Vitamin E-phytosterol oleate composite liposome as well as preparation method and application thereof

Abstract

The invention discloses a vitamin E-phytosterol oleate composite liposome as well as a preparation method and application thereof, the composite liposome is prepared by taking soybean phospholipid, phytosterol oleate and vitamin E as raw materials and adopting a film ultrasonic method, and the mass ratio of the soybean phospholipid to the phytosterol oleate to the vitamin E is 20: 1: 1. Soybean lecithin and phytosterol oleate are used as main membrane materials to embed vitamin E, the vitamin E-phytosterol oleate composite liposome is constructed, the doping of phytosterol oleate improves the ability of vitamin E to remove DPPH free radicals, and the phytosterol oleate and vitamin E in the liposome play a synergistic anti-oxidation role; by doping the phytosterol oleate, the lipid peroxidation resistance of the vitamin E can be improved; and the reduction capability of the vitamin E is improved by doping the phytosterol oleate. When the additive is applied to the fried instant noodles, the storage stability of the fried instant noodles is effectively improved.

Description

Technical Field

[0001] The invention relates to the field of antioxidants in food, in particular to a vitamin E-phytosterol oleate composite liposome, and also to a preparation method of the vitamin E-phytosterol oleate composite liposome and application of the vitamin E-phytosterol oleate composite liposome in fried instant noodles. Background Art

[0002] Vitamin E, also known as tocopherol or tocopherol, is one of the most effective antioxidants and has anti-inflammatory properties. It is widely used in medicine, cosmetics, food and other fields. However, vitamin E is prone to oxidation reactions and is a heat-sensitive fat-soluble vitamin. It is insoluble in water and soluble in fat-soluble solvents such as methanol, ethanol, and chloroform. Ultraviolet rays and oxygen can affect the stability of vitamin E, and its bioavailability is low, which in turn limits its development. How to overcome these obstacles and improve its water dispersibility, stability and bioavailability is the current research focus.

[0003] At present, researchers mostly use encapsulation technology to encapsulate vitamin E, and improve the water solubility, stability and bioavailability of vitamin E through encapsulation. Existing vitamin E is mainly encapsulated in the form of liposomes, but liposomes have their own defects. How to improve the stability of liposomes is of great scientific and practical significance for expanding the application of liposomes in food, health products, medicines and cosmetics industries. Summary of the invention

[0004] In view of this, the present invention provides a vitamin E-phytosterol oleate composite liposome, and also provides a preparation method of the vitamin E-phytosterol oleate composite liposome and an application of the vitamin E-phytosterol oleate composite liposome in fried instant noodles.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The vitamin E-phytosterol oleate composite liposomes of the present invention are prepared by using soybean lecithin, phytosterol oleate and vitamin E as raw materials through a thin film ultrasonic method, and the mass ratio of soybean lecithin, phytosterol oleate and vitamin E is 20:1:1.

[0006] Preferably, the method for preparing the composite liposome comprises the following contents: dissolving soybean lecithin, phytosterol oleate and vitamin E in an organic solvent, and performing rotary evaporation in the dark to form a thin film; washing the thin film with a phosphate buffer containing Tween-80 to form a liposome suspension, and subjecting the liposome suspension to probe ultrasound in an ice-water bath for 4 minutes to obtain vitamin E-phytosterol oleate composite liposomes.

[0007] Preferably, the organic solvent is a mixture of n-hexane and anhydrous ethanol in a volume ratio of 1:1.

[0008] Preferably, the concentration of Tween-80 in the phosphate buffer is 10 g / L, and the pH of the phosphate buffer is 7.4.

[0009] Preferably, the probe ultrasound is turned on for 5 s, turned off for 5 s, and repeated for 4 min.

[0010] The invention also provides application of vitamin E-phytosterol oleate composite liposome in fried instant noodles, and the storage stability of fried instant noodles is improved by adding the composite liposome into the fried instant noodles.

[0011] Compared with the prior art, the present invention uses soybean lecithin and phytosterol oleate as the main membrane materials to embed vitamin E, and constructs vitamin E-phytosterol oleate composite liposomes. According to experiments, the incorporation of phytosterol oleate improves the ability of vitamin E to scavenge DPPH free radicals, and plays a synergistic antioxidant role with vitamin E in the liposome; the incorporation of phytosterol oleate can increase the anti-lipid peroxidation ability of vitamin E; the incorporation of phytosterol oleate improves the reducing ability of vitamin E. The composite liposome of the present invention is applied to fried instant noodles, and research data show that the composite liposome can effectively inhibit the rancidity and oxidation of oil in fried instant noodles, improve the storage stability of fried instant noodles, and extend the shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the particle size distribution diagram of the composite liposome of the present invention.

[0013] Figure 2 is a transmission electron micrograph of the composite liposome of the present invention.

[0014] Figure 3 It is the clearance rate of the composite liposome of the present invention in clearing free radicals.

[0015] Figure 4 It is the anti-lipid peroxidation ability of the composite liposome of the present invention.

[0016] Figure 5 is the reducing ability of the composite liposome of the present invention.

[0017] Figure 6 The invention relates to the determination of the acid value of the composite liposome used in fried instant noodles.

[0018] Figure 7 The invention relates to the determination of the antioxidant value of the composite liposome used in fried instant noodles. DETAILED DESCRIPTION

[0019] The present invention is described in detail below in conjunction with the accompanying drawings. It should be pointed out that the reagents and experimental instruments used in the present invention are all commercially available reagents or instruments commonly used in existing laboratories.

[0020] The present invention provides a vitamin E-phytosterol oleate composite liposome, wherein soybean lecithin and phytosterol oleate are used as membrane materials to embed vitamin E, and the mass ratio of soybean lecithin, phytosterol oleate and vitamin E is 20:1:1. The specific preparation method is as follows: Soybean lecithin, phytosterol oleate and vitamin E were dissolved in an organic solvent (mixed by volume of n-hexane-anhydrous ethanol in a ratio of 1:1), and then rotary evaporated in the dark to form a thin film on the inner wall of the flask; the film was washed with phosphate buffer (pH=7.4) containing Tween-80 to form a liposome suspension, and the liposome suspension was probe-ultrasonicated in an ice-water bath (i.e., on for 5 s, off for 5 s, repeated for 4 min) to obtain vitamin E-phytosterol oleate complex liposomes. Example

[0021] The vitamin E-phytosterol oleate complex liposomes described in this embodiment are prepared by the following steps: accurately weigh 0.2 g soybean lecithin, 0.01 g phytosterol oleate, and 0.01 g vitamin E, dissolve in 10 mL of a mixed solvent of n-hexane-anhydrous ethanol (volume ratio 1:1), and remove the solvent by rotary evaporation at 40° C. in the dark; weigh 0.1 g Tween-80 and dissolve in 20 mL of phosphate buffer (PH=7.4), pour the buffer into a rotary evaporation flask for membrane washing to form a liposome suspension, and then probe-ultrasound in an ice-water bath (180 W, 5 s on, 5 s off, 4 min) to obtain vitamin E-phytosterol ester complex liposomes.

[0022] Example 2 Physical properties and characterization of vitamin E-phytosteryl oleate complex liposomes 1. Particle size distribution, zeta potential and encapsulation efficiency of composite liposomes The particle size and distribution, zeta potential and encapsulation efficiency of the vitamin E-phytosterol oleate composite liposomes of the present invention are shown in Tables 1 and Figure 1 From Table 1 and Figure 1 It can be seen that the average particle size of the composite liposome is 134.0 ± 12.5 nm, which is nanoscale and more conducive to digestion and absorption. The PDI value of the composite liposome is 0.34 ± 0.01, showing a single peak morphology and normal distribution, indicating that the composite liposome vesicles are evenly distributed and have good dispersibility. The average zeta potential of the composite liposome is -23.6 ± 1.2 mV, indicating that the electrostatic repulsion between liposome vesicles is relatively large, which can effectively avoid the aggregation and fusion of liposomes, and the liposome system is stable. The encapsulation efficiency of the composite liposome is 93 ± 0.45%, indicating that the composite liposome vesicles have a large volume and a good encapsulation effect.

[0023] Table 1 2. The microscopic morphology of vitamin E-phytosterol oleate composite liposomes was observed using a transmission electron microscope. Figure 2 .Depend on Figure 2 It can be seen that the vitamin E-phytosterol oleate composite liposomes are approximately spherical, with a particle size of about 130 nm, uniform distribution, regular shape, and no obvious signs of collapse and agglomeration, indicating that the vitamin E-phytosterol oleate composite liposomes of the present invention are uniformly distributed and have good microscopic morphological characteristics.

[0024] Example 3 Determination of the Antioxidant Capacity of Vitamin E-Phytosterol Ester Complex Liposomes 1. Determination of the ability of vitamin E-phytosterol ester complex liposomes to scavenge free radicals DPPH is a stable free radical that turns purple when dissolved in ethanol. When DPPH reacts with hydrogen provided by antioxidants, it causes a change in the color of the solution. The ethanol solution of DPPH has a maximum absorbance at 517 nm, so the change in absorbance at 517 nm is used to measure the ability to scavenge DPPH free radicals.

[0025] The determination method is as follows: blank liposomes (Liposomes, L), phytosterol oleate ester-loaded liposomes (Phytosterol oleate ester-loaded liposomes, L-POE), vitamin E liposomes (Vitamin E-loaded liposomes, L-VE), vitamin E-phytosterol oleate ester complex liposomes (Liposomes co-encapsulating Vitamin E and phytosterol oleate ester, L-VE-POE), and free vitamin E (Vitamin E, VE) were prepared respectively.

[0026] Accurately pipette 2 mL of sample into a test tube, add 4 mL of DPPH ethanol solution (0.1 mmol / L) and mix with it. After placing in the dark for 30 min, take the supernatant and measure its absorbance at 517 nm. Use blank liposomes instead of sample as blank and measure the absorbance value A. 517 Blank, ethanol was used instead of DPPH as a control. The calculation formula for DPPH free radical scavenging rate is as follows: DPPH free radical scavenging rate (%) = [1-(A 517 Sample-A 517 Control) / A 517 blank] ×100.

[0027] Depend on Figure 3It can be seen that phytosterol oleate significantly enhanced the ability of vitamin E liposomes to scavenge DPPH free radicals, and the incorporation of phytosterol oleate and vitamin E in liposomes played a synergistic antioxidant role.

[0028] 2. Determination of Anti-lipid Peroxidation Ability of Liposome Composites Malondialdehyde is one of the main products of lipid peroxidation in the sample, and its content can reflect the degree of lipid peroxidation in the sample. The present invention uses malondialdehyde as an indicator to investigate the anti-lipid peroxidation ability of the composite liposome, and the specific determination method is: prepare L, L-POE, L-VE, and L-VE-POE respectively, accurately pipette 5 mL of TBA solution into a test tube, and then accurately pipette 0.5 mL of the sample and mix it evenly with the TBA solution. Seal the mouth of the test tube with plastic wrap, poke a few small holes, heat in a boiling water bath for 30 min under light-proof conditions, take out and cool quickly, and centrifuge at 5000 r / min for 5 min. After centrifugation, measure the absorbance A of the supernatant at 532 nm.

[0029] The calculation formula of MDA is as follows: MDA content (ng / mg) = (A × 4.15 × 1000) / W; Where, 4.15 is the conversion factor of malondialdehyde per milliliter of lipid, μg / mL; W is the lipid content per unit volume, mg / mL.

[0030] TBA solution: accurately weigh 0.375 g thiobarbituric acid (TBA) and 15 g trichloroacetic acid (TCA), add them into 100 mL 0.25 mol / L hydrochloric acid solution, and dissolve them under slight heat.

[0031] The anti-lipid peroxidation ability of the composite liposome of the present invention is shown in Figure 4 .Depend on Figure 4 It can be seen that the incorporation of phytosterol oleate enhances the anti-lipid peroxidation ability of vitamin E liposomes to a certain extent. The enhancement of anti-lipid peroxidation ability may be attributed to the interaction between phytosterol oleate and phospholipids, which improves the ability of lipid membrane to resist oxidation induced by external free radicals.

[0032] 3. Determination of reducing ability of liposome complexes Antioxidants are substances with reducing effects. They provide electrons to free radicals, thereby interrupting the chain reaction of free radicals to achieve the purpose of anti-oxidation. 3+ Reduction to Fe 2+ , Fe 2+ Further with Fe 3+A Prussian blue is generated, and the maximum absorption wavelength of this substance is 700 nm. Therefore, the reducing ability of the sample can be evaluated by the absorbance value at 700 nm. The specific principle is as follows: K3Fe(CN)6+ Sample ® K4Fe(CN)6+ Sample Oxide K4Fe(CN)6+Fe3+®Fe4[Fe(CN)6]3 The reducing ability was determined as follows: L, L-POE, L-VE, L-VE-POE, and VE were prepared respectively, 1 mL of sample was accurately drawn, 1 mL of potassium ferrocyanide (1% w / w) was added, the mixture was reacted in a water bath at 50°C for 20 min, 1 mL of trichloroacetic acid (10% w / w) was added, the mixture was cooled to room temperature and centrifuged at 5000 r / min for 10 min, 2 mL of supernatant was drawn, 0.4 mL of ferric chloride (0.1% w / w) and 2 mL of distilled water were added, the mixture was allowed to stand for 10 min, and the absorbance was measured at 700 nm. The results are shown in Table 1. Figure 5 .

[0033] Depend on Figure 5 It can be seen that the incorporation of phytosterol oleate greatly improves the reducing ability of vitamin E liposomes.

[0034] Example 4 Application of Vitamin E-phytosterol Ester Complex Liposomes in Fried Instant Noodles 1. Preparation of fried instant noodles The composite liposomes of the present invention are applied to instant noodles. Raw material pretreatment: Flour is sieved through a 100-mesh sieve and set aside; Dough preparation: 1000 g of flour is poured into a dough mixing bowl, 1.5% salt, 35% water, (0, 1%, 2%) composite liposomes (all based on the mass of flour) and the like are added to the flour, poured into a food mixer and evenly mixed for 15 minutes, and stirred until the flour becomes snowflake noodles. Ripening: Wrap the prepared noodles with plastic wrap and let stand at room temperature for 20 minutes. Rolling: Put the matured dough into a noodle machine and press it into uniform sheets. After repeated sheeting (about 6 times), it is cut into noodles with a width of 2 mm and a thickness of 0.7 mm by a machine. Frying: Put the noodles into a mold, fry them in a 150°C oil pan for 90 seconds, remove them, and cool them. The product was stored for a total of 36 days, and the acid value and peroxide value were measured every 6 days. The results are shown in Table 1. Figure 6 and Figure 7 .

[0035] 2. Extraction of oil from fried instant noodles First, use a grinder to crush the fried instant noodles into uniform fine particles, take the crushed fried instant noodles fine particles and put them into the Soxhlet fat extraction device, then add an appropriate amount of petroleum ether extraction solvent, heat and reflux for 12 hours. Finally, collect and combine all the extracts in a flask, place it in a rotary evaporator with a water temperature not higher than 45°C (under negative pressure conditions of 0.08MPa~0.1MPa) for desolventizing, and take the residual liquid oil as a sample for peroxide value and acid value determination.

[0036] 3. Determination of acid value of fried instant noodles Take a clean 250 mL conical flask and weigh the prepared oil sample with a balance. The mass m is in grams. Add 50 mL to 100 mL of ether-isopropanol mixture and 3 to 4 drops of phenolphthalein indicator, and shake thoroughly to dissolve the sample. Then use a graduated burette filled with standard titration solution to manually titrate the sample solution. When the sample solution begins to turn slightly red and there is no obvious fading within 15 seconds, it is the end point of the titration. Stop the titration immediately and record the milliliters of standard titration solution consumed in this titration. This value is V .

[0037] Blank test Take another clean 250 mL conical flask, accurately add the same volume and type of organic solvent mixture and indicator as used in the sample determination, and shake to mix. Then use a graduated burette filled with standard titration solution to titrate. When the solution begins to turn slightly red and there is no obvious fading within 15 seconds, it is the end point of the titration. Stop the titration immediately. Record the number of milliliters of standard titration solution consumed in this titration. This value is V 0.

[0038] The acid value calculation formula is as follows X AV

[0039] Where: X AV ——Acid value, in milligrams per gram (mg / g); V ——The volume of standard titration solution consumed in the sample determination, in milliliters (mL); V 0 ——The volume of the standard titration solution consumed in the corresponding blank determination, in milliliters (mL); c ——The molar concentration of the standard titration solution, in moles per liter (mol / L); 56.1 – the molar mass of potassium hydroxide in grams per mole (g / mol); m ——Weighing weight of oil sample, in grams (g).

[0040] Depend on Figure 6 It can be seen that the addition of composite liposomes slows down the rate of rancidity of fried instant noodles.

[0041] 4. Determination of peroxide value of fried instant noodles Avoid measuring the sample under direct sunlight. Weigh 2g~3g (accurate to 0.001g) of the prepared sample, place it in a 250mL iodine volumetric flask, add 30mL of chloroform-glacial acetic acid mixture, and shake gently to completely dissolve the sample. Accurately add 1.00mL of saturated potassium iodide solution, plug the bottle cap tightly, and shake gently for 0.5 min, and place it in a dark place for 3 min. Take it out and add 100mL of water. After shaking well, immediately titrate the precipitated iodine with sodium thiosulfate standard solution (when the estimated peroxide value is 0.15 g / 100 g or less, use 0.002 mol / L standard solution; when the estimated peroxide value is greater than 0.15 g / 100 g, use 0.01 mol / L standard solution). When the titration reaches a light yellow color, add 1mL of starch indicator, continue titrating and shake vigorously until the blue color of the solution disappears as the end point. Perform a blank test at the same time. The volume of 0.01 mol / L sodium thiosulfate solution consumed in the blank test is V 0 shall not exceed 0.1mL.

[0042] When the peroxide value is expressed as the mass fraction of peroxide equivalent to iodine, the peroxide value calculation formula is as follows X 1=

[0043] Where: X 1——Peroxide value, in grams per 100 grams (g / 100g); V ——The volume of sodium thiosulfate standard solution consumed by the sample, in milliliters (mL); V 0 ——The volume of sodium thiosulfate standard solution consumed in the blank test, in milliliters (mL); c ——The concentration of sodium thiosulfate standard solution, in moles per liter (mol / L); 0.1269 – the mass of iodine equivalent to 1.00 mL of sodium thiosulfate standard titration solution [c(Na2S2O3)=1.000 mol / L], expressed in grams per milligram (g / m mol); m ——mass of the sample, in grams (g); 100——Conversion factor converted to 100 g of sample.

[0044] Depend on Figure 7 It can be seen that the addition of composite liposomes helps to delay the oxidation of fried instant noodles.

[0045] During storage, oils and fried foods usually undergo auto-oxidation and free radical reactions, which lead to oxidation and rancidity, and thus lose their edible value. Figure 6 and Figure 7 It can be seen that the composite liposome of the present invention can effectively delay the induction period of the oxidation reaction of oil, inhibit the rancidity and oxidation of oil in fried instant noodles, improve the storage stability of fried instant noodles, and extend the shelf life. The invention has great potential for application in the food industry.

Claims

1. A vitamin E-phytosterol oleate complex liposome, characterized in that: The composite liposome is prepared by using soybean lecithin, phytosterol oleate and vitamin E as raw materials through thin film ultrasonic method, and the mass ratio of soybean lecithin, phytosterol oleate and vitamin E is 20:1:

1.

2. The vitamin E-phytosterol oleate complex liposome according to claim 1, characterized in that: The preparation method of the composite liposome comprises the following contents: dissolving soybean lecithin, phytosterol oleate and vitamin E in an organic solvent, and performing rotary evaporation in the dark to form a film; washing the film with a phosphate buffer containing Tween-80 to form a liposome suspension, and subjecting the liposome suspension to probe ultrasonication in an ice water bath for 4 minutes to obtain vitamin E-phytosterol oleate composite liposomes.

3. The vitamin E-phytosterol oleate complex liposome according to claim 2, characterized in that: The organic solvent is prepared by mixing n-hexane and anhydrous ethanol in a volume ratio of 1:

1.

4. The vitamin E-phytosterol oleate complex liposome according to claim 2, characterized in that: The concentration of Tween-80 in the phosphate buffer is 10 g / L, and the pH of the phosphate buffer is 7.

4.

5. The vitamin E-phytosterol oleate complex liposome according to claim 2, characterized in that: The probe ultrasound was turned on for 5 seconds and off for 5 seconds, and this was repeated for 4 minutes.

6. Use of the vitamin E-phytosterol oleate complex liposomes according to any one of claims 1 to 5 in fried instant noodles.

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