Preparation method and application of Rana sylvatica egg oil

Abstract

The present application provides a method for preparing forest frog egg oil and its application, the preparation method comprising the steps of crushing, microwave-assisted extraction, β-cyclodextrin embedding to remove cholesterol, etc. The preparation method of the present application can effectively remove the cholesterol from forest frog egg oil while maintaining its antioxidant and lipid-lowering activities, and the time required is short and the cost is low.

Description

Technical Field

[0001] The present application belongs to the field of deep processing of health products and traditional Chinese medicines. Specifically, the present application provides a preparation method of forest frog egg oil and its application. Background Art

[0002] Rana sylvaticum is a traditional health food and medicine in my country. Its meat and oviduct (Rana sylvaticum oil) have high medicinal and economic value, but its eggs are not fully utilized in the processing of Rana sylvaticum products and are even discarded as waste in many cases.

[0003] In recent years, with the in-depth research on forest frog related products, attempts have been made to process forest frog eggs into forest frog egg oil products. Studies have shown that it contains a large amount of high-value fatty acids, estradiol, fat-soluble vitamins and other ingredients. It has antioxidant, blood lipid regulation, anti-depression and other functions, and has high value for the treatment and health care of patients with cardiovascular and cerebrovascular diseases, hyperlipidemia and other groups.

[0004] Although Rana oleifera oil has the above-mentioned medical and health functions, its cholesterol content is very high, more than 10 times that of egg yolk, a recognized high-cholesterol food. Long-term use as a health product is a great health hazard. Therefore, removing cholesterol from Rana oleifera oil and maintaining the effective ingredients in Rana oleifera oil as much as possible is an important part of optimizing the preparation method of Rana oleifera oil. Summary of the invention

[0005] To solve the above problems:

[0006] On the one hand, the present application provides a method for preparing Rana sylvatica egg oil, the preparation method comprising:

[0007] (1) crushing the Rana sylvatica eggs and sieving to obtain Rana sylvatica egg powder;

[0008] (2) Add 10 g of Rana sylvatica egg powder into 80-100 mL of anhydrous ethanol and mix well;

[0009] (3) 400-1000w microwave-assisted extraction for 2-5 hours;

[0010] (4) Allow to settle naturally for 0.5-2 hours and filter to obtain the supernatant;

[0011] (5) Repeat steps (3) and (4) once;

[0012] (6) combining the supernatants obtained in the two (4) steps, and removing ethanol by vacuum distillation to obtain Rana sylvatica egg oil;

[0013] (7) adding 5 g of Rana sylvatica egg oil to 8-10 mL of protease solution, mixing, and subjecting the mixture to an enzymatic reaction for 10-60 minutes to obtain an enzymatic reaction product;

[0014] (8) adding 10-15 mL of ethanol and 3-7 g of β-cyclodextrin to the enzymatic reaction product obtained in step (7); (9) mixing for 2-3 hours, centrifuging, and taking the upper layer of oil as the Rana sylvatica oil product.

[0015] Furthermore, the method comprises:

[0016] (1) crushing the Rana sylvatica eggs and passing them through a 40-mesh sieve to obtain Rana sylvatica egg powder;

[0017] (2) Add 10 g of Rana sylvatica egg powder into 95-100 mL of anhydrous ethanol and mix well;

[0018] (3) 500-700w microwave-assisted extraction for 3-4 hours;

[0019] (4) Allow to settle naturally for 1 hour and filter to obtain the supernatant;

[0020] (5) Repeat steps (3) and (4) once;

[0021] (6) combining the supernatants obtained in the two (4) steps, and removing ethanol by vacuum distillation to obtain Rana sylvatica egg oil;

[0022] (7) At 25°C, 5 g of Rana sylvatica egg oil was added to 8 mL of 600 U / mL protease solution, mixed, and subjected to enzymatic hydrolysis at 40°C for 30 minutes to obtain an enzymatic hydrolysis product;

[0023] (8) adding 15 mL of ethanol and 5 g of β-cyclodextrin to the enzymatic reaction product obtained in step (7);

[0024] (9) Mixing at 40° C. for 2-3 hours, centrifuging, and taking the upper layer of oil as the Rana sylvatica oil product.

[0025] Furthermore, the protease is trypsin or bromelain.

[0026] Furthermore, in step (9), mixing is carried out at 40° C. for 2 hours.

[0027] Furthermore, in step (9), centrifugation is performed at 3200 rpm for 15 minutes.

[0028] Furthermore, the frog eggs in step (1) are eggs of Rana chensinensis.

[0029] Furthermore, in step (3), 600W microwave-assisted extraction was used for 3.3 hours;

[0030] On the other hand, the present application provides Rana sylvatica egg oil prepared using the above method.

[0031] On the other hand, the present application provides Rana sylvaticum egg oil microcapsules, which are prepared from the Rana sylvaticum egg oil.

[0032] Furthermore, the Rana sylvatica egg oil microcapsules are prepared according to the following method:

[0033] Weigh 70 g of maltodextrin and 45 g of oligofructose and dissolve them in 700 ml of deionized water;

[0034] The mixture was stirred at 40 degrees Celsius for 30 minutes, and the resulting solution was thoroughly mixed with 30 g of the above-mentioned Rana sylvatica egg oil, 0.3 g of monoglyceride, 0.6 g of sucrose ester, and 2 g of soybean lecithin. After high-speed shearing at 10,000 r / min for 10 minutes, the mixture was homogenized 3 times in a high-pressure homogenizer at a homogenization pressure of 600 bar to form a stable emulsion.

[0035] The emulsion was spray dried with an air inlet temperature of 170 degrees Celsius, an air outlet temperature of 90 degrees Celsius, and a rate of 6 ml / min to obtain Rana sylvatica egg oil microcapsule powder.

[0036] Those skilled in the art will appreciate that the parameters such as mass and volume in the claims and the content of the invention are only used as examples, and that the method that is appropriately scaled up is still within the scope of protection of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The DPPH radical scavenging rate results of the protease-improved cholesterol removal method (in the figure, "original Rana oil" refers to the Rana oil without cholesterol removal in Example 2; "decholesterolized" refers to the Rana oil after cholesterol removal in Example 2);

[0038] Figure 2 The results of estradiol content in the cholesterol removal method improved by protease (in the figure, "original Rana oil" refers to the Rana oil without cholesterol removal in Example 2; "decholesterolized" refers to the Rana oil after cholesterol removal in Example 2);

[0039] Figure 3 The removal rate of the protease-modified cholesterol removal method changes over time (the "original frog oil" in the figure refers to the direct removal method in Example 2). DETAILED DESCRIPTION

[0040] Example 1 Basic Experimental Methods

[0041] Determination of DPPH free radical scavenging rate of Rana sylvatica egg oil:

[0042] Prepare 0.5 mg / mL of Rana sylvatica egg oil solution / suspension as the sample to be tested; take 2.5 mL of the liquid sample to be tested, mix it with 2.5 mL of 0.1 mM DPPH anhydrous ethanol solution (sample) or 2.5 mL of anhydrous ethanol (control), and react at 25° C. in the dark for 30 min; measure the absorbance of the sample reaction solution, the control reaction solution and the reference reaction solution (2.5 mL of anhydrous ethanol plus 2.5 mL of 0.1 mM DPPH anhydrous ethanol solution) at 517 nm with a spectrophotometer; repeat three groups.

[0043] DPPH free radical scavenging rate % = (1-(A 样品 -A 对照 ) / A 参比 )×100%.

[0044] Cholesterol content determination:

[0045] Prepare cholesterol standard solution (0.001 g / mL);

[0046] Saponification sample: Take 0.2g of Rana sylvatica egg oil, add 7mL of anhydrous ethanol and 3mL of 60% KOH solution, and mix well; after saponification and condensation reflux at 60 degrees Celsius for 1 hour, add 5mL of anhydrous ethanol;

[0047] Extraction: The saponified product was extracted three times with 10 mL of n-hexane, and the organic phase was washed to neutrality and then dehydrated with anhydrous sodium sulfate;

[0048] After concentration, the solution was measured using Shimadzu HPLC (model 2010, C18 column).

[0049] Estradiol content determination:

[0050] Use Huamei Bio's estradiol quantification kit (ELISA method) to quantify the estradiol content in the sample. Determination of fatty acid content:

[0051] The fatty acid content was tested by the School of Food Science and Engineering of Jilin University using gas chromatographic analysis; the DHA and EPA contents were further tested by Qingdao Pony Testing Co., Ltd.

[0052] Embodiment 2 Rana tanshinii egg oil preparation process design

[0053] Through cooperative research with Jilin University, the applicant has determined the extraction method of frog egg oil and the cholesterol removal method:

[0054] (1) crushing the Rana sylvatica eggs and passing them through a 40-mesh sieve to obtain Rana sylvatica egg powder;

[0055] (2) Add 10 g of Rana sylvatica egg powder into 95-100 mL of anhydrous ethanol and mix well;

[0056] (3) 500-700w microwave-assisted extraction for 3-4 hours;

[0057] (4) Allow to settle naturally for 1 hour and filter to obtain the supernatant;

[0058] (5) Repeat steps (3) and (4) once;

[0059] (6) The supernatants obtained in the two (4) steps are combined, and the ethanol is removed by vacuum distillation to obtain Rana sylvatica egg oil.

[0060] In order to remove cholesterol from Rana affine oil, the cooperative unit studied silica gel adsorption, activated carbon adsorption, β-cyclodextrin embedding, combined precipitation and other methods, and finally determined the process of removing cholesterol by combined precipitation:

[0061] (A) At 25°C, 3 g of β-cyclodextrin was dissolved in 10 mL of ethanol solution, wherein the ethanol solution consisted of ethanol and distilled water in a volume ratio of 7:3; 5 g of Rana sylvatica egg oil was added thereto.

[0062] (B) Mix at 40°C for 3 hours and centrifuge at 3200 rpm for 15 minutes. The upper layer of oil is the decholesterolized Rana sylvatica egg oil.

[0063] The extraction rate of Rana sylvatica egg oil is about 19-22%, and the cholesterol removal rate is more than 75%. After removing cholesterol, the unsaturated fatty acid content is basically stable, and the saturated fatty acid content is reduced to a certain extent. DPPH clearance rate determination and mouse hyperlipidemia model animal experiments show that the product has obvious antioxidant and lipid-lowering activities.

[0064] Example 3 Optimization of decholesterolization process

[0065] The process in Example 2 does effectively reduce the cholesterol content in Rana sylvatica egg oil, but the applicant found that the antioxidant activity of the product was significantly reduced after removing cholesterol, and the estradiol content had been reduced to about one-tenth of the original level, basically reaching the detection limit of the kit, which is very unfavorable to the cardiovascular and cerebrovascular protection and antiplatelet aggregation effects of the product.

[0066] The basic principle of removing cholesterol by β-cyclodextrin combined with precipitation is that its hydrophilic exterior and hydrophobic interior can encapsulate cholesterol to form a structure that is insoluble in both oil and water. However, the presence of structures such as lipoproteins in frog oil complicates the encapsulation process, and while removing lipids such as cholesterol, it also takes away some proteins, fatty amide compounds, estradiol and other components with antioxidant activity. The applicant tried to use high-concentration proteases to quickly cleave the protein part of some lipoproteins to change the encapsulation tendency.

[0067] The applicant tried bromelain, trypsin, ficin, papain, and aminopeptidase (Xiasheng, food grade product), and the specific method was as follows:

[0068] (A) At 25°C, 5 g of Rana sylvatica egg oil was added to 8 mL of 600 U / mL enzyme solution, mixed and subjected to enzymatic hydrolysis at 40°C for 30 minutes to obtain an enzymatic hydrolysis product;

[0069] (B) adding 15 mL of ethanol and 5 g of β-cyclodextrin to the enzymatic reaction product obtained in step (A);

[0070] (C) Mix at 40°C for 3 hours, centrifuge at 3200 rpm for 15 minutes, and take the upper oil sample.

[0071] The results are as follows Figure 1 and Figure 2 As shown, bromelain and trypsin, which are preferred for enzymatic hydrolysis of the carboxyl side of basic amino acids and aromatic amino acids, can effectively restore the antioxidant activity and estradiol content lost during the cholesterol removal process in a short time enzymatic hydrolysis treatment, wherein the antioxidant activity is slightly improved compared with the original forest frog egg oil product (possibly due to certain antioxidant short peptides produced during enzymatic hydrolysis), while the cholesterol removal rate is maintained above 70%.

[0072] Due to the stability of product properties, the applicant did not consider the solution of composite enzymatic hydrolysis.

[0073] Example 4 Further Optimization of Decholesterolization Process

[0074] The applicant expected that the results of Example 2 were due to changes in the embedding process after lipoprotein enzymatic hydrolysis. Theoretically, the cholesterol removal efficiency should also be improved at this time. The applicant further tried to shorten the mixing time of step (C).

[0075] The results are as follows Figure 3 As shown, bromelain or trypsin-assisted cholesterol removal can effectively shorten the removal time, and the maximum removal effect is basically achieved in about 2 hours, which saves about half the time compared with direct removal of original frog oil.

[0076] In addition, the short removal time also brought about further improvement in the estradiol content, and the estradiol that was insoluble in water and slightly soluble in ethanol was further protected. The results are shown in Table 1.

[0077] Table 1 Antioxidant effect and estradiol content of different products

[0078]

[0079]

[0080] Main fatty acid content of "Example 4 Bromelain 2h" product

[0081] Fatty acid name Content w / v% Hexadecanoic acid 17.337 Octadecanoic acid 2.385 Linoleic acid 25.862 Linolenic acid 7.262 11,14,17-cis-eicosatrienoic acid 2.699 EPA 0.36 DHA 4.25

[0082] The distribution of major fatty acids is basically the same as that of the cholesterol-free product in Example 2.

Claims

1. A method for preparing Rana sylvatica egg oil, characterized in that: The preparation method comprises: (1) crushing the Rana sylvatica eggs and passing them through a 40-mesh sieve to obtain Rana sylvatica egg powder; (2) Add 10 g of Rana sylvatica egg powder into 95-100 mL of anhydrous ethanol and mix well; (3) 500-700w microwave-assisted extraction for 3-4 hours; (4) Allow to settle naturally for 1 hour and filter to obtain the supernatant; (5) Repeat steps (3) and (4) once; (6) combining the supernatants obtained in the two (4) steps, and removing ethanol by vacuum distillation to obtain a crude product of Rana sylvatica egg oil; (7) adding 5 g of crude Rana sylvatica egg oil to 8 mL of 600 U / mL trypsin or bromelain solution at 25° C., mixing, and performing enzymatic hydrolysis at 40° C. for 30 minutes to obtain an enzymatic hydrolysis product; (8) adding 15 mL of ethanol and 5 g of β-cyclodextrin to the enzymatic reaction product obtained in step (7); (9) Mixing at 40° C. for 2-3 hours, centrifuging, and taking the upper layer of oil as the Rana sylvatica egg oil product.

2. The method according to claim 1, wherein the mixing in step (9) is carried out at 40°C for 2 hours.

3. The method according to claim 1, wherein the step (9) is performed by centrifugation at 3200 rpm for 15 minutes.

4. The method according to claim 1, wherein the frog eggs in step (1) are eggs of Rana chensinensis.

5. The method according to claim 1, wherein step (3) uses 600W microwave-assisted extraction for 3.3 hours.

6. The Rana sylvatica egg oil prepared using the method described in any one of claims 1 to 5.

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