Phytosterol ester emulsion, its preparation method and application

Abstract

The application provides a phytosterol ester emulsion and a preparation method and application thereof, and the method comprises the following steps: S1, respectively preparing an emulsifier solution and a stabilizer solution, the emulsifier is sodium caseinate or egg albumin, the mass concentration of the emulsifier solution is 0.5-2%, and the stabilizer is high methoxyl pectin, sodium carboxymethyl cellulose or propylene glycol alginate, and the mass concentration of the stabilizer solution is 0.5-2%; S2, mixing the emulsifier solution with phytosterol ester, the mass ratio of the emulsifier solution to the phytosterol ester is 80:20-95:5, and the intermediate emulsion is obtained through high-speed shearing and high-pressure homogenization; and S3, mixing the intermediate emulsion with the stabilizer solution, the mass ratio of the intermediate emulsion to the stabilizer solution is 1:2-2:1, adjusting pH, and high-speed shearing to obtain the phytosterol ester emulsion. The application improves the dispersibility and stability of phytosterol ester in water, so that the phytosterol ester can be conveniently applied in liquid beverages, and the phytosterol ester has high stability and bioavailability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food, cosmetics, medicine and feed, and particularly relates to a phytosterol ester emulsion, a preparation method and application thereof. BACKGROUND

[0002] Phytosterols are a kind of natural sterols widely existing in various plant oils, nuts and other plant foods, mainly including β-sitosterol, stigmasterol, campesterol and brassicasterol. Studies have shown that phytosterols have various biological activities such as lowering cholesterol, antioxidant, anticancer and anti-inflammatory, and have a very broad application prospect. Phytosterol esters are products of phytosterols and fatty acids or triglycerides (edible oil) through esterification or transesterification, and their oil solubility is significantly higher than that of phytosterols, and they are more easily applied to oil and fat and oil-rich systems (such as vegetable oil, margarine, etc.).

[0003] In comparison, more products are mainly in water phase, and phytosterol esters have the characteristics of water insolubility, which seriously restricts their application in these product systems. Liquid beverages are a kind of food that can satisfy thirst, supplement nutrition and provide energy, and are widely loved by consumers. Liquid beverages mainly have the following advantages: 1. Convenient to drink, easy to digest and absorb, without additional brewing; 2. Rapidly satisfy the thirst of the human body, quickly replenish water. Phytosterol esters have important biological activities such as lowering cholesterol and lowering blood lipids, and the development of liquid beverages rich in phytosterol esters has a broad application prospect. However, liquid beverages generally face the problems of poor dispersibility and storage stability during processing. Phytosterol esters are insoluble in water and have poor dispersibility, which makes it difficult to add them to liquid beverages. In addition, the bioavailability (bioavailability) of phytosterol esters is low, which is not conducive to the digestion and absorption of the body. Therefore, how to realize the application of phytosterol esters in water phase and water-containing systems, improve their addition amount and improve their bioavailability is a problem that needs to be solved at present.

[0004] CN116195652A discloses a preparation method of phytosterol ester microcapsules, which uses gum arabic and resistant starch or cyclodextrin composition as wall material, sucrose ester and monoglyceride as emulsifier, and adopts emulsification-spray drying method to prepare phytosterol ester microcapsules. The content of core material in the prepared microcapsule product is 33.3%-50%. CN103404854A discloses a phytosterol ester microcapsule prepared by using gum arabic and malt dextrin as wall material and adopting spray drying method. The content (or loading capacity) of phytosterol ester in the microcapsule is up to 65%. Although microencapsulation can greatly improve the water dispersibility of phytosterol ester, the content of phytosterol ester is low. In addition, although the phytosterol ester microcapsule can be in a dispersed state in a short term after reconstitution, it is difficult to exist in a liquid form for a long time. CN110192651A discloses a preparation method of nanoemulsion loaded with phytosterol ester by using soybean protein isolate. In the method, phytosterol ester dissolved in ethyl acetate is added dropwise into an ultrasonic aqueous solution containing soybean protein isolate under high-speed shearing for dispersion. Ethyl acetate is removed by rotary evaporation, and nano-particle dispersion liquid is formed after ultrasonic treatment, thereby preparing nanoemulsion loaded with phytosterol ester. The loading capacity of phytosterol ester in the emulsion is 9.1%-16.7%. Although the existing technology can improve the dispersibility of phytosterol ester in water to a certain extent, the bioavailability (bioaccessibility) is not improved, and there are generally low loading capacity and poor stability. In addition, the existing phytosterol ester emulsion is easily destroyed under external environment, that is, the ability to resist external environment is weak, and becomes unstable during actual product processing. Therefore, it is necessary to make further research to solve the above technical problems. SUMMARY

[0005] In view of the above technical problems, the present application provides a preparation method of phytosterol ester emulsion, which improves the dispersibility and stability of phytosterol ester in water, so as to facilitate the application in liquid beverages.

[0006] The present application also provides a phytosterol ester emulsion prepared by the above method, which breaks through the limitation of water insolubility of phytosterol ester, and has high hydrolysis rate and bioavailability. Compared with intermediate emulsion, the storage stability of the phytosterol ester emulsion is better, the ability to resist external environment is enhanced, and the phytosterol ester emulsion can exist in a liquid form for a long time, which is beneficial to subsequent processing, transportation and storage.

[0007] The present application also provides an application of the above phytosterol ester emulsion in the technical field of food, medicine, cosmetics and feed processing, especially in liquid beverages.

[0008] The edible protein and polysaccharide used in the present application can make the emulsion have better storage stability and processing stability and a long shelf life through electrostatic repulsion and steric hindrance. The advantages of the protein emulsifier and the polysaccharide stabilizer are fully utilized to combine the two to form a phytosterol ester emulsion, so that the phytosterol ester emulsion is more easily applied to the production and processing of food, medicine, cosmetics and feed. In addition, the use of the technology minimizes the contact between the phytosterol ester and the external environment, and has higher stability and bioavailability than free phytosterol ester.

[0009] In the prior art, the preparation of the phytosterol ester emulsion in the present application has not been found, and the research on the phytosterol ester emulsion in improving stability and bioavailability (or bioaccessibility) has not been found.

[0010] The phytosterol ester emulsion prepared by the method has a large loading capacity (≥84%), a high encapsulation rate, good processing stability, high storage stability, can exist in a liquid form for a long time, and has significantly improved bioavailability, and can be applied in the fields of food, medicine, cosmetics and feed.

[0011] The present application achieves the above technical purpose through the following technical means.

[0012] A preparation method of a phytosterol ester emulsion, comprising the following steps:

[0013] Step S1, respectively preparing an emulsifier solution and a stabilizer solution, the emulsifier being sodium caseinate (CN) or egg white protein (OVA), the mass concentration of the emulsifier solution being 0.5-2%, the stabilizer being high methoxyl pectin (HMP), sodium carboxymethyl cellulose (CMC) or propylene glycol alginate (PGA), and the mass concentration of the stabilizer solution being 0.5-2%;

[0014] Step S2, mixing the emulsifier solution with phytosterol ester, the mass ratio of the emulsifier solution to the phytosterol ester being 80:20-95:5, and performing high-speed shearing and high-pressure homogenization to obtain an intermediate emulsion;

[0015] Step S3, mixing the intermediate emulsion with the stabilizer solution, the mass ratio of the intermediate emulsion to the stabilizer solution being 1:2-2:1, adjusting the pH, and performing high-speed shearing to obtain the phytosterol ester emulsion.

[0016] In the above scheme, the emulsifier in step S1 is sodium caseinate (CN).

[0017] In the above scheme, the stabilizer in step S1 is sodium carboxymethyl cellulose (CMC) or propylene glycol alginate (PGA).

[0018] In the above scheme, the stabilizer in step S1 is sodium carboxymethyl cellulose (CMC) or propylene glycol alginate (PGA).

[0019] In the above scheme, the mass ratio of the emulsifier solution to the phytosterol ester in step S2 is 85:15-90:10.

[0020] In the above scheme, the high-speed shearing condition in step S2 is 10000-20000 rpm shearing for 2-4 min.

[0021] In the above scheme, the high-pressure homogenization pressure in step S2 is 20-40 MPa, and the cycle number is 2-4 times.

[0022] In the above scheme, the pH in step S3 is adjusted to 2.5-4.5; and the high-speed shearing condition in step S3 is 10000-20000 rpm shearing for 2-4 min.

[0023] A phytosterol ester emulsion obtained according to the preparation method of the phytosterol ester emulsion.

[0024] The application of a phytosterol ester emulsion, according to the application of the phytosterol ester emulsion in the fields of food, medicine, cosmetics and feed.

[0025] In the above scheme, the application of the phytosterol ester emulsion in liquid beverages.

[0026] The method is simple to operate, convenient and easy to implement, the involved protein and polysaccharide are edible ingredients, have a wide source, low price, good nutritional and functional properties and are biodegradable.

[0027] Compared with the prior art, the present application has the following advantages:

[0028] 1. The present application selects sodium caseinate and egg white protein as the emulsifier, the mass concentration of the emulsifier solution is 0.5-2%, the mass ratio of the emulsifier solution to the phytosterol ester is 80:20-95:5; high methoxyl pectin (HMP), carboxymethyl cellulose sodium (CMC) or alginate propylene glycol ester (PGA) is used as the stabilizer, the mass concentration of the stabilizer solution is 0.5-2%, and the mass ratio of the intermediate emulsion to the stabilizer solution is 1:2-2:1; the phytosterol ester emulsion prepared according to the above formula has the advantages of good water dispersibility, large loading capacity, high encapsulation rate, good storage stability, strong resistance to external environment, long-term stable existence in liquid form and long shelf life.

[0029] 2. The phytosterol ester emulsion prepared by the present application has a higher degree of hydrolysis in intestinal digestion and is more easily absorbed by the body, so that the bioavailability is high.

[0030] 3. The phytosterol ester emulsion prepared by the present application can be directly applied in liquid beverages. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1Preparation flow chart of the present application.

[0032] Figure 2 Emulsifying activity and emulsion stability of emulsifiers. Wherein, OVA is ovalbumin, CN is sodium caseinate, EAI is emulsifying activity, ESI is emulsion stability.

[0033] Figure 3 Microscope and appearance pictures of intermediate emulsion. Wherein, Figure 3 (A) is the microscope picture of intermediate emulsion prepared with ovalbumin and sodium caseinate as emulsifiers, Figure 3 (B) is the appearance picture of intermediate emulsion prepared with ovalbumin and sodium caseinate as emulsifiers at day 0 and day 7.

[0034] Figure 4 Microscope pictures of intermediate emulsion of emulsifier solution and phytosterol ester at different mass ratios.

[0035] Figure 5 Appearance and microscope changes of phytosterol ester emulsion. Wherein, Figure 5 (A) is the appearance picture of phytosterol ester emulsion prepared with sodium caseinate as emulsifier, without or with carboxymethyl cellulose sodium, propylene glycol alginate or high methoxyl pectin as stabilizer at day 0 and day 7, Figure 5 (B) is the microscope picture of phytosterol ester emulsion prepared with sodium caseinate as emulsifier, without or with carboxymethyl cellulose sodium, propylene glycol alginate or high methoxyl pectin as stabilizer at day 0 and day 7.

[0036] Figure 6 Effects of storage time and temperature on particle size, potential and PDI of emulsion. Wherein, Figure 6 (A) is the change of particle size of phytosterol ester emulsion prepared with sodium caseinate as emulsifier, without or with PGA as stabilizer at 4℃, Figure 6 (B) is the change of potential of phytosterol ester emulsion prepared with sodium caseinate as emulsifier, without or with PGA as stabilizer at 4℃, Figure 6 (C) is the change of polydispersity index of phytosterol ester emulsion prepared with sodium caseinate as emulsifier, without or with PGA as stabilizer at 4℃, Figure 6 (D) is the change of particle size of phytosterol ester emulsion prepared with sodium caseinate as emulsifier, without or with PGA as stabilizer at 15℃, Figure 6 (E) is the change of potential of phytosterol ester emulsion prepared with sodium caseinate as emulsifier, without or with PGA as stabilizer at 15℃, Figure 6 (F) is the change of polydispersity index of phytosterol ester emulsion prepared with sodium caseinate as emulsifier, without or with PGA as stabilizer at 15℃.

[0037] Figure 7 The appearance and microscopic changes of the emulsion at different storage times and temperatures. Among them, Figure 7 (A) is the appearance change of the phytosteryl esters emulsion prepared with sodium caseinate as emulsifier and without or with propylene glycol alginate as stabilizer at 4℃, Figure 7 (B) is the microscopic change of the phytosteryl esters emulsion prepared with sodium caseinate as emulsifier and without or with propylene glycol alginate as stabilizer at 4℃, Figure 7 (C) is the appearance change of the phytosteryl esters emulsion prepared with sodium caseinate as emulsifier and without or with propylene glycol alginate as stabilizer at 15℃, Figure 7 (D) is the microscopic change of the phytosteryl esters emulsion prepared with sodium caseinate as emulsifier and without or with propylene glycol alginate as stabilizer at 15℃.

[0038] Figure 8 The particle size, PDI, potential and microscopic changes of the emulsion under different heating methods. Among them, Figure 8 (A) is the particle size of the phytosteryl esters emulsion prepared with sodium caseinate as emulsifier and without or with propylene glycol alginate as stabilizer under different temperature treatments, Figure 8 (B) is the polydispersity index of the phytosteryl esters emulsion prepared with sodium caseinate as emulsifier and without or with propylene glycol alginate as stabilizer under different temperature treatments, Figure 8 (C) is the potential of the phytosteryl esters emulsion prepared with sodium caseinate as emulsifier and without or with propylene glycol alginate as stabilizer under different temperature treatments, Figure 8 (D) is the microscopic picture of the phytosteryl esters emulsion prepared with sodium caseinate as emulsifier and without or with propylene glycol alginate as stabilizer under different temperature treatments.

[0039] Figure 9 The particle size, PDI, potential, appearance and microscopic changes of the emulsion under different freezing times. Among them, Figure 9 (A) is the particle size of the phytosteryl esters emulsion prepared with sodium caseinate as emulsifier and without or with propylene glycol alginate as stabilizer under different freezing times; Figure 9 (B) is the polydispersity index of the phytosteryl esters emulsion prepared with sodium caseinate as emulsifier and without or with propylene glycol alginate as stabilizer under different freezing times; Figure 9 (C) is the potential of the phytosteryl esters emulsion prepared with sodium caseinate as emulsifier and without or with propylene glycol alginate as stabilizer under different freezing times; Figure 9(D) is the appearance picture of phytosteryl esters emulsion prepared with sodium caseinate as emulsifier, without or with propylene glycol alginate as stabilizer under different freezing time; 9(E) is the micrograph of phytosteryl esters emulsion prepared with sodium caseinate as emulsifier, without or with propylene glycol alginate as stabilizer under different freezing time.

[0040] Figure 10 The changes of emulsion particle size and potential during gastrointestinal digestion stage. Among them, Figure 10 (A) is the particle size change of phytosteryl esters emulsion prepared with sodium caseinate as emulsifier, without or with propylene glycol alginate as stabilizer under simulated gastric digestion; Figure 10 (B) is the potential change of phytosteryl esters emulsion prepared with sodium caseinate as emulsifier, without or with propylene glycol alginate as stabilizer under simulated gastric digestion; Figure 10 (C) is the particle size change of phytosteryl esters emulsion prepared with sodium caseinate as emulsifier, without or with propylene glycol alginate as stabilizer under simulated intestinal digestion; Figure 10 (D) is the potential change of phytosteryl esters emulsion prepared with sodium caseinate as emulsifier, without or with propylene glycol alginate as stabilizer under simulated intestinal digestion.

[0041] Figure 11 The micrograph changes of emulsion during gastrointestinal digestion stage. Among them, Figure 11 (A) is the micrograph change of phytosteryl esters emulsion prepared with sodium caseinate as emulsifier, without or with propylene glycol alginate as stabilizer under simulated gastric digestion; Figure 11 (B) is the micrograph change of phytosteryl esters emulsion prepared with sodium caseinate as emulsifier, without or with propylene glycol alginate as stabilizer under simulated intestinal digestion.

[0042] Figure 12 The hydrolysis rate, bioavailability of phytosteryl esters and corresponding liquid chromatogram during gastrointestinal digestion stage. Among them, Figure 12 (A) is the liquid chromatogram of phytosteryl esters emulsion prepared with sodium caseinate as emulsifier, without or with propylene glycol alginate as stabilizer under simulated gastric digestion; Figure 12 (B) is the hydrolysis rate of phytosteryl esters of phytosteryl esters emulsion prepared with sodium caseinate as emulsifier, without or with propylene glycol alginate as stabilizer under simulated gastric digestion; Figure 12 (C) is the liquid chromatogram of phytosteryl esters emulsion prepared with sodium caseinate as emulsifier, without or with propylene glycol alginate as stabilizer under simulated intestinal digestion; Figure 12 (D) is the hydrolysis rate of phytosteryl esters of phytosteryl esters emulsion prepared with sodium caseinate as emulsifier, without or with propylene glycol alginate as stabilizer under simulated intestinal digestion.Figure 1 (E) represents the bioavailability of phytosterol ester emulsions prepared with sodium caseinate as emulsifier and without or with propylene glycol alginate as stabilizer under simulated gastrointestinal digestion. Detailed Implementation

[0043] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0044] like Figure 2 As shown, the preparation method of the phytosterol ester emulsion of the present invention includes the following steps:

[0045] Step S1: Add the protein to phosphate buffer to obtain an emulsifier solution; add the polysaccharide to phosphate buffer to obtain a stabilizer solution;

[0046] Step S2: Mix the emulsifier solution with phytosterol esters, and then homogenize under high speed and high pressure to obtain phytosterol ester intermediate emulsion;

[0047] Step S3: Mix the phytosterol ester intermediate emulsion with the stabilizer solution, adjust the pH, and perform high-speed shearing to obtain the phytosterol ester emulsion.

[0048] In step S1, the emulsifier is sodium caseinate or ovalbumin, preferably sodium caseinate.

[0049] In step S1, the concentration of the emulsifier solution is 0.5% to 2%.

[0050] In step S1, the stabilizer is high methoxyl pectin (HMP), sodium carboxymethyl cellulose (CMC), or propylene glycol alginate (PGA), preferably CMC or PGA.

[0051] In step S1, the concentration of the stabilizer solution is 0.5% to 21%.

[0052] In step S2, the mass ratio of the emulsifier solution to the phytosterol ester is 80:20 to 95:5, preferably 85:15 to 90:10.

[0053] In step S2, the high-speed shearing condition is 10,000 to 20,000 rpm for 2 to 4 minutes.

[0054] In step S2, the pressure of the high-pressure homogenization is 20-40 MPa, and the number of cycles is 2-4.

[0055] In step S3, the mass ratio of the intermediate emulsion to the stabilizer solution is 1:2-2:1.

[0056] In step S3, the pH is 2.5-4.5.

[0057] In step S3, the high-speed shearing condition is 10000-20000 rpm shearing for 2-4 min.

[0058] The method is simple to operate, convenient and easy to implement, the proteins and polysaccharides involved are edible ingredients, which are widely available, low in price, good in nutritional function and biodegradable.

[0059] Determination of particle size, potential and PDI: dilute the phytosteryl ester emulsion 500 times, and place the diluted solution in an EP tube; use a Litesizer TM 500 laser particle size analyzer to measure the particle size, Zeta potential and PDI, the particle size and PDI are measured by using a disposable sample cell, the temperature is 15℃, the measurement angle is backscattering 185°, and the sample volume is 1mL; the Zeta-potential measurement selects an Omega sample cell II, the temperature is 15℃, and the sample volume is 0.7mL.

[0060] Inverted fluorescence microscopic observation: take 1mL of the phytosteryl ester emulsion in an EP tube, add 10μL of Nile red to the emulsion for dyeing, dye for 20min, then take 10μL on a glass slide, cover with a cover glass, and stand for 15min; use a CTR4000B inverted fluorescence microscope (Leica Instruments Co., Ltd., Germany) with an eyepiece of 10x and an objective lens of 40x to observe the microstructure.

[0061] Loading rate (%) = mass of phytosteryl ester in emulsion / total mass of solids in emulsion x 100 Formula (1)

[0062] Encapsulation rate (%) = (total mass of phytosteryl ester in emulsion - mass of unencapsulated phytosteryl ester) / total mass of phytosteryl ester in emulsion x 100 Formula (2)

[0063] Example 1: Investigation of emulsifier emulsification activity and emulsion stability

[0064] Prepare 1% ovalbumin solution (OVA) and sodium caseinate solution (CN) respectively, then mix them with soybean oil at a mass ratio of 3:1, emulsify at 16000r / min for 1min, take 50μL of the emulsion and drop it into 5mL of 0.1% sodium dodecyl sulfate solution, shake well, and then measure the absorbance at 500nm, repeat 3 times. Calculate the emulsification activity index (EAI) and emulsion stability index (ESI) of the two proteins according to formulas (3) and (4).

[0065]

[0066]

[0067] In formula (3), A0 is the absorbance of the emulsion that is rapidly diluted after emulsification; c is the volume fraction of the oil phase (value is 0.15); c is the concentration of various emulsifiers (g / mL); L is the optical path length of the cuvette (10). -2 m).

[0068] In formula (4), A t The absorbance is the value of the emulsion after standing for t min and then diluting.

[0069] EAI characterizes the emulsifying ability of an emulsifier. Figure 2 (A) shows that both emulsifier solutions have the ability to rapidly adsorb onto the oil surface to form an interfacial layer. Comparatively, sodium caseinate of the same concentration has a higher EAI, indicating stronger emulsifying ability. ESI characterizes the ability of an emulsion to maintain stability. A higher value indicates stronger emulsion stability. Emulsifier type (B) shows that sodium caseinate has better emulsifying stability compared to ovalbumin. This indicates that emulsions prepared using sodium caseinate as an emulsifier have better stability.

[0070] Example 2: Determination of Emulsifier Type

[0071] A 1% (w / w) solution of ovalbumin and a sodium caseinate solution were prepared. The two emulsifier solutions were then mixed with phytosterol esters (Xi'an Haisifu Biotechnology Co., Ltd., LowChol). TM Phytosterol esters (-S) were mixed at a mass ratio of 80:20, sheared at 10000 rpm for 4 min, and homogenized four times at 20 MPa to obtain two intermediate emulsions. Their particle size, potential, and PDI were measured, and appearance images were taken. After being diluted five times, microscopic images were taken under an optical microscope.

[0072] Table 1. Particle size, potential, and PDI of intermediate emulsions

[0073] Particle size (nm) Zeta potential (mV) PDI (%) Ovalbumin Sodium caseinate 6783±432a -28.4±1.2a 37.3±2.2a Figure 3 4885±157b -32.3±0.5b 31.3±1.5b

[0074] Note: Data labeled with different letters (a, b) in the same column showed significant differences (p<0.05).

[0075] Particle size, particle size distribution index (PDI), and zeta potential are important characterization methods for emulsions. Smaller droplet sizes indicate better emulsion stability. A lower PDI indicates a more uniform droplet distribution, and vice versa. A zeta potential greater than 20 mV generally indicates that the emulsion possesses sufficient electrostatic repulsion and is unlikely to aggregate into large droplets.

[0076] From Table 1, it can be seen that the particle size and PDI of the intermediate emulsion in the sodium caseinate group were significantly smaller than those in the ovalbumin group, and the zeta potentials of the intermediate emulsions in the two groups were both greater than 20 mV. However, the PDI was greater than 30%, indicating that the droplet dispersion was relatively uneven, which may be related to the high content of phytosteryl ester and the presence of unencapsulated phytosteryl ester. Figure 3 (A), it can be seen that unencapsulated large oil droplets exist in both intermediate emulsions, which is related to the limited emulsifying activity of the two proteins and the high content of phytosteryl ester. In comparison, there are fewer unencapsulated large oil droplets in the sodium caseinate group, which is related to its better emulsifying activity. Mass ratio (B), it can be seen that after the two intermediate emulsions were stored at 15℃ for 7 days, obvious creaming phenomenon occurred, indicating that the stability of the emulsion stabilized by a single emulsifier is poor. Overall, the stability of sodium caseinate is better than that of ovalbumin, so sodium caseinate is selected as the emulsifier.

[0077] Example 3: Determination of the mass ratio of emulsifier solution to phytosteryl ester

[0078] A 1% sodium caseinate solution was prepared, and the sodium caseinate solution and phytosteryl ester (Xi'an Haishifu Biological Technology Co., Ltd., LowChol-S phytosteryl ester) were mixed in a mass ratio of 80:20, 85:15 and 95:5, respectively, sheared at 20000 rpm for 2 min, and homogenized at 40 MPa for 2 times to obtain intermediate emulsions with different phytosteryl ester contents. The particle size, zeta potential and PDI were measured and micrographs were taken. TM

[0079] Table 2 Particle size, zeta potential and PDI of intermediate emulsions prepared by mixing emulsifier solution and phytosteryl ester at different mass ratios

[0080] Particle size (nm) Zeta potential (mV) PDI (%) Figure 4 80：20 4852±273a -32.8±0.4a 31.2±2.2a 85：15 3097±74b -32.3±0.1a 27.2±1.7b 95：5 2987±154b -32.7±0.7a 26.7±1.7b

[0081] Note: There is a significant difference (p<0.05) between the data marked with different letters (a, b) in the same column.

[0082] The particle size, zeta potential and PDI of the intermediate emulsions prepared by mixing emulsifier solution and phytosteryl ester at different mass ratios are shown in Table 2. When the mass ratio was 80:20, the particle size and PDI of the prepared intermediate emulsion were relatively large. This may be due to the high content of phytosteryl ester, and the sodium caseinate was not enough to fully emulsify and stabilize the phytosteryl ester, resulting in a larger particle size (greater than 4000 nm) of the intermediate emulsion. Wall material type ​It can be seen that there are large oil droplets of unencapsulated phytosterol ester in the intermediate emulsion with mass ratio of 80:20. When the content of phytosterol ester is reduced (mass ratio of 85:15), these indicators are significantly improved, and the droplets are uniformly dispersed, indicating that the emulsifier is sufficient to emulsify phytosterol ester at this time. When the content of phytosterol ester continues to decrease, there is no significant change in particle size, PDI and microscopic picture. In summary, sodium caseinate solution and phytosterol ester are selected in a mass ratio of 85:15 for the preparation of phytosterol ester intermediate emulsion.

[0083] Example 4: Investigation of the type of stabilizer

[0084] Prepare 1% high methoxyl pectin (HMP) solution, sodium carboxymethyl cellulose (CMC) solution and propylene glycol alginate (PGA) solution respectively. Mix 1% sodium caseinate solution and phytosterol ester (Xi'an Haishifu Biological Technology Co., Ltd., Low Chol TM -S phytosterol ester) in a mass ratio of 85:15, shear at 10000 rpm for 2 min, and homogenize at 20 MPa for 4 times to obtain an intermediate emulsion. Mix the intermediate emulsion with the three stabilizer solutions in a mass ratio of 1:1, and adjust the pH to 3.5 with 1M NaOH and 1M HCl. Shear at 10000 rpm for 2 min to obtain phytosterol ester emulsion. By comparing particle size, zeta potential, PDI, micrograph and appearance picture, investigate their short-term stability at 15℃ for 7 days.

[0085] Table 3 Particle size, potential and PDI of phytosterol ester emulsion

[0086] Particle size (nm) Zeta potential (mV) PDI (%) CN CN-CMC 2956±76.3b -31.2±0.7c 26.8±1.2b -30.2 ± 0.5 bc 3319±31.1a CN-PGA 26.5±1.5b CN-HMP 3304±87.1a -13.1±1.1a 27.1±0.2b Figure 5 3199±83.6a -29.1±0.6b 29.8±0.6a

[0087] Note: There is a significant difference (p<0.05) between the data of the same column marked with different letters (a, b, c).

[0088] As can be seen from Table 3, the particle size of the emulsion increases after adding the stabilizer, which is due to the adsorption of the stabilizer on the surface of the emulsifier, and the increase of the thickness of the interface layer. The PDI of the emulsion prepared by the three kinds of stabilizers is less than 30%, and the droplets are uniformly dispersed. The absolute value of the potential of the CN-PGA group emulsion is low, which is related to the charged nature of propylene glycol alginate.

[0089] The stability of the emulsion is related to factors such as viscosity, particle size, temperature and pH. From the results in Table 3, it can be seen that the stability of the emulsion is related to the viscosity of the stabilizer solution. The stability of the emulsion prepared by the CN-PGA group is the best, and the stability of the emulsion prepared by the CMC group is the worst. Wall material typeIt was found that, except for CN-CMC and CN-PGA, the intermediate emulsion and CN-HMP group emulsions all exhibited varying degrees of emulsification. Among them, the intermediate emulsion showed the worst stability. This is related to the viscosity of the emulsion and the interfacial layer. According to Stokes' law, the addition of stabilizers can increase the viscosity of the emulsion, thereby reducing the upward flow of oil droplets. The electrostatic deposition of stabilizers on the surface of emulsifiers can effectively reduce their emulsification and aggregation. In addition, the electrostatic combination of emulsifiers and stabilizers increases the thickness of the adsorption layer, providing more electrostatic and steric repulsion forces, which is the result of the combined effect of inter-droplet repulsion and viscosity.

[0090] Taking into account factors such as particle size, potential, and stability, CN-CMC and CN-PGA are more suitable for preparing phytosterol ester emulsions.

[0091] Example 5: Encapsulation efficiency of phytosterol ester emulsions

[0092] Accurately weigh 20g of phytosterol esters (Xi'an Haisifu Biotechnology Co., Ltd., LowChol). TM A phytosterol ester intermediate emulsion was prepared by adding 20 mL of petroleum ether (boiling point 60-90℃) and stirring at 100 rpm for 5 min. The mixture of petroleum ether and intermediate emulsion was then placed in a 50 mL centrifuge tube and centrifuged at 5000 rpm for 10 min. 10 mL of the upper petroleum ether solution was collected in a round-bottom cake and evaporated by rotary evaporation at 40℃. The mixture was then dried in a 120℃ oven until constant weight was achieved. The mass of the collected phytosterol esters (i.e., the mass of unencapsulated phytosterol esters) was recorded, and the encapsulation rate of the phytosterol esters in the intermediate emulsion was calculated.

[0093] Accurately weigh 20g of CN-CMC group phytosterol esters (Xi'an Haisifu Biotechnology Co., Ltd., LowChol) TM A CN-CMC emulsion containing phytosterol esters (-S) was prepared by adding 20 mL of petroleum ether (boiling point 60-90℃) and stirring at 100 rpm for 5 min. The mixture of petroleum ether and CN-CMC emulsion was then placed in a 50 mL centrifuge tube and centrifuged at 5000 rpm for 10 min. 10 mL of the supernatant petroleum ether solution was collected and placed in a round-bottom cake. The cake was then rotary evaporated at 40℃ and dried at constant weight in a 120℃ oven. The mass of the collected phytosterol esters (i.e., the mass of unencapsulated phytosterol esters) was recorded, and the encapsulation rate and loading of phytosterol esters in the CN-CMC emulsion were calculated.

[0094] Accurately weigh 20g of CN-PGA group phytosterol esters (Xi'an Haisifu Biotechnology Co., Ltd., LowChol) TMThe CN-HMP phytosterol ester emulsion was added with 20 mL petroleum ether (boiling point 60-90 °C) and stirred at 100 rpm for 5 min; then the mixture of petroleum ether and CN-HMP emulsion was placed in a 50 mL centrifuge tube and centrifuged at 5000 rpm for 10 min. 10 mL of the supernatant petroleum ether solution after centrifugation was taken to a round-bottom flask and rotary evaporated at 40 °C, and the mass of the collected phytosterol ester (i.e. the mass of unencapsulated phytosterol ester) was recorded. The encapsulation efficiency and loading capacity of phytosterol ester in the CN-HMP group emulsion were calculated.

[0095] Accurately weigh 20 g of CN-HMP group phytosterol ester (Xi'an Haishifu Biological Technology Co., Ltd., Low Chol TM The CN-HMP phytosterol ester emulsion was added with 20 mL petroleum ether (boiling point 60-90 °C) and stirred at 100 rpm for 5 min; then the mixture of petroleum ether and CN-HMP emulsion was placed in a 50 mL centrifuge tube and centrifuged at 5000 rpm for 10 min. 10 mL of the supernatant petroleum ether solution after centrifugation was taken to a round-bottom flask and rotary evaporated at 40 °C, and the mass of the collected phytosterol ester (i.e. the mass of unencapsulated phytosterol ester) was recorded. The encapsulation efficiency and loading capacity of phytosterol ester in the CN-HMP group emulsion were calculated.

[0096] Table 4 Loading capacity and encapsulation efficiency of CN and its emulsion combined with different stabilizers

[0097] Loading amount (%) Encapsulation efficiency (%) CN CN-PGA 94 90.0±1.1c CN-CMC 89 99.2±0.5b CN-HMP 89 99.5±0.3a Figure 6 89 98.9±1.3b

[0098] Note: There are significant differences (p < 0.05) between the data marked with different letters (a, b, c) in the same column.

[0099] As can be seen from Table 4, the encapsulation efficiency of the intermediate emulsion prepared with sodium caseinate as the emulsifier can reach 90%, and the addition of the three stabilizers can all improve the encapsulation efficiency of the emulsion. Among them, the CN-CMC group emulsion has a higher encapsulation efficiency, followed by the CN-PGA group, which may be related to the thickness of the emulsion interface film. In comparison, the intermediate emulsion has the thinnest interface layer, poor stability, and the lowest encapsulation efficiency.

[0100] Example 6: Storage stability of phytosterol ester emulsion

[0101] Prepare a 1% sodium caseinate solution and a 1% propylene glycol alginate solution, respectively. Mix the sodium caseinate solution with phytosterol ester (Xi'an Haishifu Biological Technology Co., Ltd., Low Chol TMThe CN intermediate emulsion was prepared by mixing the 1% casein sodium solution and phytosteryl esters (S-phytosteryl esters) at a mass ratio of 85:15, shearing at 20 000 rpm for 2 min, and homogenizing at 40 MPa for 2 times. The intermediate emulsion was mixed with the propylene glycol alginate solution at a mass ratio of 1:1, and the pH was adjusted to 3.5 by using 1 M NaOH and 1 M HC1. The CN-PGA phytosteryl ester emulsion was obtained by shearing at 20 000 rpm for 2 min. The stability of the emulsions was investigated by determining the particle size, zeta potential, PDI, micrographs, and appearance pictures during long-term storage at 4 °C and 15 °C.

[0102] As shown in Figs. 4A and 4B, the emulsions were stored at 4 °C and 15 °C for 56 days, respectively. The particle size and PDI increased with time. After storage at 4 °C for 56 days, the particle size and PDI of the intermediate emulsion increased from 2905 nm and 26% to 15537 nm and 35.8%, respectively, and the particle size increased to 8.7 times the original value. The particle size of the CN-PGA phytosteryl ester emulsion increased to 1.7 times the original value, and the change in particle size was small. After storage at 15 °C for 56 days, the particle size of the intermediate emulsion increased to 85 times the original value, and the PDI increased to 46%. The particle size of the CN-PGA phytosteryl ester emulsion increased to 2.7 times the original value. The zeta potential of the two groups of emulsions changed little at different temperatures and storage times. Figure 7 As shown in Figs. 5A and 5B, the CN intermediate emulsion showed obvious creaming at the top of the emulsion after storage at 4 °C for 14 days. As shown in Figs. 6A and 6B, flocculation and coalescence occurred between the droplets. As shown in Figs. 7A and 7B, the intermediate emulsion showed obvious oil-water separation after storage at 15 °C for 14 days, and the droplets coalesced and broke down seriously. In contrast, the CN-PGA phytosteryl ester emulsion did not produce flocculation after storage at 4 °C for 56 days, and no emulsion layer or stratification occurred, indicating good stability. Similarly, the CN-PGA phytosteryl ester emulsion showed only a small amount of droplet creaming after storage at 15 °C for 56 days, and the overall stability was good. In summary, the CN-PGA stable phytosteryl ester emulsion remained stable after storage at 4 °C and 15 °C for 56 days, indicating that the CN-PGA phytosteryl ester emulsion had good storage stability.

[0103] Figure 7 (A) shows that the CN intermediate emulsion showed obvious creaming at the top of the emulsion after storage at 4 °C for 14 days. (B) shows that flocculation and coalescence occurred between the droplets. (C and D) show that the intermediate emulsion showed obvious oil-water separation after storage at 15 °C for 14 days, and the droplets coalesced and broke down seriously. In contrast, the CN-PGA phytosteryl ester emulsion did not produce flocculation after storage at 4 °C for 56 days, and no emulsion layer or stratification occurred, indicating good stability. Similarly, the CN-PGA phytosteryl ester emulsion showed only a small amount of droplet creaming after storage at 15 °C for 56 days, and the overall stability was good. In summary, the CN-PGA stable phytosteryl ester emulsion remained stable after storage at 4 °C and 15 °C for 56 days, indicating that the CN-PGA phytosteryl ester emulsion had good storage stability. Figure 7 Figure 8

[0104] Example 7: Thermal treatment stability of phytosteryl ester emulsion

[0105] A 1% casein sodium solution was mixed with phytosteryl esters (Xi'an Haishifu Biological Technology Co., Ltd., Low Chol TM ​​​CN-PGA phytosterol ester emulsion was prepared by mixing the intermediate emulsion and the alginate propylene glycol ester solution at a mass ratio of 1:1, adjusting the pH to 3.5 with 1 M NaOH and 1 M HC1, and shearing at 10,000 rpm for 2 min. 8 mL of the CN-PGA phytosterol ester emulsion and the intermediate emulsion were respectively placed in 10 mL cryogenic tubes, and the emulsions were treated under different heat treatment conditions such as pasteurization (63°C / 30 min), preheating (80°C / 30 min), boiling (100°C / 10 min), and sterilization (121°C / 15 min). After cooling to room temperature, the particle size, zeta potential, PDI, microscopic pictures, and appearance images of the emulsions were determined.

[0106] In actual production and processing, in order to achieve the ideal shelf life of the product, it is usually subjected to heat treatment, such as pasteurization or preheating and boiling. Therefore, it is extremely important to determine the effect of heat treatment on the stability of the emulsion.

[0107] As can be seen from Figure 8 (A), compared with the untreated group, the particle size of the intermediate emulsion was significantly increased (p<0.05) after the three heat treatments, and the particle size of the sodium caseinate group was the largest, increasing from 2842 nm to 16463 nm. As can be seen from Figure 9 (D), after heat treatment, flocculation and coalescence occurred between the droplets of the intermediate emulsion, which was caused by the denaturation of protein during heat treatment, forming large protein aggregates, and the protein could not completely coat the phytosterol ester, resulting in instability. After heat treatment, the hydrophobic sites of the denatured protein molecules in different emulsion droplets were attracted to each other, so that heat-induced flocculation was the main reason for the increase in the particle size of the intermediate emulsion.

[0108] Compared with the intermediate emulsion group, the particle size of the CN-PGA group after heat treatment increased slightly, the droplets were uniformly dispersed, and the stability was good. This was mainly due to the adsorption of polysaccharides on the surface of the protein-stable droplets, which increased the electrostatic and steric repulsion of the emulsion by increasing the thickness and charge of the interface, thereby having strong temperature stability. In addition, heat treatment did not cause a change in the zeta potential of the emulsion, because the heating process did not cause significant changes in the composition or structure of the interface. In summary, the CN-PGA phytosterol ester emulsion has good thermal stability.

[0109] Example 8: Freeze-thaw stability of phytosterol ester emulsion

[0110] A 1% sodium caseinate solution was mixed with phytosterol ester (Xi'an Haishifu Biological Technology Co., Ltd., LowChol TMCN-PGA phytosterol esters were mixed at a mass ratio of 85:15, sheared at 20,000 rpm for 2 min, and homogenized twice at 40 MPa to obtain an intermediate emulsion. The intermediate emulsion was mixed with propylene glycol alginate solution at a mass ratio of 1:1, and the pH was adjusted to 3.5 using 1M NaOH and 1M HCl. The mixture was sheared at 10,000 rpm for 2 min to obtain a CN-PGA phytosterol ester emulsion. 8 mL of both the CN-PGA phytosterol ester emulsion and the intermediate emulsion were placed in 10 mL cryovials and frozen at -20℃ for 0–10 h. One vial was removed from each group every 2 h, and after thawing in a 37℃ water bath for 1 h. Particle size, potential, PDI, and microscopic images were measured, and images of the appearance were taken at different freeze-thaw times.

[0111] To prevent product quality deterioration, frozen storage is often necessary. The stability of emulsions after freeze-thaw cycles is crucial. Figure 9 (AC) analysis shows that after 2 hours of freeze-thaw cycles, the particle size of the intermediate emulsion increased from 2300 nm to over 10000 nm (>4.3 times). With increasing freezing time, the particle size increased, and the PDI (particulate density) also gradually increased, resulting in uneven droplet distribution. After 10 hours of freeze-thaw cycles, the particle size of the intermediate emulsion increased by 14.7 times. In contrast, after 10 hours of freezing, the particle size of the CN-PGA phytosterol ester emulsion increased only 1.7 times, from 3400 nm to 5900 nm. This difference is mainly attributed to the fact that the intermediate emulsion is stabilized solely by protein emulsification, has a thin and easily ruptured interfacial film, and the ice crystals generated during freezing disrupt the interfacial film, leading to the precipitation of phytosterol esters.

[0112] Depend on Figure 10 (D&E) results show that the intermediate emulsion exhibits significant demulsification after freezing at -18℃ for 2 hours and then thawing. The droplets gradually increase in size with increasing freeze-thaw time. Compared to the intermediate emulsion, the CN-PGA phytosterol ester emulsion maintains relative stability after freeze-thaw treatment. This is because the polysaccharides coated on the protein-coated droplet surface provide sufficient steric and electrostatic repulsion, and the thicker interfacial film is less susceptible to ice crystal damage, thus maintaining the emulsion's freeze-thaw stability.

[0113] Example 9: Digestive properties of phytosterol ester emulsions

[0114] (1) Particle size and potential changes

[0115] Weigh out 7.5g of phytosterol esters (Xi'an Haisifu Biotechnology Co., Ltd., LowChol). TM-S phytosterol esters) were mixed with 100 mL of 1% sodium caseinate solution and stirred at 1000 rpm for 5 min to obtain a physically mixed sample (without high-pressure homogenization) with the same phytosterol ester content as the CN-PGA phytosterol ester emulsion. The fresh intermediate emulsion prepared in Example 4 was diluted 2 times to make it have the same phytosterol ester content as the CN-PGA phytosterol ester emulsion. The blank control group sample was an aqueous solution at pH 7.

[0116] A gastrointestinal digestion model was established to simulate the human digestive tract environment. The standardized in vitro digestion method (INFOGEST) was used to study the digestion of the physical mixture group, intermediate emulsion, and CN-PGA phytosterol ester emulsion in the gastrointestinal tract. The physical mixture group and the intermediate emulsion group had the same content of phytosterol esters and emulsifiers, but the former was not subjected to high-pressure homogenization.

[0117] Simulated gastric digestion for 2 hours was performed, with 0.5 mL samples taken at 0, 60, and 120 min to measure the particle size and potential changes of the intermediate emulsion and the CN-PGA phytosterol ester emulsion. Similarly, simulated intestinal digestion for 4 hours was performed, with 0.5 mL samples taken at 0, 60, 120, 180, and 240 min to measure the particle size and potential changes of the intermediate emulsion and the CN-PGA phytosterol ester emulsion.

[0118] Depend on Figure 11 It was observed that the particle size of the intermediate emulsion continuously increased during the gastric stage, reaching a maximum of 18 μm at the end of gastric digestion. This is mainly because the outermost layer of the intermediate emulsion is sodium caseinate, and the pH during the gastric stage is 3, close to the isoelectric point of proteins. The emulsion's charge is close to 0, reducing electrostatic repulsion. Furthermore, pepsin hydrolyzes the protein, leading to demulsification and the aggregation of phytosterol esters, thus increasing the particle size. In contrast, the outermost layer of the CN-PGA group emulsion is polysaccharide. At pH 3, the opposite charge between proteins and polysaccharides provides sufficient electrostatic repulsion to prevent emulsion flocculation, resulting in uniform dispersion and smaller particle size.

[0119] After the intermediate emulsion enters the intestine, it completely breaks down, exposing the phytosterol esters. Under the action of pancreatic lipase and cholesterol esterase, some of the phytosterol esters are hydrolyzed into phytosterols, resulting in a smaller particle size, with the smallest particle size at the end of digestion being 15 μm. In a simulated intestinal fluid environment at pH 7, the CN-PGA phytosterol ester emulsion shows that proteins and polysaccharides carry the same charge, and the potential gradually increases, causing the polysaccharides to gradually dissociate from the protein surface. Under the action of pancreatic proteins, the proteins gradually decompose, and the phytosterol esters enter the digestive fluid and continuously undergo hydrolysis in contact with pancreatic lipase and cholesterol esterase, resulting in a continuously decreasing particle size, with the final particle size being approximately 2 μm. These results indicate that compared to the intermediate emulsion, the CN-PGA phytosterol ester emulsion has better stability in a gastric environment at pH 3, enabling targeted release of phytosterol esters in the intestine.

[0120] (2) Microstructure changes of phytosterol ester emulsions

[0121] At 0, 60, 120 min, 1 mL was sampled at regular intervals during the simulated gastric digestion process, and the microstructure changes of phytosterol ester in the physical mixing group, the intermediate emulsion and the water-dispersible phytosterol ester (Xi'an Haishifu Biological Technology Co., Ltd., LowChol TM -S phytosterol ester) emulsion were observed by inverted fluorescence. Similarly, at 0, 60, 120, 180, 240 min, 1 mL was sampled at regular intervals during the simulated intestinal digestion for 4 h, and the microstructure changes of phytosterol ester in the physical mixing group, the intermediate emulsion and the CN-PGA phytosterol ester emulsion were observed by inverted fluorescence.

[0122] Gastric digestion stage: The phytosterol ester in the physical mixing group sample was partially floated on the solution during the digestion process due to its high melting point and poor water dispersibility, and the phytosterol ester in the solution was aggregated together. As can be seen from Figure 12 (A), the phytosterol ester was aggregated into flakes. After the intermediate emulsion entered the gastric digestion stage, the droplets were flocculated due to the low electrostatic repulsion, and the droplets were gradually aggregated and broken emulsion as the digestion proceeded, and the phytosterol ester was released. At the end of the gastric digestion, the phytosterol ester was aggregated into large oil droplets. The CN-PGA group emulsion was uniformly distributed between the droplets during the gastric stage, and no obvious flocculation and aggregation phenomenon was observed.

[0123] Intestinal digestion stage: As the digestion time proceeded, the phytosterol ester droplets in the physical mixing group gradually decreased. This was because the phytosterol ester was gradually hydrolyzed into phytosterol under the action of cholesterol esterase and pancreatic lipase. At the end of the intestinal digestion, there were still a large amount of unhydrolyzed phytosterol ester. After the intermediate emulsion entered the intestinal digestion stage, the phytosterol ester was almost completely broken emulsion into the digestion solution. As the digestion time proceeded, the content of phytosterol ester gradually decreased. After the CN-PGA phytosterol ester emulsion entered the intestinal stage, the emulsifier and the stabilizer were dissociated due to the same charge at pH 7, resulting in flocculation between the droplets; During the whole digestion process, the droplets were small and no large droplets were observed. Compared with the physical mixing and intermediate emulsion groups, the content of unhydrolyzed phytosterol ester in the CN-PGA group phytosterol ester emulsion digestion solution was less at the end of the digestion.

[0124] (3) Hydrolysis rate of phytosterol ester in emulsion

[0125] During the simulation of gastric digestion, 0.5 mL was taken at 0, 60, 120 min, respectively, and the digestion solution was placed in a 1 mL EP tube, 0.5 mL chloroform was added, vortexed for 5 min, and the chloroform was extracted from the unhydrolyzed phytosterol esters and hydrolysis products phytosterols in the digestion solution, and centrifuged. 200 μL of chloroform layer was taken in a centrifuge tube, and nitrogen was blown to obtain the phytosterol and unhydrolyzed phytosterol ester samples produced during digestion. Similarly, during the intestinal digestion stage, 0.5 mL was taken at 0, 60, 120, 180, 240 min, respectively; the same treatment was performed as above to obtain the phytosterol and unhydrolyzed phytosterol ester samples produced during intestinal digestion. The foregoing samples were redissolved with anhydrous ethanol / n-hexane (1:1, v / v), filtered through an organic filter membrane, and used for high performance liquid chromatography analysis.

[0126] A Japan Shimadzu LC-20AD high performance liquid chromatograph equipped with a ZAM4000 evaporative light detector was used for analysis, and Symmetry C 18 A chromatographic column (5 μm, 4.6 x 150 mm, Waters) was used at a column temperature of 35 °C. The mobile phase was methanol / isopropanol (6:4, v / v) at a flow rate of 1 mL / min. The evaporative light detector used nitrogen as the carrier gas, the drift tube temperature was 70 °C, and the pressure was 0.5 MPa.

[0127] The content of phytosterols was calculated by external standard method. The hydrolysis rate of phytosterol esters was calculated according to formula (5).

[0128]

[0129] As can be seen from Figure 12 (B), during the gastric stage, phytosterol esters in the three groups of samples hardly hydrolyzed, and the hydrolysis rate was low. Figure 12 As can be seen from (A), at the end of gastric digestion, the peak area of phytosterol esters in the chromatogram of the intermediate emulsion was large, and there was a large amount of phytosterol esters, while the phytosterol esters in the physical mixing and CN-PGA groups were less. This is because the sodium caseinate in the outermost layer of the intermediate emulsion is hydrolyzed by pepsin in the gastric juice, and the emulsion is broken, and the phytosterol esters are dispersed in the digestion solution. The phytosterol esters in the physical mixing group are not well dispersed because they float on the upper layer of the digestion solution, so the peak area of the esters in the liquid phase is low. The phytosterol ester emulsion of CN-PGA can exist stably under acidic conditions, and no obvious emulsion breaking occurs, so less phytosterol esters are detected.

[0130] In the intestinal stage, the amount of phytosteryl ester dispersed in the digestive juice increased with the increase of digestion time in the physical mixing group. The hydrolysis rate increased continuously under the action of enzymes, but the increase was not significant after 60 min of digestion because of the limited amount of phytosteryl ester dispersed in the digestive juice. In the intermediate emulsion group, the phytosteryl ester in the sample entered the intestinal stage was completely demulsified, and part of the phytosteryl ester was aggregated, which increased the particle size and reduced the contact area with the enzyme. The increase in the hydrolysis rate of phytosteryl ester was not significant after 60 min of digestion. In the CN-PGA group, the emulsifier and stabilizer were dissociated after the emulsion entered the intestinal stage. With the progress of protein hydrolysis, phytosteryl ester was slowly released, and the particle size was small, which increased the contact area with the enzyme, so the hydrolysis rate increased significantly with time.

[0131] In summary, the hydrolysis rate of phytosteryl ester in the CN-PGA group was the highest among the three groups of samples, and the targeted release and slow release in the intestine could be achieved.

[0132] (4) Bioavailability of phytosteryl ester

[0133] Phytosteryl ester can only be utilized by the body after being hydrolyzed into phytosterol, so its hydrolysis rate can represent the bioavailability (or bioaccessibility). The amount of phytosterol in the digestive juice at the end of digestion was calculated according to formula (6) using the phytosterol standard curve prepared in "(3) Hydrolysis rate of phytosteryl ester in emulsion".

[0134]

[0135] From ​ (E), it can be seen that the bioavailability of phytosteryl ester in the physical mixing group was only 11%, while the bioavailability in the CN stabilized intermediate emulsion was comparable. In contrast, the bioavailability of the CN-PGA emulsion was 26%, which increased by 100.6% compared with the CN intermediate emulsion group.

[0136] Example 10: Preparation of phytosteryl ester liquid beverage

[0137] The intermediate emulsion and phytosterol ester emulsion prepared in Example 8 were selected and mixed with hawthorn juice beverage, concentrated orange juice beverage, concentrated coconut juice beverage, yogurt, fruit juice vinegar beverage, lactic acid bacteria beverage, soy milk beverage, milk beverage, peanut milk beverage, cola beverage, and green tea beverage according to the proportions shown in Table 5. The mixtures were stirred at 500 rpm for 10 minutes, bagged and sealed, and sterilized at 100℃ for 10 minutes. Subsequently, they were stored in a 25℃ constant temperature incubator for 6 months, with the appearance observed every half month to examine the uniformity of dispersion and the presence of suspended matter and sediment. Table 5 shows that the liquid beverages formed by the intermediate emulsion and hawthorn juice beverage, concentrated orange juice beverage, concentrated coconut juice beverage, yogurt, fruit juice vinegar beverage, lactic acid bacteria beverage, soy milk beverage, milk beverage, peanut milk beverage, cola beverage, and green tea beverage showed varying degrees of suspended matter within one or two months, indicating uneven dispersion. In contrast, the phytosterol ester emulsion, when combined with hawthorn juice, concentrated orange juice, concentrated coconut juice, yogurt, fruit vinegar, lactic acid bacteria, soy milk, cow's milk, peanut milk, cola, and green tea, remained uniformly dispersed after four months of storage, with no suspended matter or sediment. This indicates that the phytosterol ester emulsion prepared by this technology can be directly and conveniently applied to liquid beverages, possessing advantages such as good water dispersibility, strong stability, and high bioavailability.

[0138] Table 5. Main formulations and storage status of phytosterol ester liquid beverages after 6 months.

[0139]

[0140]

[0141] It should be understood that although this specification is described according to various embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0142] The detailed descriptions listed above are merely specific illustrations of feasible embodiments of the present invention and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a phytosterol ester emulsion, characterized in that, Includes the following steps: Step S1: Prepare emulsifier solution and stabilizer solution separately. The emulsifier is sodium caseinate, and the mass concentration of the emulsifier solution is 0.5-2%. The stabilizer is propylene glycol alginate, and the mass concentration of the stabilizer solution is 0.5-2%. Step S2: Mix the emulsifier solution with phytosterol esters at a mass ratio of 85:15 to 90:10, and then homogenize under high speed and high pressure to obtain an intermediate emulsion. Step S3: Mix the intermediate emulsion with the stabilizer solution at a mass ratio of 1:2 to 2:1, adjust the pH to 2.5 to 4.5, and perform high-speed shearing to obtain the phytosterol ester emulsion.

2. The method for preparing the phytosterol ester emulsion according to claim 1, characterized in that, In step S2, the high-speed shearing conditions are 10,000 to 20,000 rpm for 2 to 4 minutes.

3. The method for preparing the phytosterol ester emulsion according to claim 1, characterized in that, In step S2, the pressure of high-pressure homogenization is 20~40 MPa, and the number of cycles is 2~4.

4. The method for preparing the phytosterol ester emulsion according to claim 1, characterized in that, In step S3, the high-speed shearing conditions are 10,000 to 20,000 rpm for 2 to 4 minutes.

5. A phytosterol ester emulsion, characterized in that, The phytosterol ester emulsion is obtained by the preparation method according to any one of claims 1 to 4.

6. An application of a phytosterol ester emulsion, characterized in that, Application of the phytosterol ester emulsion according to claim 5 in the preparation of food, pharmaceuticals, cosmetics and feed.

7. The application of the phytosterol ester emulsion according to claim 6, characterized in that, Application of the phytosterol ester emulsion in liquid beverages.

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