A testosterone inducing solution of male wood frog, testosterone-enriched male wood frog polypeptide and preparation method and application thereof

Abstract

The present application belongs to the field of biological health care products and food processing technology, and relates to a testosterone inducing liquid of Rana spinosa, testosterone-enriched Rana spinosa polypeptide, and a preparation method and application thereof. The present application carries out multi-disciplinary and multi-factor intervention experiments on Rana spinosa in the breeding period, explores the estrus information substance of Rana spinosa, finds an effective method for stimulating the estrus of Rana spinosa in the breeding period by using the information substance of female frogs, and facilitates the production and application of raw materials, thereby creating a new path for the development of functional products of Rana spinosa. Through artificial intervention and induction, the testosterone content of Rana spinosa in the breeding period is synchronously increased, and the testosterone level is more than twice that of the control group. The present application further provides a production process for extracting polypeptide from Rana spinosa, including the steps of raw material storage, enzymolysis, fermentation, centrifugation, drying, and the like, and finally obtaining a testosterone-enriched polypeptide product. The product can be applied to the fields of food, health care products, and medicine, and has the potential to improve the economic value of Rana spinosa.

Description

Technical Field

[0001] This invention belongs to the field of biological health products and food processing technology, specifically relating to a testosterone-inducing fluid for male forest frogs, testosterone-rich polypeptides from male forest frogs, their preparation methods, and applications. Background Technology

[0002] Testosterone (also known as testosterone, testosterone, or testosterone) is a steroid hormone secreted by the testes of male animals or the ovaries of female animals. The adrenal glands also secrete small amounts of testosterone. It plays a role in maintaining muscle strength and mass, maintaining bone density and strength, regulating gene expression and cell metabolism, improving sexual function, and maintaining a youthful state. In humans, testosterone levels follow a low-high-low curve with age. Therefore, biological testosterone supplementation is crucial for maintaining peak levels and raising existing low levels.

[0003] The forest frog, a distinctive frog species mainly distributed in Northeast my country, possesses high nutritional value and broad application prospects, encompassing the food, health, and pharmaceutical industries. Currently, the forest frog market primarily focuses on female frog oil, used in pharmaceuticals and health products. Research and development of products featuring male forest frogs lags behind, with the market primarily focused on food. Current research indicates that male forest frogs have testosterone levels more than 30 times higher during the breeding season than in the non-breeding season. Furthermore, product development is not limited by the "food and medicine homology" concept of forest frog oil, as raw materials are widely available, abundant, and inexpensive. This presents the necessary conditions for developing characteristic products featuring male forest frogs, aligning with the rapid development of the health industry and indicating a promising future. For male frog populations, achieving synchronized estrus and peak sex hormone levels requires artificial intervention and induction to meet the demands of mass production. However, the current lack of effective artificial intervention and induction methods to enhance testosterone levels in male forest frogs limits the development of male forest frog products. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a testosterone-inducing solution for male forest frogs, prepared by the following method:

[0005] After rinsing the female forest frogs during their breeding season with clean water, place them in a container filled with purified water. Place an ultrasonic generator in the container and use an ultrasonic frequency of 28-40 kHz to clean the female frogs with ultrasonic vibration for 3-10 minutes. Once the mucous membrane on the female frog's body surface is partially sloughed off, remove the female frog. The resulting washing solution is the testosterone-inducing solution for male forest frogs.

[0006] Furthermore, the density of female forest frogs placed in the container should be no less than 200 per square meter. 3 The preferred ultrasonic frequency is 40kHz.

[0007] This invention also provides a method for increasing testosterone levels in male frogs, comprising the following steps:

[0008] (1) During the forest frog breeding season, male forest frogs that are three years or older and have not yet mated for breeding are stored in a storage pond as preferred specimens. The water temperature in the pond is 9-15 degrees Celsius, and the storage density is 500-800 frogs / m². 3 The average daily light intensity is not less than 20,000 LUX, and the color temperature is not less than 3,000 K;

[0009] (2) In the storage pond adjacent to the male forest frogs, breeding female forest frogs in the egg-laying stage are placed in the pond. The estrus signaling of the female frogs and the mutual calling of the male frogs are used to stimulate the male frogs' desire to mate, so as to promote the peak testosterone content in the male frogs. As a preferred method, the density of female forest frogs should not be less than 100 / m². 3 ;

[0010] (3) While the female and male forest frogs are adjacent to each other to induce estrus, the mating calls of the male frogs are transmitted to the central nervous system through the tympanic membrane, which stimulates each other and causes the hormone threshold in the male frogs in the area to rise continuously. Then, a certain number of female frogs that have lost their eggs are put into the male frog pond to introduce a competition mechanism, stimulate the male frogs' desire to mate, and increase the testosterone level in the male frogs. As a preferred option, the ratio of female frogs put into the male frog pond is male frog: female frog = 200~250:1.

[0011] (4) Pour the above-mentioned male frog testosterone-inducing solution into the male frog pond and induce treatment for 24h~72h. The male frogs receive sexual attraction signals through their skin receptors and vomeronasal organs. Each cubic meter of inducing solution can induce 1000m 3 Male frogs in the water synchronized their estrus within a certain period of time, and their testosterone levels reached a peak, which was greater than the ideal value of the conventional control. As an optimal method, the volume ratio of the induction solution to the water in the male frog pond was 1:1000. After 48 hours of induction treatment with the induction solution, the testosterone levels in the male frogs reached their peak.

[0012] This invention also provides a testosterone-rich male forest frog polypeptide, the preparation method of which includes the following steps:

[0013] Based on the above steps (1)-(4), the following is also included:

[0014] (5) Male forest frogs whose testosterone levels have reached their peak were electrocuted in the pond, rinsed with clean water, ground into a porridge-like raw material, and placed in an enzymatic hydrolysis tank.

[0015] (6) The raw materials are diluted with purified water at a ratio of 1:4 (material to liquid volume). 2% papain is added by mass and hydrolyzed for 2-4 hours. After the hydrolysis rate is greater than 80%, the enzyme activity is inactivated. Preferably, the hydrolysis is carried out at a constant temperature of 50℃ for 2.5-3.5 hours, and then the enzyme activity is inactivated at 100℃ for 15 minutes. The enzyme activity concentration of papain is 100,000 U / g.

[0016] (7) After filtering the enzymatic hydrolysis mixture, centrifuge it, take the supernatant, concentrate it to 50%, and send it to the fermenter for fermentation using fermentation bacteria. The fermentation time is 32-40 hours.

[0017] The fermentation bacteria are one or a mixture of several of the following in equal proportions: freeze-dried Lactobacillus powder JYLP-002, freeze-dried Lactobacillus powder-Bifidobacterium BB-20, or Kluyveromyces martensii; Kluyveromyces martensii is preferred, with an inoculum size of 3.5-4.0%, and a fishy odor removal rate of greater than 80%.

[0018] (8) The fermentation broth obtained is a testosterone-rich male forest frog polypeptide solution. The peptide content in the polypeptide solution is not less than 60 mg / mL, and the testosterone content in the polypeptide solution is as high as 71 ng / g. After drying, the fermentation broth obtained is a testosterone-rich male forest frog polypeptide.

[0019] This invention applies to all subspecies of the Chinese forest frog, especially the Changbai Mountain subspecies of the Northeast forest frog. It also applies to edible frog species such as the black-spotted frog.

[0020] The beneficial effects of this invention are:

[0021] The purpose of this invention is to provide a method for preparing and developing testosterone-rich peptides from male forest frogs. This invention involves multidisciplinary and multifactorial intervention experiments on male forest frogs during their breeding season, exploring estrus signaling substances in male forest frogs, and finding an effective method to stimulate synchronous estrus in male frogs during the breeding season using female frog signaling substances, facilitating the production and application of raw materials. This creates a new pathway for the development of functional products from male forest frogs. Through artificial intervention and induction, the testosterone content in male forest frogs is synchronously increased during the breeding season, with testosterone levels more than twice that of the control group. This invention also provides a production process for extracting peptides from male forest frogs, including raw material storage, enzymatic hydrolysis, fermentation, centrifugation, and drying, ultimately yielding a testosterone-rich peptide product. Attached Figure Description

[0022] Figure 1 The curves showing testosterone levels in the enzymatic fermentation broth of adult male frogs from different months under natural conditions;

[0023] Figure 2 This is a line graph comparing induced and non-induced testosterone levels in Example 2;

[0024] Figure 3 This is a standard curve for proteins.

[0025] Figure 4 This is a bar chart showing the peptide content in the sample from Example 2. Detailed Implementation

[0026] Example 1

[0027] This embodiment provides a testosterone-inducing solution for male forest frogs, which is prepared by the following method:

[0028] After rinsing the female forest frogs in their breeding season with clean water, place them in a container filled with water, ensuring a density of at least 200 females per square meter. 3 An ultrasonic generator with an ultrasonic frequency of 40 kHz is placed in a container to clean the female frogs for 3 to 10 minutes. With the high-frequency vibration of the water, the skin and mucus of the female frogs, which are covered with a large amount of reproductive induction information (sex-inducing factors), are washed off and dispersed or dissolved in the water. After the mucus on the surface of the female frogs is partially shed, the female frogs are taken out, and the resulting washing solution is the testosterone-inducing solution for male frogs.

[0029] The obtained male frog testosterone-inducing solution (estrus-inducing solution) was poured into the male frog pond. The male frogs received the sexual attraction signal through their skin receptors and vomeronasal organs. Each cubic meter of inducing solution can induce male frogs in 1000m³ of water to estrus synchronously within 24h to 72h, and the testosterone content in their bodies reaches its peak at 48h.

[0030] Example 2

[0031] This embodiment provides a method for preparing testosterone-rich male forest frog polypeptides, including the following steps:

[0032] (1) Selection of forest frog species: Chinese forest frog Changbai Mountain subspecies. During the forest frog breeding season (April to May in Northeast China), male forest frogs that are three years old or older and have not yet mated for breeding are collected and stored in a storage pond. The water temperature in the pond is 9-15 degrees Celsius, and the storage density is 500-800 frogs / m². 3 The average daily light intensity is not less than 20,000 LUX, and the color temperature is not less than 3,000 K;

[0033] (2) In the storage pond adjacent to the male forest frogs, place breeding female forest frogs in the egg-laying stage, with a density of no less than 100 female forest frogs / m². 3 By utilizing the estrus signals transmitted by female frogs and the call-and-response between male frogs, the mating desire of male frogs is stimulated, causing the testosterone content in the male frogs to reach its peak.

[0034] (3) While the female and male forest frogs are adjacent to each other to induce estrus, the mating calls of the male frogs are transmitted to the central nervous system through the tympanic membrane, which stimulates each other and causes the hormone threshold in the male frogs in the area to rise continuously. Then, a small number of female frogs that have lost their eggs are put into the male frog pond, with the ratio of male frogs to female frogs ≈ 200:1. This introduces a competition mechanism, stimulates the male frogs' desire to mate, and increases the testosterone level in the male frogs.

[0035] (4) The testosterone-inducing solution for male forest frogs provided in Example 1 was poured into the male frog pond and treated for 24-72 hours. The male frogs received the sex-inducing signal through their skin receptors and vomeronasal organ. Each cubic meter of inducing solution can induce sex in 1000m³ of frogs.3 Male frogs in the water synchronized their estrus within a certain period of time, and their testosterone levels reached a peak, which was greater than the ideal value of the conventional control. As a preferred method, the volume ratio of the induction solution to the water in the male frog pond was 1:1000.

[0036] (5) Male forest frogs that have reached peak testosterone levels are electrocuted in the pond, rinsed with clean water, minced with a meat grinder, and quantitatively ground into a porridge-like raw material in a colloid mill and then placed in an enzymatic hydrolysis tank.

[0037] (6) The raw materials are diluted with purified water at a ratio of 1:4 (material to liquid volume). 2% papain at a mass ratio of 100,000 U / g is added for enzymatic hydrolysis for 2-4 hours. The enzymatic hydrolysis is carried out at a constant temperature of 50℃ for 2.5-3.5 hours, and then the enzyme activity is inactivated at 100℃ for 15 minutes. The hydrolysis rate is greater than 80%.

[0038] (7) After filtering the enzymatic hydrolysate through a 100-mesh filter, centrifuge the mixture, collect the supernatant, concentrate it to 50% at 80℃, and then transfer it to a fermenter for fermentation using fermenting bacteria. The fermentation time is 32-40 hours, the fermentation temperature is 38℃, and the glucose addition ratio is 1.5% by mass.

[0039] The fermentation bacteria are one or a mixture of several of the following in equal proportions: freeze-dried Lactobacillus powder JYLP-002, freeze-dried Lactobacillus powder-Bifidobacterium BB-20 produced by Shandong Zhongke Jiayi Bioengineering Co., Ltd., or Kluyveromyces martensii produced by Shandong Xinxiong Biotechnology Co., Ltd.; in this embodiment, Kluyveromyces martensii is selected, with an inoculum size of 3.5-4.0%, and a fishy odor removal rate of greater than 80%;

[0040] (8) The fermentation broth obtained is a testosterone-rich male forest frog polypeptide solution. After drying, the fermentation broth is used to obtain testosterone-rich male forest frog polypeptide.

[0041] This embodiment describes the determination of testosterone content in the enzymatic fermentation broth of male forest frogs at different stages.

[0042] (1) Experimental unit: Central Laboratory of Jilin Provincial Academy of Agricultural Specialty Products

[0043] (2) Experimental material: Chinese forest frog, Changbai Mountain subspecies male forest frog (origin: Jiaohe City, Jilin Province)

[0044] (3) Experimental instruments: Electronic balance (Sartorius Scientific Instruments Co., Ltd.); SYNERGY H1 multi-functional microplate reader (Bio Tek, USA); electric heating drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.); low-temperature centrifuge (Eppendorf); testosterone (T) quantitative detection ELISA kit (Shanghai Lunchangshuo Biotechnology Co., Ltd.)

[0045] (4) Experimental control group setup:

[0046] A: The testosterone content of the enzymatic fermentation broth of male forest frogs at different times under natural conditions was detected. Male forest frogs were sampled on February 10, April 10, June 10, August 10, October 10, and December 10. The preparation method of the enzymatic fermentation broth is as described in steps 6 and 7 of this embodiment.

[0047] B: The testosterone curve of the non-artificial intervention group (blank control group) on April 10th of the breeding season was selected as a control. The steps are as follows: steps 1, 5, 6, and 7 of this embodiment. The testosterone content of the male frogs in the testosterone induction fluid addition group (experimental group) in this embodiment was measured.

[0048] (5) Experimental methods:

[0049] ① Move the kit to room temperature for two hours to equilibrate. Take the concentrated washing solution and dilute it with distilled water at a ratio of 1:20 according to the number of tests in the batch. Mix well and set aside.

[0050] ② Remove the pre-coated plate from the sealed bag, set up one blank control well without adding any liquid; set up two wells for each calibrator, and add 50 μL of the corresponding calibrator to each well; add 50 μL of the sample to be tested directly to each of the remaining test wells.

[0051] ③ Add 50 μL of enzyme-labeled antigen to each well (except for the blank control wells), then add 50 μL of antibody to each well in the same order, mix thoroughly, cover with sealing film, and incubate at 37°C for 1 hour.

[0052] ④ Manual washing: Discard the liquid in the holes, fill each hole with washing liquid, let stand for 10 seconds and shake dry. Repeat 3 times and then pat dry.

[0053] ⑤ Add 50 μL of colorimetric reagent A and 50 μL of colorimetric reagent B to each well, shake to mix, and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well.

[0054] ⑥ Read the absorbance at 450 nm using an ELISA reader, zero the instrument using a blank control well, and then measure the absorbance of each well. Calculate the concentration using the ELISA Calc regression / fitting program.

[0055] (6) Experimental results:

[0056] ① The testosterone content of the enzymatic fermentation broth of uninduced male frogs in different months under natural conditions was detected. The results are shown in […]. Figure 1 ,Depend on Figure 1 The results showed that the testosterone levels of untreated male frogs peaked in April.

[0057] ② The results of testosterone content in the fermentation broth of male frogs in the breeding season (April) without artificial intervention in the blank control group and experimental group are shown in the figure. Figure 2In the experimental group, a testosterone-inducing solution was added, and samples were taken at different induction times (12h, 24h, 36h, 48h, 60h, 72h, 84h) to determine the testosterone content in the enzymatic fermentation broth of male frogs. Figure 2 The results showed that the initial testosterone content of the enzymatic fermentation broth of male frogs in their breeding season (April) without artificial intervention was 43 ng / g. In the experimental group with added testosterone induction solution, the testosterone content of the enzymatic fermentation broth of male frogs reached as high as 71 ng / g after 48 hours of induction treatment, and then the testosterone level tended to stabilize.

[0058] This embodiment describes the determination of peptide content in enzymatic fermentation broth.

[0059] Determination of peptide content in samples using the BCA method

[0060] ①Preparation of BCA working solution

[0061] Mix reagent A and reagent B (BCA kit Thermo Scientific) in a 50:1 ratio until thoroughly mixed. The working solution will be green.

[0062] ②Preparation of standard curve

[0063] Prepare a series of standards with known concentrations (e.g., 0, 0.02, 0.1, 0.2, 0.5, 1, 2 mg / mL) by diluting the standard protein (BSA) with PBS or deionized water. Each concentration should be tested in triplicate.

[0064] ③ Adding samples

[0065] Add the following to a 96-well plate or test tube:

[0066] Standard wells: 25 µL standard protein solution + 200 µL BCA working solution.

[0067] Sample well: 25 µL of sample to be tested + 200 µL of BCA working solution.

[0068] Blank wells: 25 µL PBS or deionized water + 200 µL BCA working solution.

[0069] Mix gently.

[0070] ④ Incubation reaction

[0071] Incubate the 96-well plate or test tube at 37°C for 30 minutes. After the reaction, the solution will change from green to purple, and the intensity of the color is directly proportional to the concentration of the protein or peptide.

[0072] ⑤ Determine absorbance

[0073] The absorbance (OD value) of each well was measured at a wavelength of 562 nm using an ELISA reader or spectrophotometer, with the blank well used for zeroing.

[0074] ⑥ Draw the standard curve

[0075] Plot a standard curve with standard protein concentration on the x-axis and absorbance (OD value) on the y-axis. Fit a linear regression equation (y = kx + b).

[0076] ⑦ Calculate sample concentration

[0077] Based on the OD value of the sample, substitute it into the standard curve equation to calculate the concentration of the polypeptide in the sample. The protein standard curve is shown below. Figure 3 As shown.

[0078] ⑧ Experimental results: The peptide content in the prepared sample was approximately 60 mg / mL.

Claims

1. A testosterone-inducing solution for male forest frogs, characterized in that: It was prepared by the following method: After rinsing the female forest frogs during their breeding season with clean water, place them in a container filled with purified water. Place an ultrasonic generator in the container and use an ultrasonic frequency of 28-40 kHz to clean the female frogs with ultrasonic vibration for 3-10 minutes. Once the mucous membrane on the female frog's body surface is partially sloughed off, remove the female frog. The resulting washing solution is the testosterone-inducing solution for male forest frogs.

2. The testosterone-inducing solution for male forest frogs according to claim 1, characterized in that: The density of female forest frogs in the container should be no less than 200 per square meter. 3 The ultrasonic frequency is 40KHz.

3. A testosterone-rich polypeptide from the male forest frog, characterized in that: The preparation method includes the following steps: (1) During the breeding season of forest frogs, male forest frogs that are three years old or older and have not yet mated to participate in breeding are stored in a storage pond; (2) In the storage pond adjacent to the male forest frog, female forest frogs in the egg-laying stage are placed in the storage pond. By using the estrus information transmission of the female frogs and the mutual calls of the male forest frogs, the male forest frogs are encouraged to mate, which causes the testosterone content in the male forest frogs to reach its peak. (3) A certain number of female frogs that have lost their eggs are put into the male frog pond, with a ratio of 200 to 250:1 between male and female frogs; to stimulate the male frogs’ desire to mate and increase the testosterone level in the male frogs. (4) Pour the male frog testosterone induction solution into the male frog pond and treat it for 24h~72h. The male frogs will synchronously go into estrus within a certain period of time and the testosterone content in their bodies will reach the peak. The male frog testosterone induction solution is the male frog testosterone induction solution described in claim 1 or 2. (5) The male forest frogs after induction were electrocuted in the pond, rinsed with clean water, ground into porridge-like raw materials, and put into an enzymatic hydrolysis tank. (6) The raw materials are diluted with purified water at a ratio of 1:4 (material to liquid volume). 2% papain is added by mass ratio and hydrolyzed for 2-4 hours. After the hydrolysis rate is greater than 80%, the enzyme activity is inactivated. (7) After filtration, the enzymatic hydrolysate is centrifuged, the supernatant is collected, concentrated to 50%, and then sent to a fermenter for fermentation using fermentation bacteria. The fermentation time is 32-40 hours. The fermentation bacteria are one or more of the following in equal proportions: freeze-dried Lactobacillus powder JYLP-002, freeze-dried Lactobacillus powder-Bifidobacterium BB-20, or Kluyveromyces martensii. (8) The obtained fermentation broth is a testosterone-rich male forest frog polypeptide solution; the obtained fermentation broth is dried to obtain testosterone-rich male forest frog polypeptide.

4. The testosterone-rich male forest frog polypeptide according to claim 3, characterized in that: In step 1, the water temperature in the pool is 9-15 degrees Celsius, and the storage density is 500-800 animals / m³. 3 The average daily light intensity should be no less than 20,000 LUX, and the color temperature should be no less than 3,000 K; in step 2, the density of female forest frogs should be no less than 100 per m². 3 .

5. The testosterone-rich male forest frog polypeptide according to claim 3, characterized in that: In step 4, the volume ratio of the induction solution to the water in the male frog pond is 1:1000; after 48 hours of induction treatment, the testosterone content in the male frogs reaches its peak.

6. The testosterone-rich male forest frog polypeptide according to claim 3, characterized in that: In step 6, the enzyme is hydrolyzed at a constant temperature of 50℃ for 2.5~3.5h, and then the enzyme activity is inactivated at 100℃ for 15min; the enzyme activity concentration of papain is 100,000 U / g.

7. A method for increasing testosterone levels in male frogs, characterized in that: Includes the following steps: (1) During the breeding season of forest frogs, male forest frogs that are three years old or older and have not yet mated to participate in breeding are stored in a storage pond; (2) In the storage pond adjacent to the male forest frog, female forest frogs in the egg-laying stage are placed in the storage pond. By using the estrus information transmission of the female frogs and the mutual calls of the male forest frogs, the male forest frogs are encouraged to mate, which causes the testosterone content in the male forest frogs to reach its peak. (3) A certain number of female frogs that have lost their eggs are put into the male frog pond, with a ratio of 200 to 250:1 between male and female frogs; to stimulate the male frogs’ desire to mate and increase the testosterone level in the male frogs. (4) Pour the male frog testosterone induction solution into the male frog pond and treat it for 24h~72h. The male frogs will synchronously go into estrus within a certain period of time and the testosterone content in their bodies will reach the peak. The male frog testosterone induction solution is the male frog testosterone induction solution described in claim 1 or 2.

Images