Refined mulberry seed oil and extraction method and application thereof
Refined mulberry seed oil with high unsaturated fatty acid content was prepared by low-moisture enzymatic hydrolysis pretreatment and high-temperature pressing process, which solved the problems of low extraction efficiency and insufficient retention of active ingredients in the existing technology. It can be applied to regulate lipid metabolism in hyperlipidemia cells and nematodes.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU UNIV OF SCI & TECH
- Filing Date
- 2026-03-30
- Publication Date
- 2026-06-09
Smart Images
Figure CN122168368A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of food science and biomedicine, and relates to a method for the efficient utilization of mulberry seeds, specifically refined mulberry seed oil and its extraction method and application. Background Technology
[0002] Mulberry seeds are a byproduct of processing mulberries into products such as mulberry wine, mulberry juice, and mulberry vinegar. In recent years, mulberries have become increasingly popular due to their medicinal and edible properties, leading to a proliferation of processed products. However, mulberry seeds are often discarded or used only as simple animal feed, resulting in low resource utilization. Nevertheless, mulberry seeds contain a high oil content (27.5%–33.0%), primarily composed of unsaturated fatty acids, with linoleic acid being the most abundant. Studies have shown that unsaturated fatty acids (especially linoleic acid) play a crucial role in alleviating lipid metabolism disorders; therefore, the efficient extraction of linoleic acid-rich mulberry seed oil from mulberry seeds has significant application value.
[0003] Pressing is the most commonly used method for extracting vegetable oils (see patent CN115651764B), which has the advantages of simple process and no solvent residue, but the oil yield is low. To overcome this problem, existing technologies have developed processes that combine pressing with other methods, such as the "aqueous solution + pressing" combined process disclosed in patent CN117625302A, which first extracts the oil by centrifugation with salt solution to obtain clear oil, and then presses the residue to combine the oil. However, this method is complex and fails to solve the problem of low retention rate of active ingredients. Peng Zhaokang et al. (Research on the ultrasonic-assisted extraction process of mulberry seed oil of Jisang No. 1 and Jisang No. 3 by response surface methodology. Forestry and Ecological Science, 2018, 33(4): 415-422.) used ultrasonic-assisted extraction process, which has a high extraction efficiency. However, this method has the disadvantage of complex solvent recovery process and high equipment cost. The method disclosed by Zhang Zhiwei et al. (Oil extraction and comprehensive development and utilization of mulberry seeds. Grain and Oil Processing, 2005(12): 50-52.) is simple to operate with room temperature solvent extraction, but the extraction time is long (usually 48~72 h) and the solvent recovery process is complicated; the supercritical fluid extraction method (see patent CN101869294A) has no solvent residue and high quality, but the equipment investment is large and the operating cost is high, making it difficult to realize industrial application; Huang Liumin et al. (The effect of heat loss on the quality and composition of soybean oil. China Oils and Fats, 2023, 48(11): 100-106.) used Soxhlet extraction to prepare soybean oil, which proved that heat damage will lead to a significant decline in oil quality and a decrease in the content of polyunsaturated fatty acids.
[0004] The nutritional value of mulberry seed oil mainly comes from its rich unsaturated fatty acids. Therefore, how to efficiently retain these heat-sensitive components during extraction is key to evaluating the quality of the extraction method. Although existing techniques each have their advantages, they still have shortcomings in the retention and enrichment of unsaturated fatty acids (especially linoleic acid). Summary of the Invention
[0005] Technical problem to be solved: In order to overcome the shortcomings of the prior art, the present invention uses a low-moisture enzymatic hydrolysis pretreatment combined with a high-temperature pressing process to maximize the retention of unsaturated fatty acid components such as linoleic acid, thereby obtaining refined mulberry seed oil; in view of this, the present invention provides refined mulberry seed oil, its extraction method and application.
[0006] Technical solution: A method for extracting refined mulberry seed oil, the method comprising the following steps:
[0007] S1. Pretreatment: Take dried mulberry seeds, sieve them to remove impurities, and obtain clean mulberry seeds;
[0008] S2. Enzymatic conditioning: Adjust the moisture content of the cleaned mulberry seeds obtained in S1 to 15%~25%, then spray the compound enzyme solution evenly to ensure that the enzyme is fully mixed with the mulberry seeds. The total amount of enzyme used is 0.1%~1.0% of the mulberry seed mass.
[0009] S3. Temperature-controlled enzymatic hydrolysis: Mulberry seeds sprayed with enzyme solution are enzymatically hydrolyzed at 40-55℃ for 5-6 hours to obtain enzymatically hydrolyzed mulberry seeds;
[0010] S4. Enzyme inactivation and drying: The enzymatically hydrolyzed mulberry seeds obtained in S3 are heated to 110-115℃ and maintained for 20-25 minutes to inactivate the enzyme activity, thus obtaining the treated mulberry seeds;
[0011] S5. High-temperature pressing: After S4 treatment, the mulberry seeds are put into a press and pressed at a temperature of 120℃~125℃ to obtain crude mulberry seed oil.
[0012] S6. Refining: Add the crude mulberry seed oil obtained in S5 to anhydrous ethanol, stir and mix evenly, filter to remove precipitated impurities, and remove the ethanol from the filtrate by rotary evaporation to obtain refined mulberry seed oil.
[0013] Preferably, the complex enzyme solution in S2 includes pectinase, xylanase and cellulase, with a mass ratio of 1:1:2.
[0014] Preferably, the enzyme-inactivating drying in S4 is carried out by hot air drying, and the moisture content of the mulberry seeds after drying is less than 5%.
[0015] The refined mulberry seed oil obtained by any of the methods described above.
[0016] Preferably, the refined mulberry seed oil contains more than 88% unsaturated fatty acids, of which more than 80% is linoleic acid.
[0017] The above-described application of refined mulberry seed oil in regulating lipid metabolism in high-lipid LO2 cells.
[0018] The above-described application of refined mulberry seed oil in regulating lipid metabolism in *C. elegans*.
[0019] The above-described application of refined mulberry seed oil in the preparation of functional foods or health products that regulate lipid metabolism.
[0020] The above-described application of refined mulberry seed oil in the preparation of drugs or pharmaceutical excipients that regulate lipid metabolism.
[0021] Beneficial effects: (1) The method described in this invention uses agricultural waste (mulberry seeds) as raw material, and obtains mulberry seed oil through low-moisture enzymatic hydrolysis pretreatment combined with high-temperature pressing. The enzyme dosage is low (0.1%~1.0%), ethanol is recycled, solvent loss is small, the process is simple, the operation is convenient, and it is easy to realize industrial-scale production; (2) The mulberry seed oil prepared by this invention has an unsaturated fatty acid content of up to 88.26%, of which the linoleic acid content is 81.6%; (3) The mulberry seed oil of this invention shows significant lipid-regulating activity in both cell and nematode models: it can improve the lipid content index and protein expression of hyperlipidemic cells, and regulate nematode-related genes and reduce lipid accumulation; (4) This invention uses mulberry seeds, a waste of silkworms, as raw material, and realizes the high-value utilization of agricultural by-products, which has good industrial application prospects and economic and social benefits. Attached Figure Description
[0022] Figure 1 shows the effect of mulberry seed oil of the present invention on lipid content in high-lipid LO2 cells, where A is total cholesterol (TC); B is triglycerides (TG); C is low-density lipoprotein cholesterol (LDL-C); and D is high-density lipoprotein cholesterol (HDL-C).
[0023] Figure 2 shows the effect of mulberry seed oil of the present invention on the expression level of intracellular lipid metabolism-related proteins. In the figure, HM, MM and LM represent the high-dose (0.78 μL / mL), medium-dose (0.39 μL / mL) and low-dose (0.19 μL / mL) treatment groups of mulberry seed oil, respectively.
[0024] Figure 3 The figure shows the effect of mulberry seed oil on the total cholesterol (TC) and triglyceride (TG) content of Caenorhabditis elegans according to the present invention.
[0025] Figure 4 The figure shows the effect of mulberry seed oil of the present invention on the gene expression of daf-16 (A) and daf-2 (B) of Caenorhabditis elegans. Detailed Implementation
[0026] Example 1: Preparation and fatty acid composition analysis of mulberry seed oil
[0027] A method for extracting refined mulberry seed oil, the method comprising the following steps:
[0028] S1. Pretreatment: Take dried mulberry seeds, sieve them to remove impurities, and obtain clean mulberry seeds;
[0029] S2. Enzymatic conditioning: Adjust the moisture content of the cleaned mulberry seeds obtained in S1 to 15%~25%, and then spray the compound enzyme solution evenly to ensure that the enzyme is fully mixed with the mulberry seeds. The total amount of enzyme used is 0.1%~1.0% of the mulberry seed mass. The compound enzyme solution includes pectinase, xylanase and cellulase, with a mass ratio of 1:1:2.
[0030] S3. Temperature-controlled enzymatic hydrolysis: Mulberry seeds sprayed with enzyme solution are enzymatically hydrolyzed at 40-55℃ for 5-6 hours to obtain enzymatically hydrolyzed mulberry seeds;
[0031] S4. Enzyme Inactivation and Drying: The enzymatically hydrolyzed mulberry seeds obtained in S3 are heated to 110-115℃ and maintained for 20-25 minutes to inactivate the enzyme activity, resulting in treated mulberry seeds. Enzyme inactivation and drying are performed using hot air drying, and the moisture content of the dried mulberry seeds is less than 5%.
[0032] S5. High-temperature pressing: After S4 treatment, the mulberry seeds are put into a press and pressed at a temperature of 120℃~125℃ to obtain crude mulberry seed oil.
[0033] S6. Refining: Add the crude mulberry seed oil obtained in S5 to anhydrous ethanol, stir and mix evenly, filter to remove precipitated impurities, and remove the ethanol from the filtrate by rotary evaporation to obtain refined mulberry seed oil.
[0034] In specific experiments, any combination of the above parameters can be selected.
[0035] The fatty acid composition of refined mulberry seed oil was analyzed using gas chromatography-mass spectrometry (GC-MS). The refined mulberry seed oil contained more than 88% unsaturated fatty acids, of which more than 80% were linoleic acid.
[0036] Table 1 shows one set of results from this embodiment, indicating that refined mulberry seed oil contains ten fatty acids, including linoleic acid, palmitic acid, stearic acid, heptadecanoic acid, arachidic acid, behenic acid, cis-9-hexadecanoic acid, oleic acid, cis-11-eicosanoic acid, and α-linolenic acid. The total unsaturated fatty acid content is 88.26%, and the linoleic acid content is 81.6%.
[0037] Table 1. Fatty acid composition analysis of mulberry seed oil
[0038]
[0039] Example 2: Effects of different extraction methods on the fatty acid composition of mulberry seed oil
[0040] Group A: Mulberry seed oil was prepared according to the method of Example 1;
[0041] Group B: Room temperature solvent extraction method. Following the method described by Zhang Zhiwei et al. (Mulberry seed oil extraction and comprehensive development and utilization. Grain and Oil Processing, 2005(12): 50-52.), mulberry seeds were dried and extracted multiple times at room temperature for 24 h using petroleum ether as solvent. The solvent was then recovered by rotary evaporation to obtain mulberry seed oil.
[0042] Group C: Ultrasonic-assisted extraction method. Following the method of Peng Zhaokang et al. (Research on the ultrasonic-assisted extraction process of Jisang No. 1 and Jisang No. 3 mulberry seed oil optimized by response surface methodology. Forestry and Ecological Science, 2018, 33(4): 415-422.), ethyl acetate was used as solvent, and extraction was carried out under the conditions of ultrasonic power of 311 W, extraction temperature of 56℃, material-liquid ratio of 28:1 (mL / g) and extraction time of 13 min. After filtration, the mulberry seed oil was concentrated under reduced pressure to obtain mulberry seed oil.
[0043] Group D: Soxhlet extraction method, referring to the method described by Gong Lili et al. (Gas Chromatography-Mass Spectrometry Analysis of Fatty Acids in Mulberry Seed Oil. Shandong Journal of Traditional Chinese Medicine, 2013, 32(6): 41-42.), using n-hexane as solvent, Soxhlet extraction was performed for 4 h under reflux, and the solvent was recovered to obtain mulberry seed oil.
[0044] Group E: Pressing method. Following the method described by Wu Yuming et al. (Study on the nutritional components of mulberry seeds. Sericultural Science, 2006, 32(1):139-141.), a hydraulic oil press was used to press the mulberry seed oil at 50°C and then filtered to obtain mulberry seed oil.
[0045] Table 2. Effects of different extraction methods on the content of unsaturated fatty acids and linoleic acid in mulberry seed oil.
[0046]
[0047] Table 1 shows that the fatty acid composition of mulberry seed oil prepared by different extraction methods varies significantly. The total unsaturated fatty acid content (88.26%) and linoleic acid content (81.6%) of the mulberry seed oil prepared by the method of this invention (Group A) are superior to the other four methods. Although room temperature solvent extraction (Group B) and ultrasound-assisted extraction (Group C) can extract under mild conditions, the total unsaturated fatty acid content is 85.3% and 83.4%, respectively, both lower than that of the method of this invention. The total unsaturated fatty acid content of Soxhlet extraction (Group D) and conventional pressing (Group E) is 83.5% and 83.0%, respectively, significantly lower than that of the method of this invention, indicating that high temperature or long-term extraction leads to the loss of unsaturated fatty acids. The results show that the method of this invention has a significant advantage in enriching unsaturated fatty acids such as linoleic acid.
[0048] Example 3: Application of mulberry seed oil in lipid metabolism in hyperlipidemic cells
[0049] (1) Measurement of intracellular lipid content
[0050] LO2 cells were harvested at a rate of 2 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and, after adhesion, were treated with different drugs for 24 h. The groups were as follows: blank group (DMEM medium containing 30% BSA, bovine serum albumin), model group (1.56 µL / mL oleic acid, OA), experimental group (mulberry seed oil, MSO), and positive control group (rapeseed oil, RSO). After incubation, cells were washed with phosphate buffered saline (PBS), digested with trypsin to collect cells, and after lysis, the intracellular levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured according to the kit instructions.
[0051] The results are as follows Figure 1 As shown, compared with the blank group, the levels of TC, TG, and LDL-C in the model group cells increased, while the level of HDL-C decreased. After intervention with mulberry seed oil, the levels of TC, TG, and LDL-C decreased significantly (P>0.05), while the level of HDL-C increased significantly (P>0.0001). The effects of rapeseed oil in the control group were comparable to those of medium and low concentrations of mulberry seed oil.
[0052] (2) Western Blot
[0053] After cell drug treatment, cells were collected and lysed in RIPA lysis buffer containing PMSF (phenylmethanesulfonyl fluoride) for 30 min on ice. The cells were then centrifuged at 12,000 rpm for 12 min at 4°C. The supernatant was collected, and protein concentration was determined using the BCA method. After adjusting the sample to an isostatic concentration, the cells were denatured at 100°C for 10 min. Equal amounts of protein were subjected to SDS-PAGE gel electrophoresis (stacking gel 80V 30 min, separating gel 120V 90 min), transferred to a PVDF membrane (360mA 90 min), blocked with 5% skim milk for 4 h, incubated overnight at 4°C with primary antibody, washed with TBST, incubated with secondary antibody for 1.5 h, washed with TBST, developed using ECL chemiluminescence, and photographed using a gel imaging system. The grayscale values of the bands were analyzed using ImageJ software.
[0054] Western Blot results are as follows: Figure 2 As shown, compared with the blank group, the expression levels of p-RAF(ser 338), MEK, ERK1 / 2 and BCL2 proteins were increased in the high-fat model group; the expression levels of these proteins were reduced after intervention with mulberry seed oil, with the most significant decrease in the 0.39 μL / mL mulberry seed oil treatment group.
[0055] Example 4: Effects of mulberry seed oil on lipid metabolism in high-lipid Caenorhabditis elegans
[0056] (1) Lipid content determination in nematodes
[0057] Synchronized *C. elegans* nematodes were cultured on NGM plates to the L4 stage. Four groups were established: a control group (NGM medium containing 0.1% DMSO), a model group (NGM medium containing 50 mM glucose), a control group (NGM medium containing RSO), and experimental groups (NGM medium containing different concentrations of MSO). After transferring the nematodes to high-glucose NGM medium for 5 days, they were transferred to the control and experimental groups for further culture, with 50 nematodes in each group. On day 10, the nematodes were collected, washed three times with M9 buffer, and the TC and TG levels in the nematodes were determined according to the kit instructions.
[0058] The effects of mulberry seed oil on the TC and TG content in nematodes are shown in Figure 3. The TC and TG content in the model group nematodes increased, while the TC and TG content in the nematodes decreased to varying degrees after intervention with mulberry seed oil (P > 0.05).
[0059] (2) Gene expression analysis of nematodes
[0060] Synchronized fluorescently labeled nematodes were cultured on NGM plates to the L4 stage, then transferred to high-glucose NGM medium for 5 days. Afterward, 50 nematodes were transferred to the control and experimental groups for further culture. On day 10, the nematodes were collected, anesthetized with 10 mM levamisole, placed on 2% agar slides, and sealed with coverslips. Observation and photography were performed under a fluorescence microscope. ImageJ software was used to analyze fluorescence intensity and evaluate lipid accumulation in the nematodes.
[0061] As shown in Figure 4, compared with the model group, mulberry seed oil intervention can lead to downregulation of daf-2 gene expression and upregulation of daf-16 gene expression with significant changes in expression levels.
Claims
1. A method for extracting refined mulberry seed oil, characterized in that, The method includes the following steps: S1. Pretreatment: Take dried mulberry seeds, sieve them to remove impurities, and obtain clean mulberry seeds; S2. Enzymatic conditioning: Adjust the moisture content of the cleaned mulberry seeds obtained in S1 to 15%~25%, then spray the compound enzyme solution evenly to ensure that the enzyme is fully mixed with the mulberry seeds. The total amount of enzyme used is 0.1%~1.0% of the mulberry seed mass. S3. Temperature-controlled enzymatic hydrolysis: Mulberry seeds sprayed with enzyme solution are enzymatically hydrolyzed at 40-55℃ for 5-6 hours to obtain enzymatically hydrolyzed mulberry seeds; S4. Enzyme inactivation and drying: The enzymatically hydrolyzed mulberry seeds obtained in S3 are heated to 110-115℃ and maintained for 20-25 minutes to inactivate the enzyme activity, thus obtaining the treated mulberry seeds; S5. High-temperature pressing: After S4 treatment, the mulberry seeds are put into a press and pressed at a temperature of 120℃~125℃ to obtain crude mulberry seed oil. S6. Refining: Add the crude mulberry seed oil obtained in S5 to anhydrous ethanol, stir and mix evenly, filter to remove precipitated impurities, and remove the ethanol from the filtrate by rotary evaporation to obtain refined mulberry seed oil.
2. The method for extracting refined mulberry seed oil according to claim 1, characterized in that, The complex enzyme solution in S2 includes pectinase, xylanase, and cellulase, with a mass ratio of 1:1:
2.
3. The method for extracting refined mulberry seed oil according to claim 1, characterized in that, The enzyme-inactivating drying method described in S4 uses hot air drying, and the moisture content of the mulberry seeds after drying is less than 5%.
4. Refined mulberry seed oil obtained by the method described in any one of claims 1-3.
5. The refined mulberry seed oil according to claim 4, characterized in that, The refined mulberry seed oil contains more than 88% unsaturated fatty acids, of which more than 80% are linoleic acid.
6. The application of the refined mulberry seed oil according to claim 5 in regulating lipid metabolism in high-lipid LO2 cells.
7. The application of the refined mulberry seed oil according to claim 5 in regulating lipid metabolism in high-lipid Caenorhabditis elegans.
8. The use of the refined mulberry seed oil according to claim 5 in the preparation of functional foods or health products that regulate lipid metabolism.
9. The use of the refined mulberry seed oil according to claim 5 in the preparation of drugs or pharmaceutical excipients for regulating lipid metabolism.