A bioactive peptide from oyster for enhancing male reproductive health

Abstract

The application discloses a kind of oyster-derived bioactive peptides for enhancing male reproductive health, and belongs to the technical field of functional peptides.The peptide product prepared by the method provided in the application has the bioactivity of improving male sperm quality (increasing sperm quantity and survival rate) and erectile dysfunction, and enhancing male reproductive health;It is convenient to eat, has no toxic side effects, and no adverse effects on volunteers and mice involved in the test were observed during the test;The oyster-derived bioactive peptides for enhancing male reproductive health are prepared from high-quality, edible oysters cultured artificially, no toxic and harmful reagents are used in the preparation process, and the added or used components are all beneficial or harmless to the body or components existing in the body / atmosphere, so the safety is very high.

Description

Technical Field

[0001] This invention relates to the field of functional peptide technology, and in particular to a bioactive peptide derived from oysters that enhances male reproductive health. Background Technology

[0002] Drug therapy is the most common way to enhance male reproductive health, but it has contraindications, side effects, or psychological barriers (such as PDE5 inhibitors). Therefore, developing a functional food that is more acceptable, has better adherence, and is more sustainable for daily use is of great importance.

[0003] Oysters are a general term for a group of bivalve mollusks belonging to the family Ostreidae. There are hundreds of known species worldwide. Among them, the larger, meatier, and sweeter varieties, suitable for eating raw or grilled, are commonly known as oysters, such as the Pacific oyster and the Kumamoto oyster. Edible oysters are highly nutritious, often called "milk of the sea," rich in all the essential amino acids and low in fat. They are also rich in vitamin B12, an essential element for maintaining a healthy nervous system and red blood cell formation, which is often deficient in vegetarians. Furthermore, they contain various minerals, including zinc, selenium, iron, copper, manganese, and phosphorus, as well as unique nutrients such as taurine, DHA, and EPA.

[0004] In addition, oyster shells, after being cleaned and dried, are a kind of traditional Chinese medicine with effects such as calming the liver, nourishing yin, suppressing yang, softening and dispersing nodules, calming the mind, and astringing. Summary of the Invention

[0005] The purpose of this invention is to provide a bioactive peptide derived from oysters that enhances male reproductive health. This food extract is characterized by high safety, good efficacy, no toxic side effects, no need for medication adherence, and high nutritional value.

[0006] To achieve the above objectives, the present invention provides a method for preparing bioactive peptides derived from oysters that enhance male reproductive health, comprising the following steps: S1. After rinsing the fresh oyster meat, crush it under low temperature and protective atmosphere, add mixed tocopherol solution and homogenize, then centrifuge in the dark and at low temperature, and take the oil phase into a light-proof centrifuge tube, fill it with inert gas, seal it and store it temporarily at -20℃. S2. Add the portion of S1 after removing the oil phase to an aqueous solution of vitamin C, adjust the pH, enzymatically hydrolyze, sterilize, freeze-dry and pulverize to obtain a powder. S3. Add the sterilized product from step S1 to the powder obtained in step S2, pack it into bags / cans, vacuum / fill with inert gas, and store it in a sealed, light-proof container.

[0007] Preferably, the fresh oyster meat in S1 is from sexually mature, live male edible oysters that are about 1 year old; the mixed tocopherol solution is made by dissolving mixed tocopherols in high-oleic vegetable oil, and the amount of mixed tocopherols added is 0.1-0.5 mL / kg of fresh oyster meat.

[0008] Preferably, the low temperature in S1 is 0°C, and the centrifugation is 8000-10000 rpm for 10-15 minutes.

[0009] Preferably, the concentration of the vitamin C aqueous solution in S2 is 2-10 mg / 100 mL, and the amount added is 1-2 times the total volume after removing the oil phase.

[0010] Preferably, in step S2, the pH is adjusted using HCl solution and / or NaOH solution to adjust the pH to 6.8-7.5.

[0011] Preferably, the enzymatic hydrolysis in S2 is carried out using flavor protease and trypsin, and the hydrolysis conditions are 37°C, protected from light, and in an inert gas atmosphere for 10-15 minutes.

[0012] Preferably, sterilization in both S2 and S3 is performed using a 0.22 μm filter membrane.

[0013] Preferably, vitamin C powder may also be added to S3.

[0014] Preferably, in step S3, the product can also be prepared into a vacuum-sealed, opaque sheet / sphere / solvent.

[0015] A bioactive peptide derived from oysters that enhances male reproductive health, prepared by the method described above.

[0016] Oysters contain a variety of nutrients and beneficial components, among which zinc, selenium, manganese, copper, vitamin B12, and EPA play important roles in protecting sperm from oxidative and inflammatory damage. Zinc and vitamin B12 are essential components / factors for the synthesis of nucleotides, playing a vital role in maintaining normal hormone levels, osmotic pressure, and nerve health. Phosphorus and DHA are important components in the formation of cell membranes. Iron is essential for ensuring an adequate oxygen supply to testicular tissue. Selenium is an important component of the mitochondrial sheath in the sperm tail, and DHA plays an important role in the membrane fluidity of the sperm head. EPA is beneficial for improving penile vascular endothelial function and maintaining erectile health. Taurine enhances sperm motility.

[0017] In addition, fresh oyster meat is rich in protein and all the essential amino acids for the human body. The gonads of sexually mature oysters contain a large number of sperm, including many components and factors necessary for human spermatogenesis. The method provided by this invention, using mild, protective atmosphere, light-proof, and relatively low-temperature conditions, can retain most of the beneficial / active components.

[0018] Therefore, the present invention provides a bioactive peptide derived from oysters that enhances male reproductive health, the specific technical effects of which are as follows: (1) The peptide product prepared by the method provided in this invention has the bioactivity of improving male sperm quality (increasing sperm count and survival rate) and erectile dysfunction, and enhancing male reproductive health; it is convenient to consume, has no toxic side effects, and no adverse effects were observed on the volunteers and mice participating in the experiment during the experiment; (2) The bioactive peptides for enhancing male reproductive health derived from oysters prepared in this invention are made from high-quality, edible artificially farmed oysters. No toxic or harmful reagents are used in the preparation process. All added or used ingredients are either beneficial or harmless to the body or are present in the body / atmosphere, and the safety is very high.

[0019] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0020] The technical solution of the present invention will be further described below through embodiments.

[0021] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention are clearly and completely described below through embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanations of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

[0023] Example 1 The specific steps for preparing a bioactive peptide derived from oysters that enhances male reproductive health are as follows: S1. Select live Pacific oysters that are about 1 year old, sexually mature, artificially cultured in water at around 17-18℃, and have undergone "sand-removal" treatment. After opening the shell, select oyster meat with a milky white or snow-white gland color. If there are sand grains, shell fragments, or other impurities, gently rinse them with running drinking water to remove them.

[0024] S2. Take 500g of fresh oil and cut it into small pieces at 0℃ under a nitrogen atmosphere. The largest piece should not exceed 0.5-0.6cm in diameter. Then add 5mL of a mixed tocopherol solution (dissolved in high-oleic vegetable oil, with a mixed tocopherol content of 2%), mix well, and homogenize at 0℃ in the dark for 10 minutes. Then transfer it to a light-protected centrifuge tube and centrifuge at 0℃ and 10000rpm for 10 minutes. Take the uppermost oil phase and transfer it to a light-protected centrifuge tube. Seal the centrifuge tube after filling it with nitrogen and store it temporarily at -20℃.

[0025] S3. Add an equal volume of vitamin C aqueous solution (5 mg / 100 mL) to the mixture after removing the oil phase. Adjust the pH of the mixture to about 7.0 with 1 M HCl and 1 M NaOH. After mixing, add 5000 U of flavor protease and 10000 U of trypsin. Stir and react for 10 minutes at 37°C in the dark under a nitrogen atmosphere. Then, quickly filter the mixture through a 0.22 μm filter membrane under a nitrogen atmosphere. Freeze-dry the filtered mixture in the dark and then pulverize it. Weigh 75 g.

[0026] S4. Take the oil phase prepared in step S2 and quickly pass it through a 0.22μm filter membrane under a nitrogen atmosphere in the dark. Quickly add the oil phase of the filter membrane and 0.5g of vitamin C powder to the product pulverized in step S3. After mixing, the bioactive peptides from oysters that enhance male reproductive health are obtained. Press them into sheets under a nitrogen atmosphere at 0℃ (if they are not easy to stick, spray with sterile distilled water until they are easy to form), and store them in a light-proof vacuum.

[0027] Example 2 The specific steps for preparing a bioactive peptide derived from oysters that enhances male reproductive health are as follows: S1. Select live Pacific oysters that are about 1 year old, sexually mature, artificially cultured in water at around 17-18℃, and have undergone "sand-removal" treatment. After opening the shell, select oyster meat with a milky white or snow-white gland color. If there are sand grains, shell fragments, or other impurities, gently rinse them with running drinking water to remove them.

[0028] S2. Take 500g of fresh oil and cut it into small pieces at 0℃ under a nitrogen atmosphere. The largest piece should not exceed 0.5-0.6cm in diameter. Then add 5mL of a mixed tocopherol solution (dissolved in high-oleic vegetable oil, with a mixed tocopherol content of 2%), mix well, and homogenize at 0℃ for 10 minutes. Then transfer it to a centrifuge tube and centrifuge at 0℃ and 10000rpm for 10 minutes. Take the uppermost oil phase into the centrifuge tube, fill the centrifuge tube with nitrogen, seal it, and temporarily store it at -20℃.

[0029] S3. Add an equal volume of vitamin C aqueous solution (5 mg / 100 mL) to the mixture after removing the oil phase. Adjust the pH of the mixture to about 7.0 with 1 M HCl and 1 M NaOH. After mixing, add 5000 U of flavor protease and 10000 U of trypsin. Stir and react at 37 °C under a nitrogen atmosphere for 10 minutes. Then, quickly filter the mixture through a 0.22 μm filter membrane under a nitrogen atmosphere. Freeze-dry the mixture after filtration and pulverize it. Weigh 75 g.

[0030] S4. Take the oil phase prepared in step S2 and quickly pass it through a 0.22μm filter membrane under a nitrogen atmosphere. Quickly add the oil phase of the filter membrane and 0.5g of vitamin C powder to the product pulverized in step S3. After mixing, the bioactive peptides from oysters that enhance male reproductive health are obtained. Press them into sheets under a nitrogen atmosphere and at 0℃ (if they are not easy to stick, spray with sterile distilled water until they are easy to form), and store them in a light-proof vacuum.

[0031] Example 3 The specific steps for preparing a bioactive peptide derived from oysters that enhances male reproductive health are as follows: S1. Select live Pacific oysters that are about 1 year old, sexually mature, artificially cultured in water at around 17-18℃, and have undergone "sand-removal" treatment. After opening the shell, select oyster meat with a milky white or snow-white gland color. If there are sand grains, shell fragments, or other impurities, gently rinse them with running drinking water to remove them.

[0032] S2. Take 500g of fresh oil and cut it into small pieces at 0℃ under a nitrogen atmosphere. The largest piece should not exceed 0.5-0.6cm in diameter. Then add 5mL of a mixed tocopherol solution (dissolved in high-oleic vegetable oil, with a mixed tocopherol content of 2%), mix well, and homogenize at 0℃ in the dark for 10 minutes. Then transfer it to a light-protected centrifuge tube and centrifuge at 0℃ and 10000rpm for 10 minutes. Take the uppermost oil phase and transfer it to a light-protected centrifuge tube. Seal the centrifuge tube after filling it with nitrogen and store it temporarily at -20℃.

[0033] S3. Add an equal volume of vitamin C aqueous solution (5 mg / 100 mL) to the mixture after removing the oil phase. Adjust the pH of the mixture to about 7.0 with 1 M HCl and 1 M NaOH. After mixing, add 5000 U of flavor protease and 10000 U of trypsin. Stir and react for 15 minutes at 37°C, in the dark, under a nitrogen atmosphere. Then, quickly filter the mixture through a 0.22 μm filter membrane under a nitrogen atmosphere. Freeze-dry the filtered mixture in the dark, pulverize it, and weigh it to 75 g.

[0034] S4. Take the oil phase prepared in step S2 and quickly pass it through a 0.22μm filter membrane under a nitrogen atmosphere in the dark. Quickly add the oil phase of the filter membrane and 0.5g of vitamin C powder to the product pulverized in step S3. After mixing, the bioactive peptides from oysters that enhance male reproductive health are obtained. Press them into sheets under a nitrogen atmosphere at 0℃ (if they are not easy to stick, spray with sterile distilled water until they are easy to form), and store them in a light-proof vacuum.

[0035] Example of effect 1 The effects of oyster-derived bioactive peptides for enhancing male reproductive health, prepared in Examples 1-3, on sperm development in mice were investigated, as follows: Twenty-four adult male ICR mice (22±2g) approximately 6 months old were randomly divided into four groups of six mice each after two weeks of acclimatization in the animal facility. Three groups were administered 1 mL of oyster-derived bioactive peptides for enhancing male reproductive health (prepared in Examples 1-3) via gavage daily between 5-6 PM (10 mg of the prepared oyster-derived bioactive peptides for enhancing male reproductive health was dissolved in 1 mL of sterile deionized water under light-protected conditions and then immediately administered via gavage). These groups were designated as Example 1-Example 3. The remaining group was administered an equal volume of sterile drinking water via gavage as a control group. All mice had free access to food and water during this period.

[0036] (1) After three consecutive months of experiments, mice were killed, and both epididymis were separated. After washing with physiological saline, the epididymis was blotted dry with filter paper and added to a test tube containing 0.5 mL of physiological saline. The epididymis was cut into small pieces and pipetted to form a suspension. The volume was adjusted to 1 mL with physiological saline to obtain a sperm suspension. After mixing, 1 μL of the sperm suspension was dropped onto a cell counting plate, and the number of sperm in the four squares was counted under a 10x microscope. When counting, the sperm on the line were counted as heads but not tails. The results are shown in Table 1.

[0037] (2) The sperm suspension prepared in (1) was analyzed by the SYBR-14 / PI method, and the sperm viability was calculated. The results are shown in Table 1.

[0038] Table 1. Statistical results of sperm count and viability.

[0039] Note: In the table This indicates that compared with the control group, P <0.05; This indicates that compared with the control group, P <0.001; This indicates that compared with the control group, P <0.0001 Example 2 The effects of the oyster-derived bioactive peptides for enhancing male reproductive health prepared in Examples 1-3 were investigated on patients with erectile dysfunction, as detailed below: With the consent of the hospital and doctors, volunteers were recruited from within the andrology hospital. The criteria were: diagnosed with erectile dysfunction, aged 25-45, weighing 60-90 kg, and with other normal physical indicators (including no history of coronary stent or open-chest / abdominal surgery within the past year). The volunteers were given detailed explanations of the raw material sources, preparation process, and reagents used in the products, along with relevant animal experimental evidence. Based on inquiries about the volunteers' lifestyles, 18 qualified volunteers were recruited, none of whom were athletes.

[0040] The participants were divided into three groups of six volunteers each. Each group included volunteers who were overweight according to their body mass index and / or who habitually slept at midnight or later and / or smoked. After dinner, the three groups of volunteers consumed oyster-derived bioactive peptides prepared according to Examples 1-3 to enhance male reproductive health. The dosage was 10g of bioactive peptide per 50kg of body weight. The peptides were consumed by chewing directly, dissolving in warm water (40-45℃) or other food or beverages, avoiding strong light exposure, and were consumed within 20 minutes.

[0041] Each group of volunteers consumed the product for three months and recorded their experiences regularly, including whether they found the taste acceptable or liked it, and how effective it felt. They received a weekly supply of oyster-derived bioactive peptides to enhance male reproductive health, and were monitored daily via phone or WeChat during dinner to ensure they had consumed the product.

[0042] Statistical results showed that all volunteers who consumed the oyster-derived bioactive peptides for enhancing male reproductive health prepared in Example 1 reported a good taste and no unpleasant odor / flavor. All six volunteers consumed it daily throughout the trial. Statistical results showed that after 3-6 weeks of consumption, they felt a significant improvement in libido. After the trial, 100% of the volunteers reported an improvement in erectile dysfunction (ED).

[0043] Among the volunteers who consumed the oyster-derived bioactive peptides for enhancing male reproductive health prepared in Example 2, all reported experiencing an unpleasant odor, albeit at different times, and not all at the same time. 50% of the volunteers dropped out of the trial midway, while the remaining three persisted for 1-2 months, reporting improvements in libido and erectile dysfunction, but the occasional unpleasant odor was still unpleasant.

[0044] Volunteers who consumed the oyster-derived bioactive peptides for enhancing male reproductive health prepared in Example 3 reported a good taste and no unpleasant odor / flavor. All six volunteers consumed the peptides daily throughout the trial. Statistical results showed that after 6-8 weeks of consumption, they felt a significant improvement in libido. At the end of the trial, 100% of the volunteers reported an improvement in erectile dysfunction (ED).

[0045] The statistical results in Table 1 indicate that the bioactive peptides for enhancing male reproductive health prepared in Examples 1-3 all have good promoting effects on male reproductive health, and no adverse reactions were observed in the volunteers and mice participating in the experiment. The bioactive peptide prepared in Example 1 showed the best effect and had no unpleasant odor, while the bioactive peptide prepared in Example 2 showed the worst effect and occasionally exhibited an unpleasant odor, significantly impacting volunteer participation. This suggests that using light-protection during preparation plays a crucial role in the promoting effect of the prepared bioactive peptides on male reproductive health and ensuring a good product experience. The effect of Example 3 was not as good as that of Example 1. This may be because the mixed enzyme had a high enzymatic hydrolysis efficiency, and the long hydrolysis time led to a significant increase in the proportion of free amino acids. Different molecular weight peptides can form a sustained-release effect in vivo, or other active ingredients may bind better to peptides with specific structures, or excessive exposure of the active ingredients may have caused certain changes, thus affecting the overall effect.

[0046] Therefore, the peptide products prepared using the method provided by this invention have bioactivity that improves male sperm quality (increases sperm count and survival rate) and erectile dysfunction, and enhances male reproductive health. They are convenient to consume, have no toxic side effects, and no adverse effects were observed on the volunteers and mice participating in the experiment. The oyster-derived bioactive peptides that enhance male reproductive health are made from high-quality, edible farmed oysters. The preparation process does not use any toxic or harmful reagents, and the added or used ingredients are all beneficial or harmless to the body or components present in the body / atmosphere, making them highly safe.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a bioactive peptide derived from oysters that enhances male reproductive health, characterized in that, The steps are as follows: S1. After rinsing the fresh oyster meat, crush it under low temperature and protective atmosphere, add mixed tocopherol solution and homogenize, then centrifuge in the dark and at low temperature, and take the oil phase into a light-proof centrifuge tube, fill it with inert gas, seal it and store it temporarily at -20℃. S2. Add the portion of S1 after removing the oil phase to an aqueous solution of vitamin C, adjust the pH, enzymatically hydrolyze, sterilize, freeze-dry and pulverize to obtain a powder. S3. Add the sterilized product from step S1 to the powder obtained in step S2, pack it into bags / cans, vacuum / fill with inert gas, and store it in a sealed, light-proof container.

2. The method for preparing a bioactive peptide derived from oysters to enhance male reproductive health according to claim 1, characterized in that: The fresh oyster meat in S1 refers to sexually mature, live, edible male oysters that are about 1 year old; the mixed tocopherol solution is made by dissolving mixed tocopherols in high-oleic vegetable oil, and the amount of mixed tocopherols added is 0.1-0.5 mL / kg of fresh oyster meat.

3. The method for preparing a bioactive peptide derived from oysters to enhance male reproductive health according to claim 1, characterized in that: In S1, the low temperature is 0℃, and the centrifugation is 8000-10000 rpm for 10-15 minutes.

4. The method for preparing a bioactive peptide derived from oysters to enhance male reproductive health according to claim 1, characterized in that: The concentration of the vitamin C aqueous solution in S2 is 2-10 mg / 100 mL, and the amount added is 1-2 times the total volume after removing the oil phase.

5. The method for preparing a bioactive peptide derived from oysters to enhance male reproductive health according to claim 1, characterized in that: In step S2, pH is adjusted using HCl solution and / or NaOH solution to a pH of 6.8-7.

5.

6. The method for preparing a bioactive peptide derived from oysters to enhance male reproductive health according to claim 1, characterized in that: The enzymatic hydrolysis in S2 is carried out using flavor protease and trypsin, and the hydrolysis conditions are 37°C, protected from light, and in an inert gas atmosphere for 10-15 minutes.

7. The method for preparing a bioactive peptide derived from oysters to enhance male reproductive health according to claim 1, characterized in that: In both S2 and S3, sterilization is performed using a 0.22 μm filter membrane.

8. The method for preparing a bioactive peptide derived from oysters to enhance male reproductive health according to claim 1, characterized in that: Vitamin C powder may also be added to S3.

9. The method for preparing a bioactive peptide derived from oysters to enhance male reproductive health according to claim 1, characterized in that: The S3 can also be prepared into vacuum-sealed, opaque sheet / sphere / solvent packaging.

10. A bioactive peptide derived from oysters that enhances male reproductive health, prepared by the method described in any one of claims 1-9.

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