阅读说明：本技术 一种改性林蛙油的生产方法 (Production method of modified wood frog oil ) 是由 房永清 刘志文 于 2021-08-31 设计创作，主要内容包括：本发明提供一种改性林蛙油的生产方法,包括净水产卵、受精卵处理、冷冻干燥、油卵分离等步骤,所得林蛙油即为改性林蛙油。本发明方法是根据林蛙胚胎发育特点,在不需要外界过多干预的情况下提取出不含性激素的纯净无杂质林蛙油,所得林蛙油已不具备传统林蛙油的形态、成分含量、药性等特点,但吸水膨胀率和气味与传统林蛙油相似,蛋白质含量与胚胎发育完成前的时间成反比,多肽、寡肽、氨基酸的含量与胚胎发育完成前的时间成正比。本发明方法不损害林蛙健康,正常产卵存活,延长了林蛙的利用周期,从传统的杀蛙取油升级为养蛙取“油”,对保护中国林蛙这一宝贵的种质资源,有序开发,合理利用,造福社会都具有重要意义。(The invention provides a production method of modified wood frog oil, which comprises the steps of water purification and egg laying, fertilized egg treatment, freeze drying, oil and egg separation and the like, and the obtained wood frog oil is the modified wood frog oil. The method is characterized in that pure forest frog oil without sex hormones is extracted without excessive external intervention according to the characteristics of the development of the embryo of the forest frog, the obtained forest frog oil does not have the characteristics of the form, the component content, the drug property and the like of the traditional forest frog oil, but the water absorption swelling rate and the smell are similar to the traditional forest frog oil, the protein content is inversely proportional to the time before the development of the embryo is finished, and the contents of polypeptide, oligopeptide and amino acid are proportional to the time before the development of the embryo is finished. The method does not damage the health of the wood frogs, enables the wood frogs to normally lay eggs and survive, prolongs the utilization period of the wood frogs, upgrades the traditional oil taking by killing the wood frogs into the oil taking by raising the wood frogs, and has important significance for protecting the valuable germplasm resource of the Chinese wood frogs, developing in order, reasonably utilizing and benefiting the society.)

1. A production method of modified wood frog oil is characterized by comprising the following steps: the method comprises the following steps:

(1) water purification and spawning:

capturing the wood frogs in the early stage of reproduction after the wood frogs are hibernated in 4-5 months every year, respectively putting male and female frogs into respective temporary storage tanks for preheating, wherein the temperature of the water in the tanks is 15-20 ℃, taking out the wood frogs after the female frogs enter a spawning period, and proportionally mixing the male and female frogs into a water purification tank to embrace spawning;

(2) fertilized egg treatment:

after the eggs are discharged out of the female frogs, timing for 10-15 minutes, taking out and cleaning, placing on a development plate at 20-24 ℃ for 8-9 hours, and quickly freezing and storing in a freezer at-18 ℃ after the fertilized eggs are developed to the blastocyst stage;

(3) and (3) freeze drying:

putting the frozen frog embryo into a freeze dryer for drying to ensure that the water content of the dried frog embryo is less than 8 percent;

(4) oil and egg separation:

separating ovum and oil of Rana temporaria chensinensis David by gauze bag separation or sieving separation or air separation or specific gravity method water separation, and removing ovum to obtain modified Rana temporaria chensinensis David oil.

2. The method for producing modified oviductus ranae according to claim 1, wherein the method comprises the following steps: in the water purification and spawning process of the step (1), the number ratio of male and female wood frogs is 1: 1.5.

3. The method for producing modified oviductus ranae according to claim 1, wherein the method comprises the following steps: in the freeze drying process in the step (3), the freeze drying parameters are as follows: the prefreezing temperature is-18 to-25 ℃, the material thickness is 1.0 to 1.5cm, the vacuum degree is 50 to 85Pa, and the drying time is 24 to 30 hours.

4. The method for producing modified oviductus ranae according to claim 1, wherein the method comprises the following steps: in the oil and egg separation process in the step (4), the yarn bag separation method comprises the following steps: putting the dried product into a gauze bag, kneading until the non-broken egg particles are completely separated from egg and oil, separating out the wood frog oil from the gauze bag in a flake shape, and leaving the egg particles in the bag to obtain the modified wood frog oil; the aperture of the yarn bag is smaller than the diameter of the dried egg, and the aperture specification of the yarn bag is preferably 40 meshes.

5. The method for producing modified oviductus ranae according to claim 1, wherein the method comprises the following steps: in the process of separating the oil and the eggs in the step (4), the sieving and separating method comprises the following steps: and rubbing the dried product in a closed container, pouring out the dried product through a 40-mesh screen for screening until the non-broken egg particles are completely separated from the egg and oil, screening the wood frog oil out, and leaving the egg particles in the screen to obtain the modified wood frog oil.

6. The method for producing modified oviductus ranae according to claim 1, wherein the method comprises the following steps: in the oil and egg separation process in the step (4), the air separation method comprises the following steps: and rubbing the dried product in a closed container, discharging the dried product by air separation by using a fan under the condition that the non-broken egg grains are completely separated from egg and oil, and removing the egg grains to obtain the wood frog oil, namely the modified wood frog oil.

7. The method for producing modified oviductus ranae according to claim 1, wherein the method comprises the following steps: in the process of separating the oil and the eggs in the step (4), the water purification and separation method by the hydrometric method comprises the following steps: kneading the dried product in a closed container until the ovum and oil are completely separated without breaking, pouring into a container filled with purified water, automatically separating ovum and oil by a specific gravity method, sinking the ovum into water, floating the oil on the upper layer, and rapidly taking out the floating oil on the upper layer by a 100-mesh screen within 30 seconds to obtain the modified oviductus Ranae.

Technical Field

The invention relates to a production method of oviductus ranae, in particular to a production method of modified oviductus ranae.

Background

Oviductus ranae is a traditional medicine and health-care food in China, and has various drug effects and health-care effects of resisting fatigue, regulating human endocrine and the like (peak and the like, 2005). The main component of the wood frog oil is protein, and Japanese scholars perform component analysis in the 30 s of the 20 th century, and find that the protein content accounts for more than 50% of the total amount of the wood frog oil. The wood frog oil has complete amino acid species and rich content. Meanwhile, the beverage contains various unsaturated fatty acids and esters, and the contents of vitamins and trace elements are also rich (Korea, etc., 2008).

The most known medicine effect of the wood frog oil is 'nourishing yin', namely the wood frog oil has special nourishing effect on women. Therefore, many scholars in China detect the content of sex steroid hormones in the oviductus ranae, and mainly detect estradiol, testosterone, progesterone, chorionic gonadotropin, vitamin Va, cholesterol and the like (Wangli, 2009). The values of sex hormone components detected in different studies are different and may be related to different detection methods and sampling. Estradiol concentrations in oviductus Ranae have been reported to be 25.03ng/g (Huxin et al, 2003), 291.81pg/g (Liuqilin et al, 1999),

in recent years, with the progress of research on efficacy of oviductus ranae, factors closely related to reproduction are considered to be sex hormones, and the content of sex hormones in oviductus ranae is also receiving attention. The sex hormone acts through corresponding receptors, has a targeting affinity effect on certain receptors, and is shown as an excitation and affinity effect on certain existing tumor cells to cause the phenomenon of increasing the tumor proliferation speed, so that the eating and popularization of the rana japonica oil are limited, and the development and application of the rana japonica oil are directly influenced.

Meanwhile, the existing oil taking mode of the wood frogs is to kill the wood frogs and take the oil, each wood frog needs to be cultivated for at least 3 years to kill and take the oil, and the method cannot be repeated and has no sustainability.

Therefore, the invention needs to invent a production method of modified wood frog oil, which upgrades the traditional method of killing the wood frog and taking the oil into the method of raising the wood frog and taking the oil, naturally embraces the wood frog to lay eggs on the premise of not damaging the wood frog, and can repeatedly produce the modified wood frog oil every year. The modified wood frog oil is a wood frog oil derivative which does not have the characteristics of the form, component proportion and drug property of the traditional wood frog oil, and under the condition of asexual hormone (or the content is extremely low and is not detected), the inherited wood frog oil takes protein as a main component, expands the contents of polypeptide, oligopeptide and amino acid, ensures that the application is safer, the nutrition is more complete, and is suitable for people of all ages and both sexes. The product can be expanded in wider space in the fields of food, medicine, health care products, cosmetics and the like, thereby having important significance for protecting the valuable germplasm resource of the Chinese wood frog, developing orderly, reasonably utilizing and benefiting the society.

Disclosure of Invention

In order to solve the technical problems, the invention provides a production method of modified rana japonica oil, which comprises the following steps:

(1) water purification and spawning:

capturing the wood frogs in the early stage of reproduction after the wood frogs are hibernated in 4-5 months every year, separating male and female, pre-heating in respective temporary storage tanks at the temperature of 15-20 ℃, taking out the wood frogs after the female frogs enter a spawning period, mixing the male and female in proportion, and allowing the wood frogs to enter a water purification tank for embracing spawning;

(2) fertilized egg treatment:

after eggs are discharged from female frogs, timing for 10-15 minutes (the fertilization rate is more than 98 percent), taking out and cleaning, placing on a development plate at 20-24 ℃ for 8-9 hours, and quickly freezing and storing in a freezer at-18 ℃ after the fertilized eggs are developed to the blastocyst stage;

(3) and (3) freeze drying:

putting the frozen frog embryo into a freeze dryer for drying, wherein the freeze drying parameters are as follows: the prefreezing temperature is-18 to-25 ℃, the material thickness is 1.0 to 1.5cm, the vacuum degree is 50 to 85Pa, and the drying time is 24 to 30 hours, so that the water content of the dried frog embryo is less than 8 percent;

(4) oil and egg separation:

separating ovum and oil of Rana temporaria chensinensis David by gauze bag separation or sieving separation or air separation or specific gravity method water separation, removing ovum to obtain modified Rana temporaria chensinensis David oil, and performing property treatment according to the use of the modified Rana temporaria chensinensis David oil.

In the water purification and spawning process of the step (1), the number ratio of male and female wood frogs is 1: 1.5.

In the oil and egg separation process in the step (4), the yarn bag separation method comprises the following steps: putting the dried product of the frog embryo into a gauze bag, kneading, separating out the oviductus ranae in a flake shape from the gauze bag by taking the standard that the ovum and oil are completely separated without being kneaded into pieces, and leaving the ovum in the bag, wherein the obtained oviductus ranae is the modified oviductus ranae; the aperture of the yarn bag is smaller than the diameter of the dried egg, and the aperture specification of the yarn bag is preferably 40 meshes. If the pore size is larger than 40 meshes and the pore size is too small, the rana japonica oil cannot be fully separated out, and the recovery rate is low; if the pore size is smaller than 40 meshes and the pore size is too large, the egg particles are easy to leak out and mix into the oviductus ranae, which affects the purity of the oviductus ranae.

In the process of separating the oil and the eggs in the step (4), the sieving and separating method comprises the following steps: and rubbing the dried frog embryo product in a closed container, pouring the non-rubbed ovum grains into a 40-mesh screen for screening until the ovum and oil are completely separated, screening the oviductus ranae, and leaving the ovum grains in the screen to obtain the oviductus ranae modified oviductus ranae.

In the oil and egg separation process in the step (4), the air separation method comprises the following steps: and rubbing the dried frog embryo product in a closed container, discharging the dried frog embryo product by air separation by using a fan under the condition that the non-broken frog grains are completely separated from egg and oil, and removing the egg grains to obtain the wood frog oil, namely the modified wood frog oil.

In the process of separating the oil and the eggs in the step (4), the water purification and separation method by the hydrometric method comprises the following steps: kneading the dried frog embryo in a closed container, pouring into a container filled with purified water with moderate water amount according to the standard that the non-kneaded ovum grains are completely separated into ovum and oil, automatically separating the ovum and oil by a specific gravity method, sinking the ovum into the water bottom with the oil floating on the upper layer, and quickly fishing out the floating oil on the upper layer within 30 seconds by using a 100-mesh screen to obtain the modified forest frog oil.

The principle of the invention is as follows:

in the conventional oil extraction period (10-12 months) of the wood frogs, the energy storage period of the eggs of the wood frogs is just the period when the female and male wood frogs have the sex hormone content gradually increased until the sex hormone level is stable in the hibernation period, and when the wood frogs enter the breeding preparation period, the sex hormone levels in the female and male wood frogs reach peak values. The oviductus ranae is coated on the surface of an egg cell, and after an egg mass is fertilized to become an independent individual after being separated from a parent, the sex hormone has completed the historical mission and is gradually decomposed and used up under the action of enzyme; meanwhile, the proteolytic enzyme starts to work to degrade the rana japonica oil protein, the degradation rate corresponds to the development period of the embryo, and the feed is prepared for the primary tadpoles in the forms of polypeptide, oligopeptide and amino acid. The research of the invention finds that when fertilized eggs develop for 8-9 hours at the temperature of 20-24 ℃, embryos develop into the blastocyst stage, the dried embryo products can not detect the existence of estrogen, and the oviductus ranae has the most abundant types of nutritional components such as protein, polypeptide, oligopeptide, amino acid and the like and the highest content, and reaches the characteristic requirements of asexual hormone, high peptide and low protein, which has important significance for the research and development of new products of oviductus ranae. When the eggs are discharged from the female frog, the male frog ejects the sperm on the egg mass, and the egg mass is stirred by hind limbs to make the eggs fully fertilized. The hatching result of the egg mass fertilized in water for 8 minutes proves that the hatching rate is more than 98 percent. Therefore, the fertilization time can be controlled within 10-15 minutes to ensure that the frog eggs are fully fertilized, and the water absorption capacity of the oviductus ranae is relatively low, thereby being beneficial to freeze drying and oil-egg separation operation and improving the collection rate of the modified oviductus ranae.

The invention has the beneficial effects that:

the method extracts the high peptide, low protein, pure and impurity-free forest frog oil without external excessive intervention on the premise of following the natural ecological law of the forest frog according to the development characteristics of the forest frog embryo. The obtained oviductus Ranae has no shape, component content and drug property of the traditional oviductus Ranae, but has similar water swelling rate and smell to the traditional oviductus Ranae, the protein content is inversely proportional to the time before the embryo development is finished, and the contents of polypeptide, oligopeptide and amino acid are proportional to the time before the embryo development is finished. The polypeptide, oligopeptide and amino acid are high in content, do not contain sex hormone, are beneficial to digestion and absorption, are suitable for most people and are suitable for people of all ages.

The method of the invention follows the natural ecological law of the wood frogs, spawns by natural embracing, does not damage the health of the wood frogs, prolongs the utilization period of the wood frogs, ensures the continuous utilization of the wood frogs, and has important significance for the sustainable development and ecological protection of the wood frogs industry.

Drawings

FIG. 1 is an HPLC chromatogram of an estradiol control according to the present invention;

FIG. 2 is an HPLC chromatogram of oviductus Ranae of the present invention;

FIG. 3 is an HPLC chromatogram of an oviductus Ranae egg of the present invention;

FIG. 4 is an HPLC chromatogram of a wood frog embryo of the present invention;

wherein the horizontal axis represents time, and the vertical axis represents the ultraviolet absorption value.

Detailed Description

The first embodiment is as follows:

the invention provides a production method of modified wood frog oil, which comprises the following steps:

(1) water purification and spawning:

in Changbai mountain areas of China, 4-5 months per year, after the wood frogs hibernate, the wood frogs enter the prophase of reproduction to be captured, the male and female frogs enter the respective temporary storage tanks to be preheated, the temperature of the water in the tanks is 15-20 ℃, after the female frogs enter the egg drop period, the female frogs and the male frogs are taken out and mixed according to the proportion of 1:1.5 of the number of the male frogs and the female frogs to enter the water purification tank to embrace and lay eggs;

(2) fertilized egg treatment:

after the female frogs are discharged, timing for 10 minutes, fishing out, cleaning, placing on a development plate at 20 ℃ for 9 hours, and quickly freezing and storing in a freezer at 18 ℃ below zero after the fertilized eggs are developed to the blastocyst stage;

(3) and (3) freeze drying:

putting the frozen frog embryo into a freeze dryer for drying, wherein the freeze drying parameters are as follows: the prefreezing temperature is-18 ℃, the material thickness is 1.0-1.5cm, the vacuum degree is 50-85 Pa, the drying time is 28 hours, and the water content of the dried frog embryo is less than 8 percent;

(4) oil and egg separation:

putting the dried product into a gauze bag with the aperture specification of 40 meshes for kneading, separating out the wood frog oil from the gauze bag in a flake shape by taking the standard that the egg grains are not kneaded to be completely separated from the oil, and keeping the egg grains in the bag with the separation rate of 96 percent, wherein the obtained wood frog oil is the modified wood frog oil.

Example two:

the invention provides a production method of modified wood frog oil, which comprises the following steps:

(1) water purification and spawning:

in Changbai mountain areas of China, 4-5 months per year, after the wood frogs hibernate, the wood frogs enter the prophase of reproduction to be captured, the male and female frogs enter the respective temporary storage tanks to be preheated, the temperature of the water in the tanks is 15-20 ℃, after the female frogs enter the egg drop period, the female frogs and the male frogs are taken out and mixed according to the proportion of 1:1.5 of the number of the male frogs and the female frogs to enter the water purification tank to embrace and lay eggs;

(2) fertilized egg treatment:

after eggs are discharged from female frogs, timing for 12 minutes, taking out and cleaning, placing on a development plate at the temperature of 22 ℃ for 8.5 hours, and quickly freezing and storing in a freezer at the temperature of-18 ℃ after fertilized eggs are developed to the blastocyst stage;

(3) and (3) freeze drying:

putting the frozen frog embryo into a freeze dryer for drying, wherein the freeze drying parameters are as follows: pre-freezing at-20 ℃, keeping the material thickness at 1.0-1.5cm, keeping the vacuum degree at 50-85 Pa, and drying for 26 hours, wherein the water content of the dried frog embryo is less than 8%;

(4) oil and egg separation:

putting the dried product into a gauze bag with the aperture specification of 40 meshes, kneading, separating out the wood frog oil from the gauze bag in a flake shape by taking the standard that the non-kneaded egg granules are completely separated from the oil, and keeping the egg granules in the bag, wherein the separation rate reaches 95%; the obtained oviductus Ranae is modified oviductus Ranae.

Example three:

the invention provides a production method of modified wood frog oil, which comprises the following steps:

(1) water purification and spawning:

in Changbai mountain areas of China, 4-5 months per year, after the wood frogs hibernate, the wood frogs enter the prophase of reproduction to be captured, the male and female frogs enter the respective temporary storage tanks to be preheated, the temperature of the water in the tanks is 15-20 ℃, after the female frogs enter the egg drop period, the female frogs and the male frogs are taken out and mixed according to the proportion of 1:1.5 of the number of the male frogs and the female frogs to enter the water purification tank to embrace and lay eggs;

(2) fertilized egg treatment:

after eggs are discharged from female frogs, timing for 15 minutes, taking out, cleaning, placing on a development plate at 24 ℃ for 8 hours, and quickly freezing and storing in a freezer at-18 ℃ after fertilized eggs are developed to the blastocyst stage;

(3) and (3) freeze drying:

putting the frozen frog embryo into a freeze dryer for drying, wherein the freeze drying parameters are as follows: pre-freezing at-24 deg.c, material thickness of 1.0-1.5cm, vacuum degree of 50-85 Pa, and drying time of 30 hr to water content of dried frog embryo lower than 8%;

(4) oil and egg separation:

kneading the dried product in a closed container, pouring into a container filled with purified water according to the standard that the eggs and oil are completely separated without being broken, automatically separating the eggs and oil by a specific gravity method, sinking the eggs into the water bottom, floating the oil on the upper layer, and quickly fishing out the floating oil on the upper layer by a 100-mesh screen within 30 seconds to obtain the modified rana japonica oil, wherein the separation rate is more than 98%.

And (3) carrying out component detection analysis on the modified rana japonica oil obtained in the first to third examples, wherein the modified rana japonica oil does not contain sex hormones such as estradiol, and other components are similar to the components of the unmodified rana japonica oil.

The invention carries out determination and analysis on estradiol in unmodified wood frog oil micro powder, wood frog egg micro powder and wood frog embryo micro powder, and the results of the content analysis on the estradiol in the unmodified wood frog oil, wood frog egg and wood frog embryo are as follows:

first, test materials

1. Test samples:

the wood frog oil micro powder, the wood frog egg micro powder and the wood frog embryo micro powder are provided by Jilin frog king bioengineering limited company. Wherein the wood frog eggs are obtained by the step (1) of the invention; the forest frog embryo is obtained by the development of the fertilized egg obtained in the steps (1) and (2) of the invention for 8-9 hours at the temperature of 20-24 ℃.

2. Reference substance

Estradiol controls were purchased from Goodyera Biotechnology Ltd.

3. Reagent

Acetone (analytically pure); methanol (chromatographically pure); acetonitrile (chromatographically pure); ultrapure water.

Second, test method

1. Preparation of reference substance and test substance

(1) Preparation of control solutions

2mg of estradiol reference substance is precisely weighed, and is dissolved in a 2mL volumetric flask with methanol to obtain a reference substance stock solution, and the reference substance stock solution is diluted to obtain 0.016mg/mL estradiol reference substance solution.

(2) Preparation of test solution

2g of test sample powder is precisely weighed, and 40mL of acetone is added into a conical flask; extracting in an ultrasonic cleaner for about 30min, and vacuum filtering to obtain filtrate; evaporating the filtrate at 45 deg.C under reduced pressure by using rotary evaporator to remove acetone as solvent; finally adding methanol to a volumetric flask with constant volume of 2mL to obtain a test solution; before HPLC analysis, the mixture was filtered through a 0.22 μm microfiltration membrane.

2. Chromatographic conditions

The measurement adopts Agilent TC-C18(250 multiplied by 4.6mm,5 mu m) chromatographic column, takes methanol-water (60:40) as mobile phase, and the flow rate is 1.0 mL/min; the detection wavelength is 225 nm; the column temperature is 30 ℃; the amount of the sample was 10. mu.l.

Third, the detection result

The chromatogram of estradiol control, oviductus Ranae, ovum oviductus Ranae and Rana temporaria chensinensis David embryo is shown in FIGS. 1-4, and the analysis of estradiol content is shown in Table 1;

TABLE 1 Wood frog oil, egg and embryo estradiol content (μ g/g)

	Oviductus Ranae	Wood frog egg	Wood frog embryo
				Estradiol	0.126	4.70	----

Fourth, result analysis

The measurement result shows that the content of estradiol in the wood frog egg is 4.70 mu g/g at most in the wood frog oil micro powder, the wood frog egg micro powder and the wood frog embryo micro powder, the content of estradiol in the wood frog egg is 0.126 mu g/g, and the estradiol is not detected in the wood frog embryo. The wood frog embryo is a mixture of modified wood frog oil and developed eggs, and the detection report shows that the wood frog embryo does not contain estradiol hormone, which means that the modified wood frog oil does not contain estradiol hormone.