Anti-glycation beverage containing white kidney bean and cherry blossom compound extract and preparation method thereof

Abstract

The invention discloses an anti-glycation beverage containing a white kidney bean and cherry blossom compound extract and a preparation method thereof, which specifically belongs to the field of biotechnology extracts; the beverage comprises components: a white kidney bean and cherry blossom compound extract, fish collagen peptides, erythritol, fish skin collagen peptides, Haematococcus pluvialis, L-arabinose, sodium hyaluronate and a solvent; the white kidney bean and cherry blossom compound extract is obtained by fermenting white kidney beans and cherry blossoms with mulberry igniarius; the white kidney bean and cherry blossom compound extract provided by the invention has excellent anti-glycation effect and antioxidant effect; the anti-glycation beverage containing the white kidney bean and cherry blossom compound extract provided by the invention has the effects of brightening, removing spots, whitening and tightening.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology extracts, and particularly relates to an anti-glycation beverage containing a white kidney bean and cherry blossom composite extract and a preparation method thereof. Background Art

[0002] As we age, our metabolism gradually slows down, causing the accumulated sugar in the body to be unable to be metabolized in time. The excess sugar will be added to the amino acid groups of proteins through non-enzymatic glycosylation reactions to form glycated proteins, also known as protein advanced glycation end products (AGEs).

[0003] Collagen and elastin are the scaffolds of the skin. Once combined with sugar molecules, the originally plump and elastic collagen and elastin will harden, break and become damaged, causing the skin to collapse, lose elasticity, and lose firmness. Wrinkles will also gradually appear. As glycosylated proteins accumulate in the skin, the skin will become dull. Excessive sugar will also increase the body's male hormones, increase oil secretion, and make the skin oily, which can easily lead to acne. In the process of melanin production, sugar will make tyrosinase more active, thereby increasing the production of melanin, and the skin will gradually become dark yellow.

[0004] In order to meet this challenge, many anti-glycation products have emerged on the market, but most of them have problems such as insignificant effects, poor taste or single ingredients. Therefore, it is particularly important to develop an anti-glycation beverage that is both effective and easy to accept.

[0005] As a food that can be used as both medicine and food, the extract of white kidney bean is rich in α-amylase inhibitors, which can inhibit the activity of α-amylase in the intestine, thereby delaying and partially blocking the absorption of carbohydrates, lowering the blood sugar level of the human body, and indirectly playing an anti-glycation role. In addition, white kidney bean is also rich in a variety of nutrients, such as protein, dietary fiber, vitamins and minerals, which have a positive effect on improving human health. Guanshan cherry blossoms have attracted much attention for their unique antioxidant and anti-glycation properties. The antioxidant substances such as total flavonoids rich in cherry blossoms can effectively remove free radicals in the body, inhibit oxidative stress reactions, and slow down the formation of AGEs, thereby achieving the purpose of anti-glycation. At the same time, cherry blossoms also have certain beauty and skin care effects, which can improve skin condition and delay aging.

[0006] Therefore, the present invention aims to develop an anti-glycation beverage containing a composite extract of white kidney bean and cherry blossom. The beverage effectively combines the anti-glycation components of white kidney bean and cherry blossom through scientific proportioning and fermentation technology, so that it can effectively remove free radicals in the body, inhibit oxidative stress response, slow down the formation of AGEs, and resist health problems caused by a high-sugar diet. Summary of the invention

[0007] In view of the deficiencies of the prior art, the object of the present invention is to provide an anti-glycation beverage that can effectively remove free radicals in the body, inhibit oxidative stress reactions, and slow down the formation of AGEs.

[0008] To achieve the above object, the present invention discloses the following technical solutions:

[0009] In a first aspect, the present invention provides an anti-glycation beverage containing a white kidney bean and cherry blossom compound extract, comprising components: a white kidney bean and cherry blossom compound extract, fish collagen peptide, erythritol, fish skin collagen peptide, Haematococcus pluvialis, L-arabinose, sodium hyaluronate and a solvent.

[0010] Preferably, the following components are included by mass percentage:

[0011]

[0012] Preferably, the solvent is water.

[0013] Preferably, the preparation method of the white kidney bean and cherry blossom composite extract comprises the following steps:

[0014] S1. Take peeled white kidney beans, crush and sieve to obtain white kidney bean powder, and take dried cherry blossoms from Guanshan Mountain and crush and sieve to obtain cherry blossom powder;

[0015] S2. The white kidney bean powder and cherry blossom powder of step (1) are mixed uniformly in a mass ratio of (2-3):1, distilled water is added, and the material-liquid mass ratio is 1:(3-5), and a composite enzyme is added at an addition amount of 200-300 mg / kg for uniform mixing and enzymolysis for 10-12 hours. After the enzymolysis is completed, the mixture is centrifuged and filtered to remove the residue, the supernatant is taken, and the supernatant is inactivated to obtain a composite liquid;

[0016] S3. scrape the activated Phellinus linteus slant with an inoculation loop, inoculate it into the seed culture medium, and culture it at 28-30°C for 3-4 days in a shaker at a speed of 150-200 r / min to obtain an acclimated composite bacterial solution;

[0017] S4. The acclimated composite bacterial solution of step S3 was inoculated into the fermentation medium at an inoculum size of 9-12%, the temperature was 28-30 ° C, the stirring speed was 150-200r / min, the ventilation volume was 1-2vvm, and the fermentation culture was 10-15d to obtain a fermentation composite bacterial solution;

[0018] S5. Take β-glucanase and add it to the fermentation composite bacterial solution obtained in step S4, and perform enzymolysis for 2-3 hours. After the enzymolysis is completed, centrifuge and filter to remove the residue, take the supernatant and spray dry to obtain the white kidney bean and cherry blossom composite extract.

[0019] Further preferably, the complex enzyme comprises β-glucanase, protease and mannanase, the β-glucanase activity is 10-15 U / mg, the protease activity is 2-5 U / mg, and the mannanase activity is 12-15 U / mg.

[0020] Further preferably, the seed culture medium is composed of a carbon source, a nitrogen source, an inorganic salt, vitamins, the composite liquid of step S2 and water, wherein the amount of the composite liquid added is 50wt%;

[0021] The fermentation medium is composed of a carbon source, a nitrogen source, an inorganic salt, vitamins, the composite liquid of step S2 and water, wherein the amount of the composite liquid added is 70 wt%.

[0022] In a second aspect, the present invention provides a method for preparing the anti-glycation beverage containing the white kidney bean and cherry blossom composite extract according to the first aspect, comprising the following steps:

[0023] (1) Weigh a certain amount of white kidney bean and cherry blossom composite extract, fish collagen peptide, erythritol, fish skin collagen peptide, Haematococcus pluvialis, L-arabinose, and sodium hyaluronate, put them into water, and stir them at a stirring rate of 20 r / min for 50 min to obtain a mixed solution;

[0024] (2) filtering the mixed solution, sterilizing and filling it after filtering to obtain the anti-glycation beverage.

[0025] Preferably, the filtration in step (2) is performed using a filter bag filter, and the pore size of the filter bag is 600 mesh.

[0026] Preferably, the sterilization is carried out by high-temperature instantaneous sterilization, and the sterilization temperature is controlled at 120° C. to sterilize the mixed solution for 5 seconds;

[0027] Preferably, the filling is aseptic filling.

[0028] In the present invention:

[0029] White kidney beans contain α-amylase inhibitors, which are substances that can inhibit the activity of α-amylase, thereby reducing the decomposition and absorption of starchy foods in the digestive tract. This mechanism helps control blood sugar levels and reduce the sharp rise in blood sugar after meals, thereby indirectly playing an anti-glycation role. At the same time, white kidney beans themselves are rich in high-quality protein, multiple amino acids, minerals (such as calcium, phosphorus, iron) and vitamins B1 and B2, etc. These ingredients may be further released and converted during the fermentation process, thereby improving the nutritional value of the fermented extract.

[0030] Guanshan cherry is rich in antioxidants such as total flavonoids, which can effectively remove free radicals in the body, inhibit oxidative stress, slow down the formation of advanced glycation end products (AGEs), and play an anti-glycation role. The total flavonoids in Guanshan cherry have a better nitrite clearance rate than the traditional antioxidant Vc, showing its excellent antioxidant properties. Ingredients such as sakura glycoside and sakurain in Guanshan cherry can better promote sugar metabolism, inhibit the accumulation of sugar in the body, and further help anti-glycation.

[0031] Morigensis can utilize the nutrients in white kidney beans and cherry blossoms for fermentation to produce rich metabolites and enzymes. These metabolites and enzymes have stronger biological activity and functionality, thereby enhancing the anti-glycation effect of the fermentation extract. During the fermentation process, Morigensis can degrade the macromolecules (such as proteins, polysaccharides, etc.) in white kidney beans and cherry blossoms into small molecules, thereby improving the utilization rate and biological activity of these components and further enhancing the nutritional value of the fermentation extract.

[0032] Beneficial effects of the present invention:

[0033] 1. The white kidney bean and cherry blossom composite extract obtained by compounding white kidney bean and cherry blossom and fermenting with Phellinus igniarius provided by the present invention has excellent anti-glycation effect and antioxidant effect;

[0034] 2. The anti-glycation beverage containing the white kidney bean and cherry blossom composite extract provided by the present invention has the effects of brightening, removing spots, whitening and tightening. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings required for use in the present application.

[0036] Figure 1 This is the change diagram of skin melanin MI value at different time points;

[0037] Figure 2 This is a graph showing the changes in skin whiteness L* value at different time points;

[0038] Figure 3 This is the change diagram of individual type angle ITA° value at different time points;

[0039] Figure 4 The graph of skin elasticity R2 value changes at different time points;

[0040] Figure 5 This is the change diagram of skin elasticity F3 / F4 value at different time points;

[0041] Figure 6 This is the change diagram of skin firming parameter F4 value at different time points;

[0042] In the figure, ** indicates that there is a very significant difference compared with before use, P < 0.010; * indicates that there is a significant difference compared with before use, 0.010 ≤ P < 0.050; ns indicates that there is no significant difference compared with before use, P > 0.050. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. However, the following embodiments are only preferred embodiments of the present invention, not all. For the sake of clarity, all features of the actual embodiments are not described.

[0044] Based on the embodiments in the implementation manner, other embodiments obtained by those skilled in the art without making any creative work shall all fall within the protection scope of the present invention.

[0045] In the present invention:

[0046] White kidney bean, also known as large white kidney bean and cotton bean, has the Latin name Phaseolus lunatus L., and was purchased from Yunnan Diancai Agricultural Products Co., Ltd. The variety was identified;

[0047] The dried cherry blossoms were selected from Prunus serrulata 'Sekiyama', Latin name Prunus serrulata, purchased from Yiwu Kuayou Trading Company, and the variety was identified;

[0048] Sanghuangporus sanghuang, accession number: GDMCC NO.5.1040, purchased from Guangdong Microbiological Culture Collection Center;

[0049] White kidney bean extract was purchased from Xi'an Tongze Biotechnology Co., Ltd.;

[0050] Guanshan cherry extract was purchased from Qingyunshan Pharmaceutical Technology Co., Ltd.;

[0051] Phellinus linteus extract was purchased from Shaanxi Bolin Biotechnology Co., Ltd.;

[0052] Haematococcus pluvialis was an extract of Haematococcus pluvialis, purchased from Fufeng Snort Biotechnology Co., Ltd.;

[0053] Fish collagen peptides were purchased from Hubei Ruibang Biotechnology Co., Ltd.;

[0054] Fish skin collagen peptides were purchased from Wissard Biotech, France;

[0055] Other raw materials are commercially available.

[0056] Preparation of white kidney bean and cherry blossom compound extract

[0057] Preparation of White Kidney Bean and Cherry Blossom Complex Extract 1-10:

[0058] Step (1), taking fresh, well-mature white kidney beans, washing them with clean water, soaking them in water at 4° C. for 10 hours, peeling off the outer skin and drying them in a drying oven, putting the dried peeled white kidney beans into a grinder, crushing them, and passing them through an 80-100 mesh sieve to obtain white kidney bean powder, taking dried Kwanzan cherry flowers, putting them into a grinder, crushing them, and passing them through an 80-100 mesh sieve to obtain cherry blossom powder;

[0059] Step (2), mixing the white kidney bean powder and cherry blossom powder in step (1) in a mass ratio of (2-3):1, adding distilled water, the material-liquid mass ratio is 1:(3-5), adding composite enzyme in an amount of 200-300 mg / kg, mixing evenly, enzymolysis for 10-12 hours, wherein the β-glucanase activity in the composite enzyme is 10-15 U / mg, the protease activity is 2-5 U / mg, and the mannanase activity is 12-15 U / mg. After the enzymolysis is completed, centrifuging at 8000 r / min for 25 minutes, filtering to remove the residue, taking the supernatant, and placing the supernatant in a 100° C. water bath to inactivate the enzyme for 20 minutes to obtain a composite liquid;

[0060] Step (3), preparing a seed culture medium: 2wt% glucose, 0.4wt% peptone, 0.1wt% KH2PO4, 0.05wt% MgSO4, 10mg / 100mL VB1, 50wt% composite solution, balance water, pH=6.5;

[0061] Step (4), preparing a fermentation medium: 1wt% corn flour, 1wt% bran, 2wt% glucose, 0.5wt% peptone, 0.5wt% yeast powder, 0.1wt% KH2PO4, 0.05wt% MgSO4, 10mg / L vitamin B1, 70wt% composite liquid, and the balance water;

[0062] Step (5), acclimatization and culture: scrape 0.5 cm 2 The activated Phellinus linteus slant strains are inoculated into the prepared seed culture medium, and cultured at 28-30°C for 3-4 days in a shaking incubator with a rotation speed of 150-200 r / min to obtain an acclimated composite bacterial solution;

[0063] Step (6), fermentation culture: taking the composite bacterial solution obtained in step (5) at an inoculum amount of 9-12% and inoculating it into a fermentation medium, the temperature is 28-30° C., the stirring speed is 150-200 r / min, the ventilation volume is 1-2 vvm, and the fermentation culture is carried out for 10-15 days to obtain a fermentation composite bacterial solution;

[0064] Step (7), adding 200 mg / kg of β-glucanase to the fermentation composite bacterial solution obtained in step (6), performing enzymolysis for 2-3 hours, wherein the β-glucanase activity is 200-300 U / mg. After the enzymolysis is completed, centrifuge at 8000 r / min for 25 minutes, filter to remove the residue, and take the supernatant for spray drying to obtain a white kidney bean and cherry blossom composite extract.

[0065] The specific preparation parameters are shown in Table 1.

[0066] Table 1 Preparation parameters of white kidney bean and cherry blossom compound extracts 1-8

[0067]

[0068]

[0069] Note: Inoculum size is the volume ratio of inoculum to culture medium.

[0070] Extract 4: The complex enzyme in step (2) lacks protease, and the remaining steps have the same parameters as those of Extract 3;

[0071] Extract 5: The complex enzyme in step (2) lacks β-glucanase, and the remaining steps have the same parameters as those of Extract 3;

[0072] Extract 6: The complex enzyme in step (2) lacks mannanase, and the remaining steps have the same parameters as those of Extract 3;

[0073] Extract 7: The inoculation amount of the composite bacterial solution after acclimation in step (6) was adjusted to 5%, and the remaining steps were the same as those of Extract 3;

[0074] Extract 8: without enzymatic hydrolysis in step (7), the residue is directly removed by centrifugal filtration, and the supernatant is spray-dried. The remaining steps are the same as those of Extract 3.

[0075] Extract 9: not shown in Table 1, white kidney bean was used as the raw material alone, and the remaining steps were the same as those of Extract 3;

[0076] Extract 10: Not shown in Table 1, the raw material used was dried cherry blossoms, and the remaining steps were the same as those of Extract 3.

[0077] Preparation of Examples

[0078] The extracts 1-10 were respectively mixed with sterile water to prepare Examples 1-10 with a content of 10 wt %, as shown in Table 2;

[0079] Commercially available white kidney bean extract, Prunus guanzanensis extract and Phellinus igniarius extract were mixed in a mass ratio of 3:1:2, and the resulting mixture was dissolved in sterile water to prepare a solution with a concentration of 10 wt%, which was recorded as Example 11, as shown in Table 2.

[0080] Table 2 Mass percentage of raw materials in Examples 1-11

[0081]

[0082] Note: “-” in the table means not added.

[0083] Performance Testing

[0084] In vitro non-enzymatic glycosylation inhibition assay

[0085] (1) Solution preparation

[0086] Sample solutions: Examples 1-11.

[0087] Phosphate buffer (0.2 mol / L, pH=7.4): Dissolve 71.64 g of disodium hydrogen phosphate in 1000 mL of water to prepare solution A; dissolve 31.21 g of sodium dihydrogen phosphate in 1000 mL of water to prepare solution B; mix 810 mL of solution A and 190 mL of solution B, and add 0.2% Proclin 300 to prepare an antibacterial phosphate buffer.

[0088] Bovine serum albumin solution (20 mg / mL): weigh 4.0 g of bovine serum albumin and dissolve it in 200 mL of phosphate buffer.

[0089] Glucose solution (200 mg / mL): Weigh 40.0 g of glucose and dissolve it in 200 mL of phosphate buffer.

[0090] (2) Experimental operation

[0091] Prepare the reagents in each tube according to Table 3, place them in a light-proof constant temperature incubator at 37°C and incubate them continuously for 10 days, and then perform the detection under the fluorescence conditions of excitation wavelength 370nm and emission wavelength 440nm.

[0092] Calculate as follows:

[0093]

[0094] Where:

[0095] A is the absorbance of the sample solution in the reaction system;

[0096] B is the absorbance in the reaction system when phosphate buffer is used instead of (bovine serum albumin solution + glucose solution);

[0097] C is the absorbance of the reaction system with phosphate buffer replacing the sample solution;

[0098] D is the absorbance in the reaction system using phosphate buffer.

[0099] Table 3

[0100] Reaction solution (ml) A B C D Phosphate buffer 0 5 5 10 Bovine serum albumin solution + glucose solution 5 5 5 0 Sample solution 5 0 0 0

[0101] (3) AGEs inhibition rate test results

[0102] The test results of non-enzymatic glycosylation end products AGEs inhibition rate of samples treated with different methods are shown in Table 4.

[0103] Table 4 Effect of different samples on the inhibition rate of AGEs

[0104] Sample solution Inhibition rate of non-enzymatic glycosylation end products AGEs (%) Example 1 93.1 Example 2 91.2 Example 3 94.5 Example 4 78.3 Example 5 71.6 Example 6 81.2 Example 7 61.7 Example 8 83.3 Example 9 51.8 Example 10 44.1 Embodiment 11 83.5

[0105] (4) Results analysis

[0106] The white kidney bean and cherry blossom composite extract provided by the present invention is obtained by compounding white kidney bean and cherry blossom and then fermenting them with Phellinus igniarius, and has excellent anti-glycation effect.

[0107] Among them, according to the results of Examples 4-6, the addition of the composite enzyme and the enzymes in the composite enzyme has a certain effect on the anti-glycation properties of the subsequent extracts when pre-treating white kidney bean powder and dried cherry pollen; according to the results of Example 7, it can be seen that the inoculation amount of the domesticated bacterial liquid has a greater influence on the anti-glycation properties of the subsequent extracts, and it also reflects that the fermentation process has a decisive effect on the anti-glycation properties of the output extracts; according to the results of Examples 9 and 10, it can be seen that the extracts obtained by using only white kidney beans or cherry blossoms as raw materials are significantly different from the effect of the composite fermentation of the two, that is, white kidney beans and cherry blossoms have a certain mutually promoting effect when used in combination; according to the results of Example 11, it can be seen that the anti-glycation effect of simply using the extracts of the raw materials in combination is not as good as the fermentation product extract obtained by using the technical scheme of the present application.

[0108] Antioxidant performance test

[0109] Determination of antioxidant capacity by DPPH method

[0110] Accurately transfer 2 mL of sample solution into a test tube, add 2 mL of 0.2 mol / L DPPH anhydrous ethanol solution, mix well, place in a dark place for 30 min, adjust to zero with anhydrous ethanol, measure the absorbance at a wavelength of 517 nm and record it as A. 标准样 . Take 2 mL of ethanol extract and 2 mL of anhydrous ethanol of different volume fractions, mix them evenly, and place them in the dark for 30 minutes. The absorbance at 517 nm is recorded as A 对照Accurately pipette 2 mL of DPPH solution and 2 mL of anhydrous ethanol, mix them evenly, and record the absorbance at a wavelength of 517 nm as A. 空白 .

[0111] The DPPH clearance rate was calculated as follows:

[0112]

[0113] Hydroxyl radical scavenging ability assay

[0114] Pipette 2 mL of ethanol extracts with different volume fractions into test tubes, add 2 mL of 6 mol / L FeSO4 and 6 mol / L H2O2, mix well, let stand for 10 min, add 2 mL of 6 mol / L salicylic acid, mix well, let stand for 30 min, and measure A at 510 nm. i , distilled water was used to replace 6 mol / L salicylic acid, and the absorbance was measured as A j , the absorbance measured by replacing the sample with distilled water is A0.

[0115] Calculate the OH radical scavenging rate as follows:

[0116]

[0117] Where:

[0118] A i Represents the absorbance of the sample group to be tested;

[0119] A j Absorbance of sample blank group;

[0120] A0 is the absorbance of the control group.

[0121] Experimental Results

[0122] Table 5 Experimental results of scavenging ability of different samples for DPPH free radicals and hydroxyl free radicals

[0123] Sample solution DPPH free radical scavenging rate / % Hydroxyl radical scavenging rate / % Example 1 83.17 77.29 Example 2 82.63 74.10 Example 3 85.28 78.34 Example 4 65.33 60.71 Example 5 68.21 64.39 Example 6 57.09 55.40 Example 7 60.29 61.42 Example 8 70.11 67.98 Example 9 47.36 44.17 Example 10 51.03 49.23 Embodiment 11 76.16 75.82

[0124] Results Analysis

[0125] The white kidney bean and cherry blossom composite extract provided by the present invention is obtained by compounding white kidney bean and cherry blossom and then fermenting them with Phellinus igniarius, and has excellent antioxidant effect.

[0126] Application Example Preparation

[0127] Step (1), weighing a certain amount of white kidney bean and cherry blossom composite extract, fish collagen peptide, erythritol, fish skin collagen peptide, Haematococcus pluvialis, L-arabinose, and sodium hyaluronate according to Table 6, adding them into water, stirring at a stirring rate of 20 r / min and a stirring time of 50 min to obtain a mixed solution;

[0128] Step (2), filtration: the mixed solution is introduced into a filter bag filter by a pump for filtration, and the filter bag has a pore size of 600 mesh;

[0129] Step (3), high temperature instantaneous sterilization: using high temperature instantaneous sterilization, the sterilization temperature is controlled at 120°C to sterilize the mixed solution for 5 seconds;

[0130] Step (4), cooling: the sterilized mixed solution is cooled to below 30°C and then aseptically filled to obtain the finished product.

[0131] Table 6 Application Example Raw Materials Mass Percentage

[0132] Application Example 1 Application Example 2 Application Example 3 White Kidney Bean and Cherry Blossom Extract 5％ 7％ 10％ Fish collagen peptides 3 2 2 Erythritol 1 0.5 1 Fish Skin Collagen Peptide 2 1 2 Haematococcus pluvialis 1 0.5 1 L-arabinose 2 0.5 1 Sodium Hyaluronate 0.03 0.01 0.02 water Replenish to 100% Replenish to 100% Replenish to 100%

[0133] Acute toxicity test

[0134] Experimental standard: The experiment was conducted in accordance with the method specified in GB 15193.3-2014 “National Food Safety Standard Acute Oral Toxicity Test”.

[0135] Animal grouping: 20 SPF SD rats, half male and half female, were divided into groups, and the individual weight values ​​of the same sex were within the range of the mean ± 20%.

[0136] Experimental method: Weigh application example 3 as the test solution. Before the test, the animals fasted overnight (about 16 hours) and had free access to water. During the formal test, the test group animals were gavaged with the test solution at 20 mL / kg body weight, and the test solution was gavaged twice within 24 hours, with an interval of about 4 hours between the two gavages. A small amount of feed was given during the period, and the animals continued to fast for about 3 hours after the first administration of the test substance. After the administration of the test substance, the appearance and disappearance time of the signs of poisoning and the time of death were observed and recorded, and the observation period was 14 days. During the observation period, animals that died of poisoning should be grossly dissected and observed with the naked eye. If abnormalities in tissues or organs are found, further histopathological examinations should be performed. Weigh them on days 0, 1, 3, 7, and 14 respectively.

[0137] Experimental results: The animals showed no abnormal symptoms during the administration of the test solution and the 14-day observation period, their weight increased normally, no animal died, and the LD50 was greater than 10g / kg body weight. At the end of the experiment, all animals were dissected and no abnormalities were found by naked eye observation.

[0138] Experimental conclusion: The test solution was tested according to GB 15193.3-2014 "National Food Safety Standard Acute Oral Toxicity Test", and the acute oral toxicity LD50 for SD rats was greater than 10g / kg body weight, which is actually non-toxic.

[0139] Human efficacy experiment

[0140] 1. Test Basis

[0141] [1] Wang Chunxiao, Zhao Hua. Evaluation of cosmetic efficacy (II) - Scientific support for moisturizing efficacy claims [J]. Surfactant Chemical Industry, 2018, 48(2): 67-72.

[0142] [2]T / GDCDCC-020-2022 Test Method for Firming Effect of Cosmetics

[0143] [3] T / CAFFCI66-2023 Test method for cosmetic repair efficacy

[0144] [4] Refer to the Technical Specifications for Safety of Cosmetics (2015 edition)

[0145] [5] Liu Weiyi, Zhou Lin, Zhao Hua. Evaluation of cosmetic efficacy (ⅩⅢ) - Consumer use test[J]. Surfactant Chemical Industry, 2021, 51(6): 485-490.

[0146] [6] Laboratory methods of Centre Testing International Ltd.

[0147] 2. Test Purpose

[0148] This test verifies the efficacy of Application Example 3 in removing spots, whitening, firming and brightening by measuring the skin melanin MI value, skin whiteness L* value, individual type angle ITA° value, skin elasticity R2 value, skin elasticity F3 / F4 value, and skin firming parameter F4 value of 30 female subjects before and after using the test samples.

[0149] 3. Test plan

[0150] 3.1 Sample Usage Plan

[0151] Sample information: Application example 3

[0152] Directions: Oral

[0153] Frequency and cycle of use: 3 bottles per day, 25 ml per bottle, 1 bottle each in the morning, noon and evening, for 28 consecutive days

[0154] How to use: Drink immediately after opening the bottle

[0155] Note: People who are allergic to fish should not eat this product

[0156] 3.2 Subject Information

[0157]

[0158] Note: *Subjects No. 12, No. 18 and No. 25 dropped out of the test due to personal reasons.

[0159] 3.3 Subject inclusion and exclusion

[0160] 3.3.1 Inclusion criteria

[0161] Female, aged 25-40 years old; with loose skin, facial elasticity parameter R2 baseline value below 0.700, dull skin, obvious spots, BMI>25; without serious systemic diseases, immunodeficiency or autoimmune diseases, and the test site has not received skin treatment, beauty treatment or other tests that may affect the results; without active allergic diseases; without using hormone drugs and immunosuppressants in the past month; the test site has not participated in other clinical trials now or in the past 30 days; can cooperate well with the trial; can read and understand all the contents of the informed consent form and voluntarily sign the informed consent form; only use designated skin care products during the trial; reduce sugar and high-calorie food intake in the diet; agree not to use any cosmetics, drugs and health products that may affect the results during the trial.

[0162] 3.3.2 Exclusion criteria

[0163] Those with skin diseases that may affect their judgment of the test results; those with highly allergic constitutions; women who are pregnant, breastfeeding, or planning to become pregnant during the trial; those with severe heart, liver, and kidney damage and severe immunodeficiency; those with mental illness, severe endocrine diseases, and those taking oral contraceptives; those with significant nutritional disorders; those who have participated in drug clinical trials or other trials within 30 days, or have systemically used drugs that may affect the test results within the past week; those who have taken oral or external beauty products that may affect the test results within 2 weeks; those who cannot cooperate with the trial; those who are considered by the researchers to be unsuitable for participation in this study.

[0164] 3.4 Environmental conditions

[0165] The test environment conditions during the entire test process were controlled as follows: temperature 21±1°C, humidity 50±10%RH.

[0166] 3.5 Test Instruments

[0167]

[0168] 3.6 Test steps

[0169]

[0170] 3.7 Data Analysis

[0171] The statistical analysis software SPSS was used to perform statistical analysis of the data and calculate the mean, standard deviation and change rate of each parameter at each time point.

[0172] Calculation formula:

[0173] Change rate = [(value after use - value before use) / value before use] × 100%

[0174] 3.8 Result determination basis

[0175] ①Firming effect

[0176] Before and after using the sample, the skin elasticity R2 value and skin elasticity F3 / F4 value increased significantly (P < 0.050) or the skin firming parameter F4 value decreased significantly (P < 0.050). If any of the above parameters is significantly improved, the test sample is deemed to have a firming effect.

[0177] ② Anti-freckle and whitening effect

[0178] If the individual type angle ITA° value significantly increases (P < 0.050) or the skin melanin MI value significantly decreases (P < 0.050) before and after using the sample, the test sample is deemed to have the effect of removing spots and beautifying the skin.

[0179] ③Brightening effect

[0180] Before and after using the sample, the skin whitening L* value increased significantly (P < 0.050), and it was determined that the test sample had a brightening effect.

[0181] 4. Test Results

[0182] Skin melanin MI value (Mexameter MX18)

[0183] Table 7 Skin melanin MI value test results

[0184] D0 D28 Mean 141 135 Standard Deviation 32 32 Rate of change / -4.26％ p-value / ＜0.001 Difference Analysis / There is a very significant difference

[0185] The changes of skin melanin MI values ​​at different time points are shown in the figure Figure 1 .

[0186] Parameter description: The smaller the measured value, the lower the skin melanin content.

[0187] Result interpretation: Compared with before using the sample, the skin melanin M value of 30 subjects after using the sample for 28 days showed a very significant difference. This shows that after using the test sample for 28 days, it has the effect of removing spots and whitening.

[0188] Skin whiteness L* value (ColorimeterCL400)

[0189] Table 8 Skin whiteness L* value test results

[0190] D0 D28 Mean 60.75 61.46 Standard Deviation 2.94 3.13 Rate of change / 1.17％ p-value / ＜0.001 Difference Analysis / There is a very significant difference

[0191] The changes in skin whiteness L* values ​​at different time points are shown in Figure 2 .

[0192] Parameter description: The larger the measurement value, the brighter the skin tone.

[0193] Result interpretation: Compared with before using the sample, the skin whitening L* value of 30 subjects after using the sample for 28 days showed a very significant difference. This shows that after using the test sample for 28 days, there is a brightening effect.

[0194] Individual type angle ITA° value (ColorimeterCL400)

[0195] Table 9 Test results of individual type angle ITA° value

[0196] D0 D28 Mean 40 42 Standard Deviation 6 7 Rate of change / 5.00％ p-value / ＜0.001 Difference Analysis / There is a very significant difference

[0197] The changes in the individual type angle ITA° values ​​at different time points are shown in Figure 3 .

[0198] Parameter description: The larger the measurement value, the lighter the skin color.

[0199] Result interpretation: Compared with before using the sample, the individual type angle ITA° values ​​of 30 subjects after using the sample for 28 days showed extremely significant differences. This shows that after using the test sample for 28 days, it has the effect of removing spots and whitening.

[0200] Skin elasticity R2 value (CutometerMPA580)

[0201] Table 10 Skin elasticity R2 value test results

[0202] D0 D28 Mean 0.586 0.614 Standard Deviation 0.078 0.062 Rate of change / 4.78％ p-value / 0.004 Difference Analysis / There is a very significant difference

[0203] Changes in skin elasticity R2 values ​​at different time points are shown in Figure 4 .

[0204] Parameter description: The larger the measurement value, the better the skin elasticity.

[0205] Result interpretation: Compared with before using the sample, the skin elasticity R2 value of 30 subjects after using the sample for 28 days showed a very significant difference. This shows that after using the test sample for 28 days, it has a firming effect.

[0206] Skin elasticity F3 / F4 value (CutometerMPA580)

[0207] Table 11 Skin elasticity F3 / F4 value test results

[0208] D0 D28 Mean 0.491 0.518 Standard Deviation 0.070 0.064 Rate of change / 5.50％ p-value / 0.002 Difference Analysis / There is a very significant difference

[0209] Changes in skin elasticity F3 / F4 values ​​at different time points are shown in Figure 5 .

[0210] Parameter description: The larger the measurement value, the better the skin elasticity.

[0211] Result interpretation: Compared with before using the sample, the skin elasticity F3 / F4 values ​​of 30 subjects after using the sample for 28 days showed a very significant difference. This shows that after using the test sample for 28 days, there is a firming effect.

[0212] Skin firmness parameter F4 value (CutometerMPA580)

[0213] Table 12 Skin firming parameter F4 value test results

[0214] D0 D28 Mean 2.292 1.992 Standard Deviation 0.584 0.517 Rate of change / -13.09％ p-value / ＜0.001 Difference Analysis / There is a very significant difference

[0215] The changes of skin firmness parameter F4 value at different time points are shown in Figure 6 .

[0216] Parameter description: The smaller the measurement value, the better the skin firmness.

[0217] Result interpretation: Compared with before using the sample, the skin firming parameter F4 value of 30 subjects after using the sample for 28 days showed a very significant difference. This shows that after using the test sample for 28 days, there is a firming effect.

[0218] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that this is only for illustration and the protection scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but these changes and modifications all fall within the protection scope of the present invention.

Claims

1. An anti-glycation beverage containing a composite extract of white kidney bean and cherry blossom, characterized in that: Including ingredients: white kidney bean and cherry blossom compound extract, fish collagen peptide, erythritol, fish skin collagen peptide, Haematococcus pluvialis, L-arabinose, sodium hyaluronate and solvent; The preparation method of the white kidney bean and cherry blossom composite extract comprises the following steps: S1. Take peeled white kidney beans, crush and sieve to obtain white kidney bean powder, and take dried cherry blossoms from Guanshan Mountain and crush and sieve to obtain cherry blossom powder; S2. The white kidney bean powder and cherry blossom powder of step (1) are mixed uniformly in a mass ratio of (2-3):1, distilled water is added, and the mass ratio of the material to the liquid is 1:(3-5), and a composite enzyme is added at an amount of 200-300 mg / kg for uniform mixing and enzymolysis for 10-12 hours. After the enzymolysis is completed, the residue is removed by centrifugation, and the supernatant is taken, and the supernatant is inactivated to obtain a composite liquid; S3. scrape the activated Phellinus linteus slant with an inoculation loop, inoculate it into the seed culture medium, and culture it at 28-30°C for 3-4 days in a shaker at a speed of 150-200 r / min to obtain an acclimated composite bacterial solution; S4. The acclimated composite bacterial solution of step S3 was inoculated into the fermentation medium at an inoculum size of 9-12%, the temperature was 28-30 ° C, the stirring speed was 150-200r / min, the ventilation volume was 1-2vvm, and the fermentation culture was 10-15d to obtain a fermentation composite bacterial solution; S5. Take β-glucanase and add it to the fermentation composite bacterial solution obtained in step S4, enzymolysis for 2-3h, centrifuge and filter to remove the residue after the enzymolysis is completed, take the supernatant and spray dry to obtain a white kidney bean cherry composite extract; The complex enzyme includes β-glucanase, protease and mannanase, wherein the β-glucanase activity is 10-15U / mg, the protease activity is 2-5U / mg, and the mannanase activity is 12-15U / mg; The seed culture medium is composed of a carbon source, a nitrogen source, an inorganic salt, vitamins, the composite liquid of step S2 and water, wherein the amount of the composite liquid added is 50wt%; The fermentation medium is composed of a carbon source, a nitrogen source, an inorganic salt, vitamins, the composite liquid of step S2 and water, wherein the amount of the composite liquid added is 70 wt %.

2. The anti-glycation beverage containing the white kidney bean and cherry blossom compound extract according to claim 1, characterized in that: Calculated by mass percentage, it includes the following components: White kidney bean and cherry blossom compound extract 5-10%; Fish collagen peptide 2-3%; Erythritol 0.5-1%; Fish skin collagen peptide 1-2%; Haematococcus pluvialis 0.5-1%; L-arabinose 0.5-2%; Sodium hyaluronate 0.01-0.03%; Solvent Residue.

3. The anti-glycation beverage containing the white kidney bean and cherry blossom compound extract according to claim 1 or 2, characterized in that: The solvent is water.

4. The method for preparing the anti-glycation beverage containing the composite extract of white kidney bean and cherry blossom according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Weigh a certain amount of white kidney bean and cherry blossom compound extract, fish collagen peptide, erythritol, fish skin collagen peptide, Haematococcus pluvialis, L-arabinose, and sodium hyaluronate, put them into water, and stir them at a stirring rate of 20 r / min for 50 min to obtain a mixed solution; (2) Filtering the mixed solution, sterilizing and filling it after filtration to obtain the anti-glycation beverage.

5. The method for preparing the anti-glycation beverage containing the composite extract of white kidney bean and cherry blossom according to claim 4, characterized in that: The filtration in step (2) is performed using a filter bag filter, and the pore size of the filter bag is 600 meshes.

6. The method for preparing the anti-glycation beverage containing the white kidney bean and cherry blossom composite extract according to claim 4, characterized in that: The sterilization is carried out by high-temperature instantaneous sterilization, and the sterilization temperature is controlled at 120° C. to sterilize the mixed solution for 5 seconds.

7. The method for preparing the anti-glycation beverage containing the white kidney bean and cherry blossom composite extract according to claim 4, characterized in that: The filling is aseptic filling.

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