A low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract, preparation method thereof, and use thereof for brightening skin tone

Abstract

The present invention discloses a low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract, a preparation method thereof, and use thereof for brightening skin tone. Flesh, placenta, or a mixture thereof of Cucumis melo L. melon is crushed and homogenized under low-oxygen and low-temperature conditions, extracted with a food-grade buffer, and subjected to centrifugation, microfiltration, ultrafiltration concentration, and diafiltration to remove small-molecule sugars, organic acids, and free salts. The pH and ionic strength of the resulting SOD-enriched liquid are adjusted to form a composite stabilization system between SOD protein and melon endogenous soluble polysaccharides, followed by low-temperature drying. The extract has low reducing sugar content, low water activity, low lipid content, and improved SOD stability in an acidic medium, contains no exogenously added prolamin or lipid-soluble coating layer, and is suitable for preparing oral nutritional products for non-therapeutic purposes of scavenging free radicals and brightening skin tone.

Description

[0001] DESCRIPTION

[0002] A Low-Reducing-Sugar, High-Stability SOD Melon Endogenous Polysaccharide-Protein Composite Extract, Preparation Method Thereof, and Use Thereof for Brightening Skin Tone

[0003] Technical Field

[0004] The present invention belongs to the technical fields of plant extracts, food processing, and oral nutritional products, and particularly relates to a low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract, a preparation method thereof, and an application thereof for brightening skin tone.

[0005] Background Art

[0006] Dull skin, uneven skin tone, and decreased radiance are associated with factors such as oxidative stress, free-radical reactions induced by ultraviolet rays and environmental stress, and activation of signaling pathways related to melanogenesis. Excessive production of reactive oxygen species can attack lipids, proteins, and DNA in skin cells, resulting in impaired cell function, collagen degradation, and abnormal melanin deposition. Oral nutritional products assist in maintaining the normal condition of the skin by supplementing antioxidant-related nutrients and have become an important direction in the development of oral beauty products.

[0007] Superoxide dismutase (SOD) is an antioxidant enzyme capable of catalyzing the dismutation of superoxide anion radicals and plays a key role in scavenging oxygen free radicals in organisms. SOD may be derived from animals, microorganisms, or plants. Melon (Cucumis melo L.) is one of the important raw material sources of plant-derived SOD, and certain melon varieties are particularly rich in SOD.DESCRIPTION

[0008] SOD protein extracts derived from Cucumis melo have been disclosed in the prior art. For example, GliSODin® is the world's first patented orally bioactive SOD product, which enables the antioxidant activity of SOD to function effectively in vivo by extracting a natural SOD-rich melon extract and combining it with gliadin. In addition, technical solutions have also been disclosed in which melon extract is compounded with vitamin E, selenium-enriched yeast, and other components for antioxidation. The above technologies can improve the application stability or oral applicability of SOD to a certain extent, but still have the following problems:

[0009] First, ordinary freeze-dried melon juice contains relatively large amounts of small-molecule sugars, organic acids, and free salts, and has high hygroscopicity, which is not conducive to the stability of hard-capsule powder. Existing plant SOD extraction methods mostly employ direct pulping, juice pressing, and freeze drying. The obtained products have high reducing sugar content and are prone to moisture absorption and caking, and are not suitable for preparing solid preparations such as hard capsules.

[0010] Second, lipid-soluble coating or fatty microencapsulation systems are not conducive to obtaining low-lipid, hydrophilically dispersible hard-capsule powders, and the relatively high lipid content limits their application in low-lipid products.

[0011] Third, a prolamin protection system, such as one using gliadin, is not suitable for consumption scenarios involving gluten sensitivity, requirements for no prolamin, or plant-based clean labels.

[0012] Fourth, using only SOD activity as a quality index makes it difficult to distinguish ordinary melon SOD extracts from stabilized extracts suitable for oral hard capsules, and there is a lack of systematic control over formulation-relatedDESCRIPTION

[0013] indicators such as reducing sugar content, water activity, and acidic-medium stability.

[0014] Summary of the Invention

[0015] To overcome the above defects in the prior art, the present invention provides a low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract, a preparation method thereof, and an application thereof for brightening skin tone, thereby solving the problems in the prior art that melon SOD extracts have high reducing sugar content and are prone to moisture absorption, rely on prolamin or lipid-soluble coatings to improve stability, are not suitable for gluten-free, low-lipid, and hard-capsule applications, and lack multi-dimensional quality control indicators.

[0016] To achieve the above purpose, the present invention provides the following technical solution:

[0017] A low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract, a preparation method thereof, and an application thereof for brightening skin tone comprise the following steps:

[0018] (1) taking the flesh, placenta, or a mixture thereof of a Cucumis melo L. melon, and crushing and homogenizing the same at 2-10°C under low-oxygen conditions to obtain a melon slurry;

[0019] (2) adding a food-grade buffer having a pH of 6.4-7.6 to the melon slurry, and extracting at 2-15°C for 0.5-3 hours to obtain a crude extract;

[0020] (3) subjecting the crude extract to low-temperature centrifugation and microfiltration to remove insoluble impurities, thereby obtaining a clarified extract;

[0021] (4) subjecting the clarified extract to ultrafiltration concentration andDESCRIPTION

[0022] diafiltration to retain components having a molecular weight greater than 10 kDa and to remove small-molecule sugars, organic acids, and free salts, thereby obtaining an SOD-enriched liquid;

[0023] (5) adjusting the pH and ionic strength of the SOD-enriched liquid, and allowing the same to stand at 2-15°C so that the SOD protein therein and the melon endogenous soluble polysaccharides form a composite stabilization system, thereby obtaining a composite stabilized liquid;

[0024] (6) subjecting the composite stabilized liquid to low-temperature drying, pulverizing, and sieving to obtain the extract.

[0025] Preferably, the low-oxygen conditions in step (1) are nitrogen protection, vacuum deoxygenation, or controlling the dissolved oxygen in the material system to be not higher than 3 mg / L.

[0026] Preferably, the food-grade buffer in step (2) is a phosphate buffer, a citrate buffer, or a combination thereof, and no gliadin, zein, hordein, secalin, or other exogenous prolamin is added to the food-grade buffer.

[0027] Preferably, the ultrafiltration concentration and diafiltration in step (4) are performed using an ultrafiltration membrane having a molecular weight cut-off of 10-30 kDa; the amount of water used for diafiltration is 2-8 times the volume of the SOD-enriched liquid; and the endpoint of diafiltration is that the soluble solids of the permeate are not higher than 0.5°Brix or the conductivity of the permeate is not higher than 3.0 mS / cm.

[0028] Preferably, in step (5), the pH is adjusted to 6.2-7.2, the ionic strength is adjusted to 10-80 mmol / L, and the standing time is 0.5-6 hours; and the mass ratio of the melon endogenous soluble polysaccharides to the total protein in the composite stabilized liquid is 0.2-3.0:!.DESCRIPTION

[0029] Preferably, the low-temperature drying in step (6) is freeze drying or low-temperature vacuum drying; the freeze drying comprises pre-freezing at -20°C to -60°C for 2-12 hours and drying under vacuum for 12-48 hours; and, during the low-temperature vacuum drying, the material temperature is not higher than 45°C.

[0030] Preferably, the low-reducing- sugar, high- stability SOD melon endogenous polysaccharide-protein composite extract contains, based on the total dry weight of the extract:

[0031] (1) SOD activity of 10,000-80,000 U / g, wherein the SOD activity is measured by the WST-1 method;

[0032] (2) total protein content of 8%-35%;

[0033] (3) melon endogenous soluble polysaccharide content of 5%-30%;

[0034] (4) reducing sugar content of not higher than 8%;

[0035] (5) moisture content of not higher than 8%;

[0036] (6) water activity aw of not higher than 0.45;

[0037] (7) lipid content of not higher than 2%;

[0038] (8) after treatment in a pH 1.2 acidic medium at 37°C for 60 minutes and neutralization, an SOD activity retention rate of not less than 35%;

[0039] wherein the extract contains no exogenously added prolamin, and contains no lipid-soluble coating layer, microencapsulation wall material, or prolamin embedding layer.

[0040] Preferably, the extract has:

[0041] (1) SOD activity of 20,000-60,000 U / g;

[0042] (2) total protein content of 10%-28%;

[0043] (3) melon endogenous soluble polysaccharide content of 8%-25%;

[0044] (4) reducing sugar content of not higher than 6%;DESCRIPTION

[0045] (5) water activity aw of not higher than 0.35;

[0046] (6) after treatment in a pH 1.2 acidic medium for 60 minutes, an SOD activity retention rate of not less than 45%;

[0047] (7) after storage at 40°C and 75% RH for 30 days, an SOD activity retention rate of not less than 65%.

[0048] Preferably, an oral nutritional composition comprises the extract and a food-acceptable excipient.

[0049] Preferably, the oral product is used, for non-therapeutic purposes, for scavenging free radicals, improving dull skin, brightening skin tone, improving skin tone uniformity, increasing skin L* value, improving ITA°, or reducing melanin index; wherein the oral product is a hard capsule, tablet, granule, powder, solid beverage, or compressed candy.

[0050] The technical effects and advantages of the low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract, the preparation method thereof, and the application thereof for brightening skin tone according to the present invention are as follows:

[0051] 1. The invention reduces the risk of oxidative inactivation of SOD during crushing, extraction, and concentration under low-oxygen and low-temperature conditions, which is beneficial for maintaining the enzymatic activity of plant-derived SOD.

[0052] 2. The invention removes small-molecule sugars, organic acids, and free salts through ultrafiltration concentration and diafiltration, reduces the reducing sugar content and hygroscopic tendency of the extract, and improves the storage stability of the powder in hard capsules, powders, and tablets.

[0053] 3. The invention utilizes the soluble polysaccharides contained in the melonDESCRIPTION

[0054] raw material itself to form an endogenous polysaccharide-protein composite stabilization system with the SOD protein; does not use lipid-soluble coating materials, hydrogenated oils, waxes, fatty microencapsulation coatings, gliadin, or other prolamins; and is suitable for gluten-free, low-lipid, and hard-capsule powder product development.

[0055] 4. The extract of the invention uses SOD activity, total protein, endogenous soluble polysaccharides, reducing sugars, water activity, lipid content, acidic-medium activity retention rate, and accelerated stability as quality control indicators, thereby distinguishing it from ordinary freeze-dried melon juice, lipid-soluble coated SOD melon powder, and prolamin-protected SOD products.

[0056] 5. The extract of the invention can be used in the development of oral nutritional products for non-therapeutic purposes of scavenging free radicals and brightening skin tone, and its application directions include improving dull skin, improving skin tone uniformity, increasing skin L* value, improving ITA°, and reducing melanin-related indicators.

[0057] Brief Description of the Drawing

[0058] FIG. 1 is a flow chart of the low-reducing-sugar, high- stability SOD melon endogenous polysaccharide-protein composite extract, the preparation method thereof, and the application thereof for brightening skin tone proposed by the present invention.

[0059] Detailed Description of the Embodiments

[0060] The technical solutions in the embodiments of the present invention will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, ratherDESCRIPTION

[0061] than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0062] It should be noted that, herein, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In addition, the terms comprise, include, or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or device. Without further limitations, an element defined by the statement comprising ... does not exclude the presence of another identical element in the process, method, article, or device comprising said element.

[0063] Referring to FIG. 1, the present invention provides a low-reducing-sugar, high- stability SOD melon endogenous polysaccharide-protein composite extract, a preparation method thereof, and an application thereof for brightening skin tone, belonging to the technical field of plant extracts and oral nutritional products. The preparation method comprises: taking flesh and / or placenta of Cucumis melo L. melon, crushing and homogenizing the same at 2-10°C under low-oxygen conditions, adding a pH 6.4-7.6 food-grade buffer, and extracting at 2-15°C; then performing low-temperature centrifugation, microfiltration, ultrafiltration concentration with a molecular weight cut-off greater than 10 kDa, and diafiltration to remove small-molecule sugars, organic acids, and free salts to obtain an SOD-enriched liquid; adjusting pH to 6.2-7.2 and ionic strength to 10-80DESCRIPTION

[0064] mmol / L, and allowing the same to stand at 2-15°C so that SOD protein and melon endogenous soluble polysaccharides form a composite stabilization system; and finally performing low-temperature drying to obtain the extract. The extract has SOD activity of 10,000-80,000 U / g, total protein of 8%-35%, endogenous polysaccharides of 5%-30%, reducing sugars of <8%, moisture of <8%, water activity of <0.45, lipid of <2%, and an SOD activity retention rate of >35% after pH 1.2 treatment for 60 minutes, and contains no exogenous prolamin or lipid-soluble coating layer. The present invention further provides an oral nutritional composition containing the extract and the use thereof in preparing a non-therapeutic oral product for scavenging free radicals, improving dull skin, brightening skin tone, improving skin tone uniformity, increasing skin L* value, improving ITA°, or reducing melanin index.

[0065] Example 1

[0066] Preparation of a low-reducing-sugar, high- stability SOD melon endogenous polysaccharide-protein composite extract.

[0067] Cucumis melo L. melons having consistent maturity and free from rot and mechanical damage were selected. The flesh and placenta parts were taken, surface impurities were removed, and the materials were cut into pieces. The raw materials were pre-cooled to 4-8°C and crushed and homogenized under nitrogen protection to obtain a melon slurry.

[0068] A food-grade phosphate buffer at pH 7.0 was added to the melon slurry, the solid-liquid ratio was controlled at 1:6, and extraction was carried out under stirring at 6-10°C for 1.5 hours to obtain a crude extract.

[0069] The crude extract was centrifuged at 4-8°C under a centrifugal force of 8,000 g for 20 minutes, and the supernatant was collected. The supernatant was filteredDESCRIPTION

[0070] through a 0.45 gm microfiltration membrane to obtain a clarified extract.

[0071] The clarified extract was concentrated through an ultrafiltration membrane having a molecular weight cut-off of 10 kDa, and diafiltered with purified water, wherein the amount of water used for diafiltration was 5 times the volume of the concentrate. The endpoint of diafiltration was controlled so that the soluble solids of the permeate were not higher than 0.5°Brix. An SOD-enriched liquid was obtained.

[0072] The SOD-enriched liquid was adjusted to pH 6.8, the ionic strength was controlled at 30-60 mmol / L, and the liquid was allowed to stand at 4-8°C for 2 hours, so that the SOD protein and the melon endogenous soluble polysaccharides formed a composite stabilization system, thereby obtaining a composite stabilized liquid.

[0073] The composite stabilized liquid was pre-frozen at -40°C for 6 hours, followed by vacuum freeze drying for 24-36 hours. After drying, the material was pulverized and passed through a 60-mesh sieve to obtain the low-reducing-sugar, high- stability SOD melon endogenous polysaccharide-protein composite extract.

[0074] Example 2

[0075] Extraction under different pH conditions.

[0076] An SOD melon extract was prepared according to the method of Example 1 , except that the pH values of the extraction buffer were controlled at 6.4, 6.8, 7.2, and 7.6, respectively. The resulting extracts were processed by the same centrifugation, microfiltration, ultrafiltration, diafiltration, composite stabilization, and freeze-drying steps.

[0077] SOD activity, total protein content, reducing sugar content, water activity, and activity retention rate in an acidic medium were used as evaluation indicators toDESCRIPTION

[0078] screen process conditions suitable for maintaining SOD activity and reducing reducing sugar content.

[0079] Example 3

[0080] Preparation under different membrane separation conditions.

[0081] An SOD melon extract was prepared according to the method of Example 1 , except that the molecular weight cut-offs of the ultrafiltration membrane were 10 kDa, 20 kDa, and 30 kDa, respectively, and purified water in amounts of 2 times, 5 times, and 8 times the volume of the SOD-enriched liquid was used for diafiltration, respectively.

[0082] The effects of membrane separation conditions on the quality of the final extract were evaluated by reducing sugar content, SOD activity retention rate, total protein content, soluble polysaccharide content, and powder hygroscopicity.

[0083] Example 4

[0084] Preparation by low-temperature vacuum drying.

[0085] A composite stabilized liquid was obtained according to the method of Example 1, except that the composite stabilized liquid was subjected to low-temperature vacuum drying while controlling the material temperature to be not higher than 45°C, and dried until the moisture content was not higher than 8%. After drying, the material was pulverized and passed through a 60-mesh sieve to obtain the low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract.

[0086] Comparative Example 1

[0087] Ordinary freeze-dried melon juice.

[0088] The same melon raw material as in Example 1 was taken. After crushing and homogenizing, the material was centrifuged and the supernatant was collected.DESCRIPTION

[0089] Without ultrafiltration for sugar removal and without endogenous polysaccharide-protein composite stabilization, the supernatant was directly freeze-dried to obtain ordinary freeze-dried melon juice.

[0090] Comparative Example 2

[0091] SOD melon extract without diafiltration.

[0092] Preparation was carried out according to the method of Example 1 , except that the clarified extract was only subjected to ultrafiltration concentration without diafiltration, and the other steps were the same.

[0093] Comparative Example 3

[0094] SOD melon extract without endogenous polysaccharide-protein composite stabilization.

[0095] Preparation was carried out according to the method of Example 1 , except that the SOD-enriched liquid was not subjected to pH adjustment, ionic-strength adjustment, and standing for composite stabilization, but was directly freeze-dried.

[0096] Detection Methods:

[0097] Determination of SOD activity: the WST-1 method was used as the primary detection method, and the NBT reduction method or pyrogallol autoxidation method may be used for methodological comparison. An activity unit is defined as the amount of enzyme required to achieve a 50% inhibition rate of the color reaction in the reaction system under the specified assay conditions.

[0098] Determination of total protein content: the Kjeldahl method, Bradford method, or BCA method may be used.

[0099] Determination of melon endogenous soluble polysaccharide content: the phenol- sulfuric acid method may be used, calculated using glucose as the standard.

[0100] Characterization of the composite stabilization system: dynamic lightDESCRIPTION

[0101] scattering, SEC-HPLC, Zeta potential, FT-IR, or the mass ratio of endogenous soluble polysaccharides to total protein may be used to characterize the composite stabilized state, and comparison may be made with a comparative example not subjected to composite stabilization treatment.

[0102] Determination of reducing sugar content: the DNS method or high-performance liquid chromatography may be used.

[0103] Determination of moisture content: direct drying or Karl Fischer titration may be used. Water activity is measured using a water activity meter.

[0104] Determination of lipid content: Soxhlet extraction or acid hydrolysis may be used.

[0105] Determination of stability in a pH 1.2 acidic medium: the extract of the present invention and samples of comparative examples were respectively placed in a pH 1.2 acidic medium and treated at 37°C for 60 minutes. After treatment, the pH was adjusted to a value suitable for SOD activity measurement, and residual SOD activity was measured by the WST-1 method to calculate the SOD activity retention rate.

[0106] Determination of accelerated stability: the extract of the present invention was sealed and placed at 40°C and 75% RH for 30 days. Samples were taken to determine SOD activity, which was compared with the initial SOD activity to calculate the activity retention rate.

[0107] Determination of melanin-related indicators: an a-MSH-induced B16-F10 melanoma cell model was used. The cells were divided into a blank control group, an a-MSH model group, an extract low-dose group, an extract medium-dose group, an extract high-dose group, and a comparative example group. Each administration group was treated at a non-cytotoxic concentration for 48-72 hours, and cellDESCRIPTION

[0108] viability, melanin production, and tyrosinase activity were detected; the expression levels of MITF, TYR, TRP-1, or TRP-2 may be further detected.

[0109] Skin color evaluation: when used for human dietary evaluation, a randomized, double-blind, placebo-controlled design may be adopted, with continuous intake for 8-12 weeks, and skin L* value, ITA°, melanin index, erythema index, VISIA image indicators, skin tone uniformity, and self-assessment scores for dullness may be measured.

[0110] Experimental Data:

[0111] Experimental Example 1

[0112] Comparison of quality indicators of extracts obtained by different processes: SOD activity was determined by the WST-1 method, total protein by the BCA method, soluble polysaccharides by the phenol-sulfuric acid method, reducing sugars by the DNS method, moisture by the 105°C drying method, aw by a water activity meter, and lipid content by Soxhlet extraction. Each group was measured in triplicate, and the results are expressed as mean ± standard deviation.

[0113] TOTAL REDUCING SOD ACTIVITY SOLUBLE POLYS. MOISTURE LIPID SAMPLE PROTEIN SUGAR AW

[0114] (U / G) (%) (%) (%) (%) (%)

[0115] Comp. Ex. 1 16,820±640 8.7±0.4 4.6±0.3 25.4±L1 5.9±0.2 0.51±0.01 0.62±0.05

[0116] Comp. Ex. 2 31,450±920 16.8±0.6 6.9±0.4 5.5±0.3 4.8±0.2 0.32±0.01 0.71±0.04

[0117] Comp. Ex. 3 24,780±810 12.3±0.5 18.4±0.7 18.2±0.8 6.H0.3 0.49±0.02 0.83±0.06

[0118] Ex. 1 43,620±l,180 21.7±0.8 15.H0.6 3.7±0.2 4.5±0.2 0.31±0.01 0.79±0.05

[0119] Ex. 2 38,740±l,060 18.4±0.7 12.5±0.5 4.5±0.2 4.3±0.2 0.29±0.01 0.74±0.04

[0120]

[0121] DESCRIPTION

[0122] TOTAL REDUCING SOD ACTIVITY SOLUBLE POLYS. MOISTURE LIPID SAMPLE PROTEIN SUGAR AW

[0123] (U / G) (%) (%) (%) (%) (%)

[0124] Ex. 3 34,280±970 19.5±0.6 10.8±0.5 4.1±0.2 5.H0.2 0.34±0.01 0.88±0.05

[0125]

[0126] The results show that, compared with ordinary freeze-dried melon juice, Examples 1 -3 have higher SOD activity, lower reducing sugar content, lower water activity, and lower lipid content, indicating that the process of the present invention can obtain a low-reducing-sugar, high- stability SOD melon extract more suitable for oral hard-capsule applications.

[0127] Experimental Example 2

[0128] Stability test in a pH 1.2 acidic medium:

[0129] Each sample was prepared as a 1 mg / mL suspension and placed in a pH 1.2 acidic medium, treated at 37°C for 30, 60, and 120 minutes, sampled, and then neutralized to pH 7.0. SOD activity was measured by the WST-1 method. Each group was measured in triplicate, and the retention rate was calculated using the activity at 0 minute as 100%.

[0130] SAMPLE 0 MIN 30 MIN 60 MIN 120 MIN

[0131] Comp. Ex. 1 100.0% 3L4±L8% 16.9±1.2% 8.6±0.7%

[0132] Comp. Ex. 2 100.0% 45.8±2.1% 29.7±L6% 15.9±L0%

[0133] Comp. Ex. 3 100.0% 48.5±2.0% 38.2±L7% 23.3±L2%

[0134] Ex. 1 100.0% 60.1±2.4% 47.8±2.1% 31.6±1.5%

[0135] Ex. 2 100.0% 56.4±2.2% 44.6±L9% 29.3±L4%

[0136]

[0137] DESCRIPTION

[0138] SAMPLE 0 MIN 30 MIN 60 MIN 120 MIN

[0139] Ex. 3 100.0% 52.8±2.0% 41.7±1.8% 25.5±1.3%

[0140]

[0141] The results show that the SOD activity retention rates of Examples 1-3 after treatment in the pH 1.2 acidic medium for 60 minutes were 41.7%-47.8%, higher than those of the comparative examples, indicating that ultrafiltration / diafiltration sugar removal and endogenous polysaccharide-protein composite stabilization can improve the activity retention rate of the SOD melon extract in an acidic environment.

[0142] Experimental Example 3

[0143] Accelerated stability test:

[0144] Each sample was sealed in an aluminum foil bag and placed at 40°C and 75% RH. Samples were taken on days 0, 30, 60, and 90 to determine SOD activity. Each group was measured in triplicate, and the retention rate was calculated using the activity on day 0 as 100%.

[0145] SAMPLE 0 DAY 30 DAYS 60 DAYS 90 DAYS

[0146] Comp. Ex. 1 100.0% 42.1±2.0% 26.5±L5% 17.6±1.1%

[0147] Comp. Ex. 2 100.0% 58.6±2.3% 42.8±L9% 3L2±L5%

[0148] Comp. Ex. 3 100.0% 5L8±2.1% 34.5±L7% 22.8±L2%

[0149] Ex. 1 100.0% 72.6±2.5% 59.4±2.2% 48.5±L9%

[0150] Ex. 2 100.0% 70.2±2.4% 56.6±2.0% 44.7±L8%

[0151] Ex. 3 100.0% 66.5±2.2% 5L4±L9% 39.8±1.6%

[0152]

[0153] The results show that, after storage at 40°C and 75% RH for 30 days, the SOD activity retention rates of Examples 1-3 remained 66.5%-72.6%, higher thanDESCRIPTION

[0154] those of the comparative examples, indicating that the extract of the present invention has good storage stability.

[0155] Experimental Example 4

[0156] Hygroscopicity and powder flowability test:

[0157] Each sample was placed in an open state at 25°C and 75% RH for 24 hours. The hygroscopic weight gain rate and angle of repose were measured, and the powder appearance was observed.

[0158] 24 H WEIGHT INITIAL ANGLE 24 H ANGLE

[0159] SAMPLE APPEARANCE GAIN (%) (°) (°)

[0160] Comp. Ex. 1 12.3±0.6 45.1±L2 52.6±L5 Obvious caking

[0161] Comp. Ex. 2 6.9±0.4 39.5±1.0 44.7±L2 Slightly hygroscopic

[0162] Comp. Ex. 3 10.4±0.5 43.6±1.1 50.3±L4 Obvious caking

[0163] Ex. 1 4.2±0.3 34.7±0.8 38.1±0.9 No obvious caking

[0164] Ex. 2 4.6±0.3 35.4±0.9 39.2±1.0 No obvious caking

[0165] Ex. 3 5.2±0.3 37.0±0.9 41.3±1.1 Slightly hygroscopic

[0166]

[0167] The results show that Examples 1-3 have lower hygroscopic weight gain rates and smaller angles of repose, and the powder flowability is superior to that of the comparative examples, making them more suitable for preparation into hard capsules, tablets, granules, or powders.

[0168] Experimental Example 5

[0169] In vitro antioxidant capacity test:

[0170] DPPH, ABTS, and ORAC methods were used to evaluate the in vitro antioxidant capacity of each sample. The DPPH and ABTS scavenging rates wereDESCRIPTION

[0171] measured at a sample concentration of 1 mg / mL, and ORAC results are expressed as pmol TE / g.

[0172] ORAC SAMPLE DPPH (%) ABTS (%)

[0173] (pMOL TE / G)

[0174] Comp. Ex. 1 37.6±L5 45.3±L8 982±45

[0175] Comp. Ex. 2 52.4±L9 59.5±2.0 1,675±68

[0176] Comp. Ex. 3 48.2±L7 55.4±L9 1,428±61

[0177] Ex. 1 63.7±2.1 71.6±2.3 2,416±82

[0178] Ex. 2 60.5±2.0 68.8±2.2 2,228±79

[0179] Ex. 3 57.8±1.9 65.7±2.1 2,064±73

[0180]

[0181] The results show that the DPPH and ABTS scavenging capacities and ORAC values of Examples 1-3 are all higher than those of the comparative examples, indicating that the SOD melon extract obtained by the process of the present invention has good in vitro antioxidant capacity.

[0182] Experimental Example 6

[0183] HaCaT cell ROS inhibition test:

[0184] Human keratinocyte HaCaT cells were used to establish an H2O2-induced oxidative stress model. The sample treatment concentrations were 25, 50, and 100 pg / mL. Cell viability was detected by the CCK-8 method, and intracellular ROS levels were detected using a DCFH-DA fluorescent probe. ROS levels were expressed with the model group as 100%.

[0185] CELL VIABILITY ROS LEVEL GROUP CONC.

[0186] (%) (MODEL=100%)

[0187]

[0188] DESCRIPTION

[0189] CELL VIABILITY ROS LEVEL GROUP CONC.

[0190] (%) (MODEL=100%)

[0191] Blank control 0 100.0±3.2 52.6±2.4

[0192] H2O2 model 0 78.4±2.7 100.0±3.5

[0193] Comp. Ex. 1 100 pg / rnL 84.5±2.9 84.6±3.1

[0194] Comp. Ex. 2 100 pg / rnL 88.2±3.0 75.8±2.8

[0195] Ex. 1 low dose 25 pg / rnL 91.6±3.1 82.5±2.9

[0196] Ex. 1 medium dose 50 pg / rnL 93.4±3.2 73.6±2.7

[0197] Ex. 1 high dose 100 pg / rnL 94.0±3.0 64.9±2.5

[0198]

[0199] The results show that Example 1 exhibited no obvious cytotoxicity within the range of 25-100 pg / mL and reduced H2O2 -induced intracellular ROS levels in HaCaT cells, indicating its potential for antioxidant protection of skin cells.

[0200] Experimental Example 7

[0201] B16-F10 melanogenesis test:

[0202] B16-F10 mouse melanoma cells were used, and melanogenesis was induced by a-MSH. Sample concentrations were set at 25, 50, and 100 pg / mL. After 72 hours of treatment, cell viability, melanin content, and tyrosinase activity were detected. Melanin content and tyrosinase activity were expressed with the a-MSH model group as 100%.

[0203] CELL MELANIN TYROSINASE GROUP CONC. VIABILITY

[0204] (MODEL=100%) (MODEL=100%) (%)

[0205]

[0206] DESCRIPTION

[0207] CELL MELANIN TYROSINASE GROUP CONC. VIABILITY

[0208] (MODEL=100%) (MODEL=100%)

[0209] (%)

[0210] Blank control 0 100.0±3.1 58.6±2.5 62.3±2.7

[0211] a-MSH model 0 100.0±3.3 100.0±3.6 100.0±3.4

[0212] Comp. Ex. 1 100 pg / mL 95.2±3.0 91.6±3.1 93.4±3.0

[0213] Comp. Ex. 2 100 pg / mL 94.5±2.8 86.9±2.9 89.2±2.8

[0214] Ex. 1 low dose 25 pg / mL 98.4±3.2 92.5±3.0 94.6±3.1

[0215] Ex. 1 medium dose 50 pg / mL 96.5±3.1 84.8±2.8 88.4±2.7

[0216] Ex. 1 high dose 100 pg / mL 93.4±2.9 76.3±2.6 80.2±2.5

[0217] Arbutin positive control 200 pM 91.3±2.8 63.8±2.4 67.4±2.3

[0218]

[0219] The results show that Example 1 did not significantly affect B16-F10 cell viability within the range of 25-100 pg / mL, and reduced a-MSH-induced melanin production and tyrosinase activity in a dose-related manner, indicating that it can be used in the development of oral nutritional products for non-therapeutic purposes of brightening skin tone, improving dullness, and improving skin tone uniformity.

[0220] Quality control requirements: the low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract prepared by the method of the present invention should meet the following quality control requirements: SOD activity measured by the WST-1 method is 10,000-80,000 U / g; total protein content is 8%-35%; melon endogenous soluble polysaccharide content is 5%-30%; reducing sugar content is not higher than 8%; moisture content is notDESCRIPTION

[0221] higher than 8%; water activity aw is not higher than 0.45; lipid content is not higher than 2%; after treatment in a pH 1.2 acidic medium at 37°C for 60 minutes and neutralization, SOD activity retention rate is not less than 35%. The extract contains no exogenously added prolamin, and contains no lipid-soluble coating layer, microencapsulation wall material, or prolamin embedding layer.

[0222] Preferably, the extract meets the following quality control requirements: SOD activity measured by the WST-1 method is 20,000-60,000 U / g; total protein content is 10%-28%; melon endogenous soluble polysaccharide content is 8%-25%; reducing sugar content is not higher than 6%; water activity aw is not higher than 0.35; after treatment in a pH 1.2 acidic medium at 37°C for 60 minutes and neutralization, SOD activity retention rate is not less than 45%; after storage at 40°C and 75% RH for 30 days, SOD activity retention rate is not less than 65%.

[0223] Application Mode:

[0224] The low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract of the present invention can be used as an active plant extract raw material for oral nutritional products, and used in hard capsules, tablets, granules, powders, solid beverages, or compressed candies.

[0225] The present invention further provides an oral nutritional composition comprising the above extract and a food-acceptable excipient. The food-acceptable excipient includes, but is not limited to, a filler (such as microcrystalline cellulose, starch, or lactose), a glidant (such as silicon dioxide or magnesium stearate), or an anti-caking agent.

[0226] When used in hard capsules, the extract may be mixed with a food-acceptable filler, glidant, or anti-caking agent, and then filled into HPMC hard capsules or gelatin hard capsules. The daily intake amount of the SOD melon endogenousDESCRIPTION

[0227] polysaccharide-protein composite extract in the hard capsule may be 5-100 mg, preferably 10-50 mg.

[0228] The oral product is a non-therapeutic product and is not used for the diagnosis, treatment, or prevention of diseases. Its uses include scavenging free radicals, improving dull skin, brightening skin tone, improving skin tone uniformity, increasing skin L* value, improving ITA°, or reducing melanin index.

[0229] Industrial Applicability:

[0230] The preparation method of the present invention uses low-temperature crushing, centrifugation, microfiltration, ultrafiltration, diafiltration, and low-temperature drying equipment available in the field of food processing. The process route is clear and can realize large-scale production. The obtained low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract has clear quality control indicators and application directions in oral nutritional products, is suitable for the development of products such as hard capsules, tablets, granules, powders, and solid beverages, and has good industrial applicability.

[0231] The above are merely specific embodiments of the present application, and the protection scope of the present application is not limited thereto. Any changes or substitutions that can be readily conceived by a person skilled in the art within the technical scope disclosed by the present application shall be covered by the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

[0232] Finally, the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent substitution, improvement, or the like made within the spirit and principles of theDESCRIPTION

[0233] present invention shall be included within the protection scope of the present invention.

Claims

CLAIMS1. A preparation method of a low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract, characterized by comprising the following steps:(1) taking the flesh, placenta, or a mixture thereof of a Cucumis melo L. melon, and crushing and homogenizing the same at 2-10°C under low-oxygen conditions to obtain a melon slurry;(2) adding a food-grade buffer having a pH of 6.4-7.6 to the melon slurry, and extracting at 2-15°C for 0.5-3 hours to obtain a crude extract;(3) subjecting the crude extract to low-temperature centrifugation and microfiltration to remove insoluble impurities, thereby obtaining a clarified extract;(4) subjecting the clarified extract to ultrafiltration concentration and diafiltration to retain components having a molecular weight greater than 10 kDa and to remove small-molecule sugars, organic acids, and free salts, thereby obtaining an SOD-enriched liquid;(5) adjusting the pH and ionic strength of the SOD-enriched liquid, and allowing the same to stand at 2-15°C so that the SOD protein therein and the melon endogenous soluble polysaccharides form a composite stabilization system, thereby obtaining a composite stabilized liquid;(6) subjecting the composite stabilized liquid to low-temperature drying, pulverizing, and sieving to obtain the extract.

2. The preparation method of the low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract according to claim 1 , characterized in that the low-oxygen conditions in step (1) are nitrogen protection, vacuum deoxygenation, or controlling the dissolved oxygen in the material system to be not higher than 3 mg / L.CLAIMS3. The preparation method of the low-reducing-sugar, high- stability SOD melon endogenous polysaccharide-protein composite extract according to claim 1, characterized in that the food-grade buffer in step (2) is a phosphate buffer, a citrate buffer, or a combination thereof, and no gliadin, zein, hordein, secalin, or other exogenous prolamin is added to the food-grade buffer.

4. The preparation method of the low-reducing-sugar, high- stability SOD melon endogenous polysaccharide-protein composite extract according to claim 1, characterized in that the ultrafiltration concentration and diafiltration in step (4) are performed using an ultrafiltration membrane having a molecular weight cut-off of 10-30 kDa; the amount of water used for diafiltration is 2-8 times the volume of the SOD-enriched liquid; and the endpoint of diafiltration is that the soluble solids of the permeate are not higher than 0.5°Brix or the conductivity of the permeate is not higher than 3.0 mS / cm.

5. The preparation method of the low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract according to claim 1 , characterized in that, in step (5), the pH is adjusted to 6.2-7.2, the ionic strength is adjusted to 10-80 mmol / L, and the standing time is 0.5-6 hours; and the mass ratio of the melon endogenous soluble polysaccharides to the total protein in the composite stabilized liquid is 0.2-3.0:l.

6. The preparation method of the low-reducing-sugar, high- stability SOD melon endogenous polysaccharide-protein composite extract according to claim 1, characterized in that the low-temperature drying in step (6) is freeze drying or low-temperature vacuum drying; the freeze drying comprises pre-freezing at -20°C to -60°C for 2-12 hours and drying under vacuum for 12-48 hours; and, during the low-temperature vacuum drying, the material temperature is not higher than 45°C.CLAIMS7. The low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract prepared by the method according to any one of claims 1-6, characterized in that, based on the total dry weight of the extract, the extract contains:(1) SOD activity of 10,000-80,000 U / g, wherein the SOD activity is measured by the WST-1 method;(2) total protein content of 8%-35%;(3) melon endogenous soluble polysaccharide content of 5%-30%;(4) reducing sugar content of not higher than 8%;(5) moisture content of not higher than 8%;(6) water activity aw of not higher than 0.45;(7) lipid content of not higher than 2%;(8) after treatment in a pH 1.2 acidic medium at 37°C for 60 minutes and neutralization, an SOD activity retention rate of not less than 35%;wherein the extract contains no exogenously added prolamin, and contains no lipid-soluble coating layer, microencapsulation wall material, or prolamin embedding layer.

8. The low-reducing-sugar, high-stability SOD melon endogenous polysaccharide-protein composite extract according to claim 7, characterized in that the extract has:(1) SOD activity of 20,000-60,000 U / g;(2) total protein content of 10%-28%;(3) melon endogenous soluble polysaccharide content of 8%-25%;(4) reducing sugar content of not higher than 6%;(5) water activity aw of not higher than 0.35;CLAIMS(6) after treatment in a pH 1.2 acidic medium for 60 minutes, an SOD activity retention rate of not less than 45%;(7) after storage at 40°C and 75% RH for 30 days, an SOD activity retention rate of not less than 65%.

9. An oral nutritional composition, characterized by comprising the extract according to claim 7 or 8 and a food-acceptable excipient.

10. Use of the extract according to claim 7 or 8 in the preparation of an oral product, characterized in that the oral product is used, for non-therapeutic purposes, for scavenging free radicals, improving dull skin, brightening skin tone, improving skin tone uniformity, increasing skin L* value, improving ITA°, or reducing melanin index; wherein the oral product is a hard capsule, tablet, granule, powder, solid beverage, or compressed candy.

Images