Preparation method of high-stability phenol-rich oil body derived from fresh pulp of idesia polycarpa

Abstract

The invention relates to the technical field of extraction of fresh idesia polycarpa fruits, in particular to a preparation method of high-stability phenol-rich oil from fresh idesia polycarpa flesh. The method comprises the following steps: adjusting the pH value of pulp of fresh idesia polycarpa fruits to 4-7, then adding a first enzyme seed and a second enzyme seed, extracting for 0.5-4 hours at the temperature of 30-50 DEG C, centrifuging, and collecting a first emulsion; the first enzyme comprises pectinase, and further comprises at least one of hemicellulase and cellulase; the second enzyme is composed of tannase and glycosidase; and washing the first emulsion with water or a sucrose aqueous solution, centrifuging, and collecting a second emulsion to obtain the oil body. Through simultaneous enzymolysis of the first enzyme and the second enzyme, free polyphenol is generated in situ around the oil body, interaction of the oil body and polyphenol is promoted, part of polyphenol, protein and pectin are combined on the surface of the oil body, part of weak-polarity polyphenol enters the oil body, and the physical and chemical stability of the oil body is synergistically improved.

Description

Technical Field

[0001] This invention relates to the field of fresh fruit extraction technology of *Vernicia fordii*, and in particular to a method for preparing highly stable phenol-rich oils from the pulp of fresh *Vernicia fordii*. Background Technology

[0002] Oil bodies are subcellular organelles in plants that store lipids. Their core is a triglyceride surrounded by a protein / phospholipid monolayer membrane. The hydrophobic ends of the phospholipids extend into the interior of the oil body, while the hydrophilic phosphate groups face outwards. The membrane proteins are mainly oil body proteins, caloproteins, and sterol proteins. The former two are embedded in the oil body and can be transported across the membrane from the endoplasmic reticulum to the oil body surface, while the latter is anchored to the oil body surface through its N-terminal hydrophobic structure. This unique natural structure utilizes steric hindrance and electrostatic forces to stabilize plant oil bodies, giving them excellent physicochemical stability and emulsifying properties. As a type of water-in-oil natural emulsifying microparticle, oil bodies can be used as a transport carrier for bioactive ingredients and flavor substances. Due to their high triglyceride content, they can be used as fat substitutes in products such as dairy imitations, meat products, mayonnaise, salad dressings, and edible films. They can also be used to develop oil gels and emulsion gels, showing potential application value in the food and cosmetic fields.

[0003] *Idesia polycarpa* Maxim. var. *vesita* Diels, also known as mountain privet, water melon, or oil grape, is a plant belonging to the genus *Idesia* in the family Salicaceae. It grows rapidly, tolerates poor soil and drought, and has a wide range of environmental adaptability. *Idesia polycarpa* fruits have high yields, with oil content in the fruit, pulp, and seeds ranging from 25-38%, 36-43%, and 20-29% (dry basis), respectively, earning it the nickname "oil depot on the tree." The oil is high in unsaturated fatty acids and linoleic acid, and also contains abundant polyphenols, tocopherols, phytosterols, and trace amounts of squalene and other active substances. Therefore, *Idesia polycarpa* has become an important woody oilseed tree species in my country, and large-scale cultivation has been carried out in Hubei, Guizhou, Sichuan, Anhui, Yunnan, Shaanxi, and other regions. Currently, the *Idesia polycarpa* industry in China is still in its initial stage, mainly focusing on oil extraction from dried fruit, but research on this natural emulsified microparticle product is rarely reported.

[0004] Currently, the main methods for preparing oil bodies include aqueous methods, solvent methods, and enzymatic methods. Patent document CN107125430A discloses a method for simultaneously preparing oil bodies and non-hydrolyzed protein compositions. This method includes the following steps: providing a pulverized oil containing oil and protein; dissolving the cell walls and / or breaking down cellulose, pectin, hemicellulose, and / or dextran within the cell walls of the pulverized oil in the presence of water and enzymes; and separating the resulting oil body from the protein composition. However, this preparation method does not address certain compounds that may affect the stability of the oil body, thus exhibiting stability defects such as easy aggregation, oxidation, or insufficient interfacial film strength. Summary of the Invention

[0005] The present invention aims to solve the above problems by providing a method for preparing an oil body derived from the fresh pulp of *Vernicia fordii* that is stable in properties and has a high phenol content.

[0006] The technical solution to the problem of this invention is to provide a method for preparing a highly stable phenol-rich oil from the fresh pulp of *Vernicia fordii*, comprising the following steps:

[0007] S1. Mix the pulp of fresh *Vernicia fordii* fruit with water to make a pulp paste;

[0008] S2. Adjust the pH of the pulp to 4-7, then add the first and second enzymes, extract at 30-50℃ for 0.5-4 hours, centrifuge, and collect the first emulsion;

[0009] The first enzyme includes pectinase, and also includes at least one of hemicellulase and cellulase;

[0010] The second enzyme consists of tanninase and glycosidase;

[0011] S3. After washing the first emulsion with water or sucrose aqueous solution, centrifuge and collect the second emulsion to obtain the oil body.

[0012] This invention provides a complete unit comprising a triglyceride core, a phospholipid-protein-pectin-polyphenol composite membrane, and a hydration layer encapsulating them, in an emulsion form. A first enzyme releases the oil body, while a second enzyme hydrolyzes bound and / or polymerized polyphenols in the raw materials into free polyphenols. Due to simultaneous enzymatic hydrolysis, the free polyphenols generated in situ in the surrounding environment interact with the oil body the instant it is released from the cells. These free polyphenols, based on their polarity and hydrophobicity, exhibit two distributions within the oil body system: some are bound at the oil body interface, while the other, weakly polar polyphenols, enter the interior of the oil body. Both enhance the physical and chemical stability of the oil body through different mechanisms. Specifically, the polyphenols adsorbed at the interface mainly bind to the components at the oil body interface through hydrogen bonds and hydrophobic interactions, enhancing the mechanical strength and density of the interfacial membrane, helping to resist aggregation and ripening between oil bodies, thereby improving the physical stability of the oil body. The polyphenols that enter the oil body are mainly hydrophobic polyphenols. They dissolve in the triglyceride core of the oil body, delaying oil oxidation, improving the oxidation stability of the oil body, preventing oxidation products from causing interface deterioration of the oil body, and indirectly helping to maintain physical stability.

[0013] In step S1 The raw material is fresh tung oil fruit. Compared with dried tung oil fruit and seeds, the moisture and pectin in fresh fruit are conducive to the formation of a stable oil system.

[0014] The amount of water used to prepare the pulp is not limited. As a preferred embodiment of the present invention, the mass ratio of fresh tung oil fruit pulp to water is 1:(1-10); preferably 1:(2-5); for example, it can be 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.

[0015] The pulping method is not limited. As a preferred method of the present invention, the pulp of fresh tung tree fruit is mixed with water and then ground to obtain pulp.

[0016] In some preferred embodiments, as a preferred embodiment of the present invention, the fresh *Vernicia fordii* fruit is pretreated before preparing the pulp, thereby reducing the activity of endogenous enzymes in the fresh *Vernicia fordii* fruit by 60% to 100%. The endogenous enzymes in fresh *Vernicia fordii* fruit include lipases, lipoxygenases, polyphenol oxidases, and peroxidases, which easily cause deterioration of oil quality and oxidize polyphenols, affecting oil stability.

[0017] Pretreatment typically involves heat treatment, but the temperature and time should not be too high to avoid damaging the natural polyphenols and triglycerides in the raw materials. Preferably, the pretreatment of fresh *Vernicia fordii* fruit is heat treatment at a temperature of 65–75°C for 10–40 minutes. This invention employs a medium-temperature inactivation method to reduce the activity of endogenous enzymes in the fresh *Vernicia fordii* fruit while preserving the polyphenols and oils. For example, the treatment temperature can be 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, or 75°C; and the treatment time can be 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, or 40 minutes.

[0018] In step S2 Adjust the pH of the fruit pulp to 4-7. For example, the pH can be 4, 4.5, 5, 5.5, 6, 6.5, or 7.

[0019] Extraction is carried out at a temperature of 30–50°C for 0.5–4 hours. For example, the extraction temperature can be 30°C, 35°C, 40°C, 45°C, or 50°C. The extraction time can be 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours, preferably 1–3 hours.

[0020] For the first enzyme, since the raw material is fresh fruit of the tung tree, pectinase is necessary, and obtaining a large amount of pectin is beneficial for the formation of oil bodies.

[0021] As a preferred embodiment of the present invention, the first enzyme is composed of pectinase, hemicellulase and cellulase. The three enzymes effectively disrupt the integrity of the fresh pulp cells of *Vernicia fordii*, thereby increasing the release of oil. At the same time, the exogenous proteins and pectin extracted simultaneously can bind with the endogenous proteins through hydrogen bonds to form a dense and thick interfacial film, which can promote the interaction between polyphenols and oil.

[0022] As a preferred embodiment of the present invention, the mass ratio of pectinase, hemicellulase, and cellulase is 2:(0.1-0.5):(0.5-1.5). For example, when the mass fraction of pectinase is 2 parts, the mass fraction of hemicellulase can be 0.1, 0.2, 0.3, 0.4, or 0.5 parts, and the mass fraction of cellulase can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, or 1.5 parts; the preferred mass ratio is 2:0.3:1.

[0023] As a preferred embodiment of the present invention, the amount of the first enzyme is 0.3wt% to 3wt% of the amount of fresh fruit pulp of the *Vernicia fordii*. For example, it can be 0.3wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, or 3wt%.

[0024] For the second enzyme, tanninase hydrolyzes tannins, releasing small phenolic acids and eliminating the negative effects of tannins (preventing excessive binding of tannins to proteins, leading to precipitation or interfacial instability). Glycosidase releases phenolic aglycones, which are adsorbed onto the oil-water interface through hydrophobicity.

[0025] As a preferred embodiment of the present invention, the mass ratio of the tanninase to the glycosidase is 1:(1-4), and the appropriate dosage ratio is used for targeted treatment of fresh *Vernicia fordii* fruit. For example, the mass ratio can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, or 1:4.

[0026] As a preferred embodiment of the present invention, the glycosidase is selected from at least one of α-glucosidase and β-glucosidase.

[0027] As a preferred embodiment of the present invention, the amount of the second enzyme is 0.1wt% to 2wt% of the amount of fresh fruit pulp of the *Vernicia fordii*. For example, it can be 0.1wt%, 0.3wt%, 0.5wt%, 0.8wt%, 1wt%, 1.2wt%, 1.5wt%, 1.8wt%, or 2wt%.

[0028] As a preferred embodiment of the present invention, the ratio of the first enzyme to the second enzyme is (1-3):1, and an appropriate ratio is beneficial for the uniform binding of the oil and polyphenols. For example, it can be 1:1, 1.5:1, 2:1, 2.5:1, or 3:1.

[0029] In this invention, the preparation method can be further adjusted to actively guide the rearrangement and fixation of polyphenols at the interface, forming a more stable composite film.

[0030] In some preferred embodiments, as a preferred embodiment of the present invention, in step S2, after every 10 to 40 minutes of extraction, the extraction system is treated at a temperature not exceeding 10°C for 20 to 40 seconds; during treatment, after every 2 to 5 seconds of sonication, the system is allowed to stand for 5 to 10 seconds, and the frequency of sonication is 15 to 35 kHz and the power density is 10 to 50 W / mL. For example, the extraction time between two treatments can be 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, or 40 min; the treatment temperature can be 10℃, 9℃, 8℃, 7℃, 6℃, 5℃, 4℃, 3℃, 2℃, 1℃, or 0℃; the total duration of each treatment can be 20 s, 25 s, 30 s, 35 s, or 40 s; the treatment can be ultrasonic for 2 s, 3 s, 4 s, or 5 s followed by standing for 5 s, 6 s, 7 s, 8 s, 9 s, or 10 s; the ultrasonic frequency can be 15 Hz, 20 Hz, 25 Hz, 30 Hz, or 35 Hz; and the power density can be 10 W / mL, 15 W / mL, 20 W / mL, 25 W / mL, 30 W / mL, 35 W / mL, 40 W / mL, 45 W / mL, or 50 W / mL.

[0031] In this invention, during the intervals of low- and medium-temperature extraction, low-frequency, low-power, intermittent ultrasonic co-enzymatic hydrolysis is assisted by low temperature. Low temperature prevents localized overheating of the extraction system, avoiding damage to raw material components and affecting enzyme activity. Low frequency and low power generate a mild cavitation effect and microfluidics without causing severe shear damage. This not only promotes enzyme-substrate contact and improves hydrolysis efficiency through cavitation, but also promotes thorough and uniform mixing of the newly released oil and polyphenols through microfluidic stirring, increasing the probability and uniformity of polyphenol adsorption to the oil interface or entry into the oil. The intermittent pauses are necessary, allowing the oil and polyphenols, propelled by ultrasound, to undergo conformational adjustment and reorientation, forming a more stable and ordered bond (this spontaneous adsorption and self-assembly process requires time; continuous ultrasonic turbulence can interfere with it). The intermittent extraction and ultrasonic treatment operations are essential to cyclically complete the release and binding of oil and polyphenols, ensuring the uniformity of the interfacial film.

[0032] In some other preferred embodiments, as a preferred embodiment of the present invention, in step S2, after extraction, the extraction system is heated to 65-75°C and held for 3-5 minutes before centrifugation. For example, the temperature can be 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, or 75°C; the time can be 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, or 5 minutes.

[0033] In this invention, moderate low-temperature short-time heating can partially unfold the surface proteins (oil proteins) of the oil body, exposing more hydrophobic groups, enhancing the hydrophobic interaction with polyphenols, and forming a more robust composite film; it can also achieve the effect of enzyme inactivation to a certain extent.

[0034] The conditions for centrifugation are not limited. As a preferred embodiment of the present invention, centrifugation is performed at 4000–9000 rpm for 10–30 min. For example, the centrifugation speed can be 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, or 9000 rpm; and the centrifugation time can be 10 min, 15 min, 20 min, 25 min, or 30 min.

[0035] In step S3 Washing removes substances that are detrimental to the stability of the oil.

[0036] As a preferred embodiment of the present invention, step S3 specifically involves dispersing the first emulsion in water or a sucrose aqueous solution with a pH of 4 to 12, washing and centrifuging at least once; then dispersing it in water or a sucrose aqueous solution with a pH of 6 to 7, washing and centrifuging at least twice to obtain the oil.

[0037] Preferably, the oil is washed at least once with a sucrose aqueous solution, which can further stabilize the oil.

[0038] In the first washing method, the pH of the water or sucrose aqueous solution can be 4, 5, 6, 7, 8, 9, 10, 11, or 12, preferably 4 to 6, and more preferably 5. When using a sucrose aqueous solution for washing, as a preferred embodiment of the present invention, the concentration of the sucrose aqueous solution does not exceed 20 wt%, for example, the concentration can be 1 wt%, 5 wt%, 10 wt%, 15 wt%, or 20 wt%. As a preferred embodiment of the present invention, the volume ratio of the emulsion to the water or sucrose aqueous solution is 1:(5 to 10), for example, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. The centrifugation conditions are not limited; as a preferred embodiment of the present invention, centrifugation is performed at 4000 to 9000 rpm for 10 to 30 minutes. For example, the centrifugation speed can be 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, or 9000 rpm; the centrifugation time can be 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes.

[0039] In the second washing method, the pH of the water or sucrose aqueous solution can be 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7, preferably 6.5. When using a sucrose aqueous solution for washing, as a preferred embodiment of the present invention, the concentration of the sucrose aqueous solution does not exceed 20 wt%, for example, the concentration can be 1 wt%, 5 wt%, 10 wt%, 15 wt%, or 20 wt%. As a preferred embodiment of the present invention, the volume ratio of the secondary emulsion to the water or sucrose aqueous solution is 1:(5-10), for example, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. The centrifugation conditions are not limited; as a preferred embodiment of the present invention, centrifugation is performed at 4000-9000 rpm for 10-30 min. For example, the centrifugation speed can be 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, or 9000 rpm; the centrifugation time can be 10 min, 15 min, 20 min, 25 min, or 30 min.

[0040] In some implementations, it also includes Step S4 The resulting emulsion is left to stand at a temperature not exceeding 4°C for 2–4 hours. The temperature can be 4°C, 3°C, 2°C, 1°C, or 0°C; the time can be 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours.

[0041] In this invention, a slow thermodynamic rearrangement and self-assembly are finally carried out at low temperature, such as allowing polyphenol molecules to form π-π stacking or hydrogen bond networks at the interface, ultimately forming a more thermodynamically stable interface structure and improving the stability of the oil.

[0042] The beneficial effects of this invention are:

[0043] 1. In this invention, the first enzyme and the second enzyme are simultaneously enzymatically hydrolyzed, generating free polyphenols in situ around the oil body, which promotes the interaction between the oil body and the polyphenols. Some polyphenols bind to proteins and pectin on the surface of the oil body, while some weakly polar polyphenols enter the interior of the oil body, synergistically enhancing the physical and chemical stability of the oil body.

[0044] 2. In some embodiments, low-frequency, low-power, intermittent ultrasonic co-enzymatic hydrolysis at a low temperature not exceeding 10°C is used to actively guide the rearrangement and fixation of polyphenols at the interface, forming a more stable composite membrane.

[0045] 3. In some embodiments, after extraction, the oil is heated moderately to allow the surface proteins to expand, exposing more hydrophobic groups, enhancing the hydrophobic interaction with polyphenols, and forming a more robust composite film.

[0046] 4. In some embodiments, slow thermodynamic rearrangement and self-assembly are carried out at low temperatures, resulting in a more thermodynamically stable interface structure. Attached Figure Description

[0047] Figure 1 These are average particle size diagrams of the oil bodies obtained in Examples 1-3;

[0048] Figure 2 These are line graphs showing the average particle size of the oil bodies prepared in Examples 1-3 at different pH values;

[0049] Figure 3 These are line graphs showing the average particle size of the oil bodies prepared in Examples 1-3 at different temperatures;

[0050] Figure 4 These are line graphs showing the average particle size of the oil bodies prepared in Examples 1-3 under different concentrations of salt ion solutions;

[0051] Figure 5 These are line graphs showing the average particle size of the oil bodies obtained in Examples 1-3 under different freeze-thaw cycles;

[0052] Figure 6 This is a photograph of the second emulsion obtained in Example 1;

[0053] Figure 7 This is a photograph of the second emulsion obtained in Example 10. Detailed Implementation

[0054] The following are specific embodiments of the present invention, and the technical solutions of the present invention will be further described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments.

[0055] Example 1

[0056] A method for preparing a highly stable phenol-rich oil from fresh pulp of *Vernicia fordii* includes the following steps:

[0057] S1. After removing the stems and calyxes from the fresh tung fruit, wash it and soak it in 70℃ hot water for 15 minutes. Then, crush the fresh tung fruit to separate the pulp. Mix 100g of pulp with 300g of water and grind it into a pulp paste.

[0058] S2. Adjust the pH of the fruit pulp to 5 with citric acid, then add 1.2g pectinase, 0.2g hemicellulase, 0.6g cellulase, 0.25g tanninase, and 0.75g β-glucosidase, and perform the following operations:

[0059] S21. Extract at 40℃ in a water bath for 30 min, then place the extraction system in an ice-water bath and sonicate at 20 kHz, 30 W / mL for 3 s, then let stand for 7 s, sonicate again for 3 s, let stand for 7 s, sonicate again for 3 s, and let stand for 7 s, for a total of 30 s. Repeat the above steps of extraction at 40℃ in a water bath followed by intermittent sonication in an ice-water bath 4 times, for a total of 122 min.

[0060] S22. Rapidly heat the extracted system to 70°C and keep it at 70°C for 4 minutes.

[0061] S23. Centrifuge the extraction system at 6000 rpm for 20 min and collect the first emulsion.

[0062] S3. The first emulsion was suspended in a 15 wt% sucrose aqueous solution with a pH of 5, at a volume ratio of 1:5. After washing, it was centrifuged at 6000 rpm for 20 min to obtain the first-wash emulsion. The first-wash emulsion was suspended in a 10 wt% sucrose aqueous solution with a pH of 6.5, at a volume ratio of 1:8. After washing, it was centrifuged at 6000 rpm for 20 min to obtain the second-wash emulsion. The second-wash emulsion was suspended in a 10 wt% sucrose aqueous solution with a pH of 6.5, at a volume ratio of 1:8. After washing, it was centrifuged at 6000 rpm for 20 min to obtain the second emulsion. The physical image is shown below. Figure 6 As shown.

[0063] S4. The second emulsion was left to stand at 4°C for 3 hours to obtain a highly stable phenol-rich oil.

[0064] Example 2

[0065] A method for preparing a highly stable phenol-rich oil from fresh pulp of *Vernicia fordii* includes the following steps:

[0066] S1. After removing the stems and calyxes from the fresh tung fruit and washing it, crush the fresh tung fruit to separate the pulp, and mix 100g of pulp with 300g of water to grind it into a pulp paste.

[0067] S2. Adjust the pH of the pulp to 5 with citric acid, then add 1.2g pectinase, 0.2g hemicellulase, 0.6g cellulase, 0.25g tanninase, and 0.75g β-glucosidase, and extract in a 40℃ water bath for 122 min. Then centrifuge at 6000rpm for 20 min and collect the first emulsion.

[0068] S3. The first emulsion is suspended in a 15 wt% sucrose aqueous solution at pH 5, with a volume ratio of 1:5 between the first emulsion and the sucrose aqueous solution. After washing, it is centrifuged at 6000 rpm for 20 min to obtain a first-wash emulsion. The first-wash emulsion is suspended in a 10 wt% sucrose aqueous solution at pH 6.5, with a volume ratio of 1:8 between the first-wash emulsion and the sucrose aqueous solution. After washing, it is centrifuged at 6000 rpm for 20 min to obtain a second-wash emulsion. The second-wash emulsion is suspended in a 10 wt% sucrose aqueous solution at pH 6.5, with a volume ratio of 1:8 between the second-wash emulsion and the sucrose aqueous solution. After washing, it is centrifuged at 6000 rpm for 20 min to obtain the second emulsion, which is the highly stable phenol-rich oil.

[0069] Example 3

[0070] This embodiment is basically the same as embodiment 2, except that step S2 is different.

[0071] Specifically: S2. Adjust the pH of the fruit pulp to 5 with citric acid, then add 1.2g pectinase, 0.2g hemicellulase, 0.6g cellulase, 0.25g tanninase, and 0.75g β-glucosidase. Ultrasonic extraction was performed for 122 minutes in a 40℃ water bath. Acoustic conditions: 20kHz, 30W / mL Then centrifuge at 6000 rpm for 20 min and collect the first emulsion.

[0072] The compositions of the oils obtained in Examples 1-3 are shown in Table 1 below.

[0073] Table 1. Example 1 Example 2 Example 3 Moisture (%) <![CDATA[57.36±0.02 c ]]> <![CDATA[78.06±0.33 a ]]> <![CDATA[68.77±0.02 b ]]> grease(%) <![CDATA[39.48±1.20 a ]]> <![CDATA[21.52±0.09 c ]]> <![CDATA[28.95±1.13 b ]]> protein(%) <![CDATA[4.318±0.119 a ]]> <![CDATA[2.189±0.014 b ]]> <![CDATA[1.872±0.0.019 c ]]> Pectin (%) <![CDATA[1.598±0.001 a ]]> <![CDATA[0.613±0.002 c ]]> <![CDATA[1.150±0.002 b ]]> Polysaccharides (%) <![CDATA[1.13±0.004 a ]]> <![CDATA[0.22±0.001 c ]]> <![CDATA[0.66±0.001 b ]]> Monosaccharides or disaccharides (%) <![CDATA[3.691±0.001 a ]]> <![CDATA[2.277±0.002 c ]]> <![CDATA[2.701±0.001 b ]]> Polyphenols (%) <![CDATA[0.488±0.001 a ]]> <![CDATA[0.366±0.000 c ]]> <![CDATA[0.382±0.001 b ]]> .

[0074] It can be seen that the oil bodies obtained in Examples 1 to 3 all have a high content of oil and polyphenols. When ultrasound is used to assist in the extraction process, the extraction yield of oil and polyphenols can be further improved. Further comparison shows that the extraction rate of oil and polyphenols is higher when ultrasound is intermittently performed in an ice-water bath than when ultrasound is performed continuously in a hot water bath.

[0075] The average particle size of the oil bodies obtained in Examples 1-3 after 0 days of storage is as follows: Figure 1 As shown, intermittent ice-water bath sonication during extraction, combined with pretreatment, thermal activation, and low-temperature settling, can reduce the average particle size of the oil and improve its stability. This may be because intermittent ice-water bath sonication avoids damage to the substrate and enzyme, and provides sufficient time for the release, binding, and recombination of oils and polyphenols, promoting the uniform formation of the interfacial film; while thermal activation and low-temperature settling are beneficial for the self-assembly of the interfacial film to form a more stable structure.

[0076] The average particle size of the oil bodies obtained in Examples 1-3 varies with pH, ​​temperature, salt ion concentration, and number of freeze-thaw cycles, respectively. Figure 2 , 3 As shown in Figures 4 and 5. Figure 2 The pH of the oil washed with sucrose aqueous solution was unstable. After adjusting the pH of the oil with a buffer solution, the average particle size of the oil changed significantly. When the pH reached alkalinity, its stability improved. Figure 3 In principle, an increase in temperature should lead to a decrease in the stability of the oil. However, for the oil obtained in Example 1, the stability actually increased when the temperature reached 90°C. This may be because the steps in Example 1 constructed a dynamic system with self-healing capabilities for the oil. When the temperature reaches the critical temperature, the newly activated surface-active substances in the oil migrate, insert, and reinforce the original interfacial film. Figure 4 When the salt ion concentration is 0, a corresponding amount of water needs to be added. At this time, the oil particle size in Example 1 does not change much, which indicates that the oil obtained in Example 1 has a stable dispersion state. In Examples 2 and 3, the oil particle size decreases, which indicates that the oil obtained in Examples 2 and 3 has a certain aggregation state and relatively low stability. It can be seen that pretreatment, thermal activation, low temperature standing and other operations can improve the stability of the oil.

[0077] Examples 4-6

[0078] Examples 4 to 6 are basically the same as Example 1, except that the amount of enzyme used is different, as shown in Table 2 below.

[0079] Table 2. Example Pectinase (g) Hemicellulase (g) Cellulase (g) Tanninase (g) β-glucosidase (g) Example 1 1.2 0.2 0.6 0.25 0.75 Example 4 1.2 0.2 0.6 0.5 1.5 Example 5 1.2 0.2 0.6 0.125 0.375 Example 6 1.8 0.3 0.9 0.25 0.75 .

[0080] Example 7

[0081] This embodiment is basically the same as embodiment 1, except that step S1 is different.

[0082] Specifically: S1. After removing the stems and calyxes from the fresh *Vernicia fordii* fruits, wash them and use... 95℃ hot water After soaking for 15 minutes, remove the fruit, crush the fresh tung tree fruit, separate the pulp, and mix 100g of pulp with 300g of water to make a pulp paste.

[0083] Example 8

[0084] This embodiment is basically the same as embodiment 1, except that step S21 is different.

[0085] Specifically: S21. Extract at 40℃ in a water bath for 30 min, then place the extraction system in an ice-water bath. At 20kHz, 30W / mL ultrasound for 30sThe extraction process under 40°C water bath followed by continuous sonication in an ice-water bath was repeated four times, for a total processing time of 122 min.

[0086] Example 9

[0087] This embodiment is basically the same as embodiment 1, except that step S22 is different.

[0088] Specifically: S22. Rapidly heat the extracted system to [temperature]. 95℃, and at 95℃ Keep warm for 4 minutes.

[0089] Example 10

[0090] This embodiment is basically the same as embodiment 1, except that step S3 is different.

[0091] Specifically: S3. Suspend the first emulsion at a pH of 5. 20wt% The volume ratio of the first emulsion to the sucrose aqueous solution was 1:5. After washing, the emulsion was centrifuged at 6000 rpm for 20 min to obtain the first-wash emulsion. The first-wash emulsion was then suspended in an atmosphere at pH 6.5. 15wt% The volume ratio of the first washing emulsion to the sucrose aqueous solution was 1:8. After washing, the emulsion was centrifuged at 6000 rpm for 20 min to obtain a second washing emulsion. The second washing emulsion was then suspended in an atmosphere at pH 6.5. 15wt% The volume ratio of the emulsion to the sucrose aqueous solution was 1:8 in the secondary washing process. After washing, the emulsion was centrifuged at 6000 rpm for 20 min to obtain the second emulsion, as shown in the figure below. Figure 7 As shown.

[0092] Example 11

[0093] This embodiment is basically the same as embodiment 2, except that the fresh tung oil fruit is pretreated before preparing the pulp.

[0094] The only difference is in step S1: S1. After removing the stem and stalk from the fresh tung fruit, wash it, soak it in 70℃ hot water for 15 minutes, take it out, crush the fresh tung fruit, separate the pulp, mix 100g of pulp with 300g of water, and grind it into pulp paste.

[0095] Example 12

[0096] This embodiment is basically the same as embodiment 2, except that ultrasonic treatment is performed during extraction.

[0097] The only difference lies in step S2: In S2, the pH of the fruit pulp is adjusted to 5 with citric acid. Then, 1.2g pectinase, 0.2g hemicellulase, 0.6g cellulase, 0.25g tanninase, and 0.75g β-glucosidase are added, followed by the following steps: extraction at 40℃ for 30 min, then the extraction system is placed in an ice-water bath and sonicated at 20kHz, 30W / mL for 3 s, followed by standing for 7 s. This process is repeated for a total of 30 s. The above steps of extraction at 40℃ and intermittent sonication in an ice-water bath are repeated four times, for a total of 122 min. Finally, the mixture is centrifuged at 6000rpm for 20 min, and the first emulsion is collected.

[0098] Example 13

[0099] This embodiment is basically the same as embodiment 2, except that a heating process is performed after extraction.

[0100] The only difference lies in step S2: In S2, the pH of the fruit pulp is adjusted to 5 with citric acid, then 1.2g pectinase, 0.2g hemicellulase, 0.6g cellulase, 0.25g tanninase, and 0.75g β-glucosidase are added, and extraction is performed at 40℃ for 122 minutes. The extracted system is then rapidly heated to 70℃ and maintained at 70℃ for 4 minutes. Finally, the extraction system is centrifuged at 6000rpm for 20 minutes, and the first emulsion is collected.

[0101] Example 14

[0102] This embodiment is basically the same as embodiment 2, except that a low-temperature static setting operation is performed at the end.

[0103] Specifically, it also includes step S4: placing the obtained second emulsion at 4°C for 3 hours to obtain a highly stable phenol-rich oil.

[0104] Example 15

[0105] This embodiment is basically the same as Embodiment 2, except that the glycosidase is different.

[0106] The only difference lies in step S2: S2. Adjust the pH of the pulp to 5 with citric acid, then add 1.2g pectinase, 0.2g hemicellulase, 0.6g cellulase, 0.25g tanninase, and 0.75g α-glucosidase, and extract in a 40℃ water bath for 122 min. Then centrifuge at 6000rpm for 20 min and collect the first emulsion.

[0107] Example 16

[0108] This embodiment is basically the same as Embodiment 2, except that the glycosidase is different.

[0109] The only difference lies in step S2: In S2, the pH of the fruit pulp is adjusted to 5 with citric acid, then 1.2g pectinase, 0.2g hemicellulase, 0.6g cellulase, 0.25g tanninase, 0.35g α-glucosidase, and 0.4g β-glucosidase are added, and the mixture is extracted in a 40℃ water bath for 122 minutes. Then, it is centrifuged at 6000rpm for 20 minutes, and the first emulsion is collected.

[0110] Comparative Example 1

[0111] This comparative example is basically the same as Example 2, except that it does not contain a second enzyme.

[0112] The only difference is in step S2: S2. Adjust the pH of the pulp to 5 with citric acid, then add 1.2g pectinase, 0.2g hemicellulase, and 0.6g cellulase, and extract in a 40℃ water bath for 122 min. Then centrifuge at 6000rpm for 20 min and collect the first emulsion.

[0113] Comparative Example 2

[0114] This comparative example is basically the same as Example 2, except that it does not contain tanninase.

[0115] The only difference lies in step S2: S2. Adjust the pH of the pulp to 5 with citric acid, then add 1.2g pectinase, 0.2g hemicellulase, 0.6g cellulase, and 1g β-glucosidase, and extract in a 40℃ water bath for 122 min. Then centrifuge at 6000rpm for 20 min and collect the first emulsion.

[0116] Comparative Example 3

[0117] This comparative example is basically the same as Example 2, except that it does not contain β-glucosidase.

[0118] The only difference lies in step S2: S2. Adjust the pH of the pulp to 5 with citric acid, then add 1.2g pectinase, 0.2g hemicellulase, 0.6g cellulase, and 1g tanninase, and extract at 40℃ for 122 minutes. Then centrifuge at 6000rpm for 20 minutes and collect the first emulsion.

[0119] Comparative Example 4

[0120] This comparative example is basically the same as Example 2, except that the first enzyme and the second enzyme are used in sequence.

[0121] The only difference lies in step S2: In S2, the pH of the pulp is adjusted to 5 with citric acid. First, 1.2g of pectinase, 0.2g of hemicellulase, and 0.6g of cellulase are added, and the mixture is extracted at 40℃ for 122 minutes. Then, 0.25g of tanninase and 0.75g of β-glucosidase are added, and the mixture is extracted at 40℃ for 122 minutes. Finally, the mixture is centrifuged at 6000rpm for 20 minutes, and the first emulsion is collected.

[0122] The average particle size of the oil bodies prepared in the examples and comparative examples after 0 days and 30 days of storage, as well as the peroxide value (POV) after 0 days and 30 days of storage, are shown in Table 3 below.

[0123] Table 3. experimental group Average particle size (nm) after 0 days Average particle size (nm) after 30 days POV (g / 100g) after 0 days POV (g / 100g) after 30 days Example 1 1585 1652 0.0515 0.0534 Example 2 2402 2754 0.0842 0.0925 Example 3 2108 2341 0.0730 0.0791 Example 4 1747 1839 0.0485 0.0506 Example 5 2020 2115 0.0533 0.0557 Example 6 1638 1726 0.0525 0.0551 Example 7 1692 1784 0.0507 0.0531 Example 8 2020 2120 0.0585 0.0613 Example 9 1856 1945 0.0500 0.0522 Example 10 1605 1688 0.0511 0.0530 Example 11 2347 2610 0.0822 0.0888 Example 12 2129 2337 0.0619 0.0653 Example 13 2238 2439 0.0810 0.0871 Example 14 2293 2570 0.0805 0.0867 Example 15 2432 2799 0.0843 0.0929 Example 16 2423 2761 0.0842 0.0927 Comparative Example 1 3712 4552 0.1301 0.1563 Comparative Example 2 3275 3893 0.1058 0.1211 Comparative Example 3 3548 4286 0.1243 0.1471 Comparative Example 4 3003 3525 0.1012 0.1140 .

[0124] As shown in Table 3, comparative examples 2 and 1-4 show that in this invention, by simultaneously enzymatically hydrolyzing the first and second enzymes, free polyphenols can be generated in situ around the oil body, promoting the interaction between the oil body and polyphenols at the interface and inside, thereby improving the physical and chemical stability of the oil body.

[0125] Furthermore, comparisons within the examples show that appropriate adjustments to the steps can further improve the physicochemical stability of the obtained oil: Comparisons between Examples 1 and 7-10, and between Examples 2 and 11-14, demonstrate that appropriate pretreatment at a suitable temperature, intermittent ultrasonication during extraction, appropriate thermal activation at a suitable temperature after extraction, and final low-temperature settling all contribute to improved physicochemical stability of the oil. Comparisons between Examples 1 and 4-6 show that appropriate amounts of the first and second enzymes are beneficial for the uniform binding of polyphenols and the oil; insufficient amounts of the second enzyme are insufficient to stabilize the oil and result in inadequate antioxidant properties.

[0126] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A method for preparing a highly stable phenol-rich oil from fresh pulp of *Vernicia fordii*, characterized in that: Includes the following steps: S1. Mix the pulp of fresh *Vernicia fordii* fruit with water to make a pulp paste; S2. Adjust the pH of the pulp to 4-7, then add the first and second enzymes, extract at 30-50℃ for 0.5-4 hours, centrifuge, and collect the first emulsion; The first enzyme includes pectinase, and also includes at least one of hemicellulase and cellulase; The second enzyme consists of tanninase and glycosidase; S3. After washing the first emulsion with water or sucrose aqueous solution, centrifuge and collect the second emulsion to obtain the oil body.

2. The method for preparing a highly stable phenol-rich oil from fresh pulp of *Vernicia fordii* according to claim 1, characterized in that: The first enzyme is composed of pectinase, hemicellulase and cellulase, and the mass ratio of pectinase, hemicellulase and cellulase is 2:(0.1-0.5):(0.5-1.5).

3. The method for preparing a highly stable phenol-rich oil from fresh pulp of *Vernicia fordii* according to claim 1 or 2, characterized in that: The amount of the first enzyme is 0.3wt% to 3wt% of the amount of fresh fruit pulp of the *Vernicia fordii*.

4. The method for preparing a highly stable phenol-rich oil from fresh pulp of *Vernicia fordii* according to claim 1, characterized in that: The glycosidase is selected from at least one of α-glucosidase and β-glucosidase; the mass ratio of the tanninase to the glycosidase is 1:(1-4).

5. A method for preparing a highly stable phenol-rich oil from fresh pulp of *Vernicia fordii* according to claim 1 or 4, characterized in that: The amount of the second enzyme is 0.1wt% to 2wt% of the amount of fresh fruit pulp of the *Vernicia fordii*.

6. The method for preparing a highly stable phenol-rich oil from fresh pulp of *Vernicia fordii* according to claim 1, characterized in that: In step S1, before preparing the pulp, the fresh tung oil fruit is pretreated at a temperature of 65-75°C for 10-40 minutes.

7. The method for preparing a highly stable phenol-rich oil from fresh pulp of *Vernicia fordii* according to claim 1, characterized in that: In step S2, after every 10–40 min of extraction, the extraction system is treated at a temperature not exceeding 10°C for 20–40 s; during treatment, after every 2–5 s of sonication, the system is allowed to stand for 5–10 s, and the sonication frequency is 15–35 kHz and the power density is 10–50 W / mL.

8. A method for preparing a highly stable phenol-rich oil from fresh pulp of *Vernicia fordii* according to claim 1 or 7, characterized in that: In step S2, after extraction, the extraction system is heated to 65-75°C and held for 3-5 minutes.

9. The method for preparing a highly stable phenol-rich oil from fresh pulp of *Vernicia fordii* according to claim 1, characterized in that: It also includes step S4, in which the obtained second emulsion is left to stand at a temperature not exceeding 4°C for 2 to 4 hours.

10. A method for preparing a highly stable phenol-rich oil from fresh pulp of *Vernicia fordii* according to claim 1 or 9, characterized in that: In step S3, the first emulsion is dispersed in water or sucrose aqueous solution with pH 4 to 12, washed and centrifuged at least once; then dispersed in water or sucrose aqueous solution with pH 6 to 7, washed and centrifuged at least twice to obtain the second emulsion.

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