Preparation method of soybean oil body composite particles with antioxidant efficacy

Abstract

The application discloses a preparation method of soybean oil body with antioxidant effect, and relates to the field of food processing and storage. The inner core of natural soybean oil body is hydrophobic triglyceride, which is lipophilic, and the outer part is covered by a phospholipid-oil body protein film, which is hydrophilic. The application utilizes the structural characteristics of natural soybean oil body with amphiphilicity, couples small-molecule compounds with antioxidant effect with soybean oil body protein, and wraps the antioxidant active substances into the inner part of the soybean oil body to prepare soybean oil body composite particles with antioxidant effect. The application improves the antioxidant capacity and storage stability of the soybean oil body.

Description

A method for preparing soybean oil complex particles with antioxidant effects Technical Field

[0001] This invention belongs to the field of green processing and manufacturing technology, specifically relating to a method for preparing soybean oil composite particles with antioxidant effects. Background Technology

[0002] Soybean oil bodies are widely present in plant seeds, providing energy for seed germination. The liquid matrix inside the oil body is composed of triglycerides (TAGs), and its surface is covered by a single-layer biofilm composed of phospholipids and endogenous proteins. This biofilm tightly encapsulates the TAGs, thus protecting them from the external environment. Soybean oil bodies are rich in polyunsaturated fatty acids, phospholipids, and tocopherols, among other bioactive substances. The surface proteins of the oil bodies are high in content and diverse in type, exhibiting physicochemical stability. They can be used as natural, high-quality emulsifiers and stabilizers in the food processing industry.

[0003] In recent years, soybean oil has been widely used in the pharmaceutical and food industries. Some studies have used the oil as a carrier to carry curcumin, which has poor water solubility but has the effects of preventing diseases and improving human health, thereby improving the bioavailability of curcumin. Other studies have added soybean oil to cookies, and the results showed that adding an appropriate amount of soybean oil can improve the hardness, chewiness, and elasticity of cookies. In addition, in the bread making process, soybean oil is used as an emulsifier. It was found that bread made with 15% soybean oil had better sensory scores, textural properties, acid value, and peroxide value than bread with other amounts of soybean oil, indicating that soybean oil has the potential to replace traditional emulsifiers in food production.

[0004] However, precisely because soybean oil is rich in various proteins on its surface and contains a variety of nutrients in its internal TAGs, it is conducive to the growth of microorganisms during storage. After extraction, it easily spoils and deteriorates at room temperature, making it unstable for storage. Preservatives such as sodium azide are often added during preservation, but these preservatives are highly toxic and cause environmental pollution. Therefore, finding a safe and healthy method to extend the storage life of soybean oil and improve its stability is particularly important. The feature of this invention is that the core TAGs of the soybean oil are lipophilically loaded with sesamol, and the surface proteins are coupled with ferulic acid. This structural feature, combined with antioxidants, enhances the antioxidant capacity and stability of the soybean oil. Summary of the Invention

[0005] The purpose of this invention is to solve the problem of poor stability of soybean oil by providing a preparation method that has antioxidant effects and improves the stability of soybean oil.

[0006] A method for preparing soybean oil complex particles with antioxidant effects, comprising the following steps:

[0007] 1) Establishment of an efficient soybean oil extraction method

[0008] a. Mix soybeans with the extract, soak, crush and stir;

[0009] b. Homogenize and filter, heat and then cool, add sucrose solution, and mix well;

[0010] c. Centrifuge and collect the upper layer of material;

[0011] d. Wash repeatedly three times and collect the upper layer of soybean oil.

[0012] 2) A method for preparing soybean oil containing antioxidant active substances

[0013] a. Disperse the antioxidant active substances and soybean oil in sterile water and stir until homogeneous;

[0014] b. Adjust the pH of the NaOH solution to 11.0 and stir well;

[0015] c. Adjust the pH to 1.0 using HCl solution and stir well;

[0016] d. Ultrasonic treatment, followed by adjusting the sample pH to neutral with NaOH solution;

[0017] e. Dialysis to remove salt ions, centrifugation, and collection of the supernatant.

[0018] 3) A method for preparing soybean oil complex particles with antioxidant effects

[0019] a. Mix the soybean oil containing antioxidant active substances as described in step 2) with small molecule compounds with antioxidant effects evenly;

[0020] b. Adjust the pH to 9.0 with NaOH, react overnight, and then adjust the pH to 7.0 with HCl;

[0021] c. Dialyze, centrifuge, and collect the upper soybean oil layer.

[0022] The antioxidant active substances mentioned in step 2) are sesamol, resveratrol, astaxanthin, etc.

[0023] Step 3) describes small molecule compounds with antioxidant properties, such as ferulic acid, gallic acid, and tannic acid.

[0024] In step 1), the ratio of soybeans to extract is 9:100.

[0025] The soybean seeds soaking time in step 1) is 18.5 h;

[0026] The heating temperature mentioned in step 1) is 75℃;

[0027] The sucrose concentration mentioned in step 1) is 25%;

[0028] In step 2), the NaOH concentration is 2 mol / L, and the pH is adjusted to 11.0;

[0029] In step 2), the HCl concentration is 4 mol / L, and the pH is adjusted to 1.0.

[0030] After adjusting the pH to 1.0 in step 2), the mixture is ultrasonically homogenized and then the pH is adjusted to neutral.

[0031] The ultrasound conditions described in step 2) are 150 W for 8 minutes;

[0032] Step 3) The pH of the soybean oil containing added small antioxidant molecules and encapsulated antioxidant active substances is adjusted to 9.0;

[0033] Step 3) describes a soybean oil concentration of 10% containing coupled antioxidant small molecules and encapsulating antioxidant active substances.

[0034] Step 3) describes a ferulic acid concentration of 15 mM.

[0035] The pH of the HCl solution described in step 3) is adjusted to 9.0;

[0036] 4) The main active ingredients of a soybean oil complex particle with antioxidant effects

[0037] a. The moisture content of soybean oil composite particles was determined by the loss on drying method and was greater than 50%;

[0038] b. The fat content of soybean oil complex particles was determined by Soxhlet extraction and was greater than 70%;

[0039] c. The total protein content of soybean oil bodies, determined using the BCA method, is greater than 15%;

[0040] d. The composition of unsaturated fatty acids in soybean oil was determined by gas chromatography and was greater than 70%;

[0041] e. The contents of sesamol, ferulic acid, and total tocopherol were determined by high performance liquid chromatography and reverse-phase high performance liquid chromatography, and were greater than 300 mg / kg.

[0042] 5) A soybean oil complex particle with antioxidant properties improves storage stability.

[0043] Weigh 1 g of the soybean oil and 1 g of the composite particles prepared in steps 1), 2), and 3), respectively, and place them in centrifuge tubes.

[0044] When sealed and placed vertically at 4°C, the soybean oil began to aggregate and break down after 7 days, gradually releasing internal triglycerides. The soybean oil composite particles remained unchanged after 30 days, still exhibiting a white, milky appearance, and the particle size did not change significantly.

[0045] This invention provides a method for preparing and applying soybean oil composite particles with antioxidant effects, comprising: 1) a green and safe method for extracting soybean oil; 2) encapsulating antioxidant active substances within the soybean oil obtained in step 1; and 3) coupling the soybean oil containing the antioxidant active substances described in step 2 with small molecule compounds with antioxidant effects to obtain soybean oil composite particles with antioxidant effects. The soybean oil composite particles with antioxidant effects prepared by this invention exhibit excellent ABTS free radical scavenging efficiency, overcoming the disadvantage of soybean oil being easily oxidized during storage, and significantly improving the antioxidant effect of soybean oil. Furthermore, the external coupling with internal loading extends the storage time of soybean oil, improving its storage stability. Attached Figure Description

[0046] Figure 1. Appearance of a soybean oil body with antioxidant properties.

[0047] Figure 2. Comparison of the antioxidant effects of soybean oil with antioxidant properties.

[0048] Figure 3. Comparison of storage stability of a soybean oil composite particle with antioxidant effects. Detailed Implementation

[0049] Example 1: Establishment of a method for soybean oil extraction

[0050] Five different methods for extracting soybean oil were compared, and the extraction rate was selected as the indicator for enzyme inactivation extraction. The optimal conditions for extracting soybean oil (SOB) were obtained by optimizing factors such as soaking time, solid-liquid ratio, crushing time, enzyme inactivation temperature, enzyme inactivation time, and sucrose concentration: soaking time 18.5 h, solid-liquid ratio 1:15, crushing time 160 s, enzyme inactivation temperature 75℃ for 30 min, and sucrose concentration 25%.

[0051] Example 2: Extraction of soybean oil

[0052] Soak soybeans in a 9:100 ratio of soybeans to deionized water for 18.5 h, then stir for 160 s. Filter the homogenate through three layers of nylon cloth to obtain raw soy milk. Heat the raw soy milk in a 75°C water bath for 30 min, then cool to room temperature. Add 25% sucrose to the raw soy milk, mix well, and centrifuge at 4°C, 12000 rpm for 20 min. Collect the supernatant. For the first wash, add 5 times the mass of deionized water, then add 25% sucrose and mix well. Centrifuge at 4°C, 12000 rpm for 20 min and collect the supernatant. Perform a second wash using the same method as the first wash, mix well, and centrifuge again under the same conditions to collect the supernatant oil "SOB".

[0053] Example 3: Construction of soybean oil bodies encapsulating sesamol

[0054] Sesamol was dispersed in 10 mL of deionized water, and 10% soybean oil was added. The mixture was stirred until it was evenly dispersed. The pH was adjusted to 11.0 with 2 mol / L NaOH solution. The mixture was stirred evenly, and the pH was adjusted to 1.0 with 4 mol / L HCl solution. The mixture was then sonicated (150 W, 8 min) and thoroughly mixed. The pH was then adjusted to neutral with 4 mol / L NaOH solution. The mixture was dialyzed for 2 h to remove salt ions from the emulsion. The emulsion was centrifuged at 4 °C and 12000 rpm for 20 min, and the supernatant was collected as “SOBL”.

[0055] Example 4: Construction of soybean oil bodies coupled with ferulic acid

[0056] SOBL was uniformly dispersed in deionized water, ferulic acid was added, and the pH of the SOBL solution was adjusted to 9.0 with 2 mol / L NaOH. The concentration of SOBL in the solution was 10% (w / v), and the concentration of ferulic acid was 15 mmol / L. The mixed solution was placed under pH 9 and reacted continuously for 12 h. After the reaction was completed, the pH was adjusted to 7.0 with 2 mol / L HCl solution. After dialysis, the dialysate was centrifuged (12000 rpm, 4℃), and the supernatant was collected to obtain soybean oil body "FA-SOBL".

[0057] Example 5: Composition determination of soybean oil complex particles

[0058] Determination of moisture content in soybean oil composite particles by loss on drying: Weigh soybean oil composite particles into a constant-weight flat weighing bottle, dry them in an oven at 105℃ until constant weight, and calculate the weight of moisture lost.

[0059] Determination of fat content in soybean oil complex particles by Soxhlet extraction: Weigh 2 g of freeze-dried soybean oil complex particle sample, add 50 mL of petroleum ether, mix well, add to the Soxhlet extractor needle, reflux at 60℃ for 8 h, filter the organic phase, remove the petroleum ether solvent using a rotary evaporator at 100℃, and calculate the fat content using the remaining crude residue in the flask.

[0060] Determination of total protein content of soybean oil complex particles by BCA method: Weigh 2 g of freeze-dried soybean oil complex particle sample, disperse it in 4% (w / v) SDS solution, mix well, centrifuge at 12000 rpm for 40 min, take the supernatant, and determine the protein content using the BCA kit.

[0061] Determination of fatty acid composition of soybean oil by gas chromatography: 2 g of freeze-dried soybean oil composite particle sample was weighed, 10 mL of n-hexane was added, and the mixture was stirred evenly in ice water for 10 min to extract fatty acids. The sample was centrifuged at 8000 rpm for 10 min, the supernatant was removed, and the oil was concentrated under a nitrogen atmosphere to obtain crude oil. The oil residue was then dissolved in 1 mL of n-heptane, followed by the addition of 10 mL of 2% sodium hydroxide methanol solution. The mixture was stirred at 80 °C for 20 min. After cooling, 5 mL of BF3-MeOH was added, and the mixture was stirred at 80 °C for 5 min. After cooling, 10 mL of n-heptane was added to wash the organic layer, and the two phases were shaken for 2 min and allowed to stand for separation. The organic layer was dried on anhydrous sodium sulfate and then transferred to a headspace vial for GC analysis.

[0062] Determination of total phenol content in soybean oil composite particles: Weigh 2 g of freeze-dried soybean oil composite particle sample and mix with 10 mL of methanol solution. Extract by ultrasonication in cold water for 10 min. Centrifuge at 8000 rpm for 10 min to recover the supernatant and obtain the extract. Mix 1 mL of the extract with 0.5 mL of FCR reagent and 5 mL of deionized water for 3 min, then add 1 mL of saturated sodium carbonate solution. Incubate in the dark at room temperature for 1 h. Centrifuge at 10000 rpm for 10 min at 4℃. Using gallic acid as a standard curve, measure the absorbance at 760 nm using a spectrophotometer and calculate the total phenol content.

[0063] The tocopherol content was determined by reverse-phase high-performance liquid chromatography (RP-HPLC): 2 g of freeze-dried soybean oil composite particles were weighed, added to 10 mL of hexane, mixed thoroughly, and extracted with cold water by ultrasonication for 10 min. The mixture was centrifuged at 8000 rpm for 10 min, the supernatant was removed, and the extraction process was repeated. The extract was dried under a nitrogen atmosphere to obtain crude oil, which was dissolved in 1 mL of isopropanol, filtered through a 0.22 μm filter membrane, and analyzed by GC.

[0064] Determination of sesamol content in soybean oil complex particles: Take 20 mg of soybean oil complex particles, add 1 mL of methanol, vortex for 30 s, sonicate for 5 min, centrifuge at 8000 rpm for 10 min at 4℃, take the supernatant and filter it through a 0.22 μm filter membrane to obtain the filtrate. Take 200 μL of the filtrate and measure the absorbance at 304 nm. Calculate the content according to the following formula based on the linear regression equation y=10.987x+0.3009.

[0065] (where A) 样 (where a is the absorbance of the sample and 'a' is the coefficient of the linear regression equation)

[0066] Determination of ferulic acid content in soybean oil composite particles: Take 1 mL of composite particles FA-SOBL and mix it with 5 mL of 10% Folin-Ciocalteu solution. After reacting for 5 min, add 4 mL of 7.5% Na2CO3 solution, mix well, and react in the dark at room temperature for 45 min. Measure the absorbance at a wavelength of 708 nm. The linear regression equation is y = 5.0036x + 0.0694. Calculate the content according to the following formula.

[0067] (where A) 样 (where a is the absorbance of the sample and 'a' is the coefficient of the linear regression equation)

[0068] Table 1. Basic Component Contents of Soybean Oil Complex Particles

[0069]

[0070] Example 6: Antioxidant evaluation of soybean oil body complex particles coupled with ferulic acid and encapsulated with sesamol

[0071] Take 0.2 g of soybean oil, add 1 ml of solvent (methanol:water = 80:20), vortex for 1 min, sonicate for 5 min to obtain FA-SOBL soybean oil suspension, then centrifuge (4 ℃, 8000 rpm, 5 min), filter the supernatant through a 0.22 μm filter membrane to obtain “FA-SOBL” sample solution.

[0072] Mix the FA-SOBL sample solution and ABTS solution at a ratio of 9:1 (volume ratio), let stand at room temperature in the dark for 6 minutes, and measure the absorbance at a wavelength of 734 nm. Perform the test in triplicate. Use the water and ABTS mixture as a blank solution and calculate the scavenging activity according to the following formula.

[0073]

[0074] Example 7: Storage stability evaluation of soybean oil body complex particles coupled with ferulic acid and encapsulated with sesamol

[0075] Weigh 1g of FA-SOBL composite particles into a centrifuge tube, seal it, and place it vertically at 4℃. Observe the appearance changes on day 0, day 1, day 7, day 14, and day 30, and measure its particle size potential.

[0076] Comparative Example 1: The difference between this comparative example and Example 4 is that this example does not contain sesamol-loaded soybean oil coupled with gallic acid.

[0077] Soybean oil was uniformly dispersed in deionized water, gallic acid was added, and the pH of the soybean oil dispersion was adjusted to 9.0 with 2 mol / L NaOH. The concentration of soybean oil in the solution was 10% (w / v), and the concentration of gallic acid was 15 mM. The mixed solution was then reacted continuously at pH 9 for 12 h. The pH was then adjusted to 7.0 with 2 mol / L HCl solution, dialyzed, and the dialysate was centrifuged (12000 rpm, 4℃). The upper layer was taken to obtain soybean oil “GA-SOB”.

[0078] Comparative Example 2: The difference between this comparative example and Example 4 is that it does not contain sesamol-loaded soybean oil coupled with ferulic acid.

[0079] Soybean oil was uniformly dispersed in deionized water, ferulic acid was added, and the pH of the soybean oil dispersion was adjusted to 9.0 with 2 mol / L NaOH. The concentration of soybean oil in the solution was 10% (w / v), and the concentration of ferulic acid was 15 mM. The mixed solution was then reacted continuously at pH 9 for 12 h. After the reaction was completed, the pH was adjusted to 7.0 with 2 mol / L HCl solution, and dialyzed. The dialysate was centrifuged (12000 rpm, 4℃), and the upper layer was taken to obtain soybean oil “FA-SOB”.

[0080] Comparative Example 3: The difference between this comparative example and Example 4 is that the soybean oil is loaded with sesamol and then coupled with gallic acid.

[0081] Sesamol was dispersed in 10 mL of deionized water, and 10% soybean oil was added. The mixture was stirred until it was evenly dispersed. The pH was adjusted to 11.0 with 2 mol / L NaOH solution and stirred evenly. The pH was then adjusted to 1.0 with 4 mol / L HCl solution and sonicated (150 W, 8 min) until it was evenly mixed. The pH was then adjusted to neutral with 4 mol / L NaOH solution. After dialyzing for 2 h to remove salt ions from the emulsion, the mixture was centrifuged at 4 °C and 12000 r / min for 20 min. The upper paste-like substance was collected as SOBL. Then, SOBL was uniformly dispersed in deionized water, gallic acid was added, and the pH of the GA-SOBL solution was adjusted to 9.0 with 2 mol / L NaOH. The concentration of GA-SOBL soybean oil in the solution was 10% (w / v), and the concentration of gallic acid was 15 mM. The mixed solution was then reacted continuously at pH 9 for 12 h. Subsequently, the pH was adjusted to 7.0 with 2 mol / L HCl solution, dialyzed, and the dialysate was centrifuged (12000 rpm, 4℃). The upper layer was taken to obtain soybean oil “GA-SOBL”.

[0082] Comparative Example 4: The difference between this comparative example and Example 4 is that the peanut oil content...

[0083] Peanut oil was extracted using the Tris-Cl sucrose method. The contents of moisture, protein, fat, unsaturated fatty acids, total phenols, and total tocopherols in the peanut oil were determined by drying loss, Soxhlet extraction, BCA method, gas chromatography, high performance liquid chromatography, reversed-phase high performance liquid chromatography, and ultraviolet spectrophotometry.

[0084] The compositional content of FA-SOBL prepared in Example 4 and peanut oil extracted in Comparative Example 4 were compared. As shown in Table 3, the basic components of FA-SOBL and peanut oil were compared. The soybean oil complex particles contain a large amount of tocopherol, which can slow down skin aging and delay aging.

[0085] Table 3 Comparison of soybean oil complex particle composition and peanut oil composition

[0086]

[0087] The antioxidant capacity of FA-SOBL prepared in Example 4 and GA-SOB, FA-SOB, and GA-SOBL prepared in Comparative Examples 1, 2, and 3 were tested. Table 4 shows that the addition of active antioxidants and small molecule compounds significantly improved the antioxidant capacity of soybean oil, and these antioxidant capacities remained significant even after 10 days. FA-SOBL exhibited the best antioxidant stability.

[0088] Table 4. ABTS radical scavenging rate (%) in the examples and comparative examples

[0089]

[0090] The storage stability of FA-SOBL prepared in Example 4 and GA-SOB, FA-SOB, and GA-SOBL prepared in Comparative Examples 1, 2, and 3 was evaluated. Table 5 shows that the addition of antioxidant active substances and small molecule compounds significantly improved the storage stability of soybean oil, with FA-SOBL exhibiting the best storage stability.

[0091] Table 5. Particle size changes (nm) of the Examples and Comparative Examples after storage at 4°C for different times.

[0092]

Claims

1. A method for preparing soybean oil composite particles with antioxidant effects, comprising the following steps: 1) Establishing a high-concentration soybean oil extraction method: a. Take an appropriate amount of soybeans, sterilize and disinfect them, add sterile water, soak overnight, crush, stir and filter to obtain raw soybean milk; b. Heat the raw soybean milk and then cool it, add sucrose, mix well, centrifuge, and collect the upper layer; c. Add sterile water and sucrose solution and wash repeatedly three times, collect the upper oil, and store at 4℃; 2) A method for preparing soybean oil composite particles with antioxidant effects: a. Encapsulate the active substance with antioxidant effect into the core of the soybean oil; b. Place the soybean oil containing the antioxidant active substance and the antioxidant small molecule compound in an aqueous solution and adjust the pH to 9.0 to react; c. Adjust the pH of the solution in step b to 7.0; dialyze to remove the free antioxidant small molecule compound to obtain soybean oil composite particles; wherein the preparation method of the soybean oil containing the antioxidant active substance is as follows: A) A) The active substance with antioxidant activity is dispersed in sterile water with soybean oil and stirred until homogeneous; B) The pH value is adjusted to 11.0 with NaOH solution and stirred until homogeneous; C) The pH value is adjusted to 1.0 with HCl solution and stirred until homogeneous; D) Ultrasonic treatment is performed, and the pH value of the sample is adjusted to neutral with NaOH solution; E) Dialysis is performed to remove salt ions, centrifugation is performed, and the upper paste-like substance is collected; The active substance with antioxidant activity in step 2) is sesamol, and the small molecule compound with antioxidant activity is ferulic acid; The soybean oil composite particles contain 0.014±0.015 mg / kg sesamol and 0.025±0.008 mg / kg ferulic acid.

2. The method for preparing soybean oil composite particles with antioxidant effects according to claim 1, characterized in that: In step 1), the soybean oil extraction method uses a soybean to sterile water ratio of 9:100.

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