A citrus seed oil rich in limonin, a defatted citrus seed meal, and their preparation methods and applications

Abstract

The present invention discloses a citrus seed oil rich in limonin and a defatted citrus seed meal and their preparation methods and applications. The present invention uses the crude oil obtained from citrus seeds to re-extract limonin from citrus seed residues to obtain a defatted citrus seed meal. At the same time, the refined finished oil is rich in limonin, has nutritional and health care effects, and has a characteristic lemon-like aroma. The present invention directly uses the crude oil as an extraction agent, with a simple process, without adding exogenous functional components, without the aid of organic solvents, being green, safe, efficient, and more suitable for industrial production. The prepared citrus seed oil rich in limonin has application prospects in medicine, health food and / or beauty products. At the same time, the defatted citrus seed meal has the application potential as a feed raw material or additive.

Description

Technical Field

[0001] The present invention belongs to the field of vegetable oil production and processing, and particularly relates to a citrus seed oil rich in limonin, a defatted citrus seed meal, and a preparation method and application thereof. Background Art

[0002] Citrus seeds are rich in 38.86% - 42.59% oil, which is higher than some other seed oils, such as cottonseed oil (18% - 22%), soybean oil (18 - 25%), and olive oil (20% - 25%). The oil is rich in unsaturated fatty acids, limonoid compounds, flavonoid compounds, vitamin E, etc. Existing studies have found that citrus seed oil can significantly reduce the blood sugar, serum triglyceride, and cholesterol of rats, and increase the content of high-density cholesterol in the serum, showing the potential for developing health-care oils.

[0003] In recent years, limonin has attracted more and more attention from scientists. Limonoid compounds are mainly present in the fruits of Rutaceae plants, and the content is relatively high in the fruit cores (seeds). Limonin has functions such as anti-cancer, analgesic and anti-inflammatory, anti-viral, antioxidant, improving cardiovascular and cerebrovascular circulation, and improving sleep. Research shows that limonoid compounds can effectively reduce blood lipids, promote the balance of intestinal flora, improve nerve excitability, improve immunity, etc., and can also inhibit cancers such as liver cancer, small intestine cancer, oral cancer, and gastric cancer caused by chemical substances, having excellent health-care functions.

[0004] At the present stage, China's oilseed resources are becoming increasingly scarce, and a large amount of oilseeds need to be imported every year, resulting in huge costs. In addition, although the living standards of the public have been greatly improved, due to factors such as unbalanced diet structure and irregular living schedules, people's physical health is easily threatened. Therefore, people are paying more and more attention to the health-care functions of oil products. Currently, health-care oils on the market include deep-sea fish oil, grape seed oil, seabuckthorn seed oil, etc., but there are relatively few health-care products made from citrus seed oil. As a by-product of citrus fruits, citrus seeds are not only rich in oil but also rich in limonoid compounds, having great research and development value. Therefore, if the functional activity of citrus seed oil can be fully explored and improved, it can not only solve the problem of green resource conversion of citrus seed waste in the citrus processing field, but also broaden the product R & D direction of the citrus processing industry, promote the adjustment of the industrial structure, and have great social and economic benefits.

[0005] At present, most of the research on citrus seeds focuses on the extraction of limonin and the separation of oil. However, if citrus seeds are only used for oil extraction, a large amount of limonoid compounds will be lost; if citrus seeds are only used for extracting limonin, the natural oil contained will be wasted seriously. In addition, among the common extraction methods, although the organic solvent method is easy to operate, the solvent demand is large, the cost is high, and the organic reagents are highly toxic, easy to remain, and have an adverse impact on the environment; the supercritical method has a higher extraction efficiency, but the equipment is complex and the cost is high, which is not suitable for large-scale production.

[0006] It can be seen that at the present stage, the green processing technology of citrus seeds is not mature, the product R & D direction is single, and there are still many problems to be improved. Therefore, it is urgent to develop a new large-scale extraction method of health care citrus seed oil with simple process, natural safety, no pollution and high extraction rate to solve the above problems. Summary of the Invention

[0007] The primary object of the present invention is to overcome the disadvantages and deficiencies of the prior art and provide a preparation method of citrus seed oil rich in limonin.

[0008] Another object of the present invention is to provide citrus seed oil rich in limonin and defatted bitter citrus seed meal obtained by the above preparation method.

[0009] A further object of the present invention is to provide the application of the above citrus seed oil rich in limonin and defatted bitter citrus seed meal.

[0010] The object of the present invention is achieved by the following technical solutions: A preparation method of citrus seed oil rich in limonin and defatted bitter citrus seed meal includes the following steps:

[0011] (1) Add a complex enzyme and water to citrus seeds, carry out ultrasonic-assisted constant-temperature enzymatic hydrolysis to obtain an enzymatic hydrolysis system; drain the water and then dry to obtain enzymatically hydrolyzed citrus seeds;

[0012] (2) Add the enzymatically hydrolyzed citrus seeds obtained in step (1) to a screw press for pressing to obtain seed oil 1 and oil residue 1; crush the oil residue 1 to obtain fine oil residue powder; take the seed oil 1 and mix it with the fine oil residue powder, add solid citric acid and mix evenly to dissolve, carry out ultrasonic treatment, and carry out extraction, and carry out the first solid-liquid separation. The obtained solid is defatted bitter citrus seed meal, and the obtained liquid is citrus seed oil 2 rich in limonin (abbreviated as seed oil 2);

[0013] (3) After preheating the seed oil 2 obtained in step (2), first add an acid solution at the same temperature, and then add distilled water at the same temperature and stir at a constant temperature, and carry out the second solid-liquid separation to obtain degummed seed oil 3; preheat the seed oil 3, add an alkali solution, stir and then carry out the third solid-liquid separation to obtain deacidified and degummed seed oil 4;

[0014] (4) Preheat the seed oil 4 obtained in step (3), add a decolorizing agent and stir, carry out the fourth solid-liquid separation to obtain decolorized, deacidified and degummed seed oil 5; heat the seed oil 5 in a water bath, then cool and crystallize, stand still, and filter to remove wax esters to obtain citrus seed oil rich in limonin.

[0015] The complex enzyme described in step (1) is preferably obtained by compounding cellulase, pectinase, acid protease and β-glucosidase in a mass ratio of 0.5-2:0.5-2:1-2:0.5-1; more preferably, it is obtained by compounding cellulase, pectinase, acid protease and β-glucosidase in a mass ratio of 0.5-1:1-2:1:1.

[0016] The described cellulase is a cellulase with an enzyme activity of 1×10 5 U / g.

[0017] The described pectinase is a pectinase with an enzyme activity of 5×10 4 U / g.

[0018] The described acid protease is an acid protease with an enzyme activity of 1×10 5 U / g.

[0019] The described β-glucosidase is a β-glucosidase with an enzyme activity of 5×10 4 U / g.

[0020] The pH value of the enzymatic hydrolysis system described in step (1) is preferably 5.0-6.5; more preferably 5.5.

[0021] The described pH value is preferably adjusted by a sodium bicarbonate solution or a citric acid solution.

[0022] The described sodium bicarbonate solution is preferably a sodium bicarbonate solution with a concentration of 0.5 mol / L.

[0023] The described citric acid solution is preferably a citric acid solution with a concentration of 0.5 mol / L.

[0024] The conditions of the enzymatic hydrolysis described in step (1) are as follows: the power is 100-600 W, the temperature is 45-60 °C, and the time is 1-3 h; preferably as follows: the power is 400-600 W, the temperature is 45-55 °C, and the time is 1-3 h; more preferably as follows: the power is 450-600 W, the temperature is 45-50 °C, and the time is 2 h.

[0025] The drying method described in step (1) is preferably sun drying or drying; more preferably blast drying at a temperature of 50-60 °C.

[0026] The drying degree described in step (1) is preferably dried to a moisture content not higher than 8%; more preferably dried to a moisture content not higher than 5%.

[0027] The temperature of the pressing described in step (2) ≤ 60 °C.

[0028] The degree of pulverization described in step (2) is preferably sifted to 80-100 mesh.

[0029] The addition amount of the solid citric acid described in step (2) is calculated as 0.06% - 0.08% by mass of the seed oil 1 (g); more preferably, it is calculated as 0.06% - 0.07% by mass of the seed oil 1 (g).

[0030] The fine oil residue powder and the seed oil 1 described in step (2) are mixed at a ratio of 1:3 - 1:10 (w / v) for extraction and enrichment of limonin.

[0031] The conditions of the ultrasonic treatment described in step (2) are as follows: the power is 400 - 600 W, the temperature is 40 - 50 °C, and the time is 0.5 - 1 h; preferably as follows: the power is 400 - 500 W, the temperature is 45 - 50 °C, and the time is 0.5 - 1 h.

[0032] The conditions of the extraction described in step (2) are preferably: the extraction temperature is 40 - 55 °C, the stirring speed is 300 - 500 r / min, and the extraction time is 1 - 2 h.

[0033] The method of the first solid-liquid separation described in step (2) is centrifugation or filtration; preferably, it is centrifugation first and then filtration.

[0034] The conditions of the centrifugation are preferably a centrifugal force of 3500 - 6500 g and a time of 3 - 8 min; more preferably a centrifugal force of 5000 - 6000 g and a time of 5 - 8 min.

[0035] The filtration is filtration through gauze.

[0036] The preheating temperature of the seed oil 2 described in step (3) is preferably 60 - 80 °C; more preferably 70 °C.

[0037] The acid solution described in step (3) is preferably a citric acid solution or a phosphoric acid solution; more preferably a citric acid solution or a phosphoric acid solution with a concentration of 0.5 mol / L.

[0038] The addition amount of the acid solution described in step (3) is preferably calculated as 0.15% - 0.20% of the volume of the seed oil 2.

[0039] The addition amount of the distilled water described in step (3) is calculated as 2% - 6% of the volume of the seed oil 2; more preferably calculated as 3% of the volume of the seed oil 2.

[0040] The conditions of the constant-temperature stirring in step (3) are: the temperature is 60 - 80 °C, the rotation speed is 80 - 120 r / min, and the time is 15 - 30 min; preferably: the temperature is 70 °C, the rotation speed is 100 r / min, and the time is 25 - 30 min.

[0041] The method of the second solid-liquid separation described in step (3) is centrifugation or filtration; preferably centrifugation.

[0042] The centrifugation conditions are as follows: the centrifugal force is 5000 - 6000g, and the time is 10 - 12 min.

[0043] The temperature for preheating the seed oil 3 in step (3) is 60 - 80°C; more preferably 70°C.

[0044] The alkali solution in step (3) is preferably a KOH solution; more preferably a KOH solution with a mass percentage concentration of 10% - 16%; most preferably a KOH solution with a mass percentage concentration of 12%.

[0045] The addition amount of the alkali solution in step (3) is calculated as 0.13 - 0.15% (v / v) of the volume of the seed oil 3; more preferably calculated as 0.14 - 0.15% (v / v) of the volume of the seed oil 3.

[0046] The conditions for adding and stirring the alkali solution in step (3) are as follows: temperature 60 - 80°C, rotation speed 80 - 120 r / min, time 15 - 30 min; preferably: temperature 70°C, rotation speed 100 r / min, time 25 - 30 min.

[0047] The method for the third solid-liquid separation in step (3) is centrifugation or filtration; preferably centrifugation.

[0048] The centrifugation conditions are as follows: the centrifugal force is 5000 - 6000g, and the time is 10 - 12 min.

[0049] The temperature for preheating in step (4) is 60 - 80°C; more preferably 70°C.

[0050] The decolorizing agent in step (4) is preferably attapulgite; more preferably activated clay.

[0051] The mass addition amount of the decolorizing agent in step (4) is calculated as 8 - 9% (w / v, g:mL) of the volume of the seed oil 4.

[0052] The conditions for stirring in step (4) are as follows: temperature 70 - 80°C, stirring speed 300 - 350 r / min, time 30 - 90 min.

[0053] The method for the fourth solid-liquid separation in step (4) is centrifugation or filtration; preferably centrifugation.

[0054] The centrifugation conditions are as follows: the centrifugal force is 5000 - 6000g, and the time is 10 - 12 min.

[0055] The temperature for water bath heating in step (4) is preferably 75 - 85°C; preferably 80°C.

[0056] The conditions of the cooling crystallization described in step (4) are preferably cooling to 4-10 °C and the crystallization time is 12-48 h.

[0057] A citrus seed oil rich in limonin is obtained by the above preparation method.

[0058] The application of the above citrus seed oil rich in limonin in medicine, health food, and beauty products.

[0059] A defatted citrus seed meal is obtained by the above preparation method.

[0060] The application of the above defatted citrus seed meal in feed raw materials or feed additives.

[0061] The present invention has the following advantages and effects compared with the prior art:

[0062] (1) The technological process of the present invention is simple and easy to operate, without using any organic solvents, reducing environmental pollution, being green and safe, having a low process production cost, and the enzymatic pretreatment of raw materials and pressing can extract and retain natural active ingredients and fatty acid components to the greatest extent. This process uses a screw press for pressing, and during the pressing process, the pressing temperature ≤ 60 °C, avoiding the adverse effects of high temperature on the oil quality.

[0063] (2) The oil yield of this process reaches 35-40%, the finished citrus seed oil is light yellow in color, has a lemon-like aroma, and the limonin content is as high as 550.11 μg / mL of oil. Compared with the limonin content of 109.05 μg / mL in non-enriched citrus seed oil, the limonin content after enrichment has increased by nearly 4 times, and the antioxidant activity of the citrus seed oil after enrichment has increased. Thus, it can be seen that the present invention can improve the utilization rate of citrus seeds and the health care value of citrus seed oil at the same time. In addition, the present invention can also obtain defatted citrus seed meal. The two products obtained by the present invention respectively have the possibility of being applied in fields such as beauty products and feed additives.

[0064] (3) Considering the non-polar property of limonin compounds, it is proposed for the first time to use citrus seed oil to extract defatted citrus seed powder to enrich limonin. Compared with other studies, without the aid of organic solvents or supercritical extraction, it is green, safe, efficient, and more suitable for large-scale production.

[0065] (4) Considering the various biological activities of limonin, the product concept of citrus seed oil rich in limonin is proposed for the first time. Contrary to the defatting of related products, on the contrary; in addition, there is no need to add exogenous functional ingredients, the cost is low, and the utilization value and economic benefits of citrus waste are improved. Description of the Drawings

[0066] Figure 1 It is a high-performance liquid chromatography diagram of a limonin standard product with a concentration of 500 μg / mL.

[0067] Figure 2 This is the high-performance liquid chromatography (HPLC) chromatogram of the citrus seed oil sample rich in limonin of the present invention.

[0068] Figure 3 This is the high-performance liquid chromatography (HPLC) chromatogram of the citrus seed oil without enriched limonin prepared in Comparative Example 1. Detailed Embodiments

[0069] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto. For those not specifying specific techniques or conditions in the embodiments, they are carried out according to the techniques or conditions described in the literature in the art. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0070] Example 1

[0071] (1) Take 40 g of citrus seeds, wash them, drain the water, and add a composite enzyme composed of cellulase (100,000 U / g), pectinase (50,000 U / g), acid protease (100,000 U / g), and β-glucosidase (50,000 U / g) in an amount of 1% (the mass percentage of the composite enzyme in the mass of citrus seeds) and mix them in a mass ratio of 1:1:1:1 and add them to the citrus seeds with seed coats. After adding 100 mL of water, adjust the pH to 5.5 with 0.5 mol / L citric acid solution, carry out ultrasonic enzymolysis at 500 W and 50 °C for 2 h, drain the water, and dry in an oven at 60 °C until the moisture content is 5%; the moisture content is measured with a moisture meter.

[0072] (2) Transfer the citrus seeds dried in step (1) to a screw press for pressing. The temperature of the press chamber is 60 °C to obtain seed oil 1 and oil residue 1; the oil residue 1 is pulverized by a pulverizer and passed through an 80-mesh sieve, and the fine powder is collected to obtain the fine powder of the oil residue.

[0073] (3) Mix the fine powder of the oil residue obtained in step (2) and seed oil 1 evenly in a ratio of 1:5 (w:v, g:mL, the same below), add 0.06% (the mass percentage of citric acid in the mass of seed oil 1) citric acid, carry out ultrasonic treatment at 500 W and 50 °C for 0.5 h, carry out extraction at 50 °C and 400 r / min for 1.5 h, and centrifuge at a centrifugal force of 5100 g for 5 min to separate to obtain seed oil 2 and defatted bitter citrus seed meal;

[0074] (4) Preheat seed oil 2 to 70 °C, add 0.5 mol / L phosphoric acid solution at an amount of 0.15% (v / v) of the oil volume. After 10 min, add isothermal distilled water (the added amount is 3% (v / v) of the oil volume), stir at 70 °C and 100 r / min for 30 min, and then centrifuge at a centrifugal force of 5100 g for 10 min to separate the colloid and the oil to obtain seed oil 3;

[0075] (5) The rapeseed oil 3 was preheated to 70 °C, and a KOH solution with a concentration of 12% (w / w) was added at an addition amount of 0.14% (v / v). It was stirred at 70 °C and 100 r / min for 30 min, and then centrifuged at a centrifugal force of 5100 g for 10 min to separate the white flocculent substance and the oil, obtaining rapeseed oil 4;

[0076] (6) Rapeseed oil 4 was preheated to 70 °C, and the revived activated clay (the activated clay was heated to 80 °C for revival before use, the same below) was added. The addition amount of the activated clay (g) was calculated according to 8% of the volume (mL) of rapeseed oil 4. It was stirred at 70 °C and 300 r / min for 60 min, and then centrifuged at a centrifugal force of 5100 g for 10 min to obtain rapeseed oil 5.

[0077] (7) Rapeseed oil 5 was heated in a water bath to 80 °C, slowly cooled to 4 °C, and left standing for 48 h, then filtered to obtain citrus rapeseed oil rich in limonin.

[0078] The content of limonin in the citrus rapeseed oil was determined by HPLC. The determination method and chromatographic conditions are as follows:

[0079] (1) Limonin stock solution: Accurately weigh 0.025 g of limonin standard product, completely dissolve it with absolute ethanol and make up the volume to 50 mL to prepare a limonin stock solution with a concentration of 500 μg / mL, and store it at 4 °C for standby.

[0080] (2) Limonin dilution solution: Take absolute ethanol and dilute the limonin stock solution to concentration gradients of 6.25, 12.5, 25, 50, 100, 150, 250 μg / mL respectively.

[0081] (3) Liquid chromatography conditions: Waters high performance liquid chromatography - 2998PDA, C18 (Waters Sunfire 4.6 * 250 mm, 5 μm), the mobile phase was pure water: acetonitrile (35%: 65%), isocratic elution, flow rate 1.0 mL / min, time 30 min, column temperature 30 °C, injection volume 10 μL, detection wavelength 215 nm.

[0082] Under the above chromatographic detection conditions, the liquid chromatograms of the standard product and the sample for determining the content of limonin (as shown in Figure 1 and Figure 2 ) were obtained, and the linear regression equation of the peak area as the ordinate y against the limonin concentration x (μg / mL) was y = 5568.2x + 15480. The results showed that limonin had a good linear relationship in the range of 6.25 - 500 μg / mL (R 2 = 0.9995).

[0083] The determination method of the DPPH free radical scavenging rate is as follows:

[0084] (1) Preparation of solutions: 600 μmol / L DPPH solution: Accurately weigh 0.023 g of DPPH powder, dissolve it in methanol, and make up the volume to 100 mL. Dilute it 10 times before use. 1 mg / mL Trolox (TE, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) standard solution: Accurately weigh 0.1 g of Trolox (TE), dissolve it in methanol and make up the volume to 100 mL.

[0085] (2) Preparation of the standard curve: Pipette 5, 10, 15, 20, 25, 30, 35, 40, 50 μL of Trolox standard solution into EP tubes respectively, and make up the volume to 1 mL. Pipette 25 μL of Trolox standard solution with different concentrations, add 200 μL of DPPH solution with a concentration of 60 μM, shake well, and let it stand and react in the dark at room temperature for 30 min. Then measure the absorbance at 515 nm. Plot the standard curve with the mass concentration of TE as the abscissa (mg / mL) and the corresponding scavenging rate as the ordinate. The standard curve equation is: y = 1211.8x + 0.6128 (R 2 = 0.9992).

[0086] (3) Determination of samples: Pipette 25 μL of the sample, add 200 μL of DPPH solution with a concentration of 60 μM, shake well, and let it stand and react in the dark at room temperature for 30 min. Then measure the absorbance at 515 nm. At the same time, measure the absorbance of the blank sample and the absorbance of the sample itself.

[0087] DPPH radical scavenging rate (Rdpph) = [1 - (A1 - A 本 ) / A0] × 100%

[0088] Where: A0 is the absorbance value of the blank sample;

[0089] A1 is the absorbance value of the sample;

[0090] A 本 is the absorbance value of the sample itself.

[0091] The method for determining the ABTS radical scavenging rate is as follows:

[0092] (1) Preparation of solutions: 7 mM ABTS: Dilute 0.384076 g of ABTS to 100 mL with distilled water. 2.45 mM potassium persulfate: Dilute 0.0662284 g of potassium persulfate to 100 mL with distilled water. Mix 7 mM ABTS and 2.45 mM potassium persulfate in a volume ratio of 1:1, and react in the dark at room temperature for 16 h to obtain the ABTS working solution; Dilute the ABTS working solution with distilled water until the OD value at 734 nm is 0.7 ± 0.02 to obtain the diluted ABTS solution.

[0093] (2) Preparation of standard curve: Pipette 5, 10, 15, 20, 25, 30, 35, 40, 50 μL of Trolox standard solution into EP tubes respectively, and make up the volume to 1 mL. Pipette 25 μL of Trolox standard solution with different concentrations respectively, add 200 μL of diluted ABTS solution, shake well, let stand and react in the dark at room temperature for 10 min, then measure the absorbance at 734 nm. Plot the standard curve with the mass concentration of ascorbic acid as the abscissa (mg / mL) and the corresponding scavenging rate as the ordinate. The standard curve equation is: y = 2.3341x - 4.0712 (R 2 = 0.9994).

[0094] (3) Determination of samples: Pipette 25 μL of samples, add 200 μL of diluted ABTS solution, shake well, let stand and react in the dark at room temperature for 10 min, then measure the absorbance at 734 nm.

[0095] ABTS radical scavenging rate (Rdpph) = [1 - (A1 - A 本 ) / A0] × 100%

[0096] Where: A0 is the absorbance value of the blank sample;

[0097] A1 is the absorbance value of the sample;

[0098] A 本 is the original absorbance value of the sample.

[0099] The method for determining antibacterial property is as follows:

[0100] (1) Preparation of LB medium

[0101] Weigh 10 g of tryptone, 5 g of yeast extract, 15 - 20 g of agar powder, 10 g of NaCl, make up the volume to 1000 mL with distilled water, adjust the pH to 7.2 - 7.4, and autoclave at 121 °C for 30 min.

[0102] (2) Activation of bacterial cells

[0103] Pick Gram-negative bacterium Escherichia coli ATCC25922 and Gram-positive bacterium Staphylococcus aureus ATCC29213 and activate them in LB medium for 12 - 18 h. The activation conditions are 30 °C and 180 r / min.

[0104] (3) Pouring plates

[0105] i. Make aseptic preparations;

[0106] ii. Place the sterilized petri dish on the table beside the flame. Hold the conical flask containing the culture medium in the right hand, pull out the cotton plug with the left hand, and quickly pass the mouth of the conical flask through the flame;

[0107] iii. Open the petri dish with the left hand to form a slit slightly larger than the mouth of the flask. Pour the culture medium (about 10 - 20 mL) in the conical flask into the petri dish with the right hand, immediately cover the lid of the petri dish with the left hand, and gently shake well;

[0108] iv. After waiting for the plate to cool and solidify, place the plate upside down, with the lid at the bottom and the bottom of the dish on top.

[0109] (4) Spreading the plate

[0110] i. Burn the spreader over the flame for a few seconds and cool for 8 - 10 s;

[0111] ii. Dilute the bacterial solution 100 - fold and take 25 μL, then drop it onto the surface of the culture medium;

[0112] iii. Use the spreader to evenly spread the bacterial solution on the surface of the culture medium. When spreading, the petri dish can be rotated to make the spreading uniform.

[0113] (5) Antibacterial operation

[0114] Place the Oxford cup on the petri dish and add 100 μL of the sample, then wrap the petri dish with sealing film. Finally, place the petri dish in an incubator at 37 °C for 24 h, measure the diameter (mm) of the antibacterial circle, and compare the antibacterial effects.

[0115] The results show that: After extraction by the above process, the oil yield of citrus seeds reaches 35.3%, the color of the finished citrus seed oil is light yellow, and it has a fresh plant aroma. The limonin content is 287.87 μg / mL of oil, the DPPH free radical scavenging rate is 91.03%, the ABTS free radical scavenging rate is 62.57%, and the antibacterial circles against Escherichia coli and Staphylococcus aureus are 9.67 mm and 10.67 mm respectively.

[0116] Example 2

[0117] (1) Take 40 g of citrus seeds, wash and drain them. Add a composite enzyme composed of cellulase (100,000 U / g), pectinase (50,000 U / g), acid protease (100,000 U / g) and β - glucosidase (50,000 U / g) in an amount of 1% (the percentage of the mass of the composite enzyme in the mass of citrus seeds), and mix them in a mass ratio of 1:1:1:1 and add them to the citrus seeds with seed coats. After adding 100 mL of water, adjust the pH = 6.5 with 0.5 mol / L citric acid solution, carry out ultrasonic enzymolysis at 550 W and 50 °C for 2 h, drain the water, and dry in an oven at 60 °C until the moisture content is 5%;

[0118] (2) The dried citrus seeds in step (1) are transported to a screw press for pressing. The temperature of the press chamber is 60 °C, obtaining citrus seed oil 6 and oil residue 2. The oil residue 2 is pulverized by a pulverizer, screened through a 80-mesh sieve, and the fine powder is collected to obtain fine oil residue powder.

[0119] (3) The fine oil residue powder obtained in step (2) and citrus seed oil 6 are mixed evenly at a ratio of 1:3 (w:v), 0.07% citric acid (the percentage of citric acid mass in the mass of citrus seed oil) is added, and ultrasonic treatment is carried out for 0.5 h under the conditions of 500 W and 45 °C, and extraction is carried out for 1.5 h under the conditions of 50 °C and 400 r / min. Centrifugation is carried out for 5 min under the condition of a centrifugal force of 5100 g to separate citrus seed oil 7 and defatted citrus seed meal.

[0120] (4) Citrus seed oil 7 is preheated to 70 °C, a 0.5 mol / L phosphoric acid solution is added at an addition amount of 0.20% (v / v). After 10 min, isothermal distilled water (the amount is 3% (v / v) of the oil amount) is added, and stirring is carried out for 30 min under the conditions of a temperature of 70 °C and a stirring speed of 100 r / min. Centrifugation is carried out for 10 min under the condition of a centrifugal force of 5100 g to separate colloid and oil, obtaining citrus seed oil 8.

[0121] (5) Citrus seed oil 8 is preheated to 70 °C, a 12% (w / w) KOH solution is added at an addition amount of 0.15% (v / v), and stirring is carried out for 30 min under the conditions of a temperature of 70 °C and a stirring speed of 100 r / min. Centrifugation is carried out for 10 min under the condition of a centrifugal force of 5100 g to separate white flocculent substances and oil, obtaining citrus seed oil 9.

[0122] (6) Citrus seed oil 9 is preheated to a temperature of 70 °C, activated bleaching earth that has been revived is added at 8.5% of its volume, the temperature is 70 °C, the stirring speed is 300 r / min, the time is 60 min, and centrifugation is carried out for 10 min under the condition of a centrifugal force of 5100 g for separation, obtaining citrus seed oil 10.

[0123] (7) Citrus seed oil 10 is heated in a water bath to 80 °C, slowly cooled to 4 °C, allowed to stand for 48 h, and filtered to obtain citrus seed oil rich in limonin.

[0124] The results show that the oil yield of this process reaches 36.6%. The color of the finished citrus seed oil is light yellow and has a lemon-like aroma. The steps and chromatographic conditions for determining the limonin content in citrus seed oil by liquid chromatography are the same as those in Example 1. After calculation, the limonin content is 550.11 μg / mL of citrus seed oil. The methods for measuring the DPPH and ABTS free radical scavenging rates of citrus seed oil are the same as those in Example 1. After calculation, the DPPH free radical scavenging rate is 94.69%, and the ABTS free radical scavenging rate is 66.29%. The antibacterial test method is the same as that in Example 1, and the antibacterial zones for Escherichia coli and Staphylococcus aureus are 10 mm and 10 mm respectively.

[0125] Example 3

[0126] (1) Take 40 g of citrus seeds, wash them, drain the water, and add a composite enzyme composed of cellulase (100,000 U / g), pectinase (50,000 U / g), acid protease (100,000 U / g), and β-glucosidase (50,000 U / g) in an amount of 1% (the mass percentage of the composite enzyme in the mass of citrus seeds). Mix them in a mass ratio of 1:2:1:1 and add them to the citrus seeds with seed coats. After adding 100 mL of water, adjust the pH to 5.5 with 0.5 mol / L citric acid solution, and carry out ultrasonic enzymolysis at 450 W and 50 °C for 2 h. Drain the water and dry in an oven at 60 °C until the moisture content is 5%.

[0127] (2) Transfer the dried citrus seeds in step (1) to a screw press for low-temperature pressing. The temperature of the press chamber is 60 °C to obtain seed oil 11 and oil residue 3. The oil residue 3 is crushed by a crusher, passed through an 80-mesh sieve, and the fine powder is collected to obtain fine oil residue powder.

[0128] (3) Mix the fine oil residue powder obtained in step (2) and seed oil 11 evenly in a ratio of 1:5 (w:v), add 0.06% (the mass percentage of citric acid in the mass of seed oil) citric acid, carry out ultrasonic treatment at 400 W and 45 °C for 0.5 h, and carry out extraction at 45 °C and 400 r / min for 1.5 h. Centrifuge at a centrifugal force of 5100 g for 5 min to separate seed oil 12 and defatted citrus seed meal.

[0129] (4) Preheat seed oil 12 to 70 °C, add 0.5 mol / L phosphoric acid solution with an amount of 0.20% of the oil volume. After 10 min, add isothermal distilled water (the volume is 3% (v / v) of the oil volume), at a temperature of 70 °C, and carry out slow constant-temperature stirring (100 r / min) for 30 min. Centrifuge at a centrifugal force of 5100 g for 10 min to separate the colloid and the oil to obtain seed oil 13.

[0130] (5) Preheat seed oil 13 to 70 °C, add a KOH solution with a mass fraction of 12% and an amount of 0.15% (v / v) of the oil volume. At a temperature of 70 °C, carry out slow constant-temperature stirring (100 r / min) for 30 min. Centrifuge at a centrifugal force of 5100 g for 10 min to separate the white flocculent substance and the oil to obtain seed oil 14.

[0131] (6) Preheat seed oil 14 to 70 °C, add reactivated activated clay at 9% of its volume. At a temperature of 70 °C, the stirring speed is 300 r / min, and the time is 60 min. Centrifuge at a centrifugal force of 5100 g for 10 min to separate to obtain seed oil 15.

[0132] (7) Heat seed oil 15 in a water bath to 80 °C, slowly cool it to 4 °C, let it stand for 48 h, and filter to obtain citrus seed oil rich in limonin.

[0133] The results showed that the oil yield of this process reached 34.9%. The color of the finished citrus seed oil was light yellow, clear and transparent, with a lemon-like aroma. The steps and chromatographic conditions for determining the limonin content in citrus seed oil by liquid chromatography were the same as those in Example 1. After calculation, the limonin content was 308.23 μg / mL of oil. The methods for determining the DPPH and ABTS free radical scavenging rates of citrus seed oil were the same as those in Example 1. After calculation, the DPPH free radical scavenging rate was 94.45%, and the ABTS free radical scavenging rate was 63.77%. The antibacterial test method was the same as that in Example 1. The antibacterial zones against Escherichia coli and Staphylococcus aureus were 9.67 mm and 10 mm respectively.

[0134] Example 4

[0135] (1) Take 40 g of citrus seeds, wash them, drain the water, and add a composite enzyme composed of cellulase (100,000 U / g), pectinase (50,000 U / g), acid protease (100,000 U / g) and β-glucosidase (50,000 U / g) in an amount of 1% (the percentage of the mass of the composite enzyme in the mass of citrus seeds). Mix and add them to the citrus seeds with seed coats in a mass ratio of 0.5:1:1:1. After adding 100 mL of water, adjust the pH to 5.0 with 0.5 mol / L citric acid solution, and carry out ultrasonic enzymolysis at 600 W and 45 °C for 2 h. Drain the water and dry in an oven at 60 °C until the moisture content is 5%.

[0136] (2) Transfer the citrus seeds dried in step (1) to a screw press for low-temperature pressing. The temperature of the press chamber is 60 °C to obtain 16 of seed oil and 4 of oil residue. The oil residue 4 is crushed by a crusher and passed through an 80-mesh sieve, and the fine powder is collected to obtain the fine powder of oil residue.

[0137] (3) Mix the fine powder of oil residue obtained in step (2) and 16 of seed oil evenly at a ratio of 1:8 (w:v), add 0.07% (the percentage of the mass of citric acid in the mass of seed oil) of citric acid, and carry out ultrasonic treatment at 400 W and 50 °C for 0.5 h, and extract at 50 °C and 350 r / min for 1.5 h. Centrifuge at a centrifugal force of 5100 g for 5 min to separate the seed oil 17 and the defatted citrus seed meal.

[0138] (4) Preheat the seed oil 17 to 70 °C, add 0.5 mol / L phosphoric acid solution at a dosage of 0.20% of the oil amount. After 10 min, add isothermal distilled water (the amount is 3% of the oil amount), keep the temperature at 70 °C, and stir slowly at a constant temperature (100 r / min) for 30 min. Centrifuge at a centrifugal force of 5100 g for 10 min to separate the colloid and the oil to obtain the seed oil 18.

[0139] (5) The camellia seed oil 18 was preheated to 70 °C, and a KOH solution with a mass fraction of 12% was added at a dosage of 0.15% (v / v). The temperature was maintained at 70 °C, and it was stirred slowly at a constant speed (100 r / min) for 30 min. After centrifuging for 10 min under a centrifugal force of 5100 g to separate the white flocculent matter and the oil, camellia seed oil 19 was obtained;

[0140] (6) The camellia seed oil 19 was preheated to 70 °C, and the revived activated clay was added at 8.5% of its volume. The temperature was 70 °C, the stirring speed was 300 r / min, and the time was 60 min. After centrifuging for 10 min under a centrifugal force of 5100 g for separation, camellia seed oil 20 was obtained.

[0141] (7) The camellia seed oil 20 was heated in a water bath to 80 °C, slowly cooled to 4 °C, and allowed to stand for 48 h, followed by filtration to obtain citrus seed oil rich in limonin.

[0142] The results showed that the oil yield of this process reached 37.4%. The color of the finished citrus seed oil was light yellow, clear and transparent, with a lemon-like aroma. The steps and chromatographic conditions for determining the limonin content in citrus seed oil by liquid chromatography were the same as those in Example 1. After calculation, the limonin content was 254.59 μg / mL of oil; the methods for determining the DPPH and ABTS free radical scavenging rates of citrus seed oil were the same as those in Example 1. After calculation, the DPPH free radical scavenging rate was 93.25%, and the ABTS free radical scavenging rate was 61.73%; the antibacterial test method was the same as that in Example 1, and the antibacterial zones for Escherichia coli and Staphylococcus aureus were 10.33 mm and 9.33 mm, respectively.

[0143] Comparative Example 1

[0144] The difference from Example 2 was that the enrichment of limonin was not carried out.

[0145] (1) 40 g of citrus seeds were taken, washed, and drained. A composite enzyme composed of cellulase (100,000 U / g), pectinase (50,000 U / g), acid protease (100,000 U / g), and β-glucosidase (50,000 U / g) was added in an amount of 1% (the percentage of the mass of the composite enzyme in the mass of the citrus seeds), and the mixture was added to the citrus seeds with seed coats in a mass ratio of 1:1:1:1. After adding 100 mL of water, the pH was adjusted to 6.5 with 0.5 mol / L citric acid solution, and ultrasonic enzymolysis was carried out at 550 W and 50 °C for 2 h. After draining, it was dried in an oven at 60 °C until the moisture content was 5%;

[0146] (2) The dried citrus seeds in step (1) were fed into a screw press for pressing. The temperature of the press chamber was 60 °C, and camellia seed oil 21 and oil residue 5 were obtained;

[0147] (3) The seed oil 21 obtained in step (2) is preheated to 70 °C, and a 0.5 mol / L phosphoric acid solution is added at an addition amount of 0.20% (v / v). After 10 min, isothermal distilled water (the amount is 3% (v / v) of the oil amount) is added, and the mixture is stirred at 70 °C and 100 r / min for 30 min, and then centrifuged at a centrifugal force of 5100 g for 10 min to separate the colloid and the oil, obtaining seed oil 22;

[0148] (4) The seed oil 22 is preheated to 70 °C, and a KOH solution with a concentration of 12% (w / w) is added at an addition amount of 0.15% (v / v). The mixture is stirred at 70 °C and 100 r / min for 30 min, and then centrifuged at a centrifugal force of 5100 g for 10 min to separate the white flocculent substance and the oil, obtaining seed oil 23;

[0149] (5) The seed oil 23 is preheated to 70 °C, and the revived activated clay is added at 8.5% of its volume. The mixture is stirred at 70 °C and a stirring speed of 300 r / min for 60 min, and then centrifuged at a centrifugal force of 5100 g for 10 min to separate, obtaining seed oil 24.

[0150] (6) The seed oil 24 is heated in a water bath to 80 °C, slowly cooled to 4 °C, and allowed to stand for 48 h, and then filtered to obtain the finished citrus seed oil.

[0151] The results show that the oil yield of this process is 36.4%. The refined oil is yellow and has a fruit kernel oil flavor. After calculation, the limonin content is 109.05 μg / mL oil (the liquid chromatogram is as Figure 3 shown); the DPPH free radical scavenging rate is 58.40%, and the ABTS free radical scavenging rate is 36.63%; the inhibition zones against Escherichia coli and Staphylococcus aureus are 8.67 mm and 8.33 mm respectively.

[0152] Comparative Example 2

[0153] The difference from Example 4 is that enzymatic hydrolysis is not carried out in the raw material pretreatment part.

[0154] (1) Take 40 g of citrus seeds, wash them, drain the water, add 100 mL of water, and then adjust the pH to 5.5 with a 0.5 mol / L citric acid solution. Ultrasonic for 2 h at 600 W and 45 °C, drain the water, and dry in an oven at 60 °C until the moisture content is 5%;

[0155] (2) The dried citrus seeds in step (1) are conveyed to a screw press for low-temperature pressing. The temperature of the press chamber is 60 °C, obtaining seed oil 25 and oil residue 6; the oil residue 6 is pulverized by a pulverizer and passed through an 80-mesh sieve, and the fine powder is collected;

[0156] (3) The fine oil residue powder obtained in step (2) and the seed oil 25 are mixed evenly at a ratio of 1:8 (w:v), 0.07% citric acid (the percentage of citric acid mass to the mass of seed oil) is added, and ultrasonic treatment is carried out at 400W and 50°C for 0.5h. Then, extraction is carried out at 50°C and 350r / min for 1.5h; centrifugation is carried out at a centrifugal force of 5100g for 5min to separate the seed oil 26 and the defatted bitter citrus seed meal;

[0157] (4) The seed oil 26 is preheated to 70°C, a 0.5mol / L phosphoric acid solution with an addition amount of 0.20% of the oil amount is added. After 10min, isothermal distilled water (the amount is 3% of the oil amount) is added. At a temperature of 70°C, constant temperature slow stirring (100r / min) is carried out for 30min, and centrifugation is carried out at a centrifugal force of 5100g for 10min to separate the colloid and the oil, obtaining the seed oil 27;

[0158] (5) The seed oil 27 is preheated to 70°C, a KOH solution with a mass fraction of 12% and an addition amount of 0.15% (v / v) of the oil amount is added. At a temperature of 70°C, constant temperature slow stirring (100r / min) is carried out for 30min, and centrifugation is carried out at a centrifugal force of 5100g for 10min to separate the white flocculent substance and the oil, obtaining the seed oil 28;

[0159] (6) The seed oil 28 is preheated to 70°C, activated bleaching earth that has been revived is added at 8.5% of its volume. At a temperature of 70°C, the stirring speed is 300r / min, and the time is 60min. Centrifugation is carried out at a centrifugal force of 5100g for 10min for separation, obtaining the seed oil 29.

[0160] (7) The seed oil 29 is heated in a water bath to 80°C, slowly cooled to 4°C, and left standing for 48h, then filtered to obtain the finished citrus seed oil.

[0161] The results show that the oil yield of this process reaches 25.1%. The finished citrus seed oil body is light yellow. After calculation, the limonin content is 124.79μg / mL of oil; the DPPH free radical scavenging rate is 77.32%, and the ABTS free radical scavenging rate is 53.72%; the antibacterial zones against Escherichia coli and Staphylococcus aureus are 9.33mm and 8.33mm respectively.

[0162] Comparative Example 3

[0163] The difference from Example 1 is that ultrasonic and magnetic stirring treatments are not carried out during the extraction and enrichment process.

[0164] (1) Take 40 g of citrus seeds, wash them, drain the water, and add a complex enzyme composed of cellulase (100,000 U / g), pectinase (50,000 U / g), acid protease (100,000 U / g), and β-glucosidase (50,000 U / g) at a ratio of 1:1:1:1 by mass, with the amount added being 1% (the mass percentage of the complex enzyme in the citrus seeds). Mix it evenly and add it to the citrus seeds with seed coats. After adding 100 mL of water, adjust the pH to 5.5 with 0.5 mol / L citric acid solution. Carry out ultrasonic enzymolysis at 500 W and 50 °C for 2 h, drain the water, and dry it in an oven at 60 °C until the moisture content is 5%.

[0165] (2) Convey the citrus seeds dried in step (1) to a screw press for pressing. The temperature of the press chamber is 60 °C to obtain 30 of seed oil and 7 of oil residue. The oil residue 7 is pulverized by a pulverizer and passed through an 80-mesh sieve, and the fine powder is collected.

[0166] (3) Mix the fine powder of the oil residue and 30 of seed oil obtained in step (2) evenly at a ratio of 1:5 (w:v), add 0.06% (the mass percentage of citric acid in the seed oil) of citric acid, and carry out static extraction at 50 °C for 2 h. Centrifuge at a centrifugal force of 5100 g for 5 min to separate 31 of seed oil and defatted citrus seed meal.

[0167] (4) Preheat 31 of seed oil to 70 °C, add 0.5 mol / L phosphoric acid solution at an amount of 0.15% (v / v) of the oil volume. After 10 min, add isothermal distilled water (the added amount is 3% (v / v) of the oil volume). Stir at 70 °C and 100 r / min for 30 min, and then centrifuge at a centrifugal force of 5100 g for 10 min to separate the colloid and the oil, obtaining 32 of seed oil.

[0168] (5) Preheat 32 of seed oil to 70 °C, add a KOH solution with a concentration of 12% (w / w) at an addition amount of 0.14% (v / v). Stir at 70 °C and 100 r / min for 30 min, and then centrifuge at a centrifugal force of 5100 g for 10 min to separate the white flocculent substance and the oil, obtaining 33 of seed oil.

[0169] (6) Preheat 33 of seed oil to 70 °C, add reactivated activated clay at 8% of its volume. Stir at 70 °C and 300 r / min for 60 min, and then centrifuge at a centrifugal force of 5100 g for 10 min to obtain 34 of citrus seed oil.

[0170] (7) Heat 34 of seed oil in a water bath to 80 °C, slowly cool it to 4 °C, let it stand for 48 h, and filter to obtain the finished citrus seed oil.

[0171] The results showed that the oil yield of this process reached 35.1%, and the color of the finished citrus seed oil was light yellow. After calculation, the limonin content was 205.59 μg / mL of oil; the DPPH free radical scavenging rate was 62.64%, and the ABTS free radical scavenging rate was 48.52%; the inhibition zones against Escherichia coli and Staphylococcus aureus were 9 mm and 9 mm respectively.

[0172] Comparative Example 4

[0173] The difference from Example 3 was that citric acid was not added during the extraction process.

[0174] (1) Take 40 g of citrus seeds, wash them, drain the water, and add a composite enzyme composed of cellulase (100,000 U / g), pectinase (50,000 U / g), acid protease (100,000 U / g), and β-glucosidase (50,000 U / g) in an amount of 1% (the percentage of the mass of the composite enzyme in the mass of the citrus seeds) and mix them in a mass ratio of 1:2:1:1 and add them to the citrus seeds with seed coats. After adding 100 mL of water, adjust the pH to 5.5 with 0.5 mol / L citric acid, and carry out ultrasonic enzymolysis at 450 W and 50 °C for 2 h, drain the water, and dry in an oven at 60 °C until the moisture content is 5%;

[0175] (2) Convey the citrus seeds dried in step (1) to a screw press for low-temperature pressing. The temperature of the press chamber is 60 °C to obtain 35 of seed oil and 8 of oil residue; the oil residue 8 is pulverized by a pulverizer and passed through an 80-mesh sieve, and the fine powder is collected to obtain fine oil residue powder;

[0176] (3) Mix the fine oil residue powder obtained in step (2) and the seed oil 35 evenly in a ratio of 1:5 (w:v), carry out ultrasonic treatment at 400 W and 45 °C for 0.5 h, and carry out extraction at 45 °C and 400 r / min for 1.5 h; centrifuge at a centrifugal force of 5100 g for 5 min to separate to obtain seed oil 36 and defatted bitter citrus seed meal;

[0177] (4) Preheat the seed oil 36 to 70 °C, add a 0.5 mol / L phosphoric acid solution with an amount of 0.20% of the oil volume. After 10 min, add isothermal distilled water (the amount is 3% (v / v) of the oil volume), at a temperature of 70 °C, stir slowly at a constant temperature (100 r / min) for 30 min, and centrifuge at a centrifugal force of 5100 g for 10 min to separate the colloid and the oil to obtain seed oil 37;

[0178] (5) Preheat the seed oil 37 to 70 °C, add a KOH solution with a mass fraction of 12% and an amount of 0.15% (v / v) of the oil volume, at a temperature of 70 °C, stir slowly at a constant temperature (100 r / min) for 30 min, and centrifuge at a centrifugal force of 5100 g for 10 min to separate the white flocculent substance and the oil to obtain seed oil 38;

[0179] (6) The rapeseed oil 38 is preheated to 70 °C, and activated clay that has been revived is added at 9% of its volume. The temperature is 70 °C, the stirring speed is 300 r / min, the time is 60 min, and it is centrifuged for 10 min under a centrifugal force of 5100 g for separation to obtain rapeseed oil 39.

[0180] (7) The rapeseed oil 39 is heated in a water bath to 80 °C, slowly cooled to 4 °C, allowed to stand for 48 h, and then filtered to obtain the finished citrus seed oil.

[0181] The results show that the oil yield of this process reaches 34.5%, and the color of the finished citrus seed oil is yellow. After calculation, the limonin content is 219.22 μg / mL of oil; the DPPH free radical scavenging rate is 63.74%, and the ABTS free radical scavenging rate is 57.17%; the inhibition zones against Escherichia coli and Staphylococcus aureus are 9 mm and 8 mm respectively.

[0182] Comparative Example 5

[0183] The difference from Example 4 is that the leaching process is carried out at room temperature.

[0184] (1) Take 40 g of citrus seeds, wash them, drain the water, and add a composite enzyme 8 composed of cellulase (100,000 U / g), pectinase (50,000 U / g), acid protease (100,000 U / g), and β-glucosidase (50,000 U / g) at a ratio of 0.5:1:1:1 by mass in an amount of 1% (the percentage of the mass of the composite enzyme in the mass of the citrus seeds) and add it to the citrus seeds with seed coats. After adding 100 mL of water, adjust the pH = 5.0 with 0.5 mol / L citric acid solution, and carry out ultrasonic enzymolysis at 600 W and 45 °C for 2 h, drain the water, and dry in an oven at 60 °C until the moisture content is 5%;

[0185] (2) Transfer the citrus seeds dried in step (1) to a screw press for low-temperature pressing. The temperature of the press chamber is 60 °C to obtain rapeseed oil 40 and oil residue 9; the oil residue 9 is pulverized by a pulverizer, passed through an 80-mesh sieve, and the fine powder is collected to obtain fine oil residue powder;

[0186] (3) The fine oil residue powder obtained in step (2) and rapeseed oil 40 are mixed evenly at a ratio of 1:8 (w:v), add 0.07% (the percentage of the mass of citric acid in the mass of rapeseed oil) citric acid, and carry out ultrasonic treatment at 400 W and 25 °C (room temperature) for 0.5 h. At room temperature, extract for 1.5 h at 350 r / min; centrifuge for 5 min under a centrifugal force of 5100 g for separation to obtain rapeseed oil 41 and defatted bitter citrus seed meal;

[0187] (4) Preheat the rapeseed oil 41 to 70 °C, add a 0.5 mol / L phosphoric acid solution with an addition amount of 0.20% of the oil amount. After 10 min, add isothermal distilled water (the amount is 3% of the oil amount), at a temperature of 70 °C, stir constantly at a slow speed (100 r / min) for 30 min, and centrifuge for 10 min under a centrifugal force of 5100 g to separate the colloid and the oil, obtaining rapeseed oil 42;

[0188] (5) Preheat the rapeseed oil 42 to 70 °C, add a KOH solution with a mass fraction of 12% and an addition amount of 0.15% (v / v) of the oil amount, at a temperature of 70 °C, stir constantly at a slow speed (100 r / min) for 30 min, and centrifuge for 10 min under a centrifugal force of 5100 g to separate the white flocculent substance and the oil, obtaining rapeseed oil 43;

[0189] (6) Preheat the rapeseed oil 43 to 70 °C, add the revived activated clay at 8.5% of its volume, at a temperature of 70 °C, with a stirring speed of 300 r / min and a time of 60 min, and centrifuge for 10 min under a centrifugal force of 5100 g for separation, obtaining rapeseed oil 44.

[0190] (7) Heat the rapeseed oil 44 in a water bath to 80 °C, slowly cool it to 4 °C, let it stand for 48 h, and filter to obtain the finished citrus seed oil.

[0191] The results show that the oil yield of this process reaches 35.8%, and the color of the finished citrus seed oil is yellow. After calculation, the limonin content is 244.55 μg / mL of oil; the DPPH free radical scavenging rate is 80.53%, and the ABTS free radical scavenging rate is 44.52%; the inhibition zones for Escherichia coli and Staphylococcus aureus are 9.67 mm and 8 mm respectively.

[0192] Comparative Example 6

[0193] The difference from Example 1 is that the composite enzyme 9 in the enzymatic hydrolysis consists of cellulase, pectinase, papain, and hemicellulase in a mass ratio of 1:1:1:1.

[0194] (1) Take 40 g of citrus seeds, wash them, drain the water, and add the composite enzyme 9 composed of cellulase (100,000 U / g), pectinase (50,000 U / g), papain (100,000 U / g), and hemicellulase (50,000 U / g) in a mass ratio of 1:1:1:1 at a ratio of 1% (the percentage of the mass of the composite enzyme in the mass of the citrus seeds), add 100 mL of water, and then adjust the pH = 5.5 with a 0.5 mol / L citric acid solution. Carry out ultrasonic enzymatic hydrolysis at 500 W and 50 °C for 2 h, drain the water, and dry in an oven at 60 °C until the moisture content is 5%;

[0195] (2) Transfer the dried citrus seeds from step (1) to a screw press for pressing. The temperature of the press chamber is 60 °C, obtaining 45 of seed oil and 10 of oil residue. The oil residue 10 is crushed by a crusher, passed through an 80-mesh sieve, and the fine powder is collected to obtain fine oil residue powder;

[0196] (3) The fine oil residue powder obtained in step (2) and the seed oil 45 are mixed evenly at a ratio of 1:5 (w:v), and 0.06% (the percentage of citric acid mass in the mass of seed oil) of citric acid is added. Ultrasonic treatment is carried out for 0.5 h under the conditions of 500 W and 50 °C, extraction is carried out for 1.5 h under the conditions of 50 °C and 400 r / min, and centrifugation is carried out for 5 min under the condition of a centrifugal force of 5100 g to separate the seed oil 46 and defatted bitter citrus seed meal;

[0197] (4) The seed oil 46 is preheated to 70 °C, 0.5 mol / L phosphoric acid with an addition amount of 0.15% (v / v) of the oil amount is added. After 10 min, isothermal distilled water (the addition amount is 3% (v / v) of the oil amount) is added. Stirring is carried out for 30 min under the conditions of 70 °C and 100 r / min, and then centrifugation is carried out for 10 min under the condition of a centrifugal force of 5100 g to separate the colloid and the oil, obtaining the seed oil 47;

[0198] (5) The seed oil 47 is preheated to 70 °C, and a 12% (w / w) KOH solution is added at an addition amount of 0.14% (v / v). Stirring is carried out for 30 min under the conditions of 70 °C and 100 r / min, and then centrifugation is carried out for 10 min under the condition of a centrifugal force of 5100 g to separate the white flocculent substance and the oil, obtaining the seed oil 48;

[0199] (6) The seed oil 48 is preheated to 70 °C, and reactivated activated clay is added at 8% of its volume. Stirring is carried out for 60 min under the conditions of 70 °C and 300 r / min, and then centrifugation is carried out for 10 min under the condition of a centrifugal force of 5100 g to obtain the seed oil 49.

[0200] (7) The seed oil 49 is heated in a water bath to 80 °C, slowly cooled to 4 °C, allowed to stand for 48 h, and filtered to obtain the finished citrus seed oil.

[0201] The results show that the oil yield of this process reaches 33.2%. The color of the finished citrus seed oil is dark yellow. After calculation, the limonin content is 235.28 μg / mL of oil; the DPPH free radical scavenging rate is 78.18%, and the ABTS free radical scavenging rate is 56.11%; the inhibition zones of Escherichia coli and Staphylococcus aureus are 8.33 mm and 9.33 mm respectively.

[0202] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of citrus seed oil rich in limonin and defatted citrus seed meal, characterized in that It includes the following steps: (1) Add a composite enzyme and water to citrus seeds, and perform ultrasonic-assisted constant-temperature enzymatic hydrolysis to obtain an enzymatic hydrolysis system; drain the water and then dry to obtain enzymatically hydrolyzed citrus seeds; (2) Add the enzymatically hydrolyzed citrus seeds obtained in step (1) to a screw press for pressing to obtain seed oil 1 and oil residue 1; crush the oil residue 1 to obtain fine oil residue powder; take the seed oil 1 and mix it with the fine oil residue powder, add solid citric acid and mix evenly to dissolve, perform ultrasonic treatment, then extract, and perform the first solid-liquid separation to obtain the solid as defatted citrus seed meal and the liquid as seed oil 2 rich in limonin; (3) After preheating the seed oil 2 obtained in step (2), first add an isothermal acid solution, and then add isothermal distilled water and stir at a constant temperature, and perform the second solid-liquid separation to obtain degummed seed oil 3; preheat the seed oil 3, add an alkali solution, stir and then perform the third solid-liquid separation to obtain deacidified and degummed seed oil 4; (4) Preheat the seed oil 4 obtained in step (3), add a decolorizing agent and stir, perform the fourth solid-liquid separation to obtain decolorized, deacidified and degummed seed oil 5; heat the seed oil 5 in a water bath, then cool and crystallize, stand still, and filter to remove wax esters to obtain citrus seed oil rich in limonin; In step (1), the mass dosage of the composite enzyme is 1% of the mass of citrus seeds; In step (1), the composite enzyme is obtained by compounding cellulase, pectinase, acid protease and β-glucosidase according to a mass ratio of 0.5-2:0.5-2:1-2:0.5-1; In step (1), the conditions for enzymatic hydrolysis are as follows: power is 100-600 W, temperature is 45-60 °C, and time is 1-3 h; In step (2), the temperature of pressing ≤ 60 °C; In step (2), the addition amount of the solid citric acid is calculated according to 0.06%-0.08% of the mass of seed oil 1; In step (2), the fine oil residue powder and the seed oil 1 are mixed at a ratio of 1:3-1:10 w / v; In step (2), the conditions for ultrasonic treatment are as follows: power is 400-600 W, temperature is 40-50 °C, and time is 0.5-1 h; In step (2), the conditions for extraction are: extraction temperature is 40-55 °C, stirring speed is 300-500 r / min, and extraction time is 1-2 h; In step (3), the acid solution is a citric acid solution or a phosphoric acid solution; In step (3), the alkali solution is a KOH solution; In step (4), the decolorizing agent is attapulgite; 2. The method for preparing citrus seed oil rich in limonin and defatted citrus seed meal according to claim 1, characterized in that: In step (1), the composite enzyme is obtained by compounding cellulase, pectinase, acid protease and β-glucosidase according to a mass ratio of 0.5-1:1-2:1:1; In step (4), the decolorizing agent is activated clay; 3. The method for preparing citrus seed oil rich in limonin and defatted citrus seed meal according to claim 1, characterized in that: In step (1), the pH value of the enzymatic hydrolysis system is 5.0-6.5; In step (3), the addition amount of the acid solution is calculated according to 0.15%-0.20% of the volume of seed oil 2; The addition amount of the distilled water described in step (3) is calculated according to 2% - 6% of the volume of the seed oil 2; The addition amount of the alkali solution described in step (3) is calculated according to 0.13 - 0.15% of the volume of the seed oil 3; The mass addition amount of the decolorizing agent described in step (4) is calculated according to 8 - 9% of the volume of the seed oil 4.

4. The preparation method of the citrus seed oil rich in limonin and the defatted citrus seed meal according to claim 3, characterized in that: The pH value of the enzymatic hydrolysis system described in step (1) is 5.5; The addition amount of the solid citric acid described in step (2) is calculated according to 0.06% - 0.07% of the mass of the seed oil 1; The addition amount of the distilled water described in step (3) is calculated according to 3% of the volume of the seed oil 2; The addition amount of the alkali solution described in step (3) is calculated according to 0.14 - 0.15% of the volume of the seed oil 3.

5. The preparation method of the citrus seed oil rich in limonin and the defatted citrus seed meal according to claim 1, characterized in that: The preheating temperature of the seed oil 2 described in step (3) is 60 - 80 °C; The conditions of the constant-temperature stirring described in step (3) are: temperature 60 - 80 °C, rotation speed 80 - 120 r / min, time 15 - 30 min; The preheating temperature of the seed oil 3 described in step (3) is 60 - 80 °C; The conditions of adding the alkali solution and stirring described in step (3) are: temperature 60 - 80 °C, rotation speed 80 - 120 r / min, time 15 - 30 min; The preheating temperature described in step (4) is 60 - 80 °C; The temperature of the water bath heating described in step (4) is 75 - 85 °C; The conditions of the cooling crystallization described in step (4) are cooling to 4 - 10 °C, and the crystallization time is 12 - 48 h.

6. The preparation method of the citrus seed oil rich in limonin and the defatted citrus seed meal according to claim 5, characterized in that: The conditions of the enzymatic hydrolysis described in step (1) are as follows: power is 400 - 600 W, temperature is 45 - 55 °C, and time is 1 - 3 h; The conditions of the ultrasonic treatment described in step (2) are as follows: power is 400 - 500 W, temperature is 45 - 50 °C, and time is 0.5 - 1 h; The preheating temperature of the seed oil 2 described in step (3) is 70 °C; The conditions of the constant-temperature stirring described in step (3) are: temperature 70 °C, rotation speed 100 r / min, time 25 - 30 min; The preheating temperature of the seed oil 3 described in step (3) is 70 °C; The conditions of adding the alkali solution and stirring described in step (3) are: temperature 70 °C, rotation speed 100 r / min, time 25 - 30 min; The preheating temperature described in step (4) is 70 °C; The temperature of the water bath heating described in step (4) is 80 °C.

7. The preparation method of the citrus seed oil rich in limonin and the defatted citrus seed meal according to claim 1, characterized in that: The drying method described in step (1) is sun drying or drying; The drying degree described in step (1) is drying to a moisture content not higher than 8%; The crushing degree described in step (2) is being able to pass through a sieve of 80 - 100 mesh; The first solid-liquid separation method described in step (2) is centrifugation or filtration; The second solid-liquid separation method described in step (3) is centrifugation or filtration; The third solid-liquid separation method described in step (3) is centrifugation or filtration; The conditions for stirring described in step (4) are: temperature 70 - 80 °C, stirring speed 300 - 350 r / min, time 30 - 90 min; The fourth solid-liquid separation method described in step (4) is centrifugation or filtration.

8. A citrus seed oil rich in limonin and a defatted citrus seed meal, characterized in that: Obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the citrus seed oil rich in limonin and the defatted citrus seed meal according to claim 8, characterized in that: The use of the citrus seed oil rich in limonin in the preparation of pharmaceuticals, health foods, and beauty products; The use of the defatted citrus seed meal in feed raw materials or feed additives.

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