Male salamander peptide-containing substance and its preparation method and application

Abstract

The present invention belongs to the field of functional food technology, and specifically relates to a male salamander peptide-containing substance, and its preparation method and application. The preparation method comprises the following steps: S1, crushing the testicles of a male giant salamander to obtain a giant salamander meat homogenate, then adding zinc-rich yeast, mixing evenly and fermenting to obtain a fermentation liquid; S2, adjusting the pH of the fermentation liquid, adding protease for enzymatic hydrolysis, and obtaining an enzymatic hydrolysis product; S3, adding activated carbon to the enzymatic hydrolysis product, stirring, decolorizing and deodorizing, and then inactivating the enzyme under vacuum. After the inactivation of the enzyme, the product is filtered through ceramics and molecular membranes in sequence, and dried to obtain a male salamander peptide-containing substance. The male salamander peptide-containing substance contains substances such as androgenetic peptide, carnosine and androgen. The present invention prepares a male salamander peptide-containing substance containing salamander peptide, carnosine and androgen by enzymatic hydrolysis of male salamander meat. The male salamander peptide-containing substance has the effects of anti-oxidation, anti-fatigue and improving male sexual function.

Description

Technical Field

[0001] The invention belongs to the technical field of functional foods, and particularly relates to a male salamander-containing peptide and a preparation method and application thereof. Background Art

[0002] Fatigue occurs when the body's physiological processes are unable to maintain their function at a specific level, or when organs are unable to maintain their intended exercise intensity. Fatigue can be categorized as central fatigue and peripheral fatigue. The WHO states that 35% of people experience fatigue, with around 60% of middle-aged men experiencing this condition. Chronic fatigue can reduce hormone secretion in men, leading to symptoms such as decreased libido and erectile dysfunction. In addition to rest, fatigue can be alleviated by promoting energy metabolism and reducing fatigue-causing substances. Bioactive peptides have proven beneficial in relieving physical fatigue, enhancing muscle strength, and maintaining and improving athletic performance.

[0003] At present, most giant salamander bioactive peptides are made from the muscles, skin, internal organs, fat, and blood of giant salamanders through enzymatic hydrolysis, separation, and other technologies. For example, patent number CN106047968 B, giant salamander active peptides and uses, is enzymatically hydrolyzed with a complex enzyme composed of marine alkaline protease and papain. The enzymatic hydrolysis products are separated by a trypsin-fixed ultrafiltration membrane separator, and then separated and purified by Sephadex LH-20 molecular sieve chromatography and high-performance liquid chromatography to obtain giant salamander active peptides. This giant salamander active peptide can effectively scavenge free radicals, enhance the body's immunity, and promote the proliferation of skin fibroblasts. It has broad application prospects in the fields of food, medicine, and cosmetics.

[0004] The composition of the polypeptide products after enzymatic hydrolysis of giant salamander collagen is very complex. Active peptides with different functions and molecular weights are obtained by using raw materials from different parts, different enzymes and enzymatic hydrolysis times, and different separation technologies. However, the effective ingredients and preparation methods that can resist fatigue and improve male sexual function have not been reported. Summary of the Invention

[0005] The purpose of the present invention is to provide a male salamander peptide-containing substance and a preparation method thereof. The male salamander peptide-containing substance containing salamander peptide-containing substance, carnosine and androgen is prepared by enzymatic hydrolysis of male salamander meat. The male salamander peptide-containing substance has the effects of anti-oxidation, anti-fatigue and improving male sexual function.

[0006] The first object of the present invention is to provide a method for preparing a male salamander-containing peptide, comprising the following steps:

[0007] S1. crushing the testicles of a male giant salamander to obtain a homogenate of giant salamander meat, and then adding zinc-enriched yeast, mixing and fermenting the mixture to obtain a fermentation liquid;

[0008] S2, adjusting the pH of the fermentation liquid obtained in S1 to 3-4, adding protease for enzymatic hydrolysis to obtain an enzymatic hydrolysis product;

[0009] S3. Add activated carbon to the enzymatic hydrolysate obtained in S2, stir to decolorize and deodorize, and then vacuum inactivate the enzyme. After the enzyme inactivation is completed, filter through ceramic and molecular membrane in sequence, and dry to obtain the male salamander-containing peptide.

[0010] Furthermore, in S1, the amount of zinc-enriched yeast added is 1.5-3% of the total mass of the giant salamander meat homogenate.

[0011] Furthermore, in S1, the fermentation temperature is 36-38°C and the fermentation time is 3-4 hours.

[0012] Furthermore, in S2, the pH of the fermentation broth is adjusted to 3-4 using acetic acid; the protease is an acidic protease, the amount of the acidic protease added is 1-1.5% of the total mass of the fermentation broth, and the enzymatic hydrolysis time is 6-7 hours.

[0013] Furthermore, in S3, the amount of activated carbon added is 1-1.5% of the total mass of the enzymatic hydrolysis product.

[0014] Furthermore, in S3, the decolorization and deodorization time is 1 hour, the vacuum enzyme inactivation temperature is 70-75°C, the time is 15 minutes, and the vacuum degree is 0.06-0.08Pa.

[0015] Furthermore, in S3, the pore size of the ceramic is 30-50 nm, and the molecular membrane is a 3000 Da membrane.

[0016] The second object of the present invention is to provide the male salamander peptide-containing substance prepared by the above method.

[0017] The third object of the present invention is to provide the use of the male salamander peptide-containing compound in the preparation of anti-fatigue products.

[0018] The fourth object of the present invention is to provide the use of the above-mentioned male salamander peptide in the preparation of products for improving male sexual function.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The invention adopts a method of peeling and removing the internal organs of a sexually mature male giant salamander, retaining the testis and beating it into a meat paste, fermenting it with zinc-rich yeast, adding acidic protease under acetic acid conditions to promote enzymatic hydrolysis and combine zinc with peptides to form zincated peptides, filtering it with a ceramic membrane and a nanofiltration membrane, and freeze-drying it to obtain a male salamander-containing peptide material. The male salamander-containing peptide material contains giant salamander tetrapeptide, giant salamander pentapeptide, carnosine and androgen, can play the role of antioxidant and anti-fatigue peptides, and improve male sexual function. DETAILED DESCRIPTION

[0021] The following will be combined with the data in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] It should be noted that the professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be purchased from the market or prepared by existing methods.

[0023] Example 1

[0024] A method for preparing a male salamander-containing peptide comprises the following steps:

[0025] S1. From July to November, sexually mature male giant salamanders are selected and cultured, and the skin and internal organs of the male giant salamanders are removed, with the testicles retained, and the meat is mashed into a paste to prepare a giant salamander meat homogenate. 2% zinc-enriched yeast is then added by weight, and the mixture is fermented at 36-38°C for 4 hours to absorb 90% of the zinc and degrade the fishy odor, thereby obtaining a fermentation liquid.

[0026] S2. Add acetic acid to the fermentation broth obtained in S1 to adjust the pH of the fermentation broth to 4, add 1.5% acid protease by mass fraction, and perform enzymatic hydrolysis for 7 hours to promote the hydrolysis of meat into peptides, which combine with zinc to form zincated peptides, thereby obtaining enzymatic hydrolysis products;

[0027] S3. Add 1.5% activated carbon by mass to the enzymatic hydrolysate obtained in S2, stir for decolorization and deodorization for 1 hour, inactivate the enzyme at 70-75°C for 15 minutes under a vacuum degree of 0.06-0.08Pa, filter the product through a ceramic filter with a pore size of 30-50nm, filter the peptide-containing substances below 3000Da through a molecular membrane filter with a 3000Da membrane, and freeze-dry in vacuum to obtain the male salamander-containing peptide substance.

[0028] Comparative Example 1

[0029] The preparation method of a female giant salamander-containing peptide is the same as that in Example 1, except that female giant salamanders are used instead of male giant salamanders.

[0030] Comparative Example 2

[0031] A method for preparing a male salamander peptide-containing substance comprises the following steps

[0032] S1. From July to November, sexually mature male giant salamanders were artificially cultured, skinned, viscera removed, testicles retained, and minced into a paste to prepare a giant salamander meat homogenate. 1.5% acidic protease was added and enzymatically hydrolyzed for 7 hours to promote the hydrolysis of the meat into peptides, which then combined with zinc to form zincated peptides, thereby obtaining an enzymatic hydrolysis product.

[0033] S2. Add 1.5% activated carbon by mass to the enzymatic hydrolysate obtained in S1, stir to decolorize and deodorize for 1 hour, inactivate the enzyme at 70-75°C for 15 minutes under a vacuum degree of 0.06-0.08Pa, filter the product through a ceramic filter with a pore size of 30-50nm, filter the peptide-containing substances below 3000Da through a molecular membrane filter with a 3000Da membrane, and freeze-dry in vacuum to obtain the male salamander-containing peptide substance.

[0034] The male salamander peptides obtained in Example 1 were separated and tested. The male salamander peptides included salamander tetrapeptide, salamander pentapeptide, carnosine and androgen. The amino acid sequence of the salamander tetrapeptide was Gln-Pro-Gly-Pro, and the amino acid sequence of the salamander pentapeptide was Gly-Pro-Gly-Pro-Pro.

[0035] The zincated peptides (less than 3000 Da, including zincated peptides of salamander tetrapeptide and salamander pentapeptide) and carnosine components of the male salamander peptide-containing material prepared in Example 1 and the female salamander peptide-containing material prepared in Comparative Example 1 were tested, as shown in Table 1. As can be seen from Table 1, the zincated peptides below 3000 Da and carnosine contents of the male salamander peptide-containing material prepared in Example 1 and the female salamander peptide-containing material prepared in Comparative Example 1 were 87-88.1% and 1.35-1.54%, respectively.

[0036] Table 1 Content of ingredients in peptide-containing substances

[0037] Zinc peptide (%) Carnosine (%) Example 1 88.1 1.54 Comparative Example 1 87.6 1.35

[0038] Determination of antioxidant and anti-fatigue effects in mice

[0039] Experimental method: The experimental animals were mice with an average weight of 25 g. 48 mice were divided into four groups and raised individually. The control group mice were fed a normal diet. The other two groups of mice were fed a normal diet and were fed with 0.2 mg / g / day of the male salamander peptide of Example 1, the female salamander peptide prepared in Comparative Example 1, and the male salamander peptide prepared in Comparative Example 2, respectively. They were fed with normal drinking water for 42 days, and the weights of the testicles, penis, liver, kidney, and thymus were weighed, and the body mass ratios were calculated as shown in Table 2. As can be seen from Table 2, the zinc-rich androdextrin peptide can increase the testis-to-body ratio and penis length of male mice compared with the blank and Comparative Examples 1 and 2, indicating that the zinc-rich androdextrin peptide has the effect of promoting the growth of sexual organs in male mice.

[0040] Table 2 Ratio of organs to body weight of mice fed with male salamander peptide (%)

[0041] liver(%) kidney(%) Thymus (%) testis(%) penis(%) Blank control 31.24 13.14 0.11 0.55 0.12 Example 1 32.29 13.32 0.13 0.67 0.16 Comparative Example 1 32.15 13.25 0.13 0.58 0.15 Comparative Example 2 32.15 13.16 0.13 0.56 0.15

[0042] The antioxidant activity in mice is shown in Table 3. As shown in Table 3, zinc-rich androstrum peptide can increase the activity of antioxidant enzymes in the liver, kidney and testis tissues of male mice and reduce the content of malondialdehyde compared with the blank and comparative examples 1 and 2, indicating that zinc-rich androstrum peptide has strong antioxidant activity.

[0043] Table 3 Antioxidant effects of mouse liver, kidney and testis

[0044]

[0045] Anti-fatigue experiment of male salamander peptide on mice

[0046] 30 minutes after the last administration of the test substance, the mice were tied with a lead wire weighing 5% of their body weight to their tails. Eight mice were then placed in a swimming pool at a depth of 30 cm and a temperature of 25±1°C. The swimming time was recorded from the start of swimming until the head was completely submerged in the water and could not emerge for 8 seconds, as shown in Table 4. As shown in Table 4, the swimming time of the mice was more than doubled compared to the blank control group, and the anti-fatigue effect of the estrone peptide in Comparative Example 1 was greater than that of the mice. Table 4 demonstrates that zinc-rich estrone peptide prolonged swimming time compared to the blank control group and Comparative Examples 1 and 2, demonstrating its anti-fatigue effect.

[0047] Table 4 Anti-fatigue index of mice

[0048] Swimming time (min) Blank control 25 Example 1 51 Comparative Example 1 32 Comparative Example 2 45

[0049] Anti-fatigue serum experiment

[0050] The mice underwent a 30-minute unloaded swim in a pool. Immediately after the 30-minute swim, blood samples were collected from the retroorbital vein to measure serum levels of liver glycogen, muscle glycogen, lactate dehydrogenase, lactate, urea nitrogen, and serum NO. Skeletal muscle and liver tissue samples were also collected for glycogen content determination, as shown in Table 5. As shown in Table 5, zinc-rich androstrum peptide increased liver and muscle glycogen content and energy levels in mice compared to the blank and control groups 1 and 2, while simultaneously reducing lactate and urea nitrogen production, reducing fatigue-inducing substances, and prolonging swimming time, demonstrating a significant anti-fatigue effect.

[0051] Table 5 Physiological indicators of mice after swimming

[0052]

[0053] Determination of sexual function in mice

[0054] In the mating behavior experiment, 48 male mice with an average weight of 25G were selected and divided into four groups. The blank control group was raised normally and given 0.2 mg / g / day of the male salamander peptide of Example 1, the female salamander peptide prepared in Comparative Example 1, and the male salamander peptide prepared in Comparative Example 2 by gavage. The mice were given the corresponding doses of the test substances for 42 consecutive days. After the end of the 42-day gavage, the male mice of the blank control group, Example 1, and Comparative Example 1 were placed in a dim and quiet room with red light (19:00-24:00). The male mice were placed in cages alone and allowed to adapt for 15 minutes.

[0055] Forty-eight male and female ICR mice with an average weight of 25G were divided equally into four groups. Estrus was induced by subcutaneous injection of 5 μg estradiol benzoate 48 h before the experiment and 5 μg progesterone 5 h before the experiment.

[0056] Each group of female and male mice were placed in the same cage, and their mating behavior was recorded for 25 minutes using a video camera. Sexual behavior parameters for each group of male mice were statistically analyzed, including the number of mounts, total number of ejaculations, and post-ejaculation interval, as shown in Table 6. As shown in Table 6, zinc-rich androstrum peptide stimulated sexual behavior in male mice and reduced the interval between ejaculations, compared to the blank and Comparative Examples 1 and 2, indicating that androstrum peptide has the effect of enhancing sexual function.

[0057] Table 6 Effects of male salamander peptide on male mice

[0058] Number of straddles Number of ejaculations Ejaculation interval / S Blank control 4.57 2.51 131.2 Example 1 15.84 6.23 35.3 Comparative Example 1 11.82 5.78 58.6 Comparative Example 2 12.35 6.19 45.2

[0059] After the mating experiment, blood samples of male mice were collected from the retroorbital vein, and serum testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured, as shown in Table 7. As shown in Table 7, the male salamander peptide prepared in Example 1 increased the levels of luteinizing hormone and follicle-stimulating hormone in the blood of male mice, and the testosterone content was increased by more than 2.4 times compared with the blank control. Compared with the blank and Comparative Examples 1 and 2, the male salamander peptide prepared in Example 1 can increase the amount of male hormones and improve their sexual function.

[0060] Table 7 Hormone content in mouse serum

[0061] T (pg / mL) FSH (ng / mL) LH (ng / mL) Blank control 802.7 21.56 2041.63 Example 1 1941.12 28.34 2486.12 Comparative Example 1 1110.53 26.12 2349.12 Comparative Example 2 1322.4 27.73 2378.95

[0062] It should be noted that when numerical ranges are mentioned in the present invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints may be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes preferred embodiments. Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0063] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing a male salamander peptide-containing substance, characterized in that: The steps include: S1. Crush the testicles of a male giant salamander to prepare a homogenate of giant salamander meat, then add zinc-enriched yeast, mix evenly, and ferment to obtain a fermentation liquid; the amount of zinc-enriched yeast added is 1.5-3% of the total mass of the giant salamander meat homogenate; S2, using acetic acid to adjust the pH of the fermentation broth obtained in S1 to 3-4, adding protease for enzymatic hydrolysis to obtain an enzymatic hydrolysis product; the protease is an acidic protease, and the amount of the acidic protease added is 1-1.5% of the total mass of the fermentation broth, and the enzymatic hydrolysis time is 6-7 hours; S3. Add activated carbon to the enzymatic hydrolysate obtained in S2, stir to decolorize and deodorize, and then vacuum inactivate the enzyme. After the enzyme inactivation is completed, filter through ceramic and molecular membrane in sequence, and dry to obtain the male salamander-containing peptide.

2. The method for preparing the male salamander peptide-containing substance according to claim 1, wherein: In S1, the fermentation temperature is 36-38° C. and the fermentation time is 3-4 hours.

3. The method for preparing the male salamander peptide-containing substance according to claim 1, wherein: In S3, the amount of activated carbon added is 1-1.5% of the total mass of the enzymatic hydrolysis product.

4. The method for preparing the male salamander peptide-containing substance according to claim 1, wherein: In S3, the decolorization and deodorization time is 1 hour, the vacuum enzyme inactivation temperature is 70-75°C, the time is 15 minutes, and the vacuum degree is 0.06-0.08Pa.

5. The method for preparing the male salamander peptide-containing substance according to claim 1, wherein: In S3, the pore size of the ceramic is 30-50 nm, and the molecular membrane is a 3000 Da membrane.

6. A male salamander peptide-containing substance prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the male salamander peptide-containing substance according to claim 6 in the preparation of anti-fatigue products.

8. Use of the male salamander peptide-containing substance according to claim 6 in the preparation of a product for improving male sexual function.

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