moisturizing effect of the final sugar isomer products is worse than that of Example 1.

[0096] (5) When comparing Example 1 with Comparative Examples 1-3, it can be seen that when corn starch milk is treated with three enzymes, namely α-amylase, pullulanase and β-amylase, the three enzymes can synergistically improve the enzymatic hydrolysis effect on starch, increase the degree of starch gelatinization, which is beneficial to subsequent saccharification and hydrolysis, and prepare a sugar isomer product with better moisturizing effect.

[0097] Test Example 2

[0098] Soothing test

[0099] Test method: Keratinocytes were used, and the relative expression level of capsaicin receptor (TRPV1) protein was detected by immunofluorescence (IF) to evaluate the soothing efficacy of glycosyl isomers.

[0100] According to 4.5×10 4 Cells were seeded into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2, 95% RH). When the cell deposition rate in the 24-well plates reached 40%-60%, the following treatments were administered: Blank control group: no capsaicin stimulation; Model control group: capsaicin stimulation; Sample group: 1% of the sugar isomer dilutions prepared in Examples 1-7 and Comparative Examples 1-3 was added, followed by capsaicin stimulation.

[0101] After drug administration, the 24-well plate was incubated in an incubator for 24 hours. After incubation, the supernatant was discarded, the plate was washed three times with PBS, the PBS was discarded, and 200 μL of goat serum was added to each well. The plate was blocked at 25°C for 60 min. The goat serum blocking solution was discarded, and 200 μL of diluted primary antibody Anti-VR1 (1:150 dilution of goat serum) was added to each well. The plate was incubated overnight at 4°C. The primary antibody was discarded, and the plate was washed three times with PBS for 5 min each time. The residual PBS on the slide was blotted dry with absorbent paper, and diluted secondary antibody Goat pAb to Rb IgG (Alexa) was added to each well. 488 (200 μL / well) (PBS 1:500 dilution), incubate at room temperature in the dark for 1 h; discard the secondary antibody, wash 3 times with PBS, 5 min each time, blot dry the PBS residue on the slide with absorbent paper, add Hochest 33342 (200 μL / well) (PBS 1:500 dilution) to each well, incubate at 25℃ for 5 min; discard Hochest 33342, wash 3 times with PBS, 5 min each time. Blot dry the PBS residue on the slide with absorbent paper, pick up the slide with a needle, add one drop of anti-quenching agent to a glass slide, and place the slide upside down on the glass slide, and seal the slide (use a cut yellow pipette tip to draw the anti-quenching agent); detect the relative expression inhibition rate of capsaicin receptor (TRPV1) for each group of samples under a fluorescence microscope, and calculate the results shown in the table below.

[0102] Table 2

[0103]

[0104]

[0105] According to the data in the table, the carbohydrate isomers obtained by the preparation process provided by the present invention can significantly downregulate the expression level of TRPV1 in keratinocytes and have a good soothing effect. Among them, the soothing effect of Example 1 is better than that of Examples 4-7 and Comparative Examples 1-3, indicating that the preparation process conditions of carbohydrate isomers used in the present invention have different degrees of influence on the soothing effect of the final extracted product.

[0106] Test Example 3

[0107] Product safety testing

[0108] Product safety testing

[0109] Twenty-eight participants were selected according to the inclusion criteria and randomly divided into seven groups (two males and two females per group) to undergo the trial. An appropriate amount of the carbohydrate isomer samples prepared in Examples 1-7 (the carbohydrate isomers were mixed with pure water to adjust the extract concentration to 5% before testing) was placed in the drug compartment of the test strip. The test strip containing the sample was then applied to the flexor surface of the subject's forearm with medical tape for 24 hours. Thirty minutes after removing the test strip, and once the indentation had disappeared, the skin reaction was observed. For those with negative results, the skin reaction was observed again at 24 and 48 hours post-test, and the results were recorded. The grading criteria for the degree of reaction are shown in the table below.

[0110]

[0111]

[0112] The results showed that the products prepared in Examples 1-7 all exhibited negative reactions at 24h and 48h after the test, with no irritation or erythema. Therefore, the carbohydrate isomer products prepared by this invention are non-irritating to the skin and show no positive reactions, proving that the product is highly safe, has low irritation, and is relatively mild.

[0113] The applicant declares that the technical solution of this invention is illustrated through the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, addition of auxiliary components, selection of specific methods, etc., are all within the protection scope and disclosure scope of this invention.

[0114] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0115] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing a saccharide isomer with strong moisturizing and soothing effects, characterized in that, The preparation method includes the following steps: Corn starch was mixed with water to prepare starch milk, which was then subjected to acid hydrolysis, neutralization, complex enzymatic hydrolysis, saccharification and cooling crystallization to prepare D-glucose crystals; D-glucose crystals were then reacted with water and glucose isomerase, and the enzyme was inactivated and concentrated to prepare carbohydrate isomers. The enzymes in the complex enzymatic hydrolysis include a combination of α-amylase, pullulanase, and β-amylase.

2. The preparation method according to claim 1, characterized in that, The mass ratio of α-amylase, pullulanase and β-amylase is (8-15):(3-6):(7-12).

3. The preparation method according to claim 1, characterized in that, The acid hydrolysis is performed at a temperature of 90-110℃ for 1-2 hours, with a pH of 1.5-2. Preferably, the neutralization involves adjusting the pH of the reaction system to 5-7.

4. The preparation method according to claim 1, characterized in that, The temperature for the compound enzymatic hydrolysis is 50-70℃, and the time is 1-3 hours. Preferably, the amount of the compound enzyme added is 0.04%-0.1% of the mass of corn starch; Preferably, the corn starch in the starch milk is 25%-45% by mass.

5. The preparation method according to claim 1, characterized in that, The amount of saccharifying enzyme added during saccharification is 0.6%-1.2% of the mass of corn starch; Preferably, the saccharification temperature is 60-65℃ and the time is 12-24h.

6. The preparation method according to claim 1, characterized in that, Before cooling and crystallization, activated carbon adsorption for impurity removal and filtration are also performed. Preferably, the cooling crystallization temperature is 20-30°C; Preferably, the cooled crystals are separated by centrifugation to obtain D-glucose crystals; Preferably, the centrifugation speed is 3000-5000 rpm and the time is 8-16 min.

7. The preparation method according to claim 1, characterized in that, The D-glucose crystals are mixed with water to prepare a D-glucose solution with a mass percentage of 10%-30%. Preferably, the amount of glucose isomerase added is 0.5%-1% of the dry weight of D-glucose crystals; Preferably, the mixed reaction system also contains Mg. 2+ ;Mg 2+ The concentration in the system is 1.5-2 mM; Preferably, the mixing reaction is carried out at a temperature of 55-65°C, a pH of 7-7.5, and a time of 2-4 hours.

8. The preparation method according to claim 1, characterized in that, After enzyme inactivation, the enzyme is further purified using ion exchange resin. Preferably, the ion exchange resin includes type D301 and / or type 732; Preferably, the concentration is performed using membrane separation concentration; the concentration temperature is 20-40℃.

9. A carbohydrate isomer prepared by the preparation method according to any one of claims 1-8.

10. The application of a carbohydrate isomer according to claim 9 in the preparation of cosmetics.

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