Citrus peel pectin, citrus peel pectin emulsion and application

Abstract

The embodiment of the invention discloses citrus peel pectin, citrus peel pectin emulsion and application. The content of the citrus peel pectin is 66-67%, the protein content is 13-14%, and the esterification degree is 72-73%; the sugar comprises the following components in percentage by mole: 58-59% of galacturonic acid, 25-26% of arabinose, 5-6% of galactose, 4-5% of rhamnose, 2-3% of glucose and 2-3% of xylose. The citrus peel pectin provided by the invention has excellent emulsibility; the citrus peel pectin emulsion has excellent emulsion stability, and shows a better encapsulation effect on LBE. The LBE emulsion obtained by encapsulating LBE by using CP has the characteristics of high encapsulation efficiency, low toxicity and high biocompatibility, and the synergistic effect of CP obviously enhances the antioxidant activity of LBE and the activity inhibition rate of tyrosinase, elastase and hyaluronidase, so that the LBE emulsion can be used as a novel anti-aging emulsion formula to be applied to cosmetics, and has a good application prospect. Good application prospects are realized.

Description

Technical Field

[0001] This invention relates to the fields of food science and engineering technology, specifically to a citrus peel pectin, a citrus peel pectin emulsion, and their applications. Background Technology

[0002] Pectin, as a natural plant component, is safe, non-toxic, has good processability and bioactivity, and is widely used in the food, cosmetic, and pharmaceutical industries. In recent years, the emulsifying properties of pectin have attracted widespread attention. Pectin can adsorb at the water-oil interface to form an interfacial film, reducing interfacial tension. It also prevents emulsion aggregation through steric hindrance and electrostatic repulsion, and increases the viscosity of the continuous phase to slow droplet movement, maintaining emulsion stability. The emulsifying properties of pectin are related to its complex structure. Pectin is mainly composed of three domains: homogalacturonic acid (HG), rhamnogalacturonic acid-I (RG-I), and RG-II. The degree of methyl esterification in the HG region, the arabinose / galactose side chain of RG-I, protein residues and acetyl groups, and molecular weight all significantly influence its emulsifying performance. Differences in the composition and structure of pectin obtained from different plant sources and extraction methods lead to variations in their emulsifying properties. However, the relationship between pectin polysaccharide structure and emulsifying properties remains unclear, limiting the construction and application of highly stable pectin emulsions.

[0003] Furthermore, pectin emulsions can serve as excellent carriers for delivering lipid-soluble bioactive substances. Studies have reported the successful encapsulation of lipid-soluble bioactive substances such as curcumin and naringin in pectin emulsions and emulsion gels, delivering them to specific intestinal sites for targeted and controlled release. Lycium barbarum alcohol extract (LBE) is an bioactive substance extracted from Lycium barbarum, possessing strong antioxidant properties. It can significantly inhibit oxidative stress and promote the scavenging of reactive oxygen species (ROS). However, LBE suffers from poor water solubility, poor stability, and low bioavailability. Summary of the Invention

[0004] Therefore, embodiments of the present invention provide citrus peel pectin, citrus peel pectin emulsion, and their applications.

[0005] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] According to a first aspect of the present invention, the present invention provides a citrus peel pectin with a sugar content of 66-67%, a protein content of 13-14%, and a degree of esterification of 72-73%; the sugar comprises, by molar percentage, 58-59% galacturonic acid, 25-26% arabinose, 5-6% galactose, 4-5% rhamnose, 2-3% glucose, and 2-3% xylose.

[0007] According to a second aspect of the present invention, the present invention provides a method for preparing citrus peel pectin as described above, wherein citrus peel is added to deionized water, pH is adjusted by adding acid, heated, centrifuged to obtain supernatant, concentrated, precipitated with alcohol, reconstituted, protein removed by Sevage method, organic reagent removed, precipitated with alcohol again, and freeze-dried to obtain the citrus peel pectin.

[0008] Further, citrus peel was added to deionized water at a ratio of 1g:20mL.

[0009] The acid is 0.01 mol / L HCl with a pH of 2;

[0010] The heating conditions are: 95℃, 2 h;

[0011] The centrifugation conditions were: 4000 rpm, 30 min;

[0012] The concentration conditions are as follows: the supernatant is concentrated to 1 / 4 of its original volume by vacuum distillation at 40°C.

[0013] The conditions for alcohol precipitation are as follows: add 4 times the volume of anhydrous ethanol to the concentrated solution obtained by concentration, let stand for 12 hours, centrifuge at 4000 rpm for 30 minutes, and remove the supernatant.

[0014] The resolution conditions are as follows: the precipitate obtained by alcohol precipitation is redissolved in deionized water of the same volume as the concentrate;

[0015] The specific steps of the Sevage method for protein removal include: mixing chloroform and n-butanol at a volume ratio of 5:1 to prepare the Sevage reagent; mixing the Sevage reagent with the reconstituted pectin solution at a volume ratio of 1:4; shaking; centrifuging; repeating several times until no protein precipitate appears; and collecting the supernatant.

[0016] The conditions for removing organic reagents are as follows: the supernatant collected by removing protein using the Sevage method is placed in cellophane with a molecular weight cutoff of 30 kDa, tied tightly, and dialyzed with live water at room temperature for 96 h.

[0017] The freeze-drying conditions were: vacuum freeze-drying at -50°C for 96 h.

[0018] According to a third aspect of the present invention, the present invention provides a citrus peel pectin emulsion, the preparation method of which includes the following steps:

[0019] (1) Dissolve the citrus peel pectin as described above in water to obtain an aqueous phase;

[0020] (2) Mix the aqueous phase and the oil phase to obtain a mixture;

[0021] (3) Homogenize the mixture using a high-speed dispersion homogenizer.

[0022] Further, in step (1), the mass-volume concentration of citrus peel pectin in the aqueous phase is 0.5%-2.0%;

[0023] In step (2), based on the total volume of the aqueous phase and the oil phase, the volume ratio of the oil phase is 20-50%, and the oil phase is wolfberry seed oil;

[0024] In step (3), the homogenization conditions are: 15000 rpm, 5 min.

[0025] Furthermore, the mass-volume concentration of citrus peel pectin in the aqueous phase is 2.0%;

[0026] The oil phase accounts for 20-30% of the volume, preferably 20%.

[0027] Furthermore, the preparation method further includes: adding wolfberry alcohol extract to the oil phase before mixing the aqueous phase and the oil phase, and finally obtaining a citrus peel pectin emulsion loaded with wolfberry alcohol extract.

[0028] Furthermore, the mass-volume concentration of the wolfberry extract in the oil phase is 1.0%;

[0029] The preparation method of the wolfberry extract is as follows: the wolfberry residue is dried, crushed, sieved, and mixed with 70% ethanol at a material-to-liquid ratio of 1g:20mL. The mixture is extracted under reduced pressure by rotary evaporation and reflux at 50℃ for 6 hours. After centrifugation, the supernatant is collected. The precipitate is extracted repeatedly. The supernatants are combined, concentrated by rotary evaporation, and then freeze-dried under vacuum.

[0030] According to a fourth aspect of the present invention, the present invention provides the use of citrus peel pectin as described above, or, as described in any of the preceding claims, citrus peel pectin emulsions in the preparation of food, health products, cosmetics or pharmaceuticals.

[0031] Furthermore, the cosmetic product includes an anti-aging lotion.

[0032] The embodiments of the present invention have the following advantages:

[0033] 1. This invention compares the structure and emulsifying properties of pectins from different plant sources, namely apple pectin (AP), citrus peel pectin (CP), grapefruit peel pectin (GP), sunflower seed pectin (SP), and beet pectin (BP), and selects citrus peel pectin as having the best emulsifying properties.

[0034] 2. This invention prepares an emulsion from citrus peel pectin, water, and wolfberry seed oil. By studying the CP concentration (c) and oil phase volume fraction (φ), the provided CP emulsion exhibits excellent emulsion stability. Further encapsulation of LBE shows superior encapsulation effect, indicating that the CP emulsion can serve as a carrier for stabilizing and delivering lipid-soluble bioactive substances.

[0035] 3. The LBE emulsion obtained by encapsulating LBE with CP in this invention not only has a high encapsulation rate, but also has the characteristics of low toxicity and high biocompatibility. Moreover, the synergistic effect of CP significantly enhances the antioxidant activity of LBE and the inhibition rate of tyrosinase, elastase and hyaluronidase activities. It can be used as a new type of anti-aging emulsion formula in cosmetics. Attached Figure Description

[0036] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0037] Figure 1 The monosaccharide composition of pectin from different plant sources provided by this invention;

[0038] Figure 2 Performance test results of emulsions prepared from pectin from different plant sources provided by this invention: A: Particle size distribution; B: Droplet morphology.

[0039] Figure 3 The results of stability testing of CP emulsions with different compositions provided by this invention at different storage times: A: Appearance, B: Particle size distribution;

[0040] Figure 4 The apparent morphology test results of CP emulsions with different compositions provided by the present invention at different storage times, A: 1 day, B: 30 days;

[0041] Figure 5 For the effect of heating on CP emulsion provided by the present invention, A: particle size distribution, B: average particle size;

[0042] Figure 6 For the effect of ionic strength on CP emulsion provided by the present invention, A: particle size distribution, B: average particle size;

[0043] Figure 7 The detection results of the encapsulation efficiency of LBE by the CP emulsion provided by the present invention;

[0044] Figure 8The effect of CP emulsions loaded with different concentrations of LBE provided by the present invention on the cytotoxicity of 3T3 cells;

[0045] Figure 9 The antioxidant activity test results of the LBE-loaded CP emulsion provided by the present invention during storage are as follows: A: DPPH free radical scavenging ability, B: hydroxyl radical (·OH) scavenging ability, C: total flavonoid content, D: total phenol content;

[0046] Figure 10 The enzyme activity inhibition test results of the LBE-loaded CP emulsion provided by the present invention are as follows: A: tyrosinase, B: elastase, C: hyaluronidase. Detailed Implementation

[0047] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] Example 1: Preparation, composition and structure of pectin

[0049] 1. Materials

[0050] Apple pomace, citrus peel, grapefruit peel, sunflower seed heads, and beet pulp were all sourced locally (Shaanxi, China). Goji berry residue and goji berry seed oil were provided by Ningxia Mohasa Co., Ltd. Monosaccharide standards were purchased from Sigma-Aladin (Shanghai, China). Tyrosinase, elastase, hyaluronidase, levodopa, and sodium hyaluronate were purchased from Shanghai Titan Technology Co., Ltd. All other reagents were of analytical grade.

[0051] 2. Preparation of pectin from different sources

[0052] Apple pomace, citrus peel, grapefruit peel, sunflower heads, and beet pulp were dried and added to deionized water at a material-to-liquid ratio of 1:20 (g / mL). The pH of the mixture was adjusted to 2 with 0.1 mmol / L hydrochloric acid. The mixture was heated at 95℃ for 2 h and centrifuged (4000 rpm, 30 min) to obtain the supernatant. After collecting the supernatant, the supernatant was concentrated to one-quarter of its original volume by vacuum rotary evaporation at 40℃. Four times the volume of anhydrous ethanol was added to the concentrate, and the mixture was allowed to stand for 12 h. The supernatant was removed by centrifugation (4000 rpm, 30 min) to obtain the bottom precipitate. The bottom precipitate was redissolved in an equal volume of deionized water to obtain the pectin reconstituted solution. Sevage's reagent was prepared by mixing chloroform and n-butanol at a volume ratio of 5:1. Sevage's reagent was mixed with the pectin reconstituted solution at a volume ratio of 1:4 and shaken thoroughly (1500 rpm, 30 min) to denature the protein. The mixture was then centrifuged (4000 rpm, 30 min). Remove protein precipitate by (min), repeat 5-6 times until no protein precipitate appears, collect the supernatant; place in cellophane (molecular weight cutoff 30 kDa) and dialyze with active water for 96 h to remove organic reagents, add four volumes of anhydrous ethanol again to obtain precipitate, freeze-dry under vacuum at -50℃ for 96 h to obtain pectin, which is then labeled AP, CP, GP, SP, and BP. The pectin extraction rate is the ratio of pectin dry weight to total pomace dry weight.

[0053] 3. Analysis of pectin composition and structure

[0054] (1) Determination of sugar content

[0055] Sugar content was determined using the phenol-sulfuric acid method. Glucose solutions with concentrations ranging from 0.2 to 1.6 mg / mL were prepared using glucose as a standard. 80% phenol and H₂SO₄ were added, and the absorbance at 490 nm was measured to plot a glucose standard curve. The absorbance of pectin samples (0.2 mg / mL, w / v) was measured using the same method, and the sugar content in the samples was calculated based on the glucose standard curve.

[0056] (2) Protein content determination

[0057] Protein content was determined using a BCA kit (Beyotime). BCA solutions with concentrations ranging from 0.025 to 0.5 mg / mL were prepared using BCA as a standard. G-250 solution was added, and the solutions were mixed thoroughly and allowed to stand for 30 min. The absorbance was measured at 562 nm to plot a BCA standard curve. The absorbance of pectin samples (0.1 mg / mL, w / v) was measured using the same method, and the protein content in the samples was calculated based on the glucose standard curve.

[0058] (3) Monosaccharide composition analysis

[0059] The monosaccharide composition of pectin was determined by gas chromatography. In short, the sample was first hydrolyzed with 2 mol / L trifluoroacetic acid (TFA) to remove excess TFA, then 0.5 mol / L Na₂CO₃ was added to ensure complete hydrolysis of the lactone. The lactone was then reduced with NaBH₄ to convert uronic acid into uronic acid lactone, which reacted with n-propylamine to generate an n-propylamine amide derivative of uronic acid. After acetylation, the monosaccharide composition was analyzed using a gas chromatograph (Shimadzu GC-2010) equipped with an Rtx-50 capillary tube (30.0 m × 0.25 mm × 0.25 mm, Restek, Bellefonte, PA, USA).

[0060] (4) Degree of esterification analysis

[0061] The degree of pectin esterification was analyzed by infrared spectroscopy. 1 mg of pectin powder was mixed with 200 mg of KBr and compressed into tablets. The esterification was then measured using a Fourier transform infrared spectrometer (Thermo Fisher Scientific, USA) in the range of 400-4000 cm⁻¹. -1 Absorption peaks were observed within the specified range. The scans were repeated 128 times, in triplicate, with KBr background absorption subtracted. 1730 cm⁻¹ -1 The characteristic absorption peak area corresponds to the number of methylated carboxyl groups, 1630 cm⁻¹ -1 and 1730 cm -1 The ratio of the absorption peak area to the total number of carboxyl groups is the degree of pectin esterification.

[0062] The basic components and structures of AP, CP, GP, SP and BP are shown in Table 1.

[0063] Table 1. Composition and structure of pectin from different plant sources

[0064]

[0065] Note: % refers to the percentage of each indicator to the total. Pectin extraction rate is the ratio of the extracted pectin mass to the total mass of pectin pomace; sugar content is the ratio of the total sugar mass in pectin to the total dry weight of pectin; degree of esterification is the ratio of the number of methylated carboxyl groups in pectin to the total number of carboxyl groups.

[0066] The results showed that the pectin extraction rates of all five plant sources were relatively high, with CP exhibiting the highest extraction rate (19.73% ± 0.78%). Polysaccharides were the main component of the five pectins, ranging from 66.62% to 78.12%. After protein removal using the Sevage method, only CP showed a relatively high protein content (13.48%), primarily composed of proteins covalently bound to pectin polysaccharides. Based on the degree of esterification, SP and BP were classified as low-methoxyl pectins, while AP, CP, and GP were classified as high-methoxyl pectins, with CP exhibiting the highest degree of esterification.

[0067] Analysis of monosaccharide composition reveals that ( Figure 1 Pectin polysaccharides mainly consist of six monosaccharides: Rha (rhamnose), Ara (arabinose), Xyl (xylose), Glc (glucose), Gal (galactose), and GalA (galacturonic acid). The monosaccharide composition of pectin from different sources shows a high content of GalA, and also contains certain amounts of Rha, Ara, and Gal, indicating that these pectins all possess HG and RG-I structural regions. Compared with other pectin monosaccharides, CP monosaccharides not only have a higher proportion of HG regions (higher GalA content), but also contain abundant RG-I regions (higher Ara content) (Table 2).

[0068] Table 2. Relative molar percentage of pectin monosaccharide composition

[0069]

[0070] Example 2: Study on the emulsifying properties of pectin

[0071] The pectin (AP, CP, GP, SP and BP) prepared in Example 1 was dissolved in water to obtain a pectin solution with a mass-volume concentration of 1% (1g / 100mL). The pectin solution was used as the aqueous phase and wolfberry seed oil was used as the oil phase. The aqueous phase and oil phase were mixed at a volume ratio of 1:1 (i.e., the volume fraction of oil phase was 50%) and homogenized using a high-speed dispersion homogenizer at a speed of 15000 rpm for 5 min to obtain an emulsion.

[0072] Determination of emulsion structural properties:

[0073] (1) Appearance

[0074] The appearance of the emulsion was observed and recorded using a camera, and the separation of the oil and water phases of the emulsion was observed visually, along with the degree of separation.

[0075] (2) Particle size determination

[0076] The particle size distribution and average particle size of the emulsion were determined using a MasterSizer 3000 laser particle size analyzer. A suitable amount of emulsion sample was slowly added dropwise and diluted with distilled water to a light-blocking ratio of 10%. The automatic testing mode was used, with the refractive indices of the oil and aqueous phases being 1.47 and 1.33, respectively. Three parallel measurements were performed, and the volume distribution curve and volume-weighted average diameter (D) were recorded. 4,3 ).

[0077] (3) Surface morphology

[0078] Take 20 μL of emulsion onto a glass slide and place it under an inverted microscope (Nikon ECLIPSE Ti2-A) in non-fluorescent mode to observe the morphology of the emulsion droplets.

[0079] The structure and stability of emulsions prepared from different pectins, such as Figure 2As shown in the figure, the particle size distribution of all fresh emulsions exhibited a unimodal distribution. CP and GP emulsions had the narrowest particle size distribution range (1-100 μm), followed by AP emulsion, while SP and BP emulsions had a relatively wider range. All emulsions exhibited spherical or near-spherical structures. After 7 days of storage, SP and BP emulsions showed obvious bimodal or even multimodal particle size distributions, with significant fusion of spherical droplets forming large, irregularly shaped droplets. This was caused by droplet aggregation, leading to emulsion instability. AP emulsion maintained a unimodal particle size distribution, but the peak value shifted significantly to the right, and the size of spherical droplets increased. CP and GP emulsions showed no significant changes in particle size distribution and droplet morphology before and after storage, exhibiting high stability. Compared to GP emulsion, CP emulsion had smaller, more uniformly distributed particles and higher stability. These findings indicate that citrus peel pectin (CP) exhibits the best emulsifying properties.

[0080] Example 3: Performance Study of CP Emulsion

[0081] In this embodiment, citrus peel pectin (CP) was prepared into a CP emulsion according to the method of Example 2, and the effects of CP concentration (c, pectin concentration in the aqueous phase) and oil phase volume fraction (φ) on the structure and stability of the CP emulsion were investigated.

[0082] (1) Rheological analysis

[0083] The rheological properties of the emulsion were determined using a rotational rheometer (Anton Paar MCR302). One mL of CP emulsion was placed on the sample stage, using a CP50 plate mount with a gap height of 1 mm. The test temperature was 25°C, and the shear rate range was set to 0.1–100 s⁻¹. -1 The change in emulsion viscosity with shear rate was monitored. The shear stress versus shear rate curve was fitted using the Power law model according to formula (1) to obtain the K and n values:

[0084] τ = Kγ n (1)

[0085] τ is the shear stress (Pa), and K is the consistency coefficient (Pa·s). n ), γ is the shear rate (s -1 ), where n is the liquidity index.

[0086] The rheological properties of CP emulsions with different compositions are shown in Table 3.

[0087] Table 3. Rheological properties of CP emulsions with different compositions

[0088]

[0089] The results showed that the rheological behavior of all CP emulsions conformed to the Power law model and exhibited a high correlation (R0). 2 (≥ 0.99). All emulsions have n values ​​of 0 < n < 1, indicating they are non-Newtonian fluids. With a fixed φ, as c increases, the emulsion K value increases and the n value decreases, indicating that the emulsion viscosity increases and the fluidity decreases. When φ ≤ 30%, as c increases, the CP polymers cross-link to form a denser network structure, increasing the viscosity of the continuous phase and slowing down the Brownian motion of oil droplets. At the same time, more CP is adsorbed on the surface of oil droplets, increasing the interfacial film thickness and thus enhancing the emulsion stability. However, when φ > 30% and the CP concentration exceeds 1%, the emulsion K value increases significantly (p ≤ 0.05) and the n value decreases significantly (p ≤ 0.05), resulting in excessively high emulsion viscosity and poor fluidity.

[0090] (2) Emulsion stability analysis

[0091] 2.1 Storage stability

[0092] The emulsion was stored at room temperature for 30 days, and the changes in the appearance and particle size of the emulsion over the storage time were monitored.

[0093] 2.2 Thermal stability

[0094] The emulsions were heat-treated at 50℃, 70℃ and 90℃ for 30 min respectively, then immediately cooled to room temperature and allowed to stand for 2 h. The change in emulsion particle size with temperature was monitored.

[0095] The results showed that freshly prepared CP emulsions exhibited good homogeneity under different CP concentrations (c) and oil phase volume fractions (φ). After 30 days of storage, when φ ≤ 30%, the stability of the CP emulsion was positively correlated with the CP concentration; when φ > 30%, neither excessively high nor low CP concentrations could stabilize the oil phase, and only the 1.0% CP emulsion did not exhibit water-oil separation and remained stable. Figure 3 A). The particle size variation of CP emulsion is consistent with its appearance. Figure 3 (B) When φ≤30%, the particle size of freshly prepared emulsions decreases with increasing CP concentration. After 30 days of storage, the particle size distribution peak of the 0.5%-1.0% CP emulsion shifts to the right, and the particle size increases. The particle size distribution of high CP concentration emulsions shows no significant change. When φ>30%, the particle size distribution range and peak value of both low CP concentration (c=0.5%) and high CP concentration (c=1.5%-2.0%) emulsions shift significantly to the right after 30 days of storage. Only the particle size of the 1% CP emulsion shows a small change.

[0096] Changes in the apparent morphology of CP emulsion over time, such as Figure 4As shown, all freshly prepared CP emulsions exhibited uniformly distributed spherical droplets. After 30 days of storage, 0.5% CP failed to stabilize the oil phase, resulting in droplet aggregation and the formation of large spherical droplets at different oil phase volume fractions. With 1% CP, droplet aggregation was significant at low oil phase concentrations (especially φ=30%), while droplet changes were not significant at 40%-50% oil phase volume fractions. When the CP concentration was higher than 1%, the emulsion droplets maintained a uniformly dispersed small spherical structure at φ≤30%. As φ increased, droplet aggregation occurred, with more pronounced changes observed in the 1.5% CP emulsion, consistent with the particle size distribution results. Figure 3 The above results indicate that CP is suitable for the preparation of low-oil-phase emulsions, and that increasing the CP concentration at higher oil-phase levels is detrimental to emulsion stability.

[0097] Based on the above results, c and φ have a significant impact on the formation and stability of CP emulsions. Emulsion systems with c2.0%-φ20%, c2.0%-φ30%, c1.0%-φ40%, and c1.0%-φ50% exhibited good storage stability. Further environmental stability measurements were conducted. Figure 5 As shown, the c1.0%-φ40% and c1.0%-φ50% emulsion systems are more sensitive to heat treatment. After heat treatment at 50℃-90℃, their particle size distribution range and peak value shift significantly to the right. Specifically, the average particle size of the c1.0%-φ50% emulsion increased from 16.53 ± 1.11 μm to 36.50 ± 1.43 μm, indicating a significant decrease in emulsion stability. In contrast, the c2.0%-φ20% and c2.0%-φ30% emulsions showed less change in particle size after heat treatment. In particular, the CP-2.0 (20%) emulsion remained a well-dispersed, small-sized droplet (D... 4,3 =7.770±0.1743), exhibiting high thermal stability.

[0098] (3) Ion stability

[0099] A 1 mol / L NaCl solution was prepared as the mother liquor. Different volumes of the NaCl mother liquor were added to the CP emulsion to obtain emulsion samples with NaCl concentrations of 0, 20, 50, 100, 250 and 500 mmol / L. After standing for 2 hours, the change in emulsion particle size with ionic strength was monitored.

[0100] The effect of ionic strength on the four emulsion systems is as follows: Figure 6As shown, with increasing ionic strength, the particle size distribution range and peak value of the c1.0%-φ50% emulsion shifted significantly to the right, with the average particle size increasing from 18.56±0.38 μm to 52.15±0.29 μm, indicating poor ionic stability. For the c1.0%-φ40% emulsion, at higher ionic strengths (≥250 mmol / L NaCl), the peak particle size distribution shifted to the right, and the average particle size increased significantly (p≤0.05), indicating decreased emulsion stability. In contrast, the c2.0%-φ30% emulsion only showed a significant increase in particle size at high ionic strengths (500 mmol / L NaCl), while the c2.0%-φ20% emulsion exhibited a uniform particle size distribution across different ionic strengths, with no significant change in average particle size (p>0.05), demonstrating excellent ionic stability.

[0101] Example 4: Preparation and Performance Study of LBE Emulsion

[0102] Lycium barbarum extract (LBE) is rich in flavonoids and polyphenols, and has strong antioxidant properties. It can be used as an effective active ingredient in the preparation of functional emulsions.

[0103] In this embodiment, LBE was prepared using the following method: Lycium barbarum residue was dried at 50℃ for 24 h, pulverized, and sieved. The powder was mixed with 70% ethanol at a ratio of 1 g: 20 mL in a flask. Extraction was carried out under reduced pressure by rotary evaporation and reflux at 50℃ for 6 h. After extraction, the mixture was centrifuged (5000 g × 10 min), and the supernatant was collected. The above steps were repeated to extract the precipitate again. The supernatants obtained from the two extractions were mixed, and the supernatant was concentrated to approximately 10 mL by rotary evaporation under vacuum at room temperature. The supernatant was then freeze-dried under vacuum at -50℃ for 48 h.

[0104] Preparation method of LBE-loaded CP emulsion (also known as LBE emulsion): CP is dissolved in water to obtain pectin solutions with different concentrations, which are the aqueous phase; LBE is dissolved in wolfberry seed oil to obtain a solution with a mass-volume concentration of 1% (1g / 100 mL), which is the oil phase; the aqueous phase and oil phase are mixed at different volume ratios and homogenized using a high-speed dispersion homogenizer at a speed of 15000 rpm for 5 min.

[0105] Encapsulation effect, toxicity and bioactivity testing of LBE emulsion:

[0106] (1) Encapsulation efficiency of CP emulsion for Lycium barbarum alcohol extract

[0107] The specific absorption wavelength of LBE was determined to be 285 nm using a full-wavelength scanning mode (200-900 nm). The absorbance of different concentrations of LBE at 285 nm was measured, and an LBE standard curve was plotted. The emulsion sample was mixed with anhydrous ethanol at a volume ratio of 1:4, centrifuged (10000 rpm × 15 min), and the supernatant was obtained. Its absorbance at 285 nm was measured, and the LBE content was calculated. The LBE encapsulation efficiency (EE) is the percentage of LBE encapsulated in the emulsion relative to the total amount of LBE added.

[0108] Four groups of CP emulsions (c1.0%-φ50%, c1.0%-φ40%, c2.0%-φ30%, c2.0%-φ20%) were used to encapsulate LBE, and their encapsulation effects were compared and analyzed. For example... Figure 7 As shown, all emulsion systems exhibited encapsulation rates exceeding 70% for LBE, indicating that CP emulsions can serve as a stable carrier for delivering lipid-soluble bioactive substances. Compared to c1.0%-φ40% and c1.0%-φ50% emulsions, c2.0%-φ20% and c2.0%-φ30% emulsions showed significantly increased encapsulation rates (p≤0.05). This is attributed to the higher stability of these two emulsion systems; the CP adsorbed at the water-oil interface effectively prevented the precipitation of LBE encapsulated in the oil phase, demonstrating good encapsulation and stabilization effects on LBE. Based on the environmental stability results, the c2.0%-φ20% emulsion system was selected for LBE encapsulation, and its cytotoxicity and bioactivity were further analyzed.

[0109] (2) Emulsion cytotoxicity analysis

[0110] LBE emulsion was prepared by loading 1% LBE into a c2.0%–φ20% CP emulsion system. The LBE emulsion was then mixed with DEME medium (containing 2% bovine serum and 1% penicillin antibiotics) at a volume ratio of 1:9 to obtain a stock LBE emulsion (containing 1 mg / mL LBE). Mouse embryonic fibroblasts (3T3 cells) were seeded into 96-well plates and cultured for 24 h. Different volumes of the stock LBE emulsion were mixed with DEME medium to achieve an LBE concentration range of 0–1000 μg / mL, added to the wells, and incubated for 24 h. MTT reagent was added, and the cells were incubated at 37°C in the dark for 4 h. The MTT reagent was removed, DMSO was added, and the cells were incubated for 30 min. Cell viability was measured at 490 nm.

[0111] The effect of LBE emulsion on 3T3 cytotoxicity, such as Figure 8 As shown, cell viability remained at 90% or higher within the concentration range of 0-1000 μg / mL. Within the concentration range of 15.62-1000 μg / mL, there was no significant difference in cytotoxicity compared to the control group (p>0.05), indicating that the LBE-loaded CP emulsion has low toxicity and high biocompatibility.

[0112] (3) Antioxidant activity analysis

[0113] 3.1 DPPH free radical scavenging rate

[0114] Mix 3 mL of 0.1 mmol / L DPPH working solution with 2 mL of 10 mg / mL sample, incubate at 37°C in the dark for 30 min, measure the absorbance at 517 nm, and calculate the DPPH free radical scavenging rate according to formula (2).

[0115]

[0116] A0 and A1 are the absorbances of the DPPH working solution without and with the sample, respectively.

[0117] 3.2 OH radical scavenging experiment

[0118] The ·OH radical scavenging rate is calculated according to formula (3).

[0119]

[0120] A0 is the absorbance of the control group with deionized water replacing the emulsion sample, A1 is the absorbance of the sample mixed with the reaction solution, and A2 is the absorbance of the sample mixed with deionized water.

[0121] 3.3 Determination of total flavonoid content

[0122] Mix 0.3 mL of 5% sodium nitrite, 0.3 mL of 10% aluminum nitrate, and 4 mL of 1 mol / L sodium hydroxide with 3 mL of the sample (10 mg / mL), and measure the absorbance at 510 nm. Prepare a rutin solution (10-100 μg / mL), measure its absorbance at 510 nm, and plot a rutin standard curve. The total flavonoid content in the sample is expressed as milligrams of rutin equivalents.

[0123] 3.4 Determination of total phenol content

[0124] Take 0.2 mL of the sample (10 mg / mL), mix it with 1 mL of 10% Folin-Ciocalteu, 0.8 mL of 7.5% sodium carbonate, and 3 mL of deionized water, and let it stand in the dark for 1 h. Measure the absorbance at 765 nm. Prepare a gallic acid solution (10-200 μg / mL), measure its absorbance at 765 nm, and plot a standard curve. The total phenol content in the sample is expressed as milligrams of gallic acid equivalents.

[0125] The in vitro antioxidant properties of LBE emulsion were tested, including:

[0126] F1 is a CP emulsion loaded with 1% LBE. Its preparation method is as follows: CP is dissolved in water to obtain a pectin solution with a mass-volume concentration of 2% (2g / 100mL), which is the aqueous phase; LBE is dissolved in one-quarter volume of wolfberry seed oil in the aqueous phase to obtain a solution with a mass-volume concentration of 1% (1g / 100mL), which is the oil phase; the aqueous phase and the oil phase are mixed and homogenized using a high-speed dispersion homogenizer at a speed of 15000 rpm for 5 min.

[0127] C1 is a CP emulsion without LBE. Its preparation method differs from F1 only in that LBE is not added.

[0128] C2 is a CP emulsion without goji berry seed oil. Its preparation method differs from F1 in that it does not contain goji berry seed oil and uses deionized water instead.

[0129] C3 is the control group where Tween80 replaces CP. The difference in its preparation method from F1 is that CP is not added and Tween80 is used instead.

[0130] The results of in vitro antioxidant performance tests of LBE emulsions with different formulations are shown in Table 4.

[0131] Table 4. In vitro antioxidant properties of LBE emulsions with different formulations

[0132]

[0133] The results showed that, due to the absence of LBE, the C1 control group had low total phenol and total flavonoid content, poor DPPH and ·OH free radical scavenging ability, and the weakest antioxidant capacity. C2 did not add LBSO to form an emulsion, but the high total phenol and total flavonoid content in LBE gave it good antioxidant activity. C3 used Tween80 as an emulsifier; this system had high total phenol and total flavonoid content, but the DPPH and ·OH scavenging rates were lower than C2, indicating that synthetic emulsifiers like Tween80 may reduce the antioxidant activity of LBE. Compared with these three control groups, the F1 emulsion had the highest total phenol and total flavonoid content, and significantly increased DPPH and ·OH scavenging ability (p≤0.05), exhibiting the highest antioxidant capacity. This may be due to the inherent anti-inflammatory and antioxidant capabilities of natural polysaccharides like pectin, and the synergistic effect of CP in the emulsion enhancing the antioxidant activity of LBE.

[0134] The antioxidant capacity of the four different emulsion formulations changed during storage at room temperature as follows: Figure 9As shown in the figure, the reactive oxygen species scavenging capacity and antioxidant activity content of the LBE-free blank control C1 remained at a low level throughout. Although the initial antioxidant activity level of the C2 control group was high, the DPPH and ·OH scavenging rates, as well as the total flavonoid and total phenolic content, decreased significantly with storage time (p≤0.05), showing the largest decrease. This is because LBE was not encapsulated in the emulsion, resulting in poor stability and easy oxidation, leading to a rapid decline in its antioxidant activity. As shown in groups C3 and F1, emulsion encapsulation can reduce LBE loss during storage and improve LBE storage stability. Compared with C3, the DPPH and ·OH scavenging rates, total flavonoid and total phenolic content of group F1 were significantly increased at different storage times, indicating that CP emulsion has a higher stabilizing ability for LBE than Tween80. Furthermore, CP can delay the loss of flavonoid and phenolic activity in LBE by inhibiting oxidation reactions, thus maintaining a high level of antioxidant activity of LBE in the emulsion throughout.

[0135] (4) Evaluation of enzyme activity inhibition

[0136] 4.1 Determination of Tyrosinase Activity Inhibition Rate

[0137] Mix 40 μL of sample (10 mg / mL) with 40 μL of tyrosinase (≥300 U / mL), add 80 μL of PBS, and incubate for 5 min. Add 40 μL of 5 mmol / L levodopa, incubate at 37℃ for 20 min, and measure the absorbance at 765 nm. Use the unadded sample group as a blank control and the CP emulsion with the same concentration of kojic acid as LBE as a positive control. Calculate the tyrosinase activity inhibition rate according to formula (4).

[0138]

[0139] A0 represents the absorbance of the blank control, and A1 represents the absorbance of the sample.

[0140] 4.2 Determination of elastase activity inhibition rate

[0141] Mix 50 μL of sample (10 mg / mL) with 25 μL of elastase solution (≥500 U / mL) and 100 μL of Trizma buffer (100 mM, pH 8), let stand for 15 min, add 25 μL of substrate, react for 10 min, and measure absorbance at 405 nm. Use the untreated group as a blank control and the CP emulsion with the same concentration of quercetin as LBE as a positive control. The elastase activity inhibition rate was calculated using the same method as the tyrosinase activity inhibition rate.

[0142] 4.3 Determination of Hyaluronidase Activity Inhibition Rate

[0143] Mix 50 μL of sample (10 mg / mL) and 10 μL of hyaluronidase (4200 U / mL) and react for 20 min. Add 20 μL of 12.5 mmol / L CaCl2 and incubate for 10 min. Add 50 μL of sodium hyaluronate (12 mg / mL) and incubate at 37℃ for 40 min. Add 10 mL of NaOH (0.9 mol / L) and 20 mL of Na2B4O7 (0.2 mol / L) and incubate for 3 min. Add 50 μL of p-dimethylaminobenzaldehyde solution and measure the absorbance at 585 nm. The blank control group was replaced with 50 μL of 80% anhydrous ethanol instead of the emulsion sample, and a CP emulsion with the same concentration of tannin as LBE was used as a positive control. The hyaluronidase activity inhibition rate was calculated in the same way as the tyrosinase activity inhibition rate.

[0144] Tyrosinase, elastase, and hyaluronidase are three main enzymes that accelerate skin aging. LBE emulsion exhibits a high inhibition rate against the activity of these three enzymes, as shown in the figure. Figure 10 As shown in Figure 3.1, F1 was a CP emulsion loaded with 1% LBE; C1 and C3 were control groups without LBE and with Tween80 replacing CP, respectively (same as 3.4); and C0 was a control group with positive control agents replacing LBE. Kojic acid, quercetin, and tannic acid were used as positive control agents in the assays of tyrosinase, elastase, and hyaluronidase activity inhibition rates, respectively. The results showed that the positive control group C0 and LBE emulsion F1 had significantly higher inhibition rates for tyrosinase (38.33% ± 1.78 and 36.08% ± 0.81), elastase (21.04% ± 1.05 and 19.36% ± 1.13), and hyaluronidase (31.20% ± 1.52 and 30.38% ± 0.98) than the control groups C1 and C3. Furthermore, there was no statistically significant difference in the inhibition rates of these three enzymes between emulsion F1 and the positive control C0 (p ​​> 0.05). This indicates that LBE can effectively inhibit the activities of tyrosinase, elastase, and hyaluronidase, and can be used as a positive control alternative in the development of an emulsion with both antioxidant and anti-enzymatic activities. Furthermore, using CP instead of the synthetic emulsifier (Tween80) in the emulsion formulation can effectively improve the enzyme activity inhibition rate of LBE.

[0145] The results showed that LBE emulsion exhibited low toxicity and high biocompatibility through in vitro cytotoxicity experiments. Furthermore, the CP in the LBE emulsion not only acts as an emulsifier and stabilizer but also possesses certain biological activity. The synergistic effect of CP significantly enhanced the antioxidant and anti-enzymatic activities (tyrosinase, elastase, and hyaluronidase) of LBE, effectively inhibiting melanin production, maintaining skin elasticity and hydration, and demonstrating anti-aging effects.

[0146] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A citrus pectin, characterized in that, The sugar content is 66-67%, the protein content is 13-14%, the esterification degree is 72-73%; in terms of molar percentage, the sugar includes 58-59% galacturonic acid, 25-26% arabinose, 5-6% galactose, 4-5% rhamnose, 2-3% glucose and 2-3% xylose.

2. A method for preparing citrus pectin according to claim 1, characterized in that, The citrus peel is added into deionized water, acid is added to adjust pH, heated, centrifuged to obtain supernatant, concentrated, alcohol precipitation, redissolved, protein is removed by Sevage method, organic reagent is removed, alcohol precipitation is performed again, and freeze-drying is performed to obtain the citrus peel pectin.

3. The preparation method of claim 2, wherein, The citrus peel is added into deionized water at a material-liquid ratio of 1 g:20 mL; The acid is 0.01 mol / L HCl, and the pH is 2; The heating condition is 95℃ for 2 h; The centrifugation condition is 4000 rpm for 30 min; The concentration condition is that the supernatant is concentrated to 1 / 4 of the original volume by vacuum distillation at 40℃; The alcohol precipitation condition is that 4 times the volume of anhydrous ethanol is added to the concentrated solution obtained by concentration, and the mixture is allowed to stand for 12 h, and then centrifuged at 4000 rpm for 30 min to remove the supernatant; The redissolution condition is that the precipitate obtained by alcohol precipitation is redissolved in deionized water with the same volume as the concentrated solution; The specific content of the Sevage method for removing protein includes that Sevage reagent is prepared by mixing chloroform and n-butanol at a volume ratio of 5:1, the Sevage reagent is mixed with the pectin redissolution solution obtained by redissolution at a volume ratio of 1:4, oscillation, centrifugation, and repeated for several times until no protein precipitate appears, and the supernatant is collected; The condition for removing organic reagent is that the supernatant collected by the Sevage method for removing protein is placed in glass paper with a molecular weight cut-off of 30 KDa, tightly tied, and dialyzed in running water at room temperature for 96 h; The freeze-drying condition is vacuum freeze-drying at-50℃ for 96 h.

4. A citrus pectin emulsion characterized in that, The preparation method comprises the following steps: (1) dissolving the citrus peel pectin of claim 1 in water to obtain an aqueous phase; (2) mixing the aqueous phase with an oil phase to obtain a mixture; (3) homogenizing the mixture using a high-speed dispersion homogenizer.

5. The citrus peel pectin emulsion of claim 4, wherein, In step (1), the mass-volume concentration of citrus peel pectin in the aqueous phase is 0.5%-2.0%; In step (2), the volume ratio of the oil phase to the total volume of the aqueous phase and the oil phase is 20-50%, and the oil phase is wolfberry seed oil; In step (3), the homogenization condition is 15000 rmp for 5 min.

6. The citrus peel pectin emulsion of claim 5, wherein, The mass-volume concentration of citrus peel pectin in the aqueous phase is 2.0%; The volume ratio of the oil phase is 20-30%, preferably 20%.

7. The citrus pectin emulsion according to claim 4, characterized in that, The preparation method further comprises: before mixing the aqueous phase with the oil phase, adding wolfberry alcohol extract to the oil phase to finally obtain a citrus peel pectin emulsion loaded with wolfberry alcohol extract.

8. The citrus peel pectin emulsion of claim 7, wherein, The mass-volume concentration of the wolfberry extract in the oil phase is 1.0%; The preparation method of the wolfberry extract is as follows: wolfberry residues are dried, crushed, sieved, mixed with 70% ethanol according to a solid-liquid ratio of 1g:20mL, extracted by rotary evaporation and condensation reflux under reduced pressure at 50°C for 6h, centrifuged, the supernatant is collected, the precipitate is repeatedly extracted, the supernatants are combined, concentrated by rotary evaporation, and vacuum freeze-dried.

9. Use of the citrus pectin of claim 1 or the citrus pectin emulsion of any one of claims 4-7 in the preparation of a food, nutraceutical, cosmetic, or pharmaceutical product.

10. Use according to claim 9, characterized in that, The cosmetic product comprises an anti-aging emulsion.

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