One-pot method for separating hawthorn kernel oil, active substances and hawthorn kernel residue

Abstract

The invention discloses a method for separating hawthorn kernel oil, active substances and hawthorn kernel residue by a one-pot method, and belongs to the technical field of food by-product development. The method comprises the steps of frying, crushing, promoting dissolution, enzymolysis, inactivation, removing residues, oil extraction, secondary oil extraction and active extracts. The invention adopts a "one-pot method" to prepare hawthorn kernel oil, active extracts and hawthorn kernel residues, and has a simple process, simple operation, low production cost, green and pollution-free, high extraction efficiency, and realizes the high-value development and utilization of the by-product hawthorn kernel, which can improve the utilization value of the hawthorn kernel and promote the upgrading of the hawthorn industry chain, and has huge economic and social benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of food by-product development, and particularly relates to a method for separating hawthorn kernel oil, active substances and hawthorn kernel residues by a one-pot method. Background Art

[0002] Hawthorn kernels account for 1 / 3 of the total weight of hawthorn and are waste in the production of hawthorn food and hawthorn slices. Tens of millions of tons of hawthorn kernel byproducts are produced every year. Hawthorn kernels are either directly discarded, polluting the environment, or resold at a low price for charcoal burning. A small amount is also directly crushed and added to animal feed, which has low added value. Therefore, it is urgent to transform hawthorn kernels into high-value-added commodities and extend the hawthorn processing industry chain.

[0003] Hawthorn kernel contains about 10% oil, of which fatty acids include linoleic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, and linolenic acid, and unsaturated fatty acids account for more than 90% of the total fatty acids. It is a high-quality edible oil that can be used to reduce high blood pressure, improve cardiovascular health, and support the digestive system. It can also be used in skin care products and cosmetics to reduce anxiety and stress and promote skin and hair health. However, hawthorn kernel contains more lignin, and its wood is hard, which affects the effective extraction of hawthorn kernel oil. At present, the main methods for extracting hawthorn kernel oil are dry distillation and supercritical extraction. These methods are expensive and have low extraction rates.

[0004] In addition, the active ingredients in hawthorn kernels are relatively complex. Among them, the most studied are flavonoid antioxidant active ingredients. For the extraction of active ingredients, the methods reported in the literature include ultrasonic-assisted method, organic solvent extraction method, etc. These methods all use organic solvent extraction. Although they improve the extraction efficiency, due to the residual solvent, they do not comply with the relevant national regulations on food additives and cannot be added to food, thus limiting their scope of application.

[0005] There is still huge room for the development of the hawthorn industry. However, most scientific research is currently separated from practical applications. They only study how to increase profits without taking into account the limiting factors of practical applications. In addition, the research and development of a single product has a relatively large input-output ratio, which is not conducive to enterprise development. Therefore, the research and development of a series of by-product hawthorn kernel products can not only improve the utilization rate of hawthorn kernels, but also facilitate large-scale production by enterprises. Its products are also expected to play an important role in food, medicine, cosmetics and other fields. Summary of the invention

[0006] The present invention provides a method for separating hawthorn kernel oil, active substances and hawthorn kernel residues in a one-pot process, comprising the following steps:

[0007] (1) Stir-frying: stir-fry the hawthorn kernel at 100-300°C for 1-10 hours;

[0008] (2) Crushing: crush the roasted hawthorn kernels and pass through a 20-100 mesh sieve;

[0009] (3) Dissolution promotion: add water at a material-liquid ratio of 1:1 to 1:10, add a dissolving agent, and soak for 1 to 12 hours;

[0010] (4) Enzymatic hydrolysis: adjust the pH to 4-8, then raise the temperature to 30-80°C; add mixed enzymes and perform enzymatic hydrolysis for 3-10 hours;

[0011] (5) Inactivation: After the enzymatic hydrolysis is completed, the temperature is raised to 90-100°C and maintained for 10-30 min, and then cooled to room temperature;

[0012] (6) Residue removal: Filter to separate the precipitate from the enzymatic hydrolyzate, and the resulting precipitate is the hawthorn kernel residue;

[0013] (7) Oil extraction: adding n-hexane to the enzymatic hydrolyzate, mixing thoroughly, extracting and separating the liquids, recovering n-hexane, and obtaining an oil phase;

[0014] (8) Secondary oil extraction: cool the lower aqueous phase at -40 to 4°C for 10 to 15 hours, and after returning to room temperature, add n-hexane, mix thoroughly, extract and separate, recover n-hexane to obtain an oil phase; combine the two oil phases to obtain hawthorn kernel oil;

[0015] (9) Active extract: The aqueous phase is concentrated and dried to obtain the active substance of hawthorn kernel.

[0016] In the above technical solution, in step (3), the dissolution agent is selected from one or more of hydrochloric acid, lactic acid, sodium hydroxide, urea, and glycine; preferably lactic acid and glycine, and the mass ratio of lactic acid to glycine is preferably 5:1.

[0017] In the above technical solution, in step (3), the amount of the dissolving agent is selected from: taking 1 kg of hawthorn kernels as a reference, adding 10 to 200 g of the dissolving agent; preferably 60 g of the dissolving agent.

[0018] In the above technical solution, in step (4), the mixed enzyme includes ligninase, cellulase, hemicellulase and pectinase, and the mass ratio thereof is 1-7:1-5:1-4:1-4; wherein the amount of ligninase used is not zero.

[0019] In the above technical solution, in step (4), the amount of the mixed enzyme is selected from: taking 1 kg of hawthorn kernels as a reference, adding 50 to 200 g of mixed enzyme; preferably 100 g of mixed enzyme.

[0020] In the above technical solution, in step (7) and step (8), the volume ratio of n-hexane to enzymatic hydrolysis solution is selected from 0.05 to 1:1.

[0021] The present invention provides hawthorn kernel oil, hawthorn kernel residue and / or hawthorn kernel active substance prepared by the above method.

[0022] The beneficial effects of the present invention are:

[0023] The present invention provides a method and technical route for the high-value development and utilization of hawthorn kernels. The output of three products, namely hawthorn kernel oil, hawthorn kernel active extract and hawthorn kernel residue, is achieved through "one-pot" extraction, thereby reducing the input-output ratio and facilitating large-scale production. It not only extends the technical chain of the hawthorn industry, but also improves the added utilization value of hawthorn kernels, and has huge economic and social benefits.

[0024] Because the extraction process does not use organic solvents, there is no organic solvent residual pollution to the active extracts and hawthorn kernel residues. The active extracts can be used as natural antioxidants and natural preservatives in the fields of food and skin care products. The extracted hawthorn kernel residues can be used for animal feed, edible fungus cultivation, biochar production, etc., achieving zero waste residue and high-value comprehensive utilization, reducing environmental pollution, creating more market value, and having broad prospects for industrial application.

[0025] The "one-pot method" is used to prepare hawthorn kernel oil, active extracts and hawthorn kernel residue. The process is simple, the operation is easy, the production cost is low, it is green and pollution-free, and the extraction efficiency is high. It realizes the high-value development and utilization of the by-product hawthorn kernel, which can improve the utilization value of hawthorn kernel and promote the upgrading of the hawthorn industry chain. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A technical roadmap for separating hawthorn kernel oil, active substances and hawthorn kernel residue from hawthorn kernels;

[0027] Figure 2 This is the GC spectrum of hawthorn kernel oil, in which the three main peaks at 30.1min, 35.8min, and 38.2min are palmitic acid, oleic acid, and linoleic acid, respectively;

[0028] Figure 3 This is the Venn diagram of active ingredients in hawthorn kernels and antioxidant targets;

[0029] Figure 4 This is the network diagram of active components and antioxidant targets of hawthorn kernel;

[0030] Figure 5 This is the protein-protein interaction (PPI) network diagram of the antioxidant activity targets of hawthorn kernel;

[0031] Figure 6These are the docking diagrams of active compounds and AKT1 molecules; among them, Figure a is the docking diagram of 8-methoxykaempferol and the target AKT, Figure b is the docking diagram of serpentine and the target AKT1, Figure c is the docking diagram of dehydrodisinconiferyl alcohol and the target AKT1, and Figure d is the docking diagram of isorhamnetin and the target AKT1. DETAILED DESCRIPTION

[0032] In the present invention, the raw material hawthorn kernel used comes from the hawthorn kernel discarded by Shandong Jianyuan Food Co., Ltd. in the production of hawthorn products. The hawthorn food undergoes several hours of boiling during the production process. The volatile oil content is determined (GB / T 30385-2013), and no volatile oil component is detected in the hawthorn kernel.

[0033] Other materials used in the present invention, if not otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0034] Example 1

[0035] The steps of separating hawthorn kernel oil, active substances and hawthorn kernel residue from hawthorn kernel are as follows:

[0036] (1) Stir-frying: stir-fry 1 kg of hawthorn kernels at 150 °C for 4 h;

[0037] (2) crushing: crush the roasted hawthorn kernels using a crusher, and then pass through a 40-mesh sieve;

[0038] (3) Dissolution promotion: add 2 kg of distilled water, 50 g of lactic acid and 10 g of glycine at a material-liquid ratio of 1:2, and soak for 12 h;

[0039] (4) Enzymatic hydrolysis: adjust the pH to 5, preheat to 50°C; add 100 g of mixed enzyme (ligninase: cellulase: hemicellulase: pectinase = 5:4:3:3), and perform enzymatic hydrolysis for 10 h;

[0040] (5) Inactivation: After the enzymatic hydrolysis is completed, heat to 100°C and maintain for 30 min, then cool to room temperature;

[0041] (6) Removing the residue: vacuum filtration to separate the precipitate from the enzymatic hydrolyzate; the obtained precipitate is the hawthorn kernel residue;

[0042] (7) Oil extraction: add 200 mL of n-hexane and mix thoroughly, extract and separate the liquid, recover the n-hexane, and obtain the oil phase;

[0043] (8) Secondary oil extraction: The lower aqueous phase was cooled at -20°C for 12 h, and after returning to room temperature, it was fully mixed with 200 mL of n-hexane, extracted and separated, and n-hexane was recovered to obtain an oil phase; the two oil phases were combined, and n-hexane was removed to obtain hawthorn kernel oil; the extraction rate of hawthorn kernel oil was 0.83%;

[0044] (9) Active extract: The aqueous phase was concentrated to dryness under reduced pressure to remove water, thereby obtaining the active substance from the hawthorn kernel. The extraction rate of the active substance from the hawthorn kernel was 4.6%.

[0045] Example 2

[0046] The steps of separating hawthorn kernel oil, active substances and hawthorn kernel residue from hawthorn kernel are as follows:

[0047] (1) Stir-frying: stir-fry 1 kg of hawthorn kernels at 180 °C for 4 h;

[0048] (2) crushing: crush the roasted hawthorn kernels using a crusher, and then pass through a 40-mesh sieve;

[0049] (3) Dissolution promotion: add 2 kg of distilled water, 50 g of lactic acid and 10 g of glycine at a material-liquid ratio of 1:2, and soak for 12 h;

[0050] (4) Enzymatic hydrolysis: adjust the pH to 5, preheat to 50°C; add 100 g of mixed enzyme (ligninase: cellulase: hemicellulase: pectinase = 1:3:3:2), and perform enzymatic hydrolysis for 10 h;

[0051] (5) Inactivation: After the enzymatic hydrolysis is completed, heat to 100°C and maintain for 30 min, then cool to room temperature;

[0052] (6) Removing the residue: vacuum filtration to separate the precipitate from the enzymatic hydrolyzate; the obtained precipitate is the hawthorn kernel residue;

[0053] (7) Oil extraction: add 200 mL of n-hexane and mix thoroughly, extract and separate the liquid, recover the n-hexane, and obtain the oil phase;

[0054] (8) Secondary oil extraction: The lower aqueous phase was cooled at -20°C for 12 h, and after returning to room temperature, it was fully mixed with 200 mL of n-hexane, extracted and separated, and n-hexane was recovered to obtain an oil phase; the two oil phases were combined, and n-hexane was removed to obtain hawthorn kernel oil; the hawthorn kernel oil extraction rate was 0.79%;

[0055] (9) Active extract: The aqueous phase was concentrated to dryness under reduced pressure to remove water, thereby obtaining the active substance from the hawthorn kernel; the extraction rate of the active substance from the hawthorn kernel was 3.5%.

[0056] Example 3

[0057] The steps of separating hawthorn kernel oil, active substances and hawthorn kernel residue from hawthorn kernel are as follows:

[0058] (1) Stir-frying: stir-fry 1 kg of hawthorn kernels at 180 °C for 4 h;

[0059] (2) crushing: crush the roasted hawthorn kernels using a crusher, and then pass through a 40-mesh sieve;

[0060] (3) Dissolution promotion: add 2 kg of distilled water, 50 g of lactic acid and 10 g of glycine at a material-liquid ratio of 1:2, and soak for 12 h;

[0061] (4) Enzymatic hydrolysis: adjust the pH to 5, preheat to 50°C; add 100 g of mixed enzyme (ligninase: cellulase: hemicellulase: pectinase = 5:4:3:3), and perform enzymatic hydrolysis for 10 h;

[0062] (5) Inactivation: After the enzymatic hydrolysis is completed, heat to 100°C and maintain for 30 min, then cool to room temperature;

[0063] (6) Removing the residue: vacuum filtration to separate the precipitate from the enzymatic hydrolyzate; the obtained precipitate is the hawthorn kernel residue;

[0064] (7) Oil extraction: add 200 mL of n-hexane and mix thoroughly, extract and separate the liquid, recover the n-hexane, and obtain the oil phase;

[0065] (8) Secondary oil extraction: The lower aqueous phase was cooled at -20°C for 12 h, and after returning to room temperature, it was fully mixed with 200 mL of n-hexane, extracted and separated, and n-hexane was recovered to obtain an oil phase; the two oil phases were combined, and n-hexane was removed to obtain hawthorn kernel oil; the hawthorn kernel oil extraction rate was 1.05%;

[0066] (9) Active extract: The aqueous phase was concentrated to dryness under reduced pressure to remove water, thereby obtaining the active substance from the hawthorn kernel. The extraction rate of the active substance from the hawthorn kernel was 5.2%.

[0067] Comparative Example 1

[0068] The steps of separating hawthorn kernel oil, active substances and hawthorn kernel residue from hawthorn kernel are as follows:

[0069] (1) Crushing: 1 kg of hawthorn kernels were crushed using a crusher and then passed through a 40-mesh sieve;

[0070] (2) Dissolution promotion: add 2 kg of distilled water, 50 g of lactic acid and 10 g of glycine at a material-liquid ratio of 1:2, and soak for 12 h;

[0071] (3) Enzymatic hydrolysis: adjust the pH to 5, preheat to 50°C; add 100 g of mixed enzyme (ligninase: cellulase: hemicellulase: pectinase = 5:4:3:3), and perform enzymatic hydrolysis for 10 h;

[0072] (4) Inactivation: After the enzymatic hydrolysis is completed, heat to 100°C and maintain for 30 min, then cool to room temperature;

[0073] (5) Removing residue: vacuum filtration to separate the precipitate from the enzymatic hydrolyzate; the obtained precipitate is the hawthorn kernel residue;

[0074] (6) Oil extraction: add 200 mL of n-hexane and mix thoroughly, extract and separate the liquid, recover the n-hexane, and obtain the oil phase;

[0075] (7) Secondary oil extraction: The lower aqueous phase was cooled at -20°C for 12 h, and after returning to room temperature, it was fully mixed with 200 mL of n-hexane, extracted and separated, and n-hexane was recovered to obtain an oil phase; the two oil phases were combined, and n-hexane was removed to obtain hawthorn kernel oil; the hawthorn kernel oil extraction rate was 0.12%;

[0076] (8) Active extract: The aqueous phase was concentrated to dryness under reduced pressure to remove water, thereby obtaining the active substance from the hawthorn kernel; the extraction rate of the active substance from the hawthorn kernel was 0.8%.

[0077] Comparative Example 2

[0078] The steps of separating hawthorn kernel oil, active substances and hawthorn kernel residue from hawthorn kernel are as follows:

[0079] (1) Stir-frying: stir-fry 1 kg of hawthorn kernels at 180 °C for 4 h;

[0080] (2) crushing: crush the roasted hawthorn kernels using a crusher, and then pass through a 40-mesh sieve;

[0081] (3) Enzymatic hydrolysis: adjust the pH to 5, preheat to 50°C; add 100 g of mixed enzyme (ligninase: cellulase: hemicellulase: pectinase = 5:4:3:3), and perform enzymatic hydrolysis for 10 h;

[0082] (4) Inactivation: After the enzymatic hydrolysis is completed, heat to 100°C and maintain for 20 min, then cool to room temperature;

[0083] (5) Removing residue: vacuum filtration to separate the precipitate from the enzymatic hydrolyzate; the obtained precipitate is the hawthorn kernel residue;

[0084] (6) Oil extraction: add 200 mL of n-hexane and mix thoroughly, extract and separate the liquid, recover the n-hexane, and obtain the oil phase;

[0085] (7) Secondary oil extraction: The lower aqueous phase was cooled at -20°C for 12 h, and after returning to room temperature, it was fully mixed with 200 mL of n-hexane, extracted and separated, and n-hexane was recovered to obtain an oil phase; the two oil phases were combined, and n-hexane was removed to obtain hawthorn kernel oil; the hawthorn kernel oil extraction rate was 0.69%;

[0086] (8) Active extract: The aqueous phase was concentrated to dryness under reduced pressure to remove water, thereby obtaining the active substance from the hawthorn kernel. The extraction rate of the active substance from the hawthorn kernel was 2.2%.

[0087] Comparative Example 3

[0088] The steps of separating hawthorn kernel oil, active substances and hawthorn kernel residue from hawthorn kernel are as follows:

[0089] (1) Stir-frying: stir-fry 1 kg of hawthorn kernels at 180 °C for 4 h;

[0090] (2) crushing: crush the roasted hawthorn kernels using a crusher, and then pass through a 40-mesh sieve;

[0091] (3) Dissolution promotion: add 2 kg of distilled water, 50 g of lactic acid and 10 g of glycine at a material-liquid ratio of 1:2, and soak for 12 h;

[0092] (4) Removing the residue: vacuum filtration to separate the precipitate from the solution; the obtained precipitate is the hawthorn kernel residue;

[0093] (5) Oil extraction: add 200 mL of n-hexane and mix thoroughly, extract and separate the liquid, recover the n-hexane, and obtain the oil phase;

[0094] (6) Secondary oil extraction: The lower aqueous phase was cooled at -20°C for 12 h, and after returning to room temperature, it was fully mixed with 200 mL of n-hexane, extracted and separated, and n-hexane was recovered to obtain an oil phase; the two oil phases were combined, and n-hexane was removed to obtain hawthorn kernel oil; the extraction rate of hawthorn kernel oil was 0.61%;

[0095] (7) Active extract: The aqueous phase was concentrated to dryness under reduced pressure to remove water, thereby obtaining the active substance from the hawthorn kernel; the extraction rate of the active substance from the hawthorn kernel was 1.8%.

[0096] Comparative Example 4

[0097] The steps of separating hawthorn kernel oil, active substances and hawthorn kernel residue from hawthorn kernel are as follows:

[0098] (1) Stir-frying: stir-fry 1 kg of hawthorn kernels at 180 °C for 4 h;

[0099] (2) crushing: crushing the roasted hawthorn kernels using a crusher;

[0100] (3) Subcritical butane extraction of hawthorn kernel oil: CBE-29L subcritical extraction research experimental device was used to extract hawthorn kernel oil; the extraction solvent was butane; the extraction temperature was 45°C; the extraction was repeated 4 times; the extraction time was 40 min each time; the extraction pressure was 0.52 MPa; the bulk density was 745.5 g / L; the hawthorn kernel oil extraction rate was 1.02%;

[0101] (4) Dissolution promotion: add 2 kg of distilled water, 50 g of lactic acid and 10 g of glycine at a material-liquid ratio of 1:2, and soak for 12 h;

[0102] (5) Enzymatic hydrolysis: adjust the pH to 5, preheat to 50°C; add 100 g of mixed enzyme (ligninase: cellulase: hemicellulase: pectinase = 5:4:3:3), and perform enzymatic hydrolysis for 10 h;

[0103] (6) Inactivation: After the enzymatic hydrolysis is completed, heat to 100°C and maintain for 30 min, then cool to room temperature;

[0104] (7) Removing the residue: vacuum filtration to separate the precipitate from the enzymatic hydrolyzate; the obtained precipitate is the hawthorn kernel residue;

[0105] (8) Active extract: The aqueous phase was concentrated to dryness under reduced pressure to remove water, thereby obtaining the active substance from the hawthorn kernel; the extraction rate of the active substance from the hawthorn kernel was 5.0%.

[0106] As can be seen from the above-mentioned implementation cases, frying has a greater impact on the extraction rate of hawthorn kernel oil. Without frying, the extraction rate of hawthorn kernel oil is greatly reduced. In addition, the frying temperature also affects the extraction rate of hawthorn kernel oil to a certain extent. The ratio of catalytic dissolution and mixed enzyme affects the extraction rate of active substances. Subcritical extraction method (prior art) can be used to obtain an extraction effect similar to the present invention, but the subcritical extraction method has high equipment cost, which is not conducive to promotion and application.

[0107] In the present invention, the hawthorn kernel residue is softer and more breathable than the unprocessed hawthorn kernel, contains rich lignin, and can be used for animal feed, edible fungus cultivation, and biochar production. The active extract of hawthorn kernel has antioxidant, antibacterial (Escherichia coli, Staphylococcus aureus, Candida albicans, etc.) and antiseptic effects, and can be used in skin care products, natural preservatives, biomedicine and other fields. The technical roadmap of the present invention is as follows Figure 1 shown.

[0108] 1. Index detection

[0109] 1. Hawthorn kernel oil composition detection

[0110] The components of the hawthorn kernel oil of Example 3 were detected by GC.

[0111] Agilent GC 7890B, FID detector; injection port 260℃; injection volume 1μL; flow rate 1mL / min; heating program: initial temperature 120℃, run 3min; 3-34min, heating rate 3℃ / min, heating to 210℃, keep 1min; 34-53min, heating rate 0.5℃ / min, heating to 219℃, keep 1min; 53-68.6min, heating rate 10℃, heating to 240℃, keep 13.5min. 37 kinds of fatty acid methyl ester mixed standards were used for comparison, and the main components were determined to be linoleic acid (64.43%), oleic acid (24.39%) and palmitic acid (4.18%), such as Figure 2 The test results of different embodiments are basically consistent.

[0112] 2. Study on the antioxidant active ingredients of active extracts

[0113] The antioxidant active ingredients were analyzed by network pharmacology and molecular docking methods. The main chemical components of hawthorn kernel were sorted out by searching the literature related to the chemical components of hawthorn kernel. The SwissADME tool was used to screen the active ingredients in the compounds according to the GI absorption principle. At the same time, the target proteins related to antioxidant in the human body were obtained from the GeneCards database. The two sets of target data were integrated and analyzed to obtain the intersection targets of antioxidant targets and target proteins of active components of hawthorn kernel, and a preliminary network diagram of the interaction between active components of hawthorn kernel and antioxidant targets was constructed using STRING. The network diagram was further visualized using Cytoscape software to construct a network diagram of hawthorn kernel active component target protein-antioxidant target protein interaction (PPI). According to the relevant information of the intersection targets, GO functional enrichment analysis and KEGG signaling pathway enrichment analysis were performed respectively, and the visualization diagram of enrichment analysis was made in combination with the microbial information platform. Combined with the enriched pathways, a network diagram of hawthorn kernel active components-antioxidant targets-in vivo pathways was made, and the active components with high scores were screened out as antioxidant functional active components. Molecular docking was performed on the antioxidant active ingredients and antioxidant targets, and the main antioxidant active ingredients in hawthorn kernel were analyzed based on the docking results.

[0114] (1) Prediction and screening of active ingredients from hawthorn seeds

[0115] By searching relevant literature, a total of 131 chemical components in hawthorn kernel were sorted out. The chemical formulas of 131 compounds were searched in the SwissADME database. Following the screening principle of GI absorption screening with at least 3 YES in High, Lipinski, Ghose, Veber, Ean, and Megge, 12 active ingredients were screened out, namely (7R, 8S)-dihydrodehydrodiferroic alcohol, 8-methoxykaempferol, serpentine, freno A, larch resin alcohol, shannan A, epicortisol, pinoresinol, dehydrobispinol, cinnaphosphaldehyde, isorhamnetin, and shannan A.

[0116] (2) Prediction of targets of hawthorn kernel-antioxidant active ingredients

[0117] After preliminary screening, 316 target proteins corresponding to the 12 active ingredients of hawthorn kernel were analyzed. There are 1224 target proteins related to human antioxidants. The intersection of these two groups of target proteins gave 106 hawthorn kernel-antioxidant target proteins. The target information was imported into Venny2.1 drawing software to make a target information Venn diagram, as shown in Figure 3 As shown, the intersection in the middle is the 106 screened hawthorn kernel-antioxidant targets.

[0118] (3) Hawthorn kernel active ingredient-target network diagram and antioxidant target prediction

[0119] The active component files and intersection target gene files of hawthorn kernel were analyzed using Cytoscape 3.10.0 software, and the hawthorn kernel active component-antioxidant target network diagram was constructed, as shown in Figure 4 As shown in the figure. The V-shaped nodes in the figure represent the hawthorn kernel; the diamond-shaped nodes represent the active ingredients of the hawthorn kernel; and the circular nodes represent the targeted proteins. The figure shows that the hawthorn kernel may exert its antioxidant effect by acting on key antioxidant-related targets such as AKT1, SRC, STAT3, ESR1, MAPK1, JUN, HIF1A, BCL2, etc.

[0120] (4) Protein-protein interaction (PPI) of hawthorn kernel antioxidant activity target

[0121] The STRING database was used to generate a preliminary protein-protein interaction (PPI) network diagram of the target protein information of the hawthorn core-antioxidant intersection. The diagram was processed with Cytoscape 3.10.0 software to obtain a more intuitive protein-protein interaction (PPI) network diagram of the target protein of the hawthorn core antioxidant activity, as shown in the figure. Figure 5As shown in the figure, the top 10 core genes with the highest degree values ​​are target proteins such as AKT1, SRC, STAT3, ESR1, and MAPK1. This shows that these 10 target proteins are closely related to other proteins and are the core targets corresponding to the antioxidant active components in the hawthorn kernel.

[0122] (5) Molecular docking and visualization of molecular docking results

[0123] Depend on Figure 5 The analysis results show that the top five target proteins with the highest degree values ​​are AKT1, SRC, STAT3, ESR1 and MAPK1, and the top four active ingredients in hawthorn kernel with the highest degree values ​​are SZH2, SZH3, SZH9 and SZH11, which are the main antioxidant active ingredients in hawthorn kernel.

[0124] The AutoDock Tools software was used to perform molecular docking on each component and the five target proteins, and the lowest binding free energy of each docking was sorted out, as shown in Table 1. The four groups of molecular docking diagrams with the largest absolute values ​​of binding free energy were also drawn, as shown in Table 1. Figure 6 shown.

[0125] Table 1 Binding free energy of the antioxidant active components of hawthorn kernel and the core targets

[0126]

[0127] The greater the absolute value of the binding free energy, the more firmly the active ingredients of hawthorn kernel bind to the antioxidant targets in the human body. If the absolute value of the binding free energy is between 4.25 and 7.0, it means that the ligand compound has binding activity with the target protein; if the absolute value of the binding free energy is >7.0, it means that the ligand compound has strong binding activity with the target protein.

[0128] Depend on Figure 6 As shown in Table 1, SZH11 (isorhamnetin) has the most binding sites with the target protein AKT1. Among the 8 data sets with the largest absolute value of binding free energy, the target points docked with isorhamnetin, the active ingredient of hawthorn kernel, are the most, and the molecular docking binding energy is the largest. Therefore, it is determined that the compound with the best antioxidant activity in hawthorn kernel is isorhamnetin. In addition, the antioxidant activity of 8-methoxykaempferol is second only to isorhamnetin, and both compounds belong to flavonoid compounds, so it is speculated that the main antioxidant component of hawthorn kernel is polyphenol methoxyflavonoid compounds.

[0129] The chemical structure of isorhamnetin is as follows:

[0130]

[0131] The chemical structure of 8-methoxykaempferol is as follows:

[0132]

[0133] Isorhamnetin, also known as 3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)benzopyran-4-one; 8-methoxykaempferol, also known as 3,5,7,4'-tetrahydroxy-8-methoxyflavone; the two compounds contain multiple hydroxyl groups in their molecular structure, which may increase its solubility in water compared to other flavonoids because hydroxyl groups can form hydrogen bonds and increase the interaction with water molecules. In addition, due to the presence of methoxy groups in its molecular structure, its solubility will increase under slightly acidic conditions. Due to the acidity of hawthorn kernel itself (pH = 3-6), water extraction can effectively extract this type of flavonoid compound.

[0134] 3. Antioxidant activity test of active extracts

[0135] The antioxidant activity of the active extracts was determined by the DPPH method.

[0136] Weigh a certain amount of DPPH and prepare a 0.02 mg / mL DPPH solution with anhydrous ethanol. Take 100 μL of solutions of different concentrations (2, 4, 6, 8 mg / mL) respectively, add 100 μL DPPH solution, mix well, place at room temperature for 30 minutes, and measure the absorbance at 517 nm with an ELISA reader. Use Vc as a positive control. The scavenging rate of the sample for DPPH free radicals is calculated using the following formula:

[0137] DPPH clearance rate = [1-(A1-A2) / A0] × 100%

[0138] Wherein, A0 represents the absorbance value of 100 μL anhydrous ethanol + 100 μL DPPH solution; A1 represents the absorbance value of 100 μL sample solution + 100 μL DPPH solution; A2 represents the absorbance value of 100 μL sample solution + 100 μL anhydrous ethanol.

[0139] The measurement results are shown in Table 2.

[0140] Table 2 Scavenging rate of active extracts on DPPH

[0141] concentration(%) Example 1 Example 2 Example 3 Comparative Example 4 Vc 0.20％ 86.19％ 87.06％ 85.51％ 85.23％ 93.77％ 0.10％ 78.62％ 86.13％ 80.78％ 78.26％ 93.79％ 0.05％ 74.55％ 77.67％ 80.37％ 76.54％ 91.14％ 0.03％ 55.50％ 64.22％ 79.71％ 69.87％ 91.25％ 0.01％ 36.19％ 23.94％ 78.47％ 59.23％ 90.71％

[0142] It can be seen from Table 2 that all active extracts have antioxidant activity, but under low concentration conditions, only Example 3 maintains a relatively high antioxidant activity.

[0143] 4. Antibacterial activity of active extracts

[0144] The growth curve method was used to determine the inhibitory effects of the aqueous active site on Escherichia coli, Staphylococcus aureus, and Candida albicans. Take 0.5 mL of the sample to be tested, add 0.5 mL of LB medium, add 40 μL of bacterial suspension to each, set up another group of 0.5 mL of sterile water and 0.5 mL of LB medium as a blank control, vortex mix, take 200 μL and add it to a 96-well plate, repeat 2 times, culture in a bacterial incubator for 24 hours, and measure the absorbance value at 600 nm.

[0145] The test results are shown in Table 3.

[0146] Table 3 Inhibition rate of active extracts on bacteria (%)

[0147]

[0148]

[0149] As shown in Table 3, the active extract of hawthorn kernel has an inhibitory effect on Escherichia coli, Staphylococcus aureus and Candida albicans, so it can be used in the fields of natural preservatives, feed additives, biomedicine, etc.

[0150] 5. Preservative effect of active extracts on fresh duck meat

[0151] The active extract of Example 3 was selected and prepared into an aqueous solution with a mass concentration of 0.3%, and a preservative sodium diacetate aqueous solution with a mass concentration of 0.3% was selected at the same time, and 30 g of fresh duck meat was added to each solution. The solution was refrigerated at 4° C. Every 2 days, the duck meat was taken out and the number of bacteria was detected by the plate coating method, as shown in Table 4; and the duck meat was subjected to a sensory evaluation, as shown in Table 5.

[0152] Table 4 Antiseptic effect of active extracts from hawthorn kernels, colony count

[0153] Colony count Blank control (water) Active extract preservative Day 1 4.25±1.71 4.88±2.85 4.86±2.54 Day 3 5.75±2.63 4.83±2.17 4.75±2.66 Day 5 109.25±27.04 <![CDATA[69.80±20.22 ** ]]> <![CDATA[77.78±35.62 ** ]]> Day 7 157.19±34.16 <![CDATA[89.46±26.33 ** ]]> <![CDATA[97.62±40.15 ** ]]>

[0154] Note: **p<0.01, relative to blank control.

[0155] Table 5 Sensory evaluation of the preservative effect of active extracts from hawthorn kernels

[0156] Sensory evaluation Blank control (water) Active extract preservative Day 1 normal normal normal Day 3 Fishy smell, no corruption smell normal normal Day 5 There is a corrupt smell normal normal Day 7 There is a corrupt smell normal Slightly corrupt smell Day 9 Serious corruption odor Slightly corrupt smell There is a corrupt smell

[0157] The above results show that hawthorn kernel extract has the effect of inhibiting bacterial proliferation and slowing down meat spoilage.

[0158] In the present invention, the high temperature during the frying process will change the chemical composition, and the wood will become fluffy, which is conducive to dissolution and extraction, and can also promote oil separation. Dissolution can achieve the decomposition of wood. Enzymatic hydrolysis is conducive to the separation of active ingredients.

[0159] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for separating hawthorn kernel oil, active substances and hawthorn kernel residues by a one-pot method, characterized in that: The steps include: (1) Stir-frying: stir-fry the hawthorn kernels at 100-300°C for 1-10 hours; (2) Crushing: crush the roasted hawthorn kernels and pass through a 20-100 mesh sieve; (3) Dissolution promotion: add water at a material-liquid ratio of 1:1-1:10, add a dissolving agent, and soak for 1-12 hours; (4) Enzymatic hydrolysis: adjust the pH to 4-8, then raise the temperature to 30-80°C; add mixed enzymes and perform enzymatic hydrolysis for 3-10 h; (5) Inactivation: After the enzymatic hydrolysis is completed, raise the temperature to 90-100°C and maintain for 10-30 min, then cool to room temperature; (6) Residue removal: Filter to separate the precipitate from the enzymatic hydrolysate. The resulting precipitate is the hawthorn kernel residue. (7) Oil extraction: add n-hexane to the enzymatic hydrolyzate, mix thoroughly, extract and separate the liquid, recover the n-hexane, and obtain the oil phase; (8) Secondary oil extraction: cool the lower aqueous phase at -40 to 4°C for 10 to 15 hours, and after returning to room temperature, add n-hexane, mix thoroughly, extract and separate, recover n-hexane, and obtain an oil phase; combine the two oil phases to obtain hawthorn kernel oil; (9) Active extract: Concentrate the aqueous phase and dry it to obtain the active substance of hawthorn kernel; In the step (3), the dissolution agent is lactic acid and glycine, and the mass ratio of lactic acid to glycine is 5:1; In the step (4), the mixed enzyme includes ligninase, cellulase, hemicellulase and pectinase, and the mass ratio thereof is 1~7: 1~5: 1~4: 1~4.

2. The method according to claim 1, characterized in that In the step (3), the amount of the dissolving agent is selected from: taking 1 kg of hawthorn kernel as a reference, adding 10-200 g of the dissolving agent.

3. The method according to claim 2, characterized in that The dosage of the dissolving agent is as follows: based on 1 kg of hawthorn kernels, 60 g of the dissolving agent is added.

4. The method according to claim 1, characterized in that In the step (4), the amount of the mixed enzyme is selected from: taking 1 kg of hawthorn kernels as a reference, adding 50-200 g of the mixed enzyme.

5. The method according to claim 4, characterized in that The dosage of the mixed enzyme is selected from: taking 1 kg of hawthorn kernel as a reference, adding 100 g of the mixed enzyme.

6. The method according to claim 1, characterized in that In the step (7), the volume ratio of n-hexane to the enzymatic hydrolysis solution is selected from 0.05 to 1:

1.

7. Hawthorn kernel oil, hawthorn kernel residue and / or hawthorn kernel active substance prepared by the method according to any one of claims 1 to 6.

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