A kind of production method of modified forest frog oil

Abstract

The present invention provides a production method of modified forest frog oil, including the steps of purifying water for spawning, treating fertilized eggs, freeze drying, separating oil and eggs, etc., and the obtained forest frog oil is the modified forest frog oil. The method of the present invention is based on the characteristics of forest frog embryonic development, and extracts pure forest frog oil without impurities without excessive external intervention. The obtained forest frog oil no longer has the characteristics of the morphology, component content, and medicinal properties of traditional forest frog oil, but the water absorption expansion rate and odor are similar to those of traditional forest frog oil, the protein content is inversely proportional to the time before the embryonic development is completed, and the content of polypeptides, oligopeptides, and amino acids is proportional to the time before the embryonic development is completed. The method of the present invention does not harm the health of forest frogs, and the eggs are laid and survived normally, and the utilization cycle of forest frogs is extended. It is upgraded from the traditional method of killing frogs to obtain oil to raising frogs to obtain "oil", which is of great significance to the protection of the precious germplasm resources of Chinese forest frogs, orderly development, rational utilization, and benefit to society.

Description

Technical Field

[0001] The invention relates to a production method of forest frog oil, in particular to a production method of modified forest frog oil. Background Art

[0002] Rana oil is a traditional Chinese medicine and health food, with multiple medicinal and health effects such as anti-fatigue and regulating human endocrine (Gao Feng et al., 2005). The main component of Rana oil is protein. In the 1930s, Japanese scholars analyzed its components and found that the protein content accounted for more than 50% of the total amount of Rana oil. Rana oil contains a full range of amino acids and rich content. It also contains a variety of unsaturated fatty acids and ester substances, and the content of vitamins and trace elements is also rich (Han Tiesuo et al., 2008).

[0003] The most well-known medicinal effect of Rana affine oil is "nourishing yin", which means that it has a special nourishing effect on women. Therefore, many domestic scholars have also tested the content of sex steroid hormones in Rana affine oil, mainly detecting estradiol, testosterone, progesterone, chorionic gonadotropin, vitamin Va and cholesterol (Wang Lili, 2009). The values ​​of sex hormone components detected in different studies are not the same, which may be related to different detection methods and sampling. The concentration of estradiol in Rana affine oil has been reported to be 25.03ng / g (Hu Xin et al., 2003), 291.81pg / g (Liu Qilin et al., 1999),

[0004] In recent years, with the progress of research on the efficacy of Rana oil, it is believed that sex hormones are closely related to reproduction, and the content of sex hormones in Rana oil has also been a focus of attention. Sex hormones act through corresponding receptors and have targeted affinity for certain receptors, which is manifested as an incentive and affinity for certain existing tumor cells, causing the phenomenon of accelerated tumor proliferation. This also limits the consumption and promotion of Rana oil, and has a direct impact on its product development and application.

[0005] At the same time, the existing method of extracting oil from forest frogs is to kill the frogs. Each forest frog needs to be cultivated for at least three years before it can be killed and extracted for oil. It cannot be repeated and is not sustainable.

[0006] Therefore, it is necessary to invent a method for producing modified frog oil, upgrading from the traditional method of killing frogs to extract oil to raising frogs to extract "oil". Under the premise of not causing damage to the frogs, the frogs can naturally lay eggs and produce modified frog oil repeatedly every year. The so-called modified frog oil is a frog oil derivative that does not have the morphology, component ratio, and medicinal properties of traditional frog oil. In the absence of sex hormones (or the content is extremely low and undetectable), the frog oil is inherited with protein as the main component, and the content of polypeptides, oligopeptides, and amino acids is expanded, making it safer to use and more nutritious, suitable for men, women, and children. Its products can expand a broader space in the fields of food, medicine, health products, cosmetics, etc., which is of great significance to the protection of the precious germplasm resources of the Chinese frog, orderly development, rational utilization, and benefit to society. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method for producing modified forest frog oil, comprising the following steps:

[0008] (1) Clean water for spawning:

[0009] Every year in April and May, the forest frogs are captured after they come out of hibernation and enter the early stage of reproduction. The male and female frogs are separated and placed in their own temporary storage ponds for preheating. The water temperature in the pond is 15-20℃. When the female frogs enter the egg-laying period, they are taken out and mixed in proportion into the clean water pond to lay eggs in pairs.

[0010] (2) Fertilized egg processing:

[0011] When the eggs are discharged from the female frog, time them for 10-15 minutes (fertilization rate > 98%), remove them, clean them, and place them on a 20-24℃ development plate for 8-9 hours. After the fertilized eggs develop to the blastocyst stage, quickly place them in a -18℃ freezer for freezing and storage.

[0012] (3) Freeze drying:

[0013] The frozen frog embryos are placed in a freeze dryer for drying, and the freeze drying parameters are: pre-freezing temperature -18°C to -25°C, material thickness 1.0-1.5cm, vacuum degree 50-85Pa, drying time 24-30 hours, so that the moisture content of the dried frog embryos is less than 8%;

[0014] (4) Oil and egg separation:

[0015] The dried product is separated from the frog eggs and oil by means of gauze bag separation, sieving separation, air separation or gravity water purification separation. The frog oil obtained by removing the frog eggs is the modified frog oil, which is then processed for properties according to its purpose.

[0016] During the water purification and spawning process in step (1), the ratio of the number of female and male forest frogs is 1:1.5.

[0017] In the step (4) of separating oil and eggs, the gauze bag separation method is as follows: placing the dried frog embryo in a gauze bag and rubbing it, with the standard of achieving complete separation of eggs and oil without crushing the eggs, the forest frog oil is precipitated out of the gauze bag in the form of fragments, and the eggs are left in the bag, and the obtained forest frog oil is the modified forest frog oil; the gauze bag aperture is smaller than the diameter of the dried eggs, and the gauze bag aperture specification is preferably 40 meshes. If the aperture number is greater than 40 meshes, the aperture is too small, then the forest frog oil cannot be fully precipitated, and the recovery rate is low; if the aperture number is less than 40 meshes, the aperture is too large, then the eggs are easily leaked out and mixed into the forest frog oil, affecting the purity of the forest frog oil.

[0018] In the oil and egg separation process of step (4), the screening separation method is as follows: the dried frog embryos are rubbed in a closed container to achieve complete separation of the eggs and oil without crushing the eggs, and then poured through a 40-mesh sieve for screening, the frog oil is screened out, and the eggs are left in the sieve, and the obtained frog oil is the modified frog oil.

[0019] In the step (4), during the oil and egg separation process, the air separation method is as follows: the dried frog embryos are rubbed in a sealed container to achieve complete separation of the eggs and oil without crushing the eggs, and then the oil is air-separated by a blower to remove the eggs and obtain the modified frog oil.

[0020] In the step (4), during the oil and egg separation process, the gravity method water purification separation method is as follows: the dried frog embryos are rubbed in a sealed container to achieve complete separation of the eggs and the oil without crushing the eggs, and then poured into a container filled with purified water with an appropriate amount of water, and the eggs and the oil are automatically separated by the gravity method, the eggs sink to the bottom of the water, and the oil floats on the upper layer, and the upper floating oil is quickly fished out using a 100-mesh sieve within 30 seconds to obtain the modified forest frog oil.

[0021] Principle of the present invention:

[0022] During the conventional period of extracting oil from the forest frog (October to December), it is the period when the forest frog is pregnant and storing energy. During this period, the content of sex hormones in male and female forest frogs gradually increases, and the level of sex hormones is stable during the hibernation period. When the forest frog enters the breeding preparation period, the level of sex hormones in female and male frogs reaches a peak. Forest frog oil is coated on the surface of the egg cell. When the egg mass is separated from the mother's fertilization and becomes an independent individual, the sex hormone has completed its historical mission and is gradually decomposed under the action of enzymes; at the same time, protease begins to work and degrades the forest frog oil protein. The degradation rate corresponds to the embryonic development period, and the opening bait is prepared for the newborn tadpoles in the form of polypeptides, oligopeptides, and amino acids. The present invention has found that when the fertilized egg develops for 8-9 hours at 20-24°C, the embryonic development enters the blastocyst stage, and the presence of estrogen can no longer be detected in the embryo dry product at this time, and the nutrients such as protein, polypeptide, oligopeptide, amino acid, etc. in the forest frog oil are the most abundant and the content is the highest, which has reached the characteristic requirements of no sex hormone, high peptide and low protein, which is of great significance for the research and development of new forest frog oil products. When the eggs are discharged from the female frog, the male frog ejaculates on the egg mass and stirs the egg mass with its hind legs to fully fertilize the eggs. The hatching results of the egg mass fertilized in water for 8 minutes show that its hatching rate is greater than 98%. Therefore, controlling the fertilization time within 10-15 minutes can ensure that the frog eggs are fully fertilized, and at this time, the water absorption of the forest frog oil is relatively small, which is conducive to freeze drying and oil and egg separation operations, and improves the collection rate of modified forest frog oil.

[0023] Beneficial effects of the present invention:

[0024] The method of the present invention extracts high-peptide, low-protein, pure and impurity-free frog oil without estrogen based on the characteristics of frog embryo development and under the premise of following the natural ecological laws of frogs, without excessive external intervention. The obtained frog oil no longer has the morphology, component content and medicinal properties of traditional frog oil, but the water absorption expansion rate and smell are similar to those of traditional frog oil. The protein content is inversely proportional to the time before embryo development is completed, while the content of polypeptides, oligopeptides and amino acids is proportional to the time before embryo development is completed. The content of polypeptides, oligopeptides and amino acids is high, and it does not contain sex hormones, which is conducive to digestion and absorption, and is suitable for most people, young and old.

[0025] The method of the invention follows the natural ecological law of the forest frog, lays eggs naturally, does not harm the health of the forest frog, prolongs the utilization cycle of the forest frog, ensures the continuous utilization of the forest frog, and is of great significance to the sustainable development of the forest frog industry and ecological protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the HPLC chromatogram of the estradiol reference substance of the present invention;

[0027] Figure 2 It is the HPLC chromatogram of Rana sylvatica oil of the present invention;

[0028] Figure 3 It is the HPLC chromatogram of Rana sylvatica eggs of the present invention;

[0029] Figure 4 It is the HPLC chromatogram of the Rana sylvatica embryo of the present invention;

[0030] Among them, the horizontal axis represents time, and the vertical axis represents the ultraviolet absorption value. DETAILED DESCRIPTION

[0031] Embodiment 1:

[0032] The present invention provides a method for producing modified forest frog oil, comprising the following steps:

[0033] (1) Clean water for spawning:

[0034] In China's Changbai Mountain region, forest frogs are captured in April and May every year after they come out of hibernation and enter the early stage of reproduction. Males and females are placed in their own temporary storage ponds for preheating, with the water temperature at 15-20°C. When the female frogs enter the egg-laying period, they are taken out and mixed in a ratio of 1:1.5 between males and females and placed in a clean water pond for spawning.

[0035] (2) Fertilized egg processing:

[0036] When the female frogs shed their eggs, they were fished out and cleaned after 10 minutes, and placed on a 20℃ development plate for 9 hours to allow the fertilized eggs to develop to the blastocyst stage, and then quickly placed in a -18℃ freezer for storage.

[0037] (3) Freeze drying:

[0038] The frozen frog embryos were placed in a freeze dryer for drying, and the freeze drying parameters were: pre-freezing temperature -18°C, material thickness 1.0-1.5 cm, vacuum degree 50-85 Pa, drying time 28 hours, and the moisture content of the dried frog embryos was <8%;

[0039] (4) Oil and egg separation:

[0040] The dried product is placed in a gauze bag with a pore size of 40 mesh and rubbed. The standard is to achieve complete separation of eggs and oil without crushing the eggs. The frog oil precipitates out of the gauze bag in the form of fragments, and the eggs remain in the bag. The separation rate is 96%. The obtained frog oil is the modified frog oil.

[0041] Embodiment 2:

[0042] The present invention provides a method for producing modified forest frog oil, comprising the following steps:

[0043] (1) Clean water for spawning:

[0044] In China's Changbai Mountain region, forest frogs are captured in April and May every year after they come out of hibernation and enter the early stage of reproduction. Males and females are placed in their own temporary storage ponds for preheating, with the water temperature at 15-20°C. When the female frogs enter the egg-laying period, they are taken out and mixed in a ratio of 1:1.5 between males and females and placed in a clean water pond for spawning.

[0045] (2) Fertilized egg processing:

[0046] When the eggs are discharged from the female frog, they are taken out and cleaned after 12 minutes, and then placed on a 22°C development plate for 8.5 hours to allow the fertilized eggs to develop to the blastocyst stage, and then quickly placed in a -18°C freezer for cryopreservation;

[0047] (3) Freeze drying:

[0048] The frozen frog embryos were placed in a freeze dryer for drying, and the freeze drying parameters were: pre-freezing temperature -20°C, material thickness 1.0-1.5 cm, vacuum degree 50-85 Pa, drying time 26 hours, and the moisture content of the dried frog embryos was <8%;

[0049] (4) Oil and egg separation:

[0050] The dried product is placed in a gauze bag with a pore size of 40 meshes and rubbed, with the standard of achieving complete separation of eggs and oil without crushing the eggs. The frog oil precipitates out of the gauze bag in the form of fragments, and the eggs remain in the bag. The separation rate is 95%; the obtained frog oil is the modified frog oil.

[0051] Embodiment three:

[0052] The present invention provides a method for producing modified forest frog oil, comprising the following steps:

[0053] (1) Clean water for spawning:

[0054] In China's Changbai Mountain region, forest frogs are captured in April and May every year after they come out of hibernation and enter the early stage of reproduction. Males and females are placed in their own temporary storage ponds for preheating, with the water temperature at 15-20°C. When the female frogs enter the egg-laying period, they are taken out and mixed in a ratio of 1:1.5 between males and females and placed in a clean water pond for spawning.

[0055] (2) Fertilized egg processing:

[0056] When the eggs are discharged from the female frog, they are taken out and cleaned after 15 minutes, and then placed on a 24℃ development plate for 8 hours to allow the fertilized eggs to develop to the blastocyst stage, and then quickly placed in a -18℃ freezer for freezing and storage;

[0057] (3) Freeze drying:

[0058] The frozen frog embryos were placed in a freeze dryer for drying, and the freeze drying parameters were: pre-freezing temperature -24°C, material thickness 1.0-1.5 cm, vacuum degree 50-85 Pa, drying time 30 hours, and the moisture content of the dried frog embryos was <8%;

[0059] (4) Oil and egg separation:

[0060] The dried product was rubbed in a sealed container until the eggs and the oil were completely separated without crushing the eggs. The product was poured into a container filled with clean water and the eggs and the oil were automatically separated using a specific gravity method. The eggs sank to the bottom of the water and the oil floated on the upper layer. The upper floating oil was quickly scooped out with a 100-mesh sieve within 30 seconds, which was the modified forest frog oil. The separation rate was >98%.

[0061] The modified Rana oil obtained in Examples 1 to 3 was subjected to component detection and analysis. The modified Rana oil did not contain sex hormones such as estradiol, and the other components were similar to those of the unmodified Rana oil.

[0062] The present invention measures and analyzes estradiol in unmodified Rana sylvaticum oil powder, Rana sylvaticum egg powder and Rana sylvaticum embryo powder, and compares and analyzes the content of estradiol in unmodified Rana sylvaticum oil, Rana sylvaticum egg and Rana sylvaticum embryo, and the results are as follows:

[0063] 1. Test Materials

[0064] 1. Test samples:

[0065] The Rana sylvaticum oil powder, Rana sylvaticum egg powder and Rana sylvaticum embryo powder are provided by Jilin Province Rana King Bioengineering Co., Ltd. The Rana sylvaticum egg is obtained by step (1) of the present invention; the Rana sylvaticum embryo is obtained by developing the fertilized eggs obtained by steps (1) and (2) of the present invention at 20-24°C for 8-9 hours.

[0066] 2. Reference

[0067] Estradiol control substance was purchased from Chengdu Aifa Biotechnology Co., Ltd.

[0068] 3. Reagents

[0069] Acetone (analytical grade); methanol (chromatographic grade); acetonitrile (chromatographic grade); ultrapure water.

[0070] 2. Test methods

[0071] 1. Preparation of reference and test products

[0072] (1) Preparation of reference solution

[0073] Take 2 mg of estradiol reference substance, accurately weigh it, make up to volume with methanol in a 2 mL volumetric flask to obtain the reference substance stock solution, and dilute it to obtain a 0.016 mg / ml estradiol reference substance solution.

[0074] (2) Preparation of test solution

[0075] Take 2g of the test sample powder, weigh it accurately, add 40mL of acetone to a conical flask; place it in an ultrasonic cleaner for extraction for about 30min, then filter to obtain the filtrate; use a rotary evaporator to evaporate the solvent acetone in the filtrate at 45°C under reduced pressure; finally, add methanol to make up to a 2mL volumetric flask to obtain the test sample solution; filter through a 0.22μm microporous filter membrane before HPLC analysis.

[0076] 2. Chromatographic conditions

[0077] The determination was performed using an Agilent TC-C18 (250×4.6mm, 5μm) chromatographic column with methanol-water (60:40) as the mobile phase, a flow rate of 1.0mL / min, a detection wavelength of 225nm, a column temperature of 30°C, and an injection volume of 10μl.

[0078] 3. Test results

[0079] The chromatograms of estradiol reference substance, Rana sylvatica oil, Rana sylvatica eggs and Rana sylvatica embryos are shown in Figure 2. Figure 1-4 As shown, the estradiol content analysis is shown in Table 1;

[0080] Table 1 Estradiol content in Rana sylvatica oil, Rana sylvatica eggs and Rana sylvatica embryos (μg / g)

[0081] Rana oil Rana sylvatica eggs Rana sylvatica embryo Estradiol 0.126 4.70 ----

[0082] 4. Results Analysis

[0083] The test results show that among the Rana affine oil powder, Rana affine egg powder and Rana affine embryo powder, the Rana affine egg has the highest estradiol content of 4.70μg / g, followed by Rana affine oil with an estradiol content of 0.126μg / g, and no estradiol was detected in Rana affine embryo. Rana affine embryo is a mixture of modified Rana affine oil and developing eggs. The fact that Rana affine embryo does not contain estradiol in the test report means that the modified Rana affine oil does not contain estradiol.

Claims

1. A modified frog oil, characterized in that: The modified forest frog oil is obtained by separating the frog embryo from oil and eggs and removing the frog eggs. The modified forest frog oil does not contain estrogen, is high in peptides, low in protein, and is pure and free of impurities. The frog embryo preparation method comprises: (1) Clean water for spawning: Males and females are mixed in proportion and placed in a clean water pool to form pairs for spawning; (2) Fertilized egg processing: When the eggs are discharged from the female frog, take them out and wash them after 10-15 minutes, then place them on a 20-24℃ development plate for 8-9 hours to allow the fertilized eggs to develop to the blastocyst stage, and then quickly put them into a -18℃ freezer for freezing and storage; (3) Freeze drying: The frozen frog embryos were placed in a freeze dryer for drying. The freeze drying parameters were as follows: pre-freezing temperature -18°C to -25°C, material thickness 1.0-1.5 cm, vacuum degree 50-85 Pa, drying time 24-30 hours, and the moisture content of the dried frog embryos was <8%. The oil and egg separation method is as follows: The dried product is separated from the frog eggs and oil by using a gauze bag, sieving, air separation or gravity water purification method, and the frog oil obtained by removing the frog eggs is the modified frog oil; The gauze bag separation method is as follows: the dried product is placed in a gauze bag and rubbed, with the standard of achieving complete separation of eggs and oil without crushing the eggs, the frog oil is precipitated out of the gauze bag in the form of fragments, and the eggs remain in the bag, and the obtained frog oil is the modified frog oil; the pore size of the gauze bag is smaller than the diameter of the dried eggs, and the pore size specification of the gauze bag is 40 meshes.

2. The method for producing modified Rana sylvatica oil according to claim 1, characterized in that: The following steps are involved: (1) Clean water for spawning: Every year in April and May, forest frogs are captured after they come out of hibernation and enter the early stage of reproduction. Male and female frogs are placed in their own temporary storage ponds for preheating. The water temperature in the pond is 15-20℃. When the female frogs enter the egg-laying period, they are taken out and mixed in proportion into the clean water pond to lay eggs in pairs. (2) Fertilized egg processing: When the eggs are discharged from the female frog, take them out and wash them after 10-15 minutes, then place them on a 20-24℃ development plate for 8-9 hours to allow the fertilized eggs to develop to the blastocyst stage, and then quickly put them into a -18℃ freezer for freezing and storage; (3) Freeze drying: The frozen frog embryos are placed in a freeze dryer for drying, and the freeze drying parameters are: pre-freezing temperature -18°C to -25°C, material thickness 1.0-1.5cm, vacuum degree 50-85Pa, drying time 24-30 hours, so that the moisture content of the dried frog embryos is less than 8%; at this time, the presence of estrogen in the dried embryos is no longer detected; (4) Oil and egg separation: The dried product is separated from the frog eggs and oil by using a gauze bag, sieving, air separation or gravity water purification method, and the frog oil obtained by removing the frog eggs is the modified frog oil; The gauze bag separation method is as follows: the dried product is placed in a gauze bag and rubbed, with the standard of achieving complete separation of eggs and oil without crushing the eggs, the frog oil is precipitated out of the gauze bag in the form of fragments, and the eggs remain in the bag, and the obtained frog oil is the modified frog oil; the pore size of the gauze bag is smaller than the diameter of the dried eggs, and the pore size specification of the gauze bag is 40 meshes.

3. The method for producing modified Rana sylvatica oil according to claim 2, characterized in that: During the water purification and spawning process in step (1), the ratio of the number of female and male forest frogs is 1:1.

5.

4. The method for producing modified Rana sylvatica oil according to claim 2, characterized in that: In the step (4) of separating the oil and the eggs, the sieving separation method is as follows: kneading the dried product in a sealed container to achieve complete separation of the eggs and the oil without crushing the eggs, pouring it through a 40-mesh sieve for sieving, the frog oil is sifted out, and the eggs are left in the sieve, and the frog oil obtained is the modified frog oil.

5. The method for producing modified Rana sylvatica oil according to claim 2, characterized in that: In the step (4) of separating the oil and the eggs, the air separation method is as follows: kneading the dried product in a sealed container to achieve complete separation of the eggs and the oil without crushing the eggs, and then using a blower to separate the oil by air separation. The Rana oil obtained by removing the eggs is the modified Rana oil.

6. The method for producing modified Rana sylvatica oil according to claim 2, characterized in that: In the step (4), during the oil and egg separation process, the specific gravity method water purification separation method is as follows: the dried product is rubbed in a closed container, with the standard of achieving complete separation of the eggs and the oil without crushing the eggs, poured into a container filled with clean water, and the eggs and the oil are automatically separated by specific gravity, the eggs sink to the bottom of the water, and the oil floats on the upper layer, and the upper floating oil is quickly scooped out with a 100-mesh sieve within 30 seconds, which is the modified forest frog oil.

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