Modified sea buckthorn fruit oil as well as preparation method and application thereof

Abstract

The invention relates to the technical field of deep processing of fructus hippophae, and discloses modified fructus hippophae oil and a preparation method and application thereof.The preparation method of the modified fructus hippophae oil comprises the steps that fructus hippophae oil and fish oil react for 8-10 h at the temperature of 48-55 DEG C under the catalytic action of Novozym 435, and liquid obtained after filtering is the modified fructus hippophae oil; wherein the mass ratio of the sea buckthorn fruit oil to the fish oil to the Novozym 435 is (1.5-1.3): 1: (0.184-0.25). The sea buckthorn fruit oil and the deep sea fish oil rich in EPA / DHA are used as substrates, and the modified sea buckthorn fruit oil rich in omega-3 fatty acid is obtained under the catalytic action of Novozym435, so that the daily requirements of consumers are met.

Description

Technical Field

[0001] The invention belongs to the technical field of deep processing of seabuckthorn fruit, and particularly relates to modified seabuckthorn fruit oil and a preparation method and application thereof. Background Art

[0002] The current market demand for seabuckthorn is increasing rapidly, among which the market demand for seabuckthorn seed oil and seabuckthorn fruit oil is strong, and the products are in short supply. In recent years, seabuckthorn fruit oil has attracted much attention from domestic and foreign researchers. At present, domestic and foreign scholars have fully studied the biological activity of seabuckthorn fruit oil.

[0003] However, the essential fatty acid content of the seabuckthorn fruit oil in the natural state, that is, the seabuckthorn fruit oil extracted by squeezing, solvent extraction, supercritical extraction, etc., is low. Omega-3 fatty acids are important essential fatty acids, and their main components are EPA and DHA. Therefore, the total content of EPA and DHA is used to evaluate the content of omega-3 fatty acids in the seabuckthorn fruit oil. However, the total content of EPA and DHA in the seabuckthorn fruit oil obtained by the prior art is relatively low, only 1.16%. The polyunsaturated fatty acids in the seabuckthorn fruit oil are converted into essential fatty acids such as α-linolenic acid and linoleic acid in the body. The pathway is complex and the amount is extremely small. Therefore, the seabuckthorn fruit oil obtained by the existing preparation method cannot effectively increase the content of essential fatty acids. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a modified seabuckthorn fruit oil and a preparation method and application thereof.

[0005] A method for preparing modified seabuckthorn fruit oil comprises the following steps:

[0006] The seabuckthorn fruit oil and fish oil are mixed, reacted at 48°C to 55°C for 8h to 10h under the catalytic effect of Novozym 435, and the precipitate is removed by filtration to obtain the modified seabuckthorn fruit oil;

[0007] The mass ratio of sea buckthorn berry oil, fish oil and Novozym 435 is 1.5-1.3:1:0.184-0.25.

[0008] The invention can maximize the amount of EPA and DHA incorporated into the seabuckthorn oil and the fish oil under the catalytic action of Novozym 435, thereby increasing the content of essential fatty acid ω-3 fatty acid in the modified seabuckthorn oil, so that the content of ω-3 fatty acid in the obtained modified seabuckthorn oil reaches 44.8%.

[0009] Preferably, the enzyme activity of Novozym 435 is 10000 PLU / g. The EPA / DHA incorporation rate of the transesterification reaction catalyzed by Novozym 435 is the highest.

[0010] Preferably, the preparation method of the seabuckthorn fruit oil is:

[0011] Squeeze seabuckthorn fruit to obtain seabuckthorn puree;

[0012] The seabuckthorn puree is mixed with a composite enzyme, subjected to ultrasonic enzymolysis, centrifuged, and the supernatant is collected to obtain the seabuckthorn fruit oil;

[0013] The mass of the complex enzyme accounts for 0.6% to 0.8% of the mass of the seabuckthorn puree.

[0014] Preferably, the complex enzyme is compounded by pectinase and cellulase in a mass ratio of 1:0.3 to 0.4.

[0015] Preferably, the conditions of the ultrasonic enzymolysis are as follows:

[0016] The temperature is 48℃~52℃, the ultrasonic power is 155W~165W, and the time is 4h~6h.

[0017] Preferably, the centrifugation is performed twice;

[0018] The first centrifugation condition is 3000r / min~4000r / min for 20min~30min;

[0019] The second centrifugation condition is 7000r / min~8000r / min for 10min~20min.

[0020] The second object of the present invention is to provide a modified seabuckthorn fruit oil prepared by the preparation method provided by the first aspect of the present invention.

[0021] The third object of the present invention is to provide a use of the modified seabuckthorn fruit oil in the preparation of antioxidant products.

[0022] The fourth object of the present invention is to provide the use of the modified seabuckthorn fruit oil in the preparation of immunity-enhancing products.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention uses seabuckthorn fruit oil and fish oil rich in EPA / DHA as substrates, and obtains modified seabuckthorn fruit oil rich in ω-3 fatty acids under the catalytic action of Novozym435, wherein the content of ω-3 fatty acids reaches 44.8%, which is significantly increased compared with the 1.16% of ω-3 fatty acids in natural seabuckthorn fruit oil in the prior art, i.e., unmodified seabuckthorn fruit oil. Thus, a seabuckthorn fruit oil rich in essential fatty acids can be obtained.

[0025] (2) The modified seabuckthorn fruit oil prepared by the present invention has improved in vitro antioxidant activity; the modified seabuckthorn fruit oil significantly increases the spleen index and the phagocytic activity of macrophages, thereby improving immunity, providing a new application range for the modified seabuckthorn fruit oil. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A is the effect of different single factors on the yield of sea buckthorn oil; A is the effect of different ultrasonic powers on the yield of sea buckthorn oil; B is the effect of different ultrasonic times on the yield of sea buckthorn oil; C is the effect of different enzymatic hydrolysis temperatures on the yield of sea buckthorn oil; D is the effect of different complex enzyme addition amounts on the yield of sea buckthorn oil.

[0027] Figure 2 DPPH of sea buckthorn fruit oil + Free radical scavenging ability results graph.

[0028] Figure 3 ABTS for Seabuckthorn Berry Oil + Figure 2. Results of free radical scavenging ability assay.

[0029] Figure 4 This is a graph showing the effects of different lipases on modified sea buckthorn fruit oil.

[0030] Figure 5 This is a graph showing the influence of different factors on the EPA / DHA incorporation rate in modified sea buckthorn fruit oil.

[0031] Figure 6 This is the main fatty acid composition diagram of modified sea buckthorn fruit oil.

[0032] Figure 7 DPPH of seabuckthorn oil, fish oil and modified seabuckthorn oil + Free radical scavenging ability results graph.

[0033] Figure 8 ABTS for sea buckthorn oil, fish oil, and modified sea buckthorn oil + Free radical scavenging ability results graph.

[0034] Fig. 9 The spleen index of mice after treatment in different groups.

[0035] Fig.10 The graph shows the phagocytic activity of mouse macrophages after treatment with different groups. DETAILED DESCRIPTION

[0036] The following will be combined with the specific implementation of the present invention to clearly and completely describe the technical solution in the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The methods described in the embodiments of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0038] Fish oil was purchased from Beijing Zhuoyuan Hangcheng Technology Development Co., Ltd.

[0039] Lipozyme TL IM, Lipozyme RM IM, and Novozym 435 were all purchased from Shanghai Yuanye Biotechnology Co., Ltd. In the following examples, the enzyme activities of Novozym 435, Lipozyme RM IM, and Lipozyme TL IM were all 10,000 PLU / g.

[0040] Male albino laboratory mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0041] Example 1

[0042] A method for preparing modified seabuckthorn fruit oil comprises the following steps:

[0043] S1. Wash the fresh seabuckthorn fruits and drain until there is no obvious moisture on the surface, then squeeze the juice at 18000r / min for 30s to obtain seabuckthorn puree.

[0044] S2. Mix the sea buckthorn puree with a composite enzyme containing 0.6% of the mass of the sea buckthorn puree, perform ultrasonic enzymolysis at 50°C and 160W for 5 hours, and then centrifuge twice; wherein, the first centrifugation is performed at 3500r / min for 15 minutes, and the impurities are filtered out and the upper clear oil is absorbed; the upper clear oil is centrifuged for a second time at 7500r / min for 20 minutes to remove plant fibers and obtain sea buckthorn fruit oil; wherein, the composite enzyme is compounded by pectinase and cellulase in a mass ratio of 1:0.4.

[0045] S3. Mix sea buckthorn berry oil and fish oil, react at 50° C. for 8 hours under the catalytic action of Novozym 435, and filter the resulting liquid to obtain modified sea buckthorn berry oil; wherein the mass ratio of sea buckthorn berry oil, fish oil and Novozym 435 is 1.5:1:0.184.

[0046] Example 2

[0047] A method for preparing modified seabuckthorn fruit oil comprises the following steps:

[0048] S1. Wash and drain the fresh seabuckthorn fruit until there is no obvious moisture on the surface, and then squeeze the juice at 18000r / min for 30s to obtain seabuckthorn puree.

[0049] S2. Mix the sea buckthorn puree with a composite enzyme of 0.8% of the mass of the sea buckthorn puree, perform ultrasonic enzymolysis at 48°C and 155W for 4 hours, and then centrifuge twice; wherein, the first centrifugation is performed at 3000r / min for 30min, and impurities are filtered out and the upper clear oil is absorbed; the upper clear oil is centrifuged for a second time at 7000r / min for 20min to remove plant fibers and obtain sea buckthorn fruit oil; wherein, the composite enzyme is compounded by pectinase and cellulase in a mass ratio of 1:0.4.

[0050] S3. Mix seabuckthorn berry oil and fish oil, react at 55° C. for 9 hours under the catalytic action of Novozym 435, and filter the resulting liquid to obtain modified seabuckthorn berry oil; wherein the mass ratio of seabuckthorn berry oil, fish oil and Novozym 435 is 1.3:1:0.25.

[0051] Example 3

[0052] A method for preparing modified seabuckthorn fruit oil comprises the following steps:

[0053] S1. Wash the fresh seabuckthorn fruits and drain until there is no obvious moisture on the surface, then squeeze the juice at 18000r / min for 30s to obtain seabuckthorn puree.

[0054] S2. Mix the sea buckthorn puree with a composite enzyme containing 0.7% of the mass of the sea buckthorn puree, and perform ultrasonic enzymolysis at 52°C and 165W for 4 hours, then centrifuge twice; wherein, the first centrifugation is performed at 4000r / min for 20min, and the impurities are filtered out, and the upper clear oil liquid is absorbed; the upper clear oil liquid is centrifuged for a second time at 8000r / min for 10min to remove the plant fibers and obtain sea buckthorn fruit oil; wherein, the composite enzyme is pectinase and cellulase in a mass ratio of 1:0.3.

[0055] S3. Mix the sea buckthorn berry oil and the fish oil, react at 55° C. for 10 hours under the catalytic action of Novozym 435, and filter the resulting liquid to obtain modified sea buckthorn berry oil; wherein the mass ratio of the sea buckthorn berry oil, the fish oil and Novozym 435 is 1.5:1:0.25.

[0056] Example 4

[0057] A method for preparing modified seabuckthorn fruit oil comprises the following steps:

[0058] S1. Wash the fresh seabuckthorn fruits and drain until there is no obvious moisture on the surface, then squeeze the juice at 18000r / min for 30s to obtain seabuckthorn puree.

[0059] S2. Mixing the sea buckthorn puree with a composite enzyme containing 0.6% of the mass of the sea buckthorn puree, and subjecting the mixture to ultrasonic enzymolysis at 50°C and 160W for 5 hours, centrifuging the mixture twice; wherein the first centrifugation is performed at 3500r / min for 15 minutes, filtering to remove impurities, and absorbing the upper clear oil liquid; the upper clear oil liquid is subjected to a second centrifugation at 7500r / min for 15 minutes to remove plant fibers and obtain the sea buckthorn fruit oil; wherein the composite enzyme is compounded by pectinase and cellulase in a mass ratio of 1:0.4.

[0060] S3, mixing seabuckthorn berry oil and fish oil, reacting at 50° C. for 8 hours under the catalytic action of Novozym 435, and filtering the resulting liquid to obtain the modified seabuckthorn berry oil; wherein the mass ratio of seabuckthorn berry oil, fish oil and Novozym 435 is 1.5:1.

[0061] Example 5

[0062] A method for preparing modified seabuckthorn fruit oil comprises the following steps:

[0063] S1. Wash the fresh seabuckthorn fruits and drain until there is no obvious moisture on the surface, then squeeze the juice at 18000r / min for 30s to obtain seabuckthorn puree.

[0064] S2. Mix the sea buckthorn puree with a composite enzyme containing 0.6% of the mass of the sea buckthorn puree, and perform ultrasonic enzymolysis at 50°C and 160W for 4 hours, then centrifuge twice; wherein, the first centrifugation is performed at 3500r / min for 20min, and impurities are filtered out, and the upper clear oil liquid is absorbed; the upper clear oil liquid is centrifuged for a second time at 7500r / min for 20min to remove plant fibers and obtain sea buckthorn fruit oil; wherein, the composite enzyme is compounded by pectinase and cellulase in a mass ratio of 1:0.4.

[0065] S3, mixing seabuckthorn berry oil and fish oil, reacting at 50°C for 8 hours under the catalytic action of Novozym 435, and filtering the resulting liquid to obtain the modified seabuckthorn berry oil; wherein the mass ratio of seabuckthorn berry oil, fish oil and Novozym 435 is 1.5:1:0.25.

[0066] Comparative Example 1

[0067] A method for preparing modified seabuckthorn fruit oil comprises the following steps:

[0068] S1. Wash and drain the fresh seabuckthorn fruit until there is no obvious moisture on the surface, and then squeeze the juice at 18000r / min for 30s to obtain seabuckthorn puree;

[0069] S2, 0.6% of the mass of seabuckthorn puree is mixed with the composite enzyme and the seabuckthorn puree, and ultrasonic enzymolysis is performed at 50°C and 160W for 5 hours, followed by two centrifugations; wherein the first centrifugation is performed at 3500r / min for 20 minutes, and the impurities are filtered out and the upper clear oil is absorbed; the upper clear oil is subjected to a second centrifugation at 7500r / min for 20 minutes to remove the plant fibers and obtain seabuckthorn fruit oil. wherein the composite enzyme is compounded by pectinase and cellulase in a mass ratio of 1:0.4.

[0070] S3, mixing seabuckthorn fruit oil and fish oil, reacting at 50°C for 8 hours under the catalytic action of Lipozyme TL IM, and filtering the resulting liquid to obtain the modified seabuckthorn fruit oil; wherein the mass ratio of seabuckthorn fruit oil, fish oil and Lipozyme TL IM is 1.5:1:0.25.

[0071] Comparative Example 2

[0072] A method for preparing modified seabuckthorn fruit oil comprises the following steps:

[0073] S1. Wash the fresh seabuckthorn fruits and drain until there is no obvious moisture on the surface, then squeeze the juice at 18000r / min for 30s to obtain seabuckthorn puree.

[0074] S2, 0.6% of the mass of the seabuckthorn puree is mixed with the seabuckthorn puree, and the mixture is subjected to ultrasonic enzymolysis at 50°C and 160W for 4 hours, followed by two centrifugations; wherein the first centrifugation is carried out at 3500r / min for 15 minutes, and impurities are removed by filtration, and the upper layer of clear oil is absorbed; the upper layer of clear oil is subjected to a second centrifugation at 7500r / min for 20 minutes to remove plant fibers and obtain seabuckthorn fruit oil. The compound enzyme is compounded by pectinase and cellulase in a mass ratio of 1:0.4.

[0075] S3, mixing seabuckthorn berry oil and fish oil, reacting at 50° C. for 8 hours under the catalytic action of Lipozyme RM IM, filtering lipase to obtain the modified seabuckthorn berry oil; wherein the mass ratio of seabuckthorn berry oil, fish oil and Lipozyme RM IM is 1.5:1:0.25.

[0076] In order to further demonstrate the effect of the modified seabuckthorn fruit oil of the present invention, the following experiments were conducted.

[0077] 1. Optimization of seabuckthorn oil extraction conditions

[0078] 1) Extraction of single factor experiment

[0079] A single factor experiment was used to investigate the effects of different ultrasonic powers: 40W, 80W, 120W, 160W, 200W; ultrasonic enzymatic hydrolysis time: 2h, 3h, 4h, 5h, 6h; enzymatic hydrolysis temperature: 30℃, 40℃, 50℃, 60℃, 70℃; total amount of complex enzyme added: 1.4%, 0.6%, 0.8%, 1.0%, 1.2% on the yield of sea buckthorn oil.

[0080] Figure 1 The effect of different factors on the yield of seabuckthorn fruit oil. Figure 1 In A, as the ultrasonic power increases, the yield of seabuckthorn fruit oil first increases and then decreases, and the yield is the highest at 160W, so 160W is selected as the optimal ultrasonic power; Figure 1 In B, as the ultrasonic enzymatic hydrolysis time increased, the yield of seabuckthorn fruit oil increased and tended to be flat after 4 hours, so 4 hours was selected as the optimal ultrasonic enzymatic hydrolysis time; Figure 1 The C of seabuckthorn fruit oil increased with the increase of enzyme loading ratio, and tended to be flat after 0.6%, so 0.6% was selected as the optimal value; Figure 1 In D, with the increase of enzymatic hydrolysis temperature, the yield of sea buckthorn oil first increased and then decreased. At 50℃, the yield of sea buckthorn oil was the highest, so 50℃ was selected as the optimal enzymatic hydrolysis temperature.

[0081] 2) Optimization of ultrasound-assisted seabuckthorn oil extraction process

[0082] Based on the results of the single factor test, according to the Box-Behnkendesign of the response surface method, abbreviated as BBD test, the ultrasonic power: A, ultrasonic enzymatic hydrolysis time: B and enzymatic hydrolysis temperature: C, which have a significant effect on the yield of seabuckthorn fruit oil, were selected as independent variables to investigate the process conditions of ultrasonic-assisted extraction of seabuckthorn fruit oil. The factors and levels of the test are shown in Table 1, the response surface test design and results are shown in Table 2, and the variance analysis results are shown in Table 3.

[0083] Table 1 Response surface design factors and levels

[0084] level A Ultrasonic power / W B Ultrasonic enzymatic hydrolysis time / h C enzymatic hydrolysis temperature / ℃ -1 120 3 30 0 160 4 40 1 200 5 50

[0085] Table 2 Response surface experimental design and results

[0086]

[0087]

[0088] Table 3 Results of variance analysis

[0089]

[0090]

[0091] 3)DPPH +Free radical scavenging ability

[0092] The seabuckthorn fruit oil extracted in Example 5 was subjected to DPPH + Free radical scavenging ability, results such as Figure 2 As shown. Figure 2 It can be seen that within the concentration range of 2mg / mL to 6mg / mL, the DPPH of seabuckthorn fruit oil + The free radical scavenging rate increases with the concentration of seabuckthorn oil. In the concentration range of 6mg / mL to 20mg / mL, the DPPH + The free radical scavenging rate gradually slowed down and the scavenging rate was close to 100%. + The free radical scavenging rate was always lower than that of vitamin C. Within the selected concentration range, DPPH + There is no significant change in the free radical scavenging rate. Therefore, sea buckthorn fruit oil has a good DPPH + Free radical scavenging ability, can act as a natural antioxidant.

[0093] 4) ABTS + Free radical scavenging ability assay

[0094] The seabuckthorn fruit oil extracted in Example 5 was subjected to ABTS + Free radical scavenging ability, results such as Figure 3 As shown. Figure 3 It can be seen that at 2mg / mL to 16mg / mL, ABTS + The free radical scavenging rate increased slowly. In the range of 16mg / mL-20mg / mL, ABTS + The free radical scavenging rate increased rapidly and was close to 100%. Within the same concentration range, the ABTS of seabuckthorn fruit oil + The free radical scavenging rate was always lower than that of vitamin C. Within the selected concentration range of vitamin C, ABTS + There was no significant change in the free radical scavenging rate. + It has a good free radical scavenging effect, indicating that sea buckthorn oil has the potential to be a natural antioxidant.

[0095] 5) Determination of acid value

[0096] The seabuckthorn fruit oil extracted in Examples 1 to 5 was subjected to acid value determination. The acid value determination was carried out according to the first method cold solvent indicator titration method in GB5009.229-2016. A clean 250 mL conical flask was taken and the prepared oil sample was weighed with a balance. Its mass m was in grams. 50 mL of ether-isopropanol mixture and 3 drops of thymephthalein indicator were added and the sample was fully shaken to dissolve. The sample solution was then manually titrated with a graduated burette containing a standard titration solution. When the sample solution initially showed a slightly reddish color and there was no obvious fading within 15 seconds, it was the end point of the titration. Stop the titration immediately and record the number of milliliters of the standard titration solution consumed by the titration. This value is V.

[0097] 6) Determination of peroxide value

[0098] The sea buckthorn fruit oil extracted in Examples 1 to 5 was subjected to peroxide value determination. The peroxide value determination was based on the first titration method in GB5009.227-2016. 2 g of the sample prepared in the experiment was weighed to an accuracy of 0.001 g, placed in a 250 mL iodine volumetric flask, and 30 mL of chloroform-glacial acetic acid mixture was added. The sample was gently shaken to completely dissolve. 1.00 mL of saturated potassium iodide solution was accurately added, the bottle cap was tightly plugged, and the mixture was gently shaken for 0.5 min and placed in a dark place for 3 min. 100 mL of water was taken out and added. After shaking, sodium thiosulfate standard solution was used immediately. When the peroxide value was estimated to be 0.15 g / 100 g or less, 0.002 mol / L standard solution was used; when the peroxide value was estimated to be greater than 0.15 g / 100 g, 0.01 mol / L standard solution was used to titrate the precipitated iodine. When the titration was to light yellow, 1 mL of starch indicator was added, and the titration was continued and strongly shaken until the blue color of the solution disappeared as the end point. At the same time, a blank test is performed. The volume V0 of 0.01 mol / L sodium thiosulfate solution consumed in the blank test shall not exceed 0.1 mL.

[0099] 7) Determination of saponification value

[0100] The seabuckthorn fruit oil extracted in Examples 1 to 5 was subjected to saponification value determination. 25.0 mL of potassium hydroxide-ethanol solution was added to the seabuckthorn fruit oil in Examples 1 to 5 with a pipette, and a boiling aid was added. The reflux condenser was connected to a conical flask, and the conical flask was placed on a heating device and slowly boiled, with occasional shaking, and the oil maintained in a boiling state for 60 minutes. For high melting point oils and samples that are difficult to saponify, boil for 2 hours. Add 0.5 mL of phenolphthalein indicator to the hot solution, and titrate with a hydrochloric acid standard solution until the pink color of the indicator just disappears. If the saponified solution is dark, use 0.5 mL to 1 mL of alkaline blue 6B solution as an indicator.

[0101] Blank test According to the above method, without adding sea buckthorn fruit oil in Examples 1 to 5, a blank test was performed with 25.0 ml of potassium hydroxide-ethanol solution. The saponification value was determined with reference to GB / T5534-2008. The iodine value was determined with reference to GB / T 5532-2008. The weighing dish containing the sample was placed in a 500 mL conical flask. The sample was dissolved by adding the volume of solvent according to the sample amount. 25 mL of Wei's reagent was accurately added with a pipette, the stopper was covered, and the conical flask was placed in a dark place after shaking. Except for the addition of the sample, a blank solution was made. The conical flask should be placed in a dark place for 1 hour. After the specified reaction time is reached, 20 mL of potassium iodide solution and 150 mL of water were added. Titrate with a calibrated sodium thiosulfate standard solution until the yellow color of iodine almost disappears. Add a few drops of starch solution and continue titrating. Shake the conical flask vigorously while titrating until the blue color just disappears.

[0102] Since the acid value, peroxide value, saponification value and iodine value of the seabuckthorn fruit oil prepared in Examples 1 to 4 are substantially consistent with those of the seabuckthorn fruit oil prepared in Example 5, the above-mentioned determination results of the seabuckthorn fruit oil prepared in Example 5 are taken as the results.

[0103] Table 4 Properties of seabuckthorn fruit oil in Example 5

[0104] Acid value mg / g Peroxide value mmoL / kg Saponification value mg / g Iodine value g / 100g 1.30±0.02 9.18±0.53 191.62±1.35 121.68±2.18

[0105] 2. Modified sea buckthorn fruit oil

[0106] 1) The in vitro antioxidant activity, spleen index, and macrophage phagocytic activity of the modified seabuckthorn fruit oils prepared in Examples 1 to 5 are consistent. Only the modified seabuckthorn fruit oil prepared in Example 5 is tested below.

[0107] Figure 4 For lipase screening comparison. Figure 4 It can be seen that within 0h to 8h, under the catalysis of Novozym 435, LipozymeRM IM and Lipozyme TL IM, the EPA / DHA content in the modified sea buckthorn fruit oil is constantly increasing, and the EPA / DHA incorporation rate of the ester exchange reaction catalyzed by Novozym 435 is the highest. As time goes by, the reaction of Novozym 435 and LipozymeRM IM has reached a dynamic equilibrium, and the ester exchange reaction of Lipozyme TL IM also tends to equilibrium after 16h. Compared with the three lipases, Novozym 435 has the highest efficiency, so Novozym 435 is selected as the optimal lipase.

[0108] 2) Single factor test of modified sea buckthorn fruit oil

[0109] Mix sea buckthorn fruit oil and fish oil with different mass ratios and Novozyme 435 in a 500mL flask. Place the flask in a constant temperature oil bath and stir the reaction for a certain period of time under nitrogen protection. Filter and remove the lipase to obtain the crude transesterification product, which is stored at -20℃ for further analysis. The recovered lipase is washed with petroleum ether, evaporated to remove the solvent and stored at low temperature. The results are as follows: Figure 5 shown.

[0110] Figure 5 This is the effect of different factors on the EPA / DHA incorporation rate in modified seabuckthorn fruit oil. Figure 5 A is the effect of reaction time on the incorporation rate of EPA / DHA in modified seabuckthorn fruit oil. As time goes by, the incorporation rate increases and reaches the highest at 8 hours. Figure 5 B is the effect of reaction temperature on the EPA / DHA incorporation rate in modified seabuckthorn fruit oil. As the reaction temperature increases, the incorporation rate increases and reaches the highest at 50°C. Figure 5 C is the effect of enzyme loading on the EPA / DHA incorporation rate in modified seabuckthorn fruit oil. With the increase of enzyme loading, the incorporation rate increases, and reaches the highest at an enzyme loading of 10%. Figure 5 D is the effect of different mass ratios on the EPA / DHA incorporation rate in modified seabuckthorn fruit oil. As the mass ratio of fish oil increases, the incorporation rate increases and reaches the highest at 1.5:1.

[0111] 3) Fatty acid analysis

[0112] Fatty acid analysis was performed using a gas chromatography system, denoted as GC, from Thermo, Trace 1300, with autosampler; mass spectrometer, from Thermo Scientific, ISQ 7000; capillary gas chromatography column: specification TG WAX 30m×0.25mm pore diameter×0.25μm; flame ionization detector denoted as FID. Fatty acid methyl esters were prepared according to the official method Ce 2-66 (2009), and the GC analysis scheme was as follows: the column oven temperature was maintained at 50℃ for 1 minute, then increased to 200℃ at a rate of 25℃ / min, and then increased to 230℃ at an increase of 5℃ / min and maintained for 15 minutes. The temperature of the injector and FID detector was set to 250℃ and 280℃. The analysis lasted for 28min. The helium flow rate was 1.0mL / min, the split ratio was 10, and the injection volume was 1μL. All fatty acids were identified by comparing the retention time with the standard solution of fatty acid methyl esters, which was recorded as FAME. All fatty acids were identified by comparing the retention time with the mixed standard of FAME. The relative content of fatty acids was calculated by area normalization. The results are shown in Figure 6 shown.

[0113] Figure 6 The main fatty acid composition diagram of modified seabuckthorn fruit oil. Figure 6 It can be seen that after the ester exchange reaction is completed, the main fatty acid components in the modified sea buckthorn fruit oil are measured, essential fatty acids: C22:5 (33.65%), C18:2 (15.99%), C22:6 (11.15%); non-essential fatty acids: C16:1 (12.33%), C16:0 (11.63%); EPA (C20:5) and DHA (C22:6) The total content is 44.8%, and EPA and DHA are the main components of ω-3 fatty acids. It can be seen that the ω-3 fatty acid content in the modified sea buckthorn fruit oil reaches 44.8%.

[0114] 4) Effect of modified seabuckthorn fruit oil on DPPH + Free Radicals and ABTS + Free radical scavenging ability

[0115] DPPH analysis of sea buckthorn berry oil, fish oil and modified sea buckthorn berry oil + Free radical scavenging ability test, the results are as follows Figure 7 DPPH of modified sea buckthorn fruit oil + The free radical scavenging ability was significantly higher than that of unmodified sea buckthorn fruit oil, p<0.05.

[0116] ABTS analysis of sea buckthorn berry oil, fish oil and modified sea buckthorn berry oil + Free radical scavenging ability test, the results are as follows Figure 8 ABTS modified sea buckthorn fruit oil + The free radical scavenging ability was significantly higher than that of unmodified sea buckthorn fruit oil, p<0.05.

[0117] pass Figure 7 and Figure 8 The results show that the modified seabuckthorn fruit of the present invention has a good antioxidant effect and can be used to prepare antioxidant products.

[0118] 5) Animal breeding, maintenance and design:

[0119] The seabuckthorn oil group was marked as SBO, the fish oil group was marked as FO, the modified seabuckthorn oil group was marked as SL, and the cyclophosphamide model group was marked as CTX.

[0120] Twenty-five 6-week-old male albino laboratory mice were housed in the Specific Pathogen-Free, Animal Center of Xinjiang Agricultural University, recorded as SPF. After 7 days of acclimation, they were randomly divided into 5 groups, namely, Ctrl, CTX, CTX+FO, CTX+SBO, and CTX+SL. Except for the Ctrl group, all other mice were injected intraperitoneally with 80 mg / kg of cyclophosphamide on days 1, 2, 3, 8, 10, 12, 15, 17, and 19, and the Ctrl group was injected with normal saline. During the experiment, mice in the Ctrl and CTX groups were given normal saline once a day, while mice in the CTX+FO, CTX+SBO, and CTX+SL groups were given 10 ml / kg of fish oil, sea buckthorn fruit oil, and modified sea buckthorn fruit oil, respectively, once a day.

[0121] Spleen index: After the mice were sacrificed, the spleen was separated and the index was calculated as follows:

[0122]

[0123] Depend on Fig. 9 It can be seen that compared with the CTX group, the use of modified sea buckthorn fruit oil can significantly increase the spleen index of mice, p<0.05. The increase in the spleen index helps promote the development of the immune system and thus improve immunity.

[0124] 5) Macrophage phagocytic activity test:

[0125] After killing the mice, macrophages were collected by intraperitoneal injection of 1 mL of cold phosphate buffered saline solution, then added to a culture dish and incubated at 37°C in a 5% CO2 incubator for 2 hours. The cells were washed once with a balanced salt solution, and the attached macrophages were separated for later use. The separated cells were washed once with a balanced salt solution, and then the macrophage suspension was added to a 96-well plate, and 5×10 4 Each well was stained with 0.1% neutral red for 2 hours and then washed twice with balanced salt solution. Then, cell lysis buffer, i.e., ethanol and 0.01% acetic acid, was added at a ratio of 1:1, 100 μL per well to lyse the cells. The absorbance was read at 550 nm using a multifunctional microplate reader.

[0126] Depend on Fig.10 It can be seen that compared with the CTX group, the use of modified seabuckthorn fruit oil can significantly increase the phagocytic activity of mouse macrophages, p<0.05. The phagocytic activity of macrophages can directly eliminate pathogens in the body, such as bacteria and viruses, thereby effectively preventing the occurrence and spread of infection and enhancing immunity.

[0127] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0128] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing modified seabuckthorn fruit oil, characterized in that: The following steps are involved: The seabuckthorn fruit oil and fish oil are mixed, reacted at 48°C to 55°C for 8h to 10h under the catalytic effect of Novozym 435, and the precipitate is removed by filtration to obtain the modified seabuckthorn fruit oil; The mass ratio of sea buckthorn berry oil, fish oil and Novozym 435 is 1.5-1.3:1:0.184-0.

25.

2. The method for preparing modified seabuckthorn fruit oil according to claim 1, characterized in that: The enzyme activity of Novozym 435 is 10000 PLU / g.

3. The method for preparing modified seabuckthorn fruit oil according to claim 1, characterized in that: The preparation method of the seabuckthorn fruit oil is: Squeeze seabuckthorn fruit to obtain seabuckthorn puree; Mixing seabuckthorn puree with complex enzyme, performing ultrasonic enzymolysis, centrifuging, taking supernatant, and obtaining the seabuckthorn fruit oil; The mass of the complex enzyme accounts for 0.6% to 0.8% of the mass of the seabuckthorn puree.

4. The method for preparing modified seabuckthorn fruit oil according to claim 3, characterized in that: The complex enzyme is prepared by compounding pectinase and cellulase in a mass ratio of 1:0.3-0.

4.

5. The method for preparing modified seabuckthorn fruit oil according to claim 3, characterized in that: The conditions of the ultrasonic enzymolysis are as follows: The temperature is 48℃~52℃, the ultrasonic power is 155W~165W, and the time is 4h~6h.

6. The method for preparing modified seabuckthorn fruit oil according to claim 3, characterized in that: The number of centrifugation is twice; The first centrifugation condition is 3000r / min~4000r / min for 20min~30min; The second centrifugation condition is 7000r / min~8000r / min for 10min~20min.

7. Modified seabuckthorn fruit oil obtained according to the preparation method according to any one of claims 1 to 6.

8. Use of the modified seabuckthorn fruit oil according to claim 7 in the preparation of antioxidant products.

9. Use of the modified seabuckthorn fruit oil according to claim 7 in preparing a product for improving immunity.

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