Process for preparing walnut oil by DES-assisted aqueous enzymatic method

Abstract

According to the process for preparing the walnut oil by the DES-assisted aqueous enzymatic method, pretreatment is carried out through DES, combination of cellulose and lignin is destroyed, grease exposure is facilitated, enzyme seed combination and process parameters are screened and optimized, and the walnut oil is prepared by the DES-assisted aqueous enzymatic method. The method is mild in condition, green, safe and high in extraction rate, and natural nutritional ingredients and bioactive ingredients in the walnut oil are reserved to the maximum extent. Compared with the traditional multi-enzyme compounding, the scheme provided by the invention can realize enzyme system compatibility and synergetic targeting hydrolysis, forms significant advantages in efficiency, stability and economy, and furthest retains the specific nutritional ingredients and physiological activity, so as to meet the ever-increasing requirements of the market on high-quality and high-added-value walnut oil products.

Description

Technical Field

[0001] This invention relates to the fields of food processing and bioenzyme application technology, specifically to a process for preparing walnut oil using a DES-assisted aqueous enzyme method. Background Technology

[0002] Walnut oil is rich in polyunsaturated fatty acids (especially linoleic acid and alpha-linolenic acid), vitamin E, phytosterols, and other active ingredients. It possesses various physiological functions, including preventing cardiovascular disease, delaying aging, anti-oxidation, and lowering blood lipids. One important physiological function is improving learning and memory. This function is closely related to its high content of omega-3 unsaturated fatty acids (alpha-linolenic acid). Studies have shown that omega-3 unsaturated fatty acids can improve learning and memory in animals. Polyunsaturated fatty acids are the main components of phospholipid membranes in the brain, helping to maintain the integrity and function of neurons. They also promote the growth of hippocampal neurons and nerves, increase synaptic membrane area and synaptic protein expression, thereby enhancing cognitive function. With increasing consumer demand for natural and healthy oils, how to efficiently and environmentally extract walnut oil while preserving its nutritional activity has become a core demand for industry development.

[0003] Currently, walnut oil extraction mainly relies on pressing and organic solvent extraction. Pressing (including hot and cold pressing) is simple, but hot pressing easily leads to the oxidation of unsaturated fatty acids and significant loss of heat-sensitive nutrients, resulting in a low oil yield. While cold pressing can better preserve nutrients, the oil yield is still low, and it has stringent requirements for raw material quality and pretreatment, leading to low production efficiency. Organic solvent extraction (commonly using n-hexane) offers a high oil yield, but it carries the risk of solvent residue, affecting food safety. Furthermore, the high-temperature desolventizing process also damages nutritional components, which is inconsistent with the trend towards green and clean production.

[0004] In recent years, the aqueous enzymatic method has attracted widespread attention as a green and mild oil extraction technology. Its principle involves using enzymes (such as pectinase, cellulase, and protease) to hydrolyze cell walls or lipid complexes, releasing the oil in a mild aqueous environment. This method offers significant advantages, including mild conditions (low temperature, normal pressure), low energy consumption, no solvent residue, simultaneous retention of heat-sensitive active ingredients in the oil, and comprehensive utilization of multiple components such as proteins. It has shown good potential in the extraction of various vegetable oils, including peanut oil, rapeseed oil, and corn germ oil. The aqueous enzymatic method offers advantages such as mild process conditions, simple operation, and low energy consumption, and it yields vegetable oils with well-preserved nutritional components, especially for producing vegetable oils rich in unsaturated fatty acids.

[0005] However, research and practice on applying aqueous enzymatic methods to walnut oil extraction are still limited. Walnut kernels have a complex composition and dense cell structure, with oils and proteins tightly bound together. Using conventional single enzymes or universal enzyme systems often fails to efficiently break down the cell walls and effectively dissociate lipoprotein complexes, resulting in unsatisfactory oil extraction rates and poor process stability. Existing research focuses on combinations of different enzymes, such as proteases with amylases and cellulases. However, these combinations have limitations. For example, amylases and cellulases are mostly acidic or near-acidic enzymes, which conflict with the optimal pH and temperature range of neutral proteases. In the process, compromises are often required, resulting in a 10-25% decrease in the activity of both enzymes and a weakening of the synergistic effect. Walnut oil bodies are mainly encapsulated by globulins and glutenins. Amylase targets starch (which accounts for only 2-5% of walnuts), and cellulase targets cellulose (which is low in the seed coat). They have no direct effect on oil release and instead consume enzyme preparations and prolong reaction time. The hydrolysis of polysaccharides by cellulase or amylase produces viscous oligosaccharides, which increases the viscosity of the system and causes severe oil-water emulsification. This requires the addition of surfactants or centrifugation to enhance demulsification, increasing the number of process steps and energy consumption.

[0006] Therefore, there is an urgent need to develop a novel aqueous enzymatic process for walnut oil extraction. This process, aided by DES (Distilled Extraction System), involves screening and optimizing enzyme combinations and process parameters to address the problems of low oil yield, poor product quality, and environmental pollution associated with traditional extraction methods. The goal is to achieve efficient, green, and high-quality extraction of walnut oil, maximizing the preservation of its unique nutrients and physiological activities to meet the growing market demand for high-quality, high-value-added walnut oil products. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a walnut oil preparation process. Through DES-assisted extraction, and by simultaneously screening and optimizing enzyme combinations and process parameters, a mild, green, and safe process is achieved, resulting in high extraction rates while maximizing the retention of natural nutrients and bioactive components in walnut oil. Compared to traditional multi-enzyme formulations, this invention achieves enzyme compatibility and synergistic targeted hydrolysis, demonstrating significant advantages in efficiency, stability, and economy.

[0008] On the one hand, the present invention provides a process for preparing walnut oil, which is prepared by DES-assisted aqueous enzymatic extraction. The DES-assisted aqueous enzymatic method involves pretreatment with DES followed by treatment with lichen neutral protease and calcinus albicans neutral protease.

[0009] Eutectic solvents (DES), as novel green solvents, are composed of hydrogen bond acceptors and hydrogen bond donors mixed in a specific ratio. DES can rapidly penetrate into the pores of cell walls, dissolving components such as pectin, hemicellulose, and some lignin, disrupting the dense network structure of the cell wall, causing cracks or even disintegration, and significantly reducing the mechanical strength of the cell wall. Bacillus licheniformis neutral protease (a metalloproteinase) primarily hydrolyzes peptide bonds with amino groups provided by hydrophobic amino acids (such as leucine and phenylalanine), while Bacillus subtilis neutral protease (a serine protease) shows a preference for peptide bonds with carboxyl groups provided by another series of hydrophobic or aromatic amino acids (such as tyrosine, phenylalanine, and tryptophan). The substrate cleavage sites of the two are significantly different and complementary. When they work together, they can attack and degrade the walnut protein network (especially the membrane proteins and storage proteins that encapsulate the oil) from different locations and in different ways, resulting in a more extensive and thorough hydrolysis than using a single enzyme. This more effectively disrupts the oil-protein complex, improving oil release efficiency and degree of hydrolysis.

[0010] Furthermore, the preparation process includes the following steps: (1) Raw material pretreatment: Remove the shells from the walnut kernels, crush them to obtain walnut powder; (2) Soaking: Soak in a mixture of DES and water at room temperature; (3) Enzymatic hydrolysis: Add enzymes to carry out enzymatic hydrolysis. (4) Termination of enzymatic hydrolysis: Adjust the pH of the hydrolysate to terminate enzyme activity; (5) Separation and oil extraction: The slurry after the enzymatic hydrolysis is terminated is separated and washed to obtain walnut oil.

[0011] Walnut shells are mainly composed of cellulose, lignin, and hemicellulose, with extremely low oil content and a very hard texture. If the shell is not removed, enzymes will be inefficiently adsorbed or consumed on the useless, large shell surface during extraction, resulting in serious waste and a significant increase in costs. Pigments, tannins, and lignin in the shell will dissolve in large quantities, making subsequent separation and purification extremely difficult. The resulting oil will be dark in color, bitter in taste, and of very poor quality. The oil is stored in oil bodies within cells, which are tightly wrapped by protein membranes and cell walls. Crushing the walnut kernels can break them down into tiny particles and tear part of the cell walls, which is conducive to the enzymatic hydrolysis reaction.

[0012] After soaking, walnut powder can penetrate cell walls, break the bond between cellulose and lignin, and make the oil more easily exposed. Water molecules will gradually penetrate into every tiny capillary of the powder, so that the entire raw material particle is fully hydrated and swollen from the inside out. Then, a compound neutral protease is added to carry out an enzymatic hydrolysis reaction.

[0013] Further, the DES-water mixture in step (2) is composed of DES and deionized water in an appropriate volume ratio, and the soaking time is 0.8-1.2 h; the DES includes a hydrogen bond donor and a hydrogen bond acceptor, the hydrogen bond acceptor is one of choline chloride, urea, and amino acids, and the hydrogen bond donor is any one or more of lactic acid, glucose, glycerol, ethylene glycol, and 1,2-propanediol.

[0014] Using too much DES will increase the difficulty and cost of subsequent washing, while using too little will not have a significant strengthening effect. Therefore, it is necessary to choose a DES-water mixture with an appropriate volume ratio.

[0015] In some methods, the highest walnut oil extraction yield was achieved when the volume ratio of DES-water mixture was 1:3; the highest extraction yield and the highest total unsaturated fatty acid content were also achieved when choline chloride was the hydrogen bond acceptor and lactic acid was the hydrogen bond donor.

[0016] Furthermore, the DES also contains a protective agent, which includes any one or more of vitamin C, vitamin E, L-cysteine, and glutathione.

[0017] In the aqueous enzymatic process, a large amount of water weakens the hydrogen bonding between DES components, causing some DES to dissociate into single ions or molecules, losing its ability to swell and penetrate the walnut raw material. Adding a protective agent can strengthen the hydrogen bond network structure of DES by forming secondary hydrogen bonds with the DES components, preventing it from dissociating in the aqueous system and maintaining the active function of DES.

[0018] In some methods, the extraction yield and content of various unsaturated fatty acids of walnut oil are highest when the DES protectant is L-cysteine.

[0019] Further, the enzymatic hydrolysis reaction in step (3) includes first grinding the soaked walnut powder into an emulsion state, adjusting the pH, and then adding a compound neutral protease. The compound neutral protease is a mixture of Bacillus licheniformis neutral protease and Bacillus subtilis neutral protease in a mass ratio of 0.8:1-1:1.2. Enzymatic hydrolysis is started for 1-3 hours.

[0020] Because the substrate cleavage sites of Bacillus licheniformis neutral protease and Bacillus subtilis neutral protease are significantly different and complementary, when added simultaneously, the two enzymes can bind to different sites on the protein molecule at the same time, cleaving simultaneously from the main chain and side chain, rapidly destroying the spatial network structure of the protein, and causing a large amount of lipid bodies to be released in a short period of time.

[0021] In some methods, the amino acid sequence of the protein encapsulated by walnut oil bodies contains approximately 8%-10% Leu (leucine) residues, and the corresponding Leu-X peptide bond is the target of lichen neutral protease. The total proportion of Arg (arginine) and Lys (lysine) residues is approximately 9%-11%, and the corresponding Arg / Lys-X peptide bond is the target of subtilis neutral protease. The distribution ratio of the two target peptide bonds in the protein is close to 1:1. Therefore, when using a 1:1 enzyme blending ratio, the number of substrate binding sites of the two enzymes is highly matched with the number of enzyme molecules, resulting in the best effect.

[0022] Furthermore, the separation and washing process includes washing the walnut oil with deionized water and anhydrous ethanol aqueous solution after centrifugation, and then placing it in a vacuum drying oven to allow the moisture and DES small molecules to evaporate.

[0023] DES is soluble in water, and during centrifugation, most of it is enriched in the middle aqueous phase. The upper oil phase is taken and washed again with deionized water to remove a small amount of DES in the oil phase. Then, taking advantage of the miscibility between ethanol and DES, the trace amount of DES adsorbed on the surface of the oil phase is further stripped off. Then, it is washed with deionized water to remove residual ethanol. Finally, it is placed in a vacuum drying oven to allow water, ethanol and small DES molecules to evaporate and be removed.

[0024] Furthermore, the total amount of enzyme added in the enzymatic hydrolysis reaction is 800-1200 U / g walnut powder, the particle size of the emulsion is 90-110 mesh, the pH is adjusted to 7-8, and the enzymatic hydrolysis is carried out by stirring in a 40-50℃ warm water bath.

[0025] An appropriate enzyme concentration ensures that the number of molecules of the two neutral proteases is completely saturated with the binding sites of the protein substrate in walnut powder, avoiding either "substrate excess (insufficient enzyme, incomplete hydrolysis)" or "enzyme excess (substrate depletion, enzyme idle and wasted)". When the amount of neutral protease added is too high, it will over-hydrolyze the protein and produce a large number of free amino acids. These amino acids are prone to Maillard reactions with oils, resulting in a darker color and off-flavor in the walnut oil. pH=7.5 is the optimal pH intersection of the two neutral proteases and a key parameter for adapting to the structure of walnut protein. The isoelectric point of walnut globulin and glutenin is about 6.0-7.0. At pH=7.5, the protein molecules are negatively charged and will disperse in the slurry due to electrostatic repulsion, increasing the contact area with enzyme molecules.

[0026] Furthermore, the particle size of the walnut powder mentioned in step (1) is 10-30 mesh.

[0027] To avoid the frictional heat generated by the high-speed operation of the grinder during a single fine grinding process, which could cause the material temperature to rise rapidly and lead to oil oxidation and rancidity, a gentle coarse crushing method is adopted first. This method only breaks down the macroscopic structure of the walnut kernel without excessively damaging the oil-encapsulating structure inside the cells. This results in less frictional heat generation and reduces the risk of oil oxidation.

[0028] In some methods, the best enzymatic hydrolysis effect is achieved when walnut kernels are ground to 20 mesh.

[0029] On the other hand, the present invention provides a walnut oil with high unsaturated fatty acid content, characterized in that it is prepared by the above-mentioned preparation process and the total unsaturated fatty acid content is ≥94%.

[0030] Unsaturated fatty acids (including oleic acid, linoleic acid, and linolenic acid) are essential fatty acids that the human body cannot synthesize on its own and must obtain from food. The total unsaturated fatty acid content is ≥94%, which means that the saturated fatty acid content in walnut oil is ≤6%, which is much lower than that of animal fats and some vegetable oils, meeting the requirements of modern healthy diets of "low saturated fat and high unsaturated fat".

[0031] In another aspect, the present invention provides the use of the walnut oil described above in the preparation of health foods or medicines that improve learning and memory abilities.

[0032] Walnut oil is rich in unsaturated fatty acids such as alpha-linolenic acid and linoleic acid, with a ratio close to 1:6 that facilitates absorption by the human body. Alpha-linolenic acid can be converted into DHA in the body, and linoleic acid can be converted into ARA. Both of these substances are important components of brain nerve cells and retinal cells, and are crucial for brain development and memory formation in infants and young children. At the same time, these unsaturated fatty acids can also maintain the integrity and fluidity of brain cell membranes, ensuring smooth transmission of nerve cell signals, thereby maintaining learning and memory abilities in adults and delaying cognitive decline in middle-aged and elderly people.

[0033] The walnut oil preparation process provided by this invention has the following beneficial effects: 1. High enzymatic hydrolysis efficiency: The two neutral proteases have no overlapping cleavage sites, which can simultaneously cleave the main chain and side chain of walnut membrane protein, forming a synergistic hydrolysis effect, with an oil extraction rate of ≥86%.

[0034] 2. Mild and stable process conditions: The optimal pH and temperature of the two enzymes are exactly the same, and there is no need to adjust the parameters step by step.

[0035] 3. Preservation of natural active ingredients: The preparation process involves no high temperature or chemical solvents, thus fully preserving the natural antioxidants in walnut oil, such as vitamin E and sterols that are not saponifiable. These ingredients can work together to protect nerve cells and enhance synaptic plasticity.

[0036] 4. Expand application scenarios: The prepared products can be directly used to prepare health foods / medicines that improve learning and memory abilities, and can also be used as high-end raw materials for infant complementary food oil and health care oil for middle-aged and elderly people.

[0037] 5. Green and Environmentally Friendly: This invention utilizes a pure biological enzymatic process, producing no acidic or alkaline wastewater or organic solvent residue. Production wastewater can be recycled after simple treatment; the residue can be used as feed raw material, achieving zero resource waste across the entire industrial chain. Compared to chemical extraction methods, this approach aligns better with environmental policies and green production trends. Attached Figure Description

[0038] Figure 1 Chromatogram of walnut oil ester components prepared by DES-assisted aqueous enzymatic method; Figure 2 Chromatogram of ester components in walnut oil prepared by cold pressing; Figure 3 This is a schematic diagram of the Morris water maze device. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate understanding of the present invention and are not intended to limit it in any way. The reagents used in this embodiment are all known products, and unless otherwise specified, they are all commercially available products.

[0040] Example 1: Preparation of walnut oil by DES-assisted aqueous enzymatic method This embodiment provides a DES-assisted aqueous enzymatic method for preparing walnut oil, and the specific operation is as follows: (1) Raw material pretreatment: The walnuts used in this embodiment were provided by Jinan Hualu Food Co., Ltd. Selected dried walnuts that are free from mold, insects, and full of kernels. Remove the shells manually or mechanically to remove the brown seed coat on the surface of the walnut kernels and obtain pure walnut kernels. Put the walnut kernels into a universal grinder to grind them, pass them through a 20-mesh standard sieve, collect the sieve material, and obtain uniform coarse walnut powder, which is then sealed for later use.

[0041] (2) Preparation of DES-water mixture: Mix choline chloride (purity ≥98%) and lactic acid (analytical grade) in a molar ratio of 1:2 and place them in a round-bottom flask. Place the stir bar in the flask containing the mixture and place it in a preheated oil bath. Connect the magnetic stirrer, turn on the stirring and heating, set the temperature to 60℃, and continue stirring for 2 hours. The mixture gradually changes from a turbid, opaque slurry to a homogeneous, transparent, colorless liquid, which is the basic DES. First, add 0.5% L-cysteine ​​by mass to the basic DES and stir continuously for 15 min to make it completely dissolved to obtain DES. Mix it with deionized water in a volume ratio of 1:3 to obtain the DES-water mixture.

[0042] (3) Soaking the slurry: Weigh 30.0 g of walnut powder and add the coarse walnut powder obtained in step (1) into a beaker at a ratio of 1:3 (g / mL, mass of coarse walnut powder: volume of mixed liquid). Slowly add deionized water and soak at room temperature (20~25℃) for 1 h. Stir slowly during the soaking period to avoid particle agglomeration and ensure that the slurry is fully soaked.

[0043] (4) Secondary grinding and enzymatic hydrolysis preparation: Transfer the soaked slurry to a homogenizer and grind it into an emulsion. Pass it through a 100-mesh standard sieve to ensure that the emulsion particle size is uniform and there are no obvious coarse particles. Transfer the ground emulsion back to the beaker, turn on the constant temperature water bath device, raise the emulsion temperature to 45°C, and keep the temperature stable. Adjust the pH value of the emulsion to 7.5 with 0.1 mol / L NaOH solution or 0.1 mol / L HCl solution and stir evenly.

[0044] (5) Enzymatic hydrolysis reaction: Prepare compound neutral protease: Weigh the two enzyme preparations at a mass ratio of 1:1 (Bacillus licheniformis neutral protease: Bacillus subtilis neutral protease) and mix them evenly; according to the ratio of 1000 U / g walnut powder, slowly add the mixed compound neutral protease to the emulsion while stirring slowly to avoid excessive local enzyme concentration; maintain the conditions of 45℃ water bath temperature and pH 7.5 and continue stirring for 2 h for enzymatic hydrolysis.

[0045] (6) Termination of enzymatic hydrolysis: After 2 hours of enzymatic hydrolysis, immediately adjust the pH of the hydrolysate to 5.0 with 0.1 mol / L HCl solution and stir for 5 minutes to completely deactivate the neutral protease of Bacillus licheniformis and the neutral protease of Bacillus subtilis, thereby terminating the enzymatic hydrolysis reaction.

[0046] (7) Separation and oil extraction: Transfer the slurry after the enzymatic hydrolysis is terminated to a high-speed centrifuge. Set the centrifugation speed to 10,000 rpm / min and centrifuge for 15-20 min. The slurry will separate into three layers: the upper layer is a light yellow transparent walnut oil layer, the middle layer is an aqueous phase (containing walnut polypeptides, small molecule proteins, and DES), and the lower layer is solid residue. Carefully collect the upper layer of walnut oil with a separatory funnel and wash it 2-3 times with a small amount of deionized water and 5% anhydrous ethanol solution to remove the small amount of residual DES. Finally, wash away the residual ethanol with deionized water. Place the washed walnut oil in a vacuum drying oven and dry it at 40℃ and -0.08 MPa for 30 min to remove trace amounts of water and ethanol, and obtain the walnut oil product.

[0047] Example 2: Soxhlet extraction of total oil from walnut powder This embodiment provides a Soxhlet extraction method for determining the total oil content of walnut powder. The specific operation is as follows: (1) Raw material pretreatment: The walnuts used in this embodiment were provided by Jinan Hualu Food Co., Ltd. Selected dried walnuts that are free from mold, insects, and full of kernels. Remove the shells manually or mechanically to remove the brown seed coat on the surface of the walnut kernels and obtain pure walnut kernels. Put the walnut kernels into a universal grinder to grind them, pass them through a 20-mesh standard sieve, collect the sieve material, and obtain uniform coarse walnut powder, which is then sealed for later use.

[0048] (2) Extraction: Weigh 30 g of walnut powder, place it in an evaporating dish, add 20 g of quartz sand, evaporate the water in a boiling water bath, and then dry it in an oven at 100℃±5℃ for 30 min. Transfer all the sample to a folded filter paper tube. Then place the filter paper tube in the extraction tube of a Soxhlet extractor, connect the lower end to a dry and clean round-bottom flask, add petroleum ether through the upper end of the condenser tube of the extractor to two-thirds of the flask's volume, heat in a water bath to continuously reflux the petroleum ether, and extract for a total of 6 h. After extraction, recover the petroleum ether by rotary evaporation to obtain the total oil in 30 g of walnut powder.

[0049] Example 3: Effects of different enzymes on the preparation of walnut oil using the DES-assisted aqueous enzymatic method. Walnut oil was prepared using the experimental method described in Example 1. Three control groups were established for proteases: neutral protease, acidic protease, and alkaline protease. Trials were performed in three parallel runs, and the average value was taken. Except for the enzyme type and the corresponding optimal hydrolysis pH, all other process parameters were consistent. The results are shown in Table 1 below. Table 1. Effects of different enzymes on walnut oil extraction rate

[0050] As shown in Table 1, the walnut oil extraction rate and unsaturated fatty acid content of neutral protease were significantly higher than those of acidic and alkaline protease groups. This is because the optimal pH (7.5) of neutral protease has better pH compatibility with the DES-water mixture, and the stable structure of DES will not be destroyed by drastic changes in the pH of the system (DES may decompose under strong acid / alkaline conditions). Furthermore, neutral protease has stronger specificity for the degradation of walnut protein and can more efficiently destroy the oil-protein binding network. The optimal pH of acidic protease is too low, which leads to partial decomposition of DES. At the same time, walnut protein is prone to denaturation and aggregation under strong acid conditions, which hinders oil release. The optimal pH of alkaline protease is too high. Although it can degrade some proteins, the stability of DES decreases under strong alkaline conditions. Therefore, neutral protease is the most effective.

[0051] Example 4: Effects of different neutral protease complex enzyme combinations on the preparation of walnut oil using the DES-assisted aqueous enzymatic method. Walnut oil was prepared using the experimental method of Example 1. Three control groups were established for the selection of complex enzyme combinations: (Bacillus licheniformis neutral protease + Bacillus subtilis neutral protease), (Aspergillus oryzae neutral protease + Aspergillus niger neutral protease), and (Bacillus licheniformis neutral protease + Aspergillus oryzae neutral protease). Three parallel groups were set up, and the average value was taken. All groups used neutral protease complexes with a mass ratio of 1:1. The total enzyme dosage was 1000 U / g walnut powder. The enzymatic hydrolysis conditions and other process parameters were consistent. The results are shown in Table 2 below. Table 2. Effects of different neutral protease complex enzyme combinations on walnut oil extraction rate

[0052] As shown in Table 2, the extraction effects of different complex enzyme combinations varied significantly, with the (Bacillus licheniformis neutral protease + Bacillus subtilis neutral protease) group exhibiting the best performance—the total walnut oil content of this combination reached 750.62 mg·g. -1 The extraction rate was 86%, and ω-3 linolenic acid (74.16 mg / g) was also obtained. -1 ) and linoleic acid (512.25 mg·g) -1 The content of the (Aspergillus oryzae neutral protease + Aspergillus niger neutral protease) group was significantly higher than that of the other two groups; the total content, extraction rate, and polyunsaturated fatty acid content of the (Bacillus licheniformis neutral protease + Aspergillus oryzae neutral protease) group were the lowest among all groups; the various indicators of the (Bacillus licheniformis neutral protease + Aspergillus oryzae neutral protease) group were between the two. In summary, the combination of (Bacillus licheniformis neutral protease + Bacillus subtilis neutral protease) is more conducive to the efficient extraction of walnut oil and polyunsaturated fatty acids (especially ω-3 group).

[0053] Example 5: Preparation of walnut oil by cold pressing This embodiment provides a cold-pressing process for preparing walnut oil, the specific operation of which is as follows: (1) Raw material pretreatment: The walnuts used in this embodiment were provided by Jinan Hualu Food Co., Ltd. Selected dried walnuts that are free from mold, insects, and full of kernels. Remove the shells manually or mechanically to remove the brown seed coat on the surface of the walnut kernels and obtain pure walnut kernels. Put the walnut kernels into a universal grinder to grind them, pass them through a 20-mesh standard sieve, collect the sieve material, and obtain uniform coarse walnut powder, which is then sealed for later use.

[0054] (2) Low temperature drying: Spread 30 g of walnut powder evenly in the tray of a low temperature oven with a thickness of ≤2 cm. Set the temperature to 45℃ and the drying time to 1.5 h. After drying, the moisture content of the walnut powder is controlled at 3%~4%.

[0055] (3) Equipment preheating and debugging: Turn on the screw cold press, start the cooling jacket (circulate 20℃ cooling water), preheat the equipment for 10 min, adjust the screw speed to 35 r / min, and set the pressing chamber temperature to ≤60℃ (monitor the pressing chamber temperature point in real time to ensure that it does not exceed 60℃ throughout the process); put the dried walnut powder into the cold press feed inlet at a uniform speed, and control the feeding rate at 1.2 kg / h to avoid the pressing chamber temperature from rising too quickly; place a stainless steel oil collection bucket at the cold press oil outlet to collect the first pressed oil (containing a small amount of water and protein impurities); collect the pressing cake at the slag outlet (which can be used for subsequent extraction of walnut protein or for making feed); after each pressing, rinse the pressing chamber with a small amount of room temperature edible oil to avoid residual impurities affecting the quality of the next batch of products.

[0056] (4) Refining of virgin oil: Transfer the virgin oil to a high-speed centrifuge, set the speed to 10,000 rpm, and centrifuge for 20 min to allow water and protein impurities in the oil to settle fully. After separation, the upper layer is a clear oil phase and the lower layer is a water-mixed layer. Collect the upper clear oil phase with a separatory funnel, add 10% of the oil phase volume of 35℃ deionized water, gently shake for 3 min, let stand for separation, discard the lower water phase, and repeat washing twice to remove residual water-soluble impurities. Transfer the washed walnut oil to a vacuum drying oven, set the temperature to 40℃ and the vacuum degree to -0.09 MPa, and dry for 30 min to remove trace amounts of water in the oil. Filter the dried walnut oil with a 0.22 μm organic filter membrane to remove trace amounts of suspended matter and obtain refined walnut oil. Put the refined walnut oil into a dry, clean brown glass bottle, seal it, and store it in a cool, dry place.

[0057] Example 6: Extraction of walnut oil using conventional compound aqueous enzyme method This embodiment uses the experimental method of Example 1 to prepare walnut oil. In the soaking process, walnut powder and deionized water are mixed at a ratio of 1:3. In the enzymatic hydrolysis reaction, papain and cellulase are combined (mass ratio of 2:1). Other process parameters are the same to obtain walnut oil.

[0058] Example 7: Quality Testing of Walnut Oil Prepared by Different Processes This embodiment provides a comparison of the quality of walnut oil obtained by different preparation processes. The oil extraction rate, total fatty acid content, and content of each fatty acid were measured in walnut oil from two different treatment groups. The specific procedures are as follows: Group 1: Walnut oil prepared using the experimental method of Example 1.

[0059] Group 2: Walnut oil prepared using the experimental method of Example 5.

[0060] Group 3: Walnut oil prepared using the experimental method of Example 6.

[0061] Weigh the final walnut oil obtained from both groups and calculate the extraction rate according to the following formula, where the total oil mass in the walnut powder is determined using the experimental method of Example 2:

[0062] Accurately weigh 0.1 g of pressed walnut oil and water-enzymatically extracted walnut oil into 15 mL stoppered test tubes. Add 2.0 mL of 1.0 mol / L KOH-ethanol solution and place in a 70℃ hot water bath with shaking for saponification for 40 min. After saponification, cool the sample to room temperature, adjust the pH to 4-5 with dilute hydrochloric acid, and extract twice with 6 mL of n-hexane, 3 mL each time. Collect and combine the organic phases, evaporate the n-hexane to dryness in a hot water bath, and make up to 10 mL with anhydrous methanol. Filter through a 0.22 μm microporous membrane, and perform fatty acid liquid chromatography detection of the filtrate using an evaporative light scattering detector.

[0063] The liquid chromatography detection conditions were as follows: Eclipse XDB-C18 column; column temperature 30℃; mobile phase: methanol / water (containing 0.1% acetic acid) 83:17 (v / v), flow rate 1.0 mL / min; carrier gas (N2) flow rate of 2.0 L / min for evaporative light scattering detector, and drift tube temperature 70℃.

[0064] Under the above liquid chromatography conditions, the mass spectrometry conditions were as follows: ion source: ESI-; capillary voltage: 3 kV; cone voltage: 40 V; source temperature: 150 °C; desolvation gas temperature: 350 °C; cone gas flow rate: 500 L / h; desolvation gas flow rate: 100 L / h. The fatty acids in walnut oil were qualitatively determined by HPLC-MS, identifying four fatty acids: linolenic acid, linoleic acid, palmitic acid, and oleic acid.

[0065] Accurately weigh linolenic acid, linoleic acid, palmitic acid, and oleic acid standards, and prepare standard solutions with anhydrous methanol at concentrations of 0.25 mg / mL, 0.50 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 4.0 mg / mL, and 8.0 mg / mL. Filter the solution through a 0.22 μm microporous membrane, prepare a standard curve under the determined chromatographic conditions, and quantitatively determine the fatty acid content of walnut oil using the external standard method. The chromatograms of ester components in walnut oil obtained by cold pressing and DES-assisted aqueous enzymatic method are shown below. Figure 1-2 The chromatograms of walnut oil esters showed significant differences among the three treatment groups. Walnut oil extracted by cold pressing and conventional hydroenzyme extraction had a higher content of non-triglyceride esters than walnut oil extracted by DES-assisted hydroenzyme extraction, and thus had a lower relative content of fatty acids.

[0066] The results of quantitative detection by liquid chromatography are shown in Table 3 below: Table 3. Walnut oil extraction rate, quantitative detection of fatty acids and precision

[0067] As shown in Table 3, the extraction rate of DES-assisted aqueous enzymatic method is significantly higher than that of conventional aqueous enzymatic method and cold pressing method. At the same time, its unsaturated fatty acid content is outstanding—the linoleic acid content of DES-assisted aqueous enzymatic method reaches 512.25 mg·g. -1 It has the highest total fatty acid content among the three methods, higher than both cold pressing and conventional enzymatic methods. Although its linolenic acid and oleic acid content is slightly lower than those of cold pressing and conventional enzymatic methods, the enrichment advantage of the core active ingredient linoleic acid is obvious. Therefore, the DES-assisted enzymatic method has the best effect.

[0068] Based on the results of fatty acid analysis in walnut oil, the ratio of unsaturated fatty acids to saturated fatty acids in walnut oil prepared by the aqueous enzymatic method and the cold pressing method is calculated as shown in Table 4 below: Table 4. Ratio of unsaturated to saturated fatty acids in walnut oil prepared by aqueous enzymatic method and cold pressing method

[0069] TUFA = Tri-unsaturated FA; DUFA = Di-unsaturated FA; MUFA = Mono-unsaturated FA; SFA = Saturated FA; UFA = Unsaturated fatty acid. As shown in Table 4, the proportion of unsaturated fatty acids (∑UFA) in the three processes shows a gradient increase: cold pressing (91.83%) < conventional aqueous enzymatic method (93.35%) < DES-assisted aqueous enzymatic method (94.98%), while the proportion of saturated fatty acids (∑SFA) decreases in the opposite direction, reflecting the enrichment advantage of the DES-assisted aqueous enzymatic method for low-fat components in walnut oil.

[0070] Example 8: Effect of different enzyme dosages on walnut oil quality This embodiment uses the experimental method of Example 1 to prepare walnut oil. During the enzymatic hydrolysis reaction, the total enzyme dosage was set to three groups: 800, 1000, and 1200 mg / L. The extraction rate of the walnut oil in the three groups was tested using the experimental method of Example 4, with other parameters remaining consistent. The results are shown in Table 5 below. Table 5. Extraction rate of walnut powder with different enzyme dosages

[0071] As shown in Table 5, in terms of extraction rate, as the enzyme dosage increased from 800 U / g to 1000 U / g, the extraction rate significantly increased from 77% to 86%. However, when the enzyme dosage was further increased to 1200 U / g, the extraction rate slightly decreased, indicating that when the enzyme dosage was 800 U / g of walnut powder, the enzyme dosage was insufficient, resulting in incomplete decomposition of the protein matrix in the walnut kernels, low degree of damage to the cell wall and protein network structure, and difficulty in fully releasing the oil encapsulated within, with a large amount of unextracted oil remaining in the residue. When the enzyme dosage was excessive (1200 U / g of walnut powder), the degradation of the protein matrix had reached saturation, and the additional enzyme could not further improve the oil release efficiency. For each fatty acid, when the enzyme dosage was increased to 1000 U / g, the content of each fatty acid increased significantly, and the total content also increased significantly. However, when the enzyme dosage was increased to 1200 U / g, due to excessive enzyme dosage, substrate saturation, enzyme molecule aggregation, and a slight decrease in unsaturated fatty acids, palmitic acid was less affected and therefore showed no significant difference. Therefore, an enzyme dosage of 1000 U / g can most fully disrupt the protein-cell wall structure of walnut powder, achieving efficient release and retention of oils and functional fatty acids, and making it easier to obtain active ingredients that are beneficial to mouse learning and memory.

[0072] Example 9: Screening of DES hydrogen bond acceptors This embodiment prepared walnut oil according to the method provided in Example 1. The hydrogen bond acceptors for the DES were different types as provided in Table 1, and the hydrogen bond donor was glycerol. The molar ratio of hydrogen bond acceptor to hydrogen bond donor was 1:2. The effect of DES prepared using different hydrogen bond acceptors on the extraction rate of walnut oil was investigated, and the results are shown in Table 6.

[0073] Table 6. Effects of different hydrogen bond donors on walnut oil extraction rate

[0074] As shown in Table 6, both the choline chloride group and the tetrabutylammonium chloride group showed relatively high contents of linolenic acid and linoleic acid. The tetrabutylammonium chloride cation has a large volume and strong hydrophobicity, making it easier to combine with nonpolar fatty acids (oleic acid), which is consistent with the result in Table 6 that the tetrabutylammonium chloride group had a higher oleic acid content. However, because the hydrogen bonding of the choline chloride group is milder, it can preferentially form hydrogen bonds with fatty acids without destroying the overall structure of the oil. Therefore, the extraction rate of choline chloride is higher, and the contents of linolenic acid and linoleic acid in the choline chloride group are higher than those in the tetrabutylammonium chloride group. In other words, the choline chloride group has a better extraction effect on linolenic acid and linoleic acid.

[0075] Example 10: Screening of DES hydrogen bond donors In this embodiment, walnut oil was prepared according to the method provided in Example 1. The hydrogen bond donors of DES were different types of hydrogen bond donors as provided in Table 2, and the hydrogen bond acceptor was choline chloride. The effect of DES prepared with different hydrogen bond donors on the extraction rate of walnut oil was investigated, and the results are shown in Table 7.

[0076] Table 7. Effects of different hydrogen bond donors on walnut oil extraction rate

[0077] According to Table 7, different hydrogen bond donors have a certain impact on the extraction rate of walnut oil. When glycerol is used as the hydrogen bond donor, the extraction amount of linolenic acid is significantly higher than that of other donors, and the linoleic acid content is also high, achieving efficient enrichment of polyunsaturated fatty acids. Among the other donors, the extraction rates of lactic acid and ethylene glycol are similar to those of glycerol, but the linoleic acid content is slightly lower. Therefore, using glycerol as the hydrogen bond donor for DES is more conducive to the extraction of polyunsaturated fatty acids (especially ω-3 group) from walnut oil.

[0078] Example 11: Effect of adding a protective agent to DES Adding a preservative can significantly improve the extraction rate of walnut oil, and the extraction effect varies depending on the preservative used. In this example, walnut oil was extracted according to the method provided in Example 1, wherein eutectic solvents were prepared using the preservatives provided in Table 3, and the addition ratios were the same as in Example 1. The effect of using different preservatives to prepare DES on the walnut oil extraction yield was investigated, and the test results are shown in Table 8.

[0079] Table 8 Effects of different preservatives on walnut oil extraction

[0080] According to Table 8, the addition of preservatives significantly improved the extraction rate and retention of functional unsaturated fatty acids in walnut oil. Without preservatives, the total content, extraction rate, and content of polyunsaturated fatty acids (linolenic acid + linoleic acid) in walnut oil were all at low levels. However, after adding preservatives, all indicators improved to varying degrees, with L-cysteine ​​showing the best effect: the total walnut oil content reached 750.62 mg / g. -1 The extraction rate was 86%, and the content of ω-3 linolenic acid and linoleic acid was significantly higher than that of vitamin C, vitamin E and glutathione (the total content and polyunsaturated fatty acid content of the glutathione group increased the least). In summary, L-cysteine ​​is a protective agent that is more conducive to the extraction of polyunsaturated fatty acids (especially ω-3) from walnut oil.

[0081] Example 12: Effects of walnut oil prepared by different processes on improving learning and memory abilities in mice. This embodiment provides the effect of walnut oil prepared using different processes on improving the learning and memory abilities of mice. The specific operation is as follows: (1) Laboratory animals and grouping Sixty healthy male ICR mice were given free access to water and food for one week to acclimatize to their environment. After one week, they were randomly divided into six groups of 10 mice each, based on their body weight: a blank control group; experimental group 1: walnut oil prepared using the experimental method of Example 1, with vitamin C as the protectant for DES and all other process parameters being the same; experimental group 2: walnut oil prepared using the experimental method of Example 1; experimental group 3: walnut oil prepared using the experimental method of Example 6; experimental group 4: walnut oil prepared using the experimental method of Example 5; and a positive control group (piracetam). The blank control group mice were administered an equal volume of deionized water daily by gavage, with each gavage volume calculated as 0.2 mL / 10 g, once daily for each group. The gavage period was four weeks. During the experiment, the animals had free access to water and food, and the animal room temperature was controlled at (25 ± 2)℃, and the humidity at (65 ± 10)%.

[0082] (2) Morris water maze test The device consists of a Morris water maze and an image monitoring system. The Morris water maze is a circular pool with a diameter of 150 cm and a height of 50 cm. According to Cartesian coordinates, the pool is divided into four quadrants: northeast, southeast, northwest, and southwest. A transparent circular platform, 29 cm high and 9 cm in diameter, is placed 33 cm from the pool wall in the center of the northeast quadrant. The platform is 1 cm below the water surface. Figure 3 As shown. The water temperature was controlled at 25 ± 3℃. A camera was mounted on top of the device and connected to a computer to synchronously track the mouse's movement in the water. When the mouse actively climbed onto the platform or reached the set training time, the computer stopped tracking, and the software automatically calculated the time required for the mouse to find the platform (i.e., the latency period). Water maze training began for the mice four weeks after oral administration of walnut oil.

[0083] ① Orientation and navigation experiment: The experiment lasted for 5 days. During training, the platform was placed in the northeast quadrant. Mice were placed into the water head-on from the midpoint of each of the four quadrants of the pool wall. The time it took for the mouse to find and climb onto the platform (i.e., the escape latency) was recorded. Each training session lasted 60 seconds. After climbing onto the platform, the mouse was allowed to stay on it for 15 seconds. If the mouse could not find the platform within 60 seconds, it was guided to the platform and allowed to stay there for 15 seconds (the latency in this case was recorded as 60 seconds). The escape latency of the first 5 days of the experiment was taken, and the average value was calculated. The results are shown in Table 9 below: Table 9. Effects of walnut oil prepared by different methods on the average latency of mice to find the platform.

[0084] As shown in Table 9, from day 1 to day 5, the escape latency of mice in each group shortened sequentially with the increase in the number of training sessions. Among them, experimental group 2 showed the best improvement effect, which was closest to the positive control. The latency on day 5 was better than other walnut oil groups, thanks to the efficient protection of unsaturated fatty acids by L-cysteine ​​and the full enrichment of active ingredients such as linoleic acid by the DES-assisted aqueous enzymatic method. The improvement effect of experimental group 1 was slightly inferior to that of experimental group 2, which shows that the protective effect of vitamin C is weaker than that of L-cysteine, but it is still better than the conventional aqueous enzymatic method (without DES assistance) and the cold pressing method. Therefore, walnut oil prepared by the DES-assisted aqueous enzymatic method has a more prominent effect on improving the learning and memory ability of mice, and its efficacy is close to that of the positive control drug piracetam.

[0085] ② Spatial exploration experiment: A spatial exploration experiment was conducted 24 hours after the last training session. The underwater platform was removed, and the number of times the mice crossed the quadrant corresponding to the platform within 60 seconds and the time spent in that quadrant were recorded by computer. The results are shown in Table 10 below: Table 10. Number of times mice crossed the platform in the final stage of the water maze and the proportion of time spent in the platform area.

[0086] Table 10 shows that in the Morris water maze spatial search test, the positive control piracetam group performed best. Among the walnut oil groups, experimental group 2 crossed the platform 2.55 times and remained for 34.21%, showing the closest improvement to the positive control. Experimental group 1 crossed the platform 2.30 times and remained for 34.85%, slightly inferior to experimental group 2. Experimental group 3 crossed the platform 2.05 times and remained for 34.00%, showing a weaker improvement than the DES-assisted group. Experimental group 4 crossed the platform 1.75 times, showing limited improvement, while the blank control group had the lowest remaining percentage. Overall, the improvement effect was ranked as follows: "piracetam group > experimental group 2 > experimental group 1 > experimental group 3 > experimental group 4 > blank control group," confirming that walnut oil prepared by the DES-assisted water-enzyme method has a more pronounced effect on improving mouse learning and memory due to its more abundant unsaturated fatty acid content, while the efficacy of cold-pressed walnut oil is relatively limited.

[0087] (3) Shuttle box test method Before the first training session, mice were placed in a shuttle box for 3 minutes to acclimatize to the environment. At the start of training, mice were placed on the dark side of the box, facing away from the door. A buzzing sound (conditioned stimulus) was given for 5 seconds. If the mouse escaped to the other side before the buzzing ended, it was considered an active avoidance response. If the mouse did not escape after the buzzing ended, an electrical stimulus (110V, 50Hz, AC) was given for 10 seconds. If the mouse escaped to the other side within 10 seconds of the electrical stimulus, it was considered a passive avoidance response. If the mouse remained on the same side after both the buzzing and electrical stimuli, it was considered an escape failure. Each mouse was trained 10 times per day, with an interval of at least 15 seconds between each training session, for 5 consecutive days. The passive avoidance response time was recorded, and the active avoidance response rate (i.e., the percentage of active avoidance responses out of the total number of shuttle runs) and the passive avoidance response time were calculated. The results are shown in Tables 11 and 12 below. Table 11 Active avoidance response rate in mouse shuttle box test

[0088] As shown in Table 11, the response rates of each group gradually increased with the number of training days. Among them, the piracetam group (positive control) showed the best improvement, with a response rate of 32.00% on day 5. In the walnut oil group, the response rate of experimental group 2 on day 5 was 30.50%, which was closest to the positive control. The response rate of experimental group 1 on day 5 was 28.50%, which was slightly lower than that of experimental group 2. The response rate of experimental group 3 on day 5 was 26.00%, which was weaker than that of the DES-assisted group. The response rate of experimental group 4 on day 5 was not significantly different from that of the blank control group, and the improvement effect was limited.

[0089] Table 12 Passive avoidance reaction time in mouse shuttle box test

[0090] Table 12 shows that with the increase of training sessions, the passive avoidance response time of mice in each walnut oil group shortened, and was lower than that of the blank control group. Among them, the positive control piracetam group showed the best effect. The improvement trend of experimental groups 1-4 (experimental group 2 > experimental group 1 > experimental group 3 > experimental group 4) was consistent with the results of the water maze and active avoidance response, which further confirms that walnut oil prepared by the DES-assisted water-enzyme method has a more prominent effect on improving learning and memory in mice due to its more complete enrichment of unsaturated fatty acids.

[0091] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A process for preparing walnut oil using a DES-assisted aqueous enzymatic method, characterized in that, Pretreatment was performed using DES, followed by treatment with lichen neutral protease and calomel neutral protease.

2. The preparation process as described in claim 1, characterized in that, Includes the following steps: (1) Raw material pretreatment: Remove the shells from the walnut kernels, crush them to obtain walnut powder; (2) Soaking: Soak in a mixture of DES and water at room temperature; (3) Enzymatic hydrolysis: Add enzymes to carry out enzymatic hydrolysis. (4) Termination of enzymatic hydrolysis: Adjust the pH of the hydrolysate to terminate enzyme activity; (5) Separation and oil extraction: The slurry after the enzymatic hydrolysis is terminated is separated and washed to obtain walnut oil.

3. The preparation process according to claim 2, characterized in that, The DES-water mixture in step (2) is composed of DES and deionized water in an appropriate volume ratio, and the soaking time is 0.8-1.2 h. The DES includes a hydrogen bond donor and a hydrogen bond acceptor. The hydrogen bond acceptor is one of choline chloride, L-proline, tetrabutylammonium chloride, and L-aspartic acid. The hydrogen bond donor is any one or more of lactic acid, glucose, glycerol, ethylene glycol, and 1,2-propanediol.

4. The preparation process according to claim 3, characterized in that, The DES also contains a protective agent, which includes one or more of vitamin C, vitamin E, L-cysteine, and glutathione.

5. The preparation process according to claim 2, characterized in that, The enzymatic hydrolysis reaction in step (3) involves first grinding the soaked walnut powder into an emulsion state, adjusting the pH, and then adding a compound neutral protease. The compound neutral protease is a mixture of Bacillus licheniformis neutral protease and Bacillus subtilis neutral protease in a mass ratio of 0.8:1-1:1.

2. Enzymatic hydrolysis is started for 1-3 hours.

6. The preparation process according to claim 2, characterized in that, The separation and washing process in step (5) includes washing the walnut oil with deionized water and anhydrous ethanol aqueous solution after centrifugation, and then placing it in a vacuum drying oven to allow the moisture and DES small molecules to evaporate.

7. The preparation process according to claim 5, characterized in that, The total amount of enzyme added in the enzymatic hydrolysis reaction is 800-1200 U / g walnut powder, the particle size of the emulsion is 90-110 mesh, the pH is adjusted to 7-8, and the enzymatic hydrolysis is carried out by stirring in a 40-50℃ warm water bath.

8. The preparation process according to claim 2, characterized in that, The walnut powder in step (1) has a particle size of 10-30 mesh.

9. A walnut oil with high unsaturated fatty acid content, characterized in that, The product is prepared using the aqueous enzymatic method according to any one of claims 1-8, and the total unsaturated acid content is ≥94%.

10. The use of the walnut oil as described in claim 9 in the preparation of health foods or medicines that improve learning and memory abilities.

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