Male wood frog testosterone induction liquid, male wood frog polypeptide rich in testosterone as well as preparation method and application of male wood frog polypeptide

Abstract

The invention belongs to the technical field of biological health care products and food processing, and relates to male wood frog testosterone induction liquid, male wood frog polypeptide rich in testosterone and a preparation method and application of the male wood frog polypeptide. According to the method, multidisciplinary and multi-factor intervention experiments are carried out on the male wood frogs in the breeding period, the estrus information substances of the male wood frogs are explored, an effective method for stimulating the male wood frogs in the breeding period to be estrus synchronously by utilizing the information substances of the female wood frogs and facilitating production and application of raw materials is found, and a brand new path is created for functional product development of the male wood frogs. Through manual intervention and induction, the testosterone content of male wood frogs is synchronously increased in the breeding period, and the testosterone level is more than two times that of a control group. The invention also provides a production process flow for extracting polypeptide from male wood frogs, which comprises the steps of raw material storage, enzymolysis, fermentation, centrifugation, drying and the like, and finally a polypeptide product rich in testosterone is obtained. The product can be applied to the fields of food, health care products and medicines, and has the potential of improving the economic value of male wood frogs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological health products and food processing, and particularly relates to a testosterone induction liquid for male Rana chensinensis, a male Rana chensinensis polypeptide rich in testosterone, and a preparation method and application thereof. Background Art

[0002] Testosterone, also known as testis hormone, testosterone or androsterone, is a steroid hormone secreted by the testes of male animals or the ovaries of female animals. The adrenal gland also secretes a small amount of testosterone, which has the functions of maintaining muscle strength and mass, maintaining bone density and strength, regulating gene expression and cell metabolism, improving sexual function and maintaining a youthful state. As humans age, the testosterone content in the body shows a low-high-low curve change. To maintain the peak time of the content level in the body and the increase of the low level, the supplementation of biogenic testosterone is of great significance.

[0003] Rana chensinensis is a characteristic frog species mainly distributed in the northeastern region of China, with high nutritional value and broad application prospects. Its application fields include food, health care, medicine and other industries. At present, the Rana chensinensis market mainly focuses on female frog oil, which is used as drugs and health products. However, the current product development research on male Rana chensinensis is still in a lagging state, mainly as food. According to the current research results, the testosterone hormone level of male Rana chensinensis during the breeding period is more than 30 times that of the non-breeding period, and the product development is not restricted by the "medicinal and edible homology" of frog oil. The raw material source is wide, the supply is sufficient, and the price is low, meeting the necessary conditions for developing characteristic products of male Rana chensinensis, meeting the needs of the rapid development of the large health industry, and having broad prospects. For the male frog population, to make the population in estrus synchronously and make the sex hormone level reach the peak, artificial intervention and induction must be carried out to meet the requirements of batch production of raw materials. However, there is currently a lack of effective artificial intervention and induction methods to increase the testosterone hormone level of male Rana chensinensis, which restricts the development of male Rana chensinensis products. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a testosterone induction liquid for male Rana chensinensis, which is prepared by the following preparation method:

[0005] After the female Rana chensinensis in the breeding period after egg laying is rinsed with clean water, it is put into a container filled with pure water, and an ultrasonic generator is placed in the container. The ultrasonic frequency is 28-40 KHz, and the female frog is ultrasonically vibrated and cleaned for 3-10 minutes. After seeing that the mucosal part on the surface of the female frog has partially fallen off, the female frog is fished out, and the obtained washing liquid is the testosterone induction liquid for male Rana chensinensis.

[0006] Further, the placement density of female Rana chensinensis in the container is not less than 200 per m 3 , and the ultrasonic frequency is preferably 40 KHz.

[0007] The present invention also provides a method for increasing the testosterone content in male frogs, which includes the following steps:

[0008] (1) During the breeding season of forest frogs, male forest frogs over three years old that have not mated and participated in breeding are centrally stored in a storage pond. Preferably, the water temperature in the pond is 9 - 15 degrees Celsius, the storage density is 500 - 800 frogs / m 3 , the average daily illumination is not less than 20000 LUX, and the color temperature is not less than 3000 K;

[0009] (2) In the storage pond adjacent to the male forest frogs, breeding female forest frogs in the egg - laying period are placed. By using the estrus information conduction of female frogs and the mutual calling of male forest frogs, the mating desire of male forest frogs is stimulated, so that the testosterone content in male forest frogs reaches the peak value. Preferably, the placement density of female forest frogs is not less than 100 frogs / m 3 ;

[0010] (3) While the male and female forest frogs are adjacent and induced to be in estrus, the courtship calls of male frogs are conducted to the central nervous system through the tympanic membrane and mutually stimulate, continuously increasing the hormone threshold in the male frogs in the area. Then, a certain amount of female frogs after egg - laying are put into the male frog pond, introducing a competition mechanism to stimulate the mating desire of male frogs and increase the testosterone level in male frogs. Preferably, the ratio of male frogs to female frogs put into the male frog pond is 200 - 250:1;

[0011] (4) Pour the above - provided testosterone induction liquid for male forest frogs into the male frog pond, and conduct induction treatment for 24 h - 72 h. Male frogs receive sexual induction signals through body surface receptors and vomeronasal organs. Each cubic meter of induction liquid can make male frogs in 1000 m 3 of water body be in estrus synchronously within a certain time, and the testosterone content in their bodies reaches the peak value, which is greater than the ideal value of the conventional control. Preferably, the volume ratio of the induction liquid to the water body in the male frog pond is 1:1000; when the induction liquid is used for induction treatment for 48 h, the testosterone content in male frogs reaches the peak value.

[0012] The present invention also provides a male forest frog polypeptide rich in testosterone, and the preparation method includes the following steps:

[0013] On the basis of the above steps (1) - (4), it further includes:

[0014] (5) After the testosterone content in male frogs reaches the peak value, the male frogs are electrocuted to death in the pond, fished out, rinsed with clean water, ground into a porridge - like raw material, and put into an enzymatic hydrolysis tank;

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

[0016] (7) After the enzymatic hydrolysis mixture is filtered, it enters a centrifuge for centrifugation. The supernatant is taken, concentrated to 50%, and then sent to a fermentation tank for fermentation using fermentation bacteria for 32 - 40 h.

[0017] The fermentation bacteria mentioned above are one or several of freeze - dried Lactobacillus powder JYLP - 002, freeze - dried lactic acid bacteria powder - Bifidobacterium BB - 20, or Kluyveromyces marxianus in equal proportions; preferably Kluyveromyces marxianus, and the inoculation amount of Kluyveromyces marxianus is 3.5 - 4.0%, and the fishy smell removal rate is greater than 80%.

[0018] (8) The obtained fermentation broth is a male Rana chensinensis polypeptide solution rich in testosterone. The peptide content in the polypeptide solution is not less than 60 mg / mL, and the measured testosterone content in the polypeptide solution is as high as 71 ng / g; the obtained fermentation broth is dried to obtain male Rana chensinensis polypeptide rich in testosterone.

[0019] The content of the present invention is applicable to all subspecies of Rana chensinensis, especially applicable to the Changbai Mountain subspecies of Rana dybowskii. The content of the present invention is also applicable to edible frog species such as Rana nigromaculata.

[0020] The present invention further provides the application of male Rana chensinensis polypeptide rich in testosterone, including being used for preparing male Rana chensinensis polypeptide drinks, male Rana chensinensis polypeptide health products, male Rana chensinensis polypeptide instant lozenges, and male Rana chensinensis polypeptide wine.

[0021] The present invention further provides the application of male Rana chensinensis polypeptide rich in testosterone for preparing health products for maintaining muscle strength and quality, or maintaining bone density and strength, or regulating gene expression and cell metabolism, or improving sexual function.

[0022] The beneficial effects of the present invention:

[0023] The purpose of the present invention is to provide a preparation method and product development of male Rana chensinensis polypeptide rich in testosterone. The present invention has carried out multi - disciplinary and multi - factor intervention experiments on male Rana chensinensis during the breeding period, explored the estrus information substances of male Rana chensinensis, and found an effective method to stimulate the synchronous estrus of male frogs during the breeding period using female frog information substances, which is convenient for raw material production and application, creating a new path for the development of functional products of male Rana chensinensis. Through artificial intervention and induction, the testosterone content of male Rana chensinensis is synchronously increased during the breeding period, and the testosterone level is more than twice that of the control group. The present invention also provides a production process for extracting polypeptides from male Rana chensinensis, including steps such as raw material reserve, enzymatic hydrolysis, fermentation, centrifugation, and drying, and finally obtaining a polypeptide product rich in testosterone. This product can be applied in the fields of food, health products, and medicine, and has the potential to increase the economic value of male Rana chensinensis. Description of the Drawings

[0024] Figure 1 It is the testosterone detection value curve of the enzymatic hydrolysis fermentation broth of adult male frogs in different months under natural conditions;

[0025] Figure 2 Line graph comparing induced and non-induced testosterone levels in Example 2;

[0026] Figure 3 Protein standard curve;

[0027] Figure 4 Bar graph of peptide content in the sample of Example 2. Detailed implementation method

[0028] Example 1,

[0029] This example provides a testosterone induction solution for male forest frogs, which is prepared by the following method:

[0030] After rinsing the female forest frogs in the breeding period after egg laying with clean water, put them into a container filled with water. The placement density of female forest frogs in the container is not less than 200 per m 3 , put an ultrasonic generator in the container, with an ultrasonic frequency of 40KHz, and clean the female frogs for 3 - 10 minutes; with the high-frequency vibration of the water body, the epidermal mucosa and mucus of the female frogs attached with a large amount of breeding induction information (sexual induction factors) are washed off, dispersed or dissolved in the water. After seeing partial shedding of the mucosal part on the surface of the female frogs, fish out the female frogs, and the obtained washing solution is the testosterone induction solution for male forest frogs.

[0031] Pour the obtained testosterone induction solution (estrus induction solution) for male forest frogs into the male frog pond. The male frogs receive sexual induction signals through the body surface receptors and vomeronasal organs. Each cubic meter of induction solution can make the male frogs in 1000m 3 The water body of male frogs is in synchronous estrus within 24h - 72h, and the testosterone content in the body reaches the peak at 48h.

[0032] Example 2,

[0033] This example provides a preparation method for polypeptides rich in testosterone in male forest frogs, including the following steps:

[0034] (1) Select the Changbai Mountain subspecies of Rana chensinensis as the frog variety. During the breeding season of forest frogs (April to May in Northeast China), concentrate the male forest frogs over three years old that have not paired for breeding and store them in a storage pond. The water temperature in the pond is 9 - 15 degrees, and the storage density is 500 - 800 per m 3 , and the average daily light intensity is not less than 20000 LUX, and the color temperature is not less than 3000K;

[0035] (2) Put the breeding female forest frogs in the oviposition period into the storage pond adjacent to the male forest frogs. The placement density of female forest frogs is not less than 100 per m 3 , and use the estrus information conduction of female frogs and the mutual chirping of male forest frogs to stimulate the pairing desire of male forest frogs and promote the testosterone content in male forest frogs to reach the peak;

[0036] (3) While the male and female forest frogs are adjacent to each other and in the state of courtship-induced estrus, the courtship calls of the male frogs are conducted to the central nervous system through the tympanic membranes, and they stimulate each other, continuously increasing the hormone threshold in the male frogs in the area. Then, a small amount of female frogs after egg-laying is put into the male frog pool, with the ratio of male frogs to female frogs being approximately 200:1, introducing a competition mechanism to stimulate the male frogs' desire to pair and increasing the testosterone level in the male frogs.

[0037] (4) Pour the testosterone induction liquid for male forest frogs provided in Example 1 into the male frog pool and conduct the induction treatment for 24h - 72h. The male frogs receive the sex induction signals through the body surface receptors and vomeronasal organs. For every cubic meter of the induction liquid, the male frogs in 1000m 3 of water body will be in synchronous estrus within a certain period of time, and the testosterone content in their bodies reaches the peak value, which is greater than the ideal value of the conventional control. Preferably, the volume ratio of the induction liquid to the water body in the male frog pool is 1:1000.

[0038] (5) The male forest frogs after the testosterone content reaches the peak value are electrocuted to death in the pool, fished out, rinsed with clean water, ground into a porridge-like raw material with a meat grinder in a quantitative manner, and then put into an enzymatic hydrolysis tank.

[0039] (6) The raw material is diluted with pure water according to the volume ratio of the material to the liquid of 1:4, and papain with a mass ratio of 2% and 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°C for 2.5 - 3.5h, and then the enzyme activity is inactivated at 100°C for 15min. The enzymatic hydrolysis rate is greater than 80%.

[0040] (7) The enzymatically hydrolyzed mixture is filtered through a 100-mesh filter and then centrifuged in a centrifuge. The supernatant is taken, concentrated to 50% at 80°C, and then sent to a fermentation tank for fermentation using fermentation bacteria. The fermentation time is 32 - 40h, the fermentation temperature is 38°C, and the mass ratio of glucose added is 1.5%.

[0041] The fermentation bacteria are one or several of the freeze-dried lactobacillus powder JYLP - 002 produced by Shandong Zhongke Jiayi Bioengineering Co., Ltd., the freeze-dried lactic acid bacteria powder - bifidobacterium BB - 20, or the Kluyveromyces marxianus produced by Shandong Xinxiong Biotechnology Co., Ltd. in an equal proportion mixture. In this example, Kluyveromyces marxianus is selected, and the inoculation amount of Kluyveromyces marxianus is 3.5 - 4.0%, and the fishy smell removal rate is greater than 80%.

[0042] (8) The obtained fermentation broth is a male forest frog polypeptide solution rich in testosterone, and the obtained fermentation broth is dried to obtain male forest frog polypeptide rich in testosterone.

[0043] Determination of the testosterone content in the enzymatically hydrolyzed and fermented liquid of male forest frogs at different stages in this example

[0044] (1) Experimental unit: Central Laboratory of Jilin Academy of Agricultural and Sideline Products

[0045] (2) Experimental materials: Male Rana chensinensis of the Changbaishan subspecies (origin: Jiaohe City, Jilin Province)

[0046] (3) Experimental instruments: electronic balance (Sartorius Scientific Instruments Co., Ltd.); SYNERGY H1 multifunctional microplate reader (Bio Tek, USA); electric heating blast drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.); low-temperature centrifuge (eppendorf); testosterone (T) quantitative detection ELISA kit (Shanghai Lunchangshuo Biotechnology)

[0047] (4) Experimental control group setting:

[0048] A: The testosterone content of the enzymatic fermentation liquid of male forest frogs at different periods under natural conditions was detected. The 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 liquid was referred to steps 6 and 7 of this embodiment;

[0049] B: The testosterone curve of the group without artificial intervention on April 10 during the breeding period (blank control group) was selected as a control, and the steps were referred to steps 1, 5, 6, and 7 of this embodiment; the testosterone content of the male frog in the group with testosterone induction solution added (experimental group) in this embodiment was measured;

[0050] (5) Experimental methods:

[0051] ① Move the test kit to room temperature for two hours, take the concentrated washing solution, 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.

[0052] ② Take the pre-coated plate out of the sealed bag, set up a blank control well without adding any liquid; set up two wells for each calibrator in turn, and add 50μL of the corresponding calibrator to each well; directly add 50μL of the sample to be tested to each of the remaining test wells.

[0053] ③ Add 50 μL of enzyme-labeled antigen to each well (except the blank control well), and then add 50 μL of antibody to each well in the same order, mix thoroughly, apply sealing film, and incubate at 37°C for 1 hour.

[0054] ④ Manual plate washing: discard the liquid in the wells, fill each well with washing solution, let it stand for 10 seconds and spin dry, repeat 3 times and pat dry.

[0055] ⑤ Add 50 μL of color developer A solution and 50 μL of color developer B solution to each well, shake and mix, incubate at 37°C in the dark for 15 minutes to develop the color, and add 50 μL of stop solution to each well.

[0056] ⑥ Read the sample at 450nm using an ELISA reader, adjust the zero point using the blank control well, and then measure the absorbance of each well. Calculate the concentration using the ELISACalc regression / fitting calculation program.

[0057] (6) Experimental results:

[0058] ① Detect the testosterone content in the enzymatic hydrolysis fermentation broth of male frogs without induction treatment in different months under natural conditions. The results are shown in Figure 1 , and it can be seen from Figure 1 the results that the testosterone content of untreated male frogs reaches the peak in April.

[0059] ② The results of the testosterone content in the fermentation broth of the blank control group and the experimental group of male frogs in the breeding period (April) without artificial intervention are shown in Figure 2 . Among them, the testosterone induction solution was added to the experimental group, and samples were taken at different induction times (12h, 24h, 36h, 48h, 60h, 72h, 84h) according to the testosterone induction solution, and the testosterone content in the enzymatic hydrolysis fermentation broth of male frogs was measured. It can be seen from Figure 2 the results that the initial testosterone content in the enzymatic hydrolysis fermentation broth of male frogs in the breeding period (April) without artificial intervention is 43 ng / g. In the experimental group added with the testosterone induction solution, the measured value of the testosterone content in the enzymatic hydrolysis fermentation broth of male frogs induced for 48 h is as high as 71 ng / g, and then the testosterone level tends to be stable.

[0060] Determination of the polypeptide content in the enzymatic hydrolysis fermentation broth in this example

[0061] Use the BCA method to determine the polypeptide content in the sample

[0062] ① Prepare the BCA working solution

[0063] Mix reagent A and reagent B (BCA kit Thermo Scientific) in a ratio of 50:1 and mix well. The working solution is green.

[0064] ② Prepare the standard curve

[0065] Dilute the standard protein (BSA) with PBS or deionized water to prepare a series of standard products with known concentrations (such as 0, 0.02, 0.1, 0.2, 0.5, 1, 2 mg / mL). Set 3 replicates for each concentration.

[0066] ③ Add samples

[0067] Add the following to 96-well plates or test tubes respectively:

[0068] Standard product wells: 25 μL standard protein solution + 200 μL BCA working solution.

[0069] Sample wells: 25 μL sample to be tested + 200 μL BCA working solution.

[0070] Blank wells: 25 μL PBS or deionized water + 200 μL BCA working solution.

[0071] Mix gently.

[0072] ④ Incubation reaction

[0073] Place the 96-well plate or test tube in an incubator at 37 °C for 30 minutes. After the reaction, the solution will change from green to purple, and the color intensity is proportional to the protein or polypeptide concentration.

[0074] ⑤ Measurement of absorbance

[0075] Use a microplate reader or spectrophotometer to measure the absorbance (OD value) of each well at a wavelength of 562 nm, and zero with the blank well.

[0076] ⑥ Plot the standard curve

[0077] Use the standard protein concentration as the abscissa and the absorbance (OD value) as the ordinate to plot the standard curve. Fit the linear regression equation (y = kx + b).

[0078] ⑦ Calculate the sample concentration

[0079] According to 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 as Figure 3 shown.

[0080] ⑧ Experimental result: The peptide content in the prepared sample is about 60 mg / mL.

[0081] Example 3,

[0082] This example provides a testosterone-rich male Rana chensinensis polypeptide drink, which is added by volume ratio: 10% - 15% of the male Rana chensinensis polypeptide fermentation broth prepared in Example 2, 2% of fresh ginseng, 5% of linden honey, 2% of granulated sugar, 0.06% of taurine, and the rest is the aqueous extract of deer penis. According to the steps of mixing materials, homogenizing, canning, sterilizing, inspecting, and packaging, the finished product is obtained.

[0083] Example 4,

[0084] This example provides a testosterone-rich male Rana chensinensis polypeptide instant lozenge, whose ingredients include the male Rana chensinensis polypeptide fermentation broth prepared in Example 2, oyster peptide powder, porous starch, maltodextrin, aqueous extract of deer penis, stevioside, and icing sugar. According to the steps of mixing materials, freeze-drying, making instant lozenges, sterilizing, and packaging, the finished product is obtained.

[0085] Example 5,

[0086] This example provides a testosterone-rich male Rana chensinensis polypeptide wine. Add 2% - 10% of the dry powder of the male Rana chensinensis polypeptide prepared in Example 2 by volume ratio to 60-degree liquor. After mixing and standing for at least 30 days, filter through a 200-mesh sieve to obtain the male Rana chensinensis polypeptide wine. After canning, inspecting, and packaging, the finished product is obtained.

[0087] Example 6,

[0088] This embodiment provides a health product for maintaining muscle strength and mass, the ingredients of which include the testosterone-rich male Rana chensinensis polypeptide prepared in Example 2 and are used for maintaining the muscle strength and mass of the human body.

[0089] Example 7,

[0090] This embodiment provides a health product for maintaining bone density and strength, the ingredients of which include the testosterone-rich male Rana chensinensis polypeptide prepared in Example 2 and are used for maintaining the bone density and strength of the human body.

[0091] Example 8,

[0092] This embodiment provides a health product for regulating gene expression and cell metabolism, the ingredients of which include the testosterone-rich male Rana chensinensis polypeptide prepared in Example 2 and are used for regulating the gene expression and cell metabolism of the human body.

[0093] Example 9,

[0094] This embodiment provides a health product for enhancing sexual function, the ingredients of which include the testosterone-rich male Rana chensinensis polypeptide prepared in Example 2 and are used for enhancing the sexual function of men.

Claims

1. A male Rana chensinensis testosterone induction solution, characterized in that: It is prepared by the following preparation method: After the female Rana chensinensis in the breeding period after ovulation is rinsed with purified water, it is put into a container filled with purified water. An ultrasonic generator is placed in the container, and the ultrasonic frequency is 28 - 40 KHz. The female frogs are ultrasonically vibrated and cleaned for 3 - 10 minutes. After seeing that part of the mucous membrane on the surface of the female frogs has fallen off, the female frogs are fished out, and the obtained washing solution is the testosterone induction solution for male Rana chensinensis.

2. The testosterone induction solution for male Rana chensinensis according to claim 1, characterized in that: The placing density of female forest frogs in the container is not less than 200 frogs / m 3 , and the ultrasonic frequency is 40KHz.

3. A testosterone-rich male Rana chensinensis polypeptide, characterized in that: The preparation method includes the following steps: (1) During the breeding season of Rana chensinensis, male Rana chensinensis over three years old that have not mated and participated in breeding are centrally stored in a storage pond; (2) In the storage pond adjacent to the male Rana chensinensis, breeding female Rana chensinensis in the ovulation period are put in. By using the estrus information conduction of female frogs and the mutual chirping of male Rana chensinensis, the mating desire of male Rana chensinensis is stimulated, and the testosterone content in male Rana chensinensis is promoted to reach the peak value; (3) A certain amount of female frogs after ovulation are put into the male frog pond, and the ratio of male frogs to female frogs is 200 - 250:1; the mating desire of male frogs is stimulated, and the testosterone level in male frogs is increased; (4) Pour the testosterone induction solution for male Rana chensinensis into the male frog pond and conduct the temptation treatment for 24 h - 72 h. The male frogs are in estrus synchronously within a certain period of time, and the testosterone content in their bodies reaches the peak value; the testosterone induction solution for male Rana chensinensis is the testosterone induction solution for male Rana chensinensis described in claim 1 or 2; (5) The induced male Rana chensinensis are electrocuted to death in the pond, fished out, rinsed with purified water, ground into a porridge - like raw material, and put into an enzymatic hydrolysis tank; (6) The raw material is diluted with purified water according to the volume ratio of the raw material to the liquid of 1:4, and 2% papain is added according to the mass ratio for enzymatic hydrolysis for 2 - 4 hours. After the enzymatic hydrolysis rate is greater than 80%, the enzyme activity is inactivated; (7) The enzymatically hydrolyzed mixture is filtered and then centrifuged in a centrifuge. The supernatant is taken, concentrated to 50%, and then sent to a fermentation tank for fermentation with fermentation bacteria for 32 - 40 h; (8) The obtained fermentation broth is a male Rana chensinensis polypeptide solution rich in testosterone; the obtained fermentation broth is dried to obtain male Rana chensinensis polypeptide rich in testosterone.

4. A testosterone-rich male Rana chensinensis polypeptide according to claim 3, characterized in that: In Step 1, the water temperature in the pond is 9 - 15 degrees Celsius, the storage density is 500 - 800 per cubic meter 3 , the average daily illumination is not less than 20000 LUX, and the color temperature is not less than 3000K; in Step 2, the placement density of female Rana chensinensis is not less than 100 per cubic meter 3 .

5. A testosterone-rich male Rana chensinensis polypeptide according to claim 3, characterized in that: In step 4, the volume ratio of the induction solution to the water body in the male frog pond is 1:1000; the induction solution is used for the temptation treatment for 48 h, and the testosterone content in male frogs reaches the peak value.

6. The testosterone-rich Rana chensinensis polypeptide according to claim 3, wherein: In step 6, enzymatic hydrolysis is carried out at a constant temperature of 50 °C for 2.5 - 3.5 h, and then the enzyme activity is inactivated at 100 °C for 15 min; the enzyme activity concentration of papain is 100,000 U / g.

7. A testosterone-rich male Rana chensinensis polypeptide according to claim 3, characterized in that: In step 7, the fermentation bacteria are one or several of freeze - dried Lactobacillus powder JYLP - 002, freeze - dried Lactobacillus powder - Bifidobacterium BB - 20 or Kluyveromyces marxianus in an equal - proportion mixture.

8. Use of a male Rana chensinensis polypeptide rich in testosterone according to any one of claims 3-7, characterized in that: It is used for preparing male Rana chensinensis polypeptide drinks, male Rana chensinensis polypeptide instant lozenges or male Rana chensinensis polypeptide wine.

9. Use of a testosterone-rich male Rana chensinensis polypeptide according to any one of claims 3-7, characterized in that: It is used for preparing drugs for maintaining muscle strength and quality, or maintaining bone density and strength, or regulating gene expression and cell metabolism, or improving sexual function.

10. A method for increasing the testosterone content in male frogs, characterized in that: It includes the following steps: (1) During the breeding season of Rana chensinensis, male Rana chensinensis over three years old that have not mated and participated in breeding are centrally stored in a storage pond; (2) In the storage pond adjacent to the male Rana chensinensis, breeding female Rana chensinensis in the ovulation period are put in. By using the estrus information conduction of female frogs and the mutual chirping of male Rana chensinensis, the mating desire of male Rana chensinensis is stimulated, and the testosterone content in male Rana chensinensis is promoted to reach the peak value; (3) Put a certain amount of spawn-laying female frogs into the male frog pond, with the ratio of male frogs to female frogs being 200 - 250:1; Stimulate the pairing desire of male frogs and increase the testosterone level in male frogs; (4) Pour the male Rana chensinensis testosterone induction liquid into the male frog pond and conduct the induction treatment for 24h - 72h. The male frogs will be in estrus synchronously within a certain period of time, and the testosterone content in their bodies will reach the peak value. The male Rana chensinensis testosterone induction liquid is the male Rana chensinensis testosterone induction liquid described in claim 1 or 2.

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