Modified sea cucumber peptide as well as preparation method and application thereof in improving sexual function

Abstract

The invention relates to the technical field of sea cucumber peptides, in particular to a modified sea cucumber peptide as well as a preparation method and application thereof in improving sexual function. The method comprises the following steps: adding synthesized sea cucumber peptide into a hydrochloric acid solution, carrying out heat treatment, adjusting the pH value to be neutral, dialyzing, and freeze-drying to obtain a primary modified sample; the preparation method comprises the following steps: preparing a primary modified sample solution, adding sodium tripolyphosphate into the primary modified sample solution, and carrying out magnetic stirring reaction, dialysis and freeze drying to obtain the modified sea cucumber peptide. The foaming ability and the foaming stability of the modified sea cucumber peptide are remarkably improved, and the modified sea cucumber peptide is mixed with the Chinese yam powder to prepare the emulsion with the foaming ability and the foaming stability remarkably improved. The modified sea cucumber peptides and emulsions have the effect of obviously improving the sexual function of male mice.

Description

Technical Field

[0001] The invention relates to the technical field of sea cucumber peptides, and in particular to a modified sea cucumber peptide and a preparation method thereof and an application thereof in improving sexual function. Background Art

[0002] Sea cucumber peptides are made from fresh sea cucumbers. After protease hydrolysis and other processes, they are separated and purified to obtain small molecule peptides composed of 2 to 12 amino acids or polypeptides with larger molecular weights. The components with molecular weights less than 2000U account for more than 90%. The extracted sea cucumber peptides are active peptides with good solubility, stability, and easy absorption and digestion. Yu Yihao et al. (Study on the anti-fatigue effect and mechanism of sea cucumber peptides, Master's thesis of Jiangnan University in 2021) selected and synthesized sea cucumber peptide G (HyP) LQADY based on the results of functional analysis of 134 sea cucumber peptides. At a concentration of 50 μg / mL, it significantly improved cell viability and antioxidant capacity, significantly reduced the content and expression of IL-6 and TNF-α, reduced the phosphorylation level of NF-κB, and increased the phosphorylation level of STAT3. Zuo Aihua et al. (High-throughput HPLC-MS / MS analysis and identification and activity screening of sea cucumber oligopeptides [J]. Food Industry Science and Technology, 2020, 41(03):12-17)) used high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to analyze and identify the peptides and source proteins in sea cucumber oligopeptides. The results showed that 88 small molecule peptides were identified in sea cucumber oligopeptides, containing a large number of unreported small molecule active peptides derived from 19 sea cucumber proteins, mainly collagen, α-Ⅰ type collagen, glycine-rich collagen, type II collagen fibers and extracellular matrix protein 3. A total of 9 potential antihypertensive active peptides were obtained through online database activity screening. Further structure-activity relationship analysis revealed that the core peptide RPQYPQYPS has potential antihypertensive activity and is expected to be developed as an antihypertensive peptide. The discovery of small molecule sea cucumber peptides and their active functions through existing technologies provides a basis for the further development of these sea cucumber peptides. Summary of the invention

[0003] In view of this, the present invention provides a modified sea cucumber peptide and a preparation method thereof and a use thereof for improving sexual function

[0004] One of the purposes of the present invention is to provide a method for preparing a modified sea cucumber peptide, comprising the steps of:

[0005] (1) adding the synthesized sea cucumber peptide to a hydrochloric acid solution, wherein the final concentration of the added sea cucumber peptide is 3 wt %, and stirring the solution;

[0006] (2) heat treating the reaction solution obtained in step (1) for 10 min;

[0007] (3) adjusting the pH value of the solution obtained in step (2) to neutral, dialyzing, and freeze-drying to obtain a primary modified sample;

[0008] (4) dissolving the initial modified sample in an ammonia-ammonium chloride buffer solution at pH 8.0 to prepare an initial modified sample solution, adding sodium tripolyphosphate to the initial modified sample solution, and reacting by magnetic stirring;

[0009] (5) The reaction solution obtained in step (4) is dialyzed and freeze-dried to obtain a modified sea cucumber peptide.

[0010] In step (1) of the preparation method, the concentration of the hydrochloric acid solution is 0.05-0.15 mol / L, the final concentration of the sea cucumber peptide is 2.5-3.5 wt%, and the stirring treatment time is 1-3 h. Preferably, the concentration of the hydrochloric acid solution is 0.1 mol / L, the final concentration of the sea cucumber peptide is 3 wt%, and the stirring treatment time is 2 h.

[0011] In step (2) of the preparation method, the reaction solution obtained in step (1) is subjected to wet heat treatment for 8 to 15 minutes. Preferably, the treatment time is 10 minutes.

[0012] In step (2) of the preparation method, a wet heat treatment at a temperature of 105-120°C is used. Preferably, a closed chamber is used for the wet heat treatment (eg, a wet heat sterilizer, a pressure cooker). Preferably, the wet heat treatment temperature is 110°C.

[0013] In step (3) of the preparation method, the pH is adjusted to neutral using 0.1 mol / L NaOH solution.

[0014] In step (4) of the preparation method, the final concentration of sodium tripolyphosphate is 4-8wt%, and the reaction time is 1-2h. Preferably, the final concentration of sodium tripolyphosphate is 6wt%, and the reaction time is 1.5h.

[0015] In step (3) and step (5) of the preparation method, an SP131060 cellulose dialysis bag is used for dialysis.

[0016] One of the purposes of the present invention is to provide a modified sea cucumber peptide obtained by the preparation method.

[0017] One of the purposes of the present invention is to provide a method for preparing an emulsion, comprising the steps of: preparing a modified sea cucumber peptide and a yam polysaccharide by the preparation method, mixing them by equal weight, dissolving them in distilled water, adjusting the pH to 7.0 with a PBS solution of pH 7.4, stirring at a uniform speed, and homogenizing at 28000 r / min for 3 min.

[0018] One of the purposes of the present invention is to provide the use of the modified sea cucumber peptide prepared by the preparation method or the emulsion prepared by the preparation method according to claim 9 in the preparation of products for improving sexual function.

[0019] Beneficial effects:

[0020] The present invention obtains two modified sea cucumber peptides with improved solubility by subjecting two special sea cucumber peptides to special modification treatment steps. It is also found that the two modified sea cucumber peptides can significantly improve the emulsification ability and emulsification stability of the sea cucumber peptides. Compared with unmodified sea cucumber peptides, the foaming ability and foaming stability of the modified sea cucumber peptides are significantly improved. The present invention also mixes the two modified sea cucumber peptides with yam powder to prepare an emulsion with significantly improved foaming ability and foaming stability.

[0021] In addition, through the mouse experiments on these modified sea cucumber peptides and the emulsions prepared therefrom, it was found that the modified sea cucumber peptides and emulsions prepared by the present invention can increase the serum testosterone and NO content and the corpus cavernosum cGMP level of male mice, reduce the corpus cavernosum PDE5 level, and shorten the mouse mounting latency, indicating that these modified sea cucumber peptides and emulsions have a significant effect on improving the sexual function of male mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are the solubility results of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, Group 4) at different pH values.

[0023] Figure 2 These are the emulsification ability results of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, Group 4) at different pH values.

[0024] Figure 3 These are the emulsification stability results of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, Group 4) at different pH values.

[0025] Figure 4 The foaming results are of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, and Group 4).

[0026] Figure 5These are the foaming stability results of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, and Group 4).

[0027] Figure 6 The results of serum testosterone content in different groups of mice are shown in the figure. In the figure, "ns" means no statistical difference; "*" means p<0.05, there is a statistical difference; "**" means p<0.001, there is a statistical difference.

[0028] Figure 7 The results of serum NO content in different groups of mice are shown in the figure. In the figure, "ns" means no statistical difference; "*" means p<0.05, there is a statistical difference; "**" means p<0.001, there is a statistical difference.

[0029] Figure 8 The results of cGMP content in the cavernous body of different groups of mice are shown in the figure. In the figure, "ns" means no statistical difference; "*" means p<0.05, there is a statistical difference.

[0030] Fig. 9 The results of PDE5 content in the cavernous body of different groups of mice are shown in the figure. In the figure, "ns" means no statistical difference; "*" means p<0.05, there is a statistical difference.

[0031] Fig.10 The results of the mounting latency in the mating experiment of male mice in different groups are shown in the figure. In the figure, "ns" means no statistical difference; "*" means p<0.05, there is a statistical difference; "**" means p<0.001, there is a statistical difference. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The reagents not described separately in detail in the present invention are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0033] Example 1: Modification of sea cucumber peptide

[0034] 1. Preparation of modified sea cucumber peptide

[0035] Group 1: Add chemically synthesized sea cucumber peptide G (HyP) LQADY to a 0.1 mol / L hydrochloric acid solution, the final concentration of the added sea cucumber peptide is 3 wt%, after stirring for 2 hours, wet heat treatment of the reaction solution at 110 ° C for 10 minutes (sterilizer), adjust the pH to neutral with 0.1 mol / L NaOH solution, and dialyze for 24 hours at 4 ° C using SP131060 cellulose dialysis bag (100-500, 31 mm, 3.1 ML / CM), freeze-drying, and obtaining the initial modified sample. The initial modified sample was prepared into a 0.02 g / mL initial modified sample solution with ammonia-ammonium chloride buffer solution of pH 8.0, sodium tripolyphosphate with a final concentration of 6 wt% was added, magnetic stirring was applied, and the reaction was carried out for 1.5 hours. The sample was dialyzed for 24 hours using SP131060 cellulose dialysis bag, freeze-dried, and the modified sea cucumber peptide was obtained.

[0036] Group 2: The same modification steps were applied to the sea cucumber peptide RPQYPQYPS.

[0037] Group 3: Add chemically synthesized sea cucumber peptide RPQYPQYPS to a 0.1 mol / L hydrochloric acid solution to a final concentration of 3 wt%. After stirring for 2 h, the reaction solution was wet-heat treated at 110°C for 10 min, the pH was adjusted to neutral with 0.1 mol / L NaOH solution, and dialyzed at 4°C using an SP131060 cellulose dialysis bag (100-500, 31 mm, 3.1 ML / CM) for 24 h. The modified sample was freeze-dried to obtain a modified sample.

[0038] Group 4: The sea cucumber peptide RPQYPQYPS was prepared into a 0.02 g / mL primary modified sample solution using ammonia-ammonium chloride buffer solution at pH 8.0, and sodium tripolyphosphate was added at a final concentration of 6 wt%. The solution was stirred magnetically and reacted for 1.5 h. The sample was dialyzed using an SP131060 cellulose dialysis bag for 24 h and freeze-dried to obtain the modified sea cucumber peptide.

[0039] 2. Determination of free ammonia concentration by hydrochloric acid deamidation

[0040] Weigh 50 mg of the initial modified sample, place it in a hydrolysis tube, then add 5 mL of sulfuric acid with a final concentration of 3 mol / L, seal the hydrolysis tube with an alcohol burner, and hydrolyze at 110°C for 24 hours. After the hydrolysis is completed, the hydrolyzate is neutralized to neutrality and diluted a certain multiple, and the absorbance of the hydrolyzate dilution is measured at a wavelength of 625 nm. According to the standard curve, the total amount of glutamine residues in the sample is calculated. The determination method of total amide content refers to Wagner and Gueguen (1995). Deamidation degree % = free ammonia content in the sample / total amide content in the sample × 100%. Results The deamidation degrees of the modified sea cucumber peptides obtained in Group 1, Group 2, and Group 3 were 58.63%, 56.49%, 57.82%, and 0.65%, respectively, indicating that the sea cucumber peptides in Group 1, Group 2, and Group 3 were all successfully deamidated.

[0041] 3. Phosphorylation determination of modified sea cucumber peptides

[0042] The degree of protein phosphorylation was determined by molybdenum blue colorimetry. The specific steps are as follows:

[0043] Potassium dihydrogen phosphate is used as the standard, and placed in a 25mL colorimetric tube, 2.0mL of 5% ammonium molybdate solution is added in sequence, shake well, and after standing for a few seconds, 1.0mL of 2% sodium sulfite solution and 1.0mL of 0.5% hydroquinone solution are added respectively, shake well, add water to the scale, and after standing for 30 minutes, adjust to zero with the reference, and measure the absorbance at a wavelength of 660nm. The mass concentration of the phosphate standard solution (in terms of P) is used as the abscissa and the corresponding absorbance is used as the ordinate to make a standard curve, and the standard curve equation is obtained. Take 20mg of modified sea cucumber peptide in a beaker, add 1mL of concentrated sulfuric acid and concentrated nitric acid respectively, heat on an electric furnace until smoke, and then add 1mL of 30% hydrogen peroxide solution after cooling, and then slowly heat, repeat the above steps until there is no smoke in the bottle, and the solution is colorless, transparent or light yellow. After cooling, add 1mL of 6mol / L hydrochloric acid, heat on an electric furnace to completely decompose the acid, and transfer to a 50mL volumetric flask to make up the volume. 5 mL was taken to measure the absorbance according to the standard curve operation method, and the phosphate content (in terms of P, the same below) was calculated according to the regression curve. Results The phosphate content (in terms of P) of the modified sea cucumber peptides obtained in Group 1, Group 2, Group 3 and Group 4 were 10.52%, 10.36%, 0.05% and 3.65%, respectively, indicating that the sea cucumber peptides in Group 1, Group 2 and Group 4 were all phosphorylated.

[0044] Example 2: Study on the functional properties of sea cucumber peptides and modified sea cucumber peptides

[0045] 1. Solubility determination

[0046] The unmodified sea cucumber peptide or the lyophilized powder of the modified sea cucumber peptide was dissolved in distilled water to prepare a 0.05 mg / mL solution, and different pH values ​​were adjusted with hydrochloric acid and sodium hydroxide solution. The solution was centrifuged at 20,000 g for 15 min, and the protein content in the supernatant was determined using a Lowry kit. Solubility = protein content in the supernatant / total protein content × 100%.

[0047] The results are as follows Figure 1 As shown in the figure, the solubility of unmodified sea cucumber peptides is low, especially RPQYPQYPS. After the same modification steps, the solubility of modified sea cucumber peptides obtained in Group 1 and Group 2 increased, indicating that the above modification steps can improve the solubility of sea cucumber peptides.

[0048] 2. Determination of emulsification and emulsion stability

[0049] The emulsification determination method is as follows: weigh 1.0g of unmodified sea cucumber peptide or modified sea cucumber peptide freeze-dried powder and dissolve it in 20mL water, adjust different pH with hydrochloric acid and sodium hydroxide solution, add 20mL salad oil, adjust the internal cutting homogenizer to 28000r / min to mix the oil and water for 1min, and centrifuge it in a centrifuge for 5min at a speed of 1200r / min. The emulsification stability determination method is as follows: heat the above sample in an 80℃ water bath for 30min, cool it and centrifuge it in a centrifuge for 5min at a speed of 1200r / min.

[0050] The calculation formulas for emulsification and emulsion stability are as follows:

[0051] Emulsification capacity (%) = height of emulsified layer (mm) / total height of liquid in centrifuge tube (mm)

[0052] Emulsion stability (%) = height of liquid layer that still maintains emulsification state (mm) / original emulsion layer height (mm)

[0053] like Figure 2 , 3 As shown, it is shown that under acidic and alkaline conditions, the modified sea cucumber peptide can significantly improve the emulsification ability and emulsification stability of sea cucumber peptide. Usually, the emulsification ability is due to the introduction of phosphate groups, which causes the protein conformation to change, stretching the protein molecules, exposing a large number of hydrophobic groups, and connecting with non-polar oil molecules, while the hydrophilic groups are connected with water molecules, reducing the surface tension when the two are in contact, increasing the ability to combine oil and water, and facilitating the formation of more strong interfacial films and reducing the ability of oil droplets to aggregate.

[0054] 4. Evaluation of foamability and foam stability

[0055] The unmodified sea cucumber peptides G (HyP) LQADY, RPQYPQYPS and the modified sea cucumber peptides provided in groups 1 to 4 were used.

[0056] Weigh 1.5g of unmodified sea cucumber peptide or modified sea cucumber peptide and dissolve them in 25mL of distilled water, adjust the pH to 7.0 with pH7.4 PBS solution, and stir at a constant speed for 10min, continue to add distilled water to 50mL as the test solution, adjust the internal cutting homogenizer to 28000r / min for 3min to prepare a foaming emulsion. And record the foam volume V0 (mL) appearing on the top to represent the foaming property. After standing for 30min, record the foam volume V30 (mL) again. The calculation formulas for foaming property and foam stability are as follows: Foaming property (%) = 100% × V0 / 50; Foam stability (%) = 100% × V30 / V0.

[0057] In addition, in this experiment, the modified sea cucumber peptides provided by groups 1 to 4 were mixed with an equal weight of yam powder (Henan Jiuding Junjian Biotechnology Co., Ltd., white solid powder, the main component of which is yam polysaccharide (content is 50%)), dissolved in 25 mL of distilled water, and the pH was adjusted to 7.0 with 0.1 M PBS solution of pH 7.4, and stirred at a uniform speed for 10 minutes, and distilled water was continued to be added to 50 mL as the test solution, and the internal cutting homogenizer was adjusted to 28000 r / min for 3 minutes to prepare a foaming emulsion, which was respectively used as groups 5 to 8, and their foaming properties and foam stability were also tested.

[0058] Protein foaming and foam stability are the two most commonly used indicators for evaluating protein foaming. Foaming is a property that occurs at the liquid / gas interface and is related to the interfacial tension between the two phases. Disordered proteins are easily concentrated at the liquid / gas interface and have sufficient viscosity and strength, which is conducive to the formation of more stable foam. Figure 4 , 5 As shown in the figure, compared with the unmodified sea cucumber peptide, the foaming ability of the modified sea cucumber peptide is significantly improved (P<0.05). It can be seen that the modification treatment enhances the disorder of the molecules, so that the liquid / gas interface after dissolving in water has sufficient viscosity and strength, and the modified sea cucumber peptide foam formed is more stable. Although groups 3 and 4 can also be modified, their foaming performance and foaming stability are not as good as those of group 2 (P<0.01), indicating that the modification method of group 2 can only obtain an emulsion with better foaming performance and foaming stability. In addition, there is no significant change in the foaming performance and foaming stability of groups 5 to 8 relative to groups 1 to 4, indicating that adding yam powder to the emulsion does not affect its emulsion performance.

[0059] Example 3: Biological performance study

[0060] 1. Materials and Methods

[0061] 1. Experimental Animals

[0062] Healthy clean grade adult ICR mice, half male and half female, weighing 18-22 g, were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. The experimental animals were kept in separate cages with free access to food and water. The animal breeding laboratory met the national standard clean grade, with a temperature range of (25±1)℃, a relative humidity of 50%-60%, and indoor lighting controlled at a 12h / 12h light-dark cycle.

[0063] 2. Group experiment

[0064] Male mice were randomly divided into blank group (Blank), negative group (Neg), sea cucumber peptide 1 group (G(HyP)LQADY), sea cucumber peptide 2 group (RPQYPQYPS), group 1 (Group1), group 2 (Group2), group 3 (Group2), group 4 (Group2), group 5 (Group5), group 6 (Group6), group 7 (Group7), group 8 (Group8) according to body weight. Blank mice were gavaged with distilled water. Negative group was gavaged with yam polysaccharide at 0.5g / kg body weight per day. Sea cucumber peptide 1 group was gavaged with sea cucumber peptide G(HyP)LQADY at 0.5g / kg body weight per day. Sea cucumber peptide 2 group was gavaged with sea cucumber peptide RPQYPQYPS at 0.5g / kg body weight per day. Group 1 was gavaged with modified sea cucumber peptide G(HyP)LQADY at 0.5g / kg body weight per day. Group 2 was gavaged with modified sea cucumber peptide RPQYPQYPS at 0.5g / kg body weight per day. Group 3 was gavaged with 0.5 g / kg body weight per day of the modified sea cucumber peptide provided by Group 3. Group 4 was gavaged with 0.5 g / kg body weight per day of the modified sea cucumber peptide provided by Group 4. Group 5 was gavaged with 0.5 g / kg body weight per day of the emulsion provided by Group 5 (an emulsion of modified sea cucumber peptide G (HyP) LQADY and yam polysaccharide, the mixing mass ratio was 1:1, and the mixture was prepared into a homogenous emulsion according to the above-mentioned foaming test process). Group 6 was gavaged with 0.5 g / kg body weight per day of the emulsion provided by Group 6 (an emulsion of modified sea cucumber peptide RPQYPQYPS and yam polysaccharide, the mixing mass ratio was 1:1, and the mixture was prepared into a homogenous emulsion according to the above-mentioned foaming test process). Group 7 was gavaged with 0.5 g / kg body weight per day of the emulsion provided by Group 7 (an emulsion of modified sea cucumber peptide RPQYPQYPS and yam polysaccharide, the mixing mass ratio was 1:1, and the mixture was prepared into a homogenous emulsion according to the above-mentioned foaming test process). Group 8 was gavaged with 0.5 g / kg body weight per day of the emulsion provided by Group 8 (emulsion of modified sea cucumber peptide RPQYPQYPS and yam polysaccharide, mixed in a mass ratio of 1:1, and the mixture was prepared into a homogenized emulsion according to the above foaming test process). Each group of mice was gavaged once a day for 45 consecutive days.

[0065] 3. Mating experiment

[0066] The male mice in each group were subjected to mating experiments on the 45th day of intervention and the mounting latency of the mice was recorded. Each experiment was conducted 60 minutes after the last administration. Female mice were subcutaneously injected with 5 μg of estradiol benzoate 48 hours before the experiment, and 5 μg of progesterone was subcutaneously injected 5 hours before the experiment to induce estrus. During the mating experiment, a male mouse was first placed in the mating box (30cm×15cm×15cm), and a female mouse was immediately placed after 5 minutes of adaptation. The time from the time the mouse was put into the cage to the first mounting (i.e., the mounting latency) was recorded. The experiment was conducted in a low-light, quiet environment, and the observation time was from 20:00 to 23:00.

[0067] 4. Determination of biochemical indicators

[0068] The weight of male mice in each group was weighed 30 minutes after the last administration. Blood was collected from the eyeballs, placed in a 4°C refrigerator for 3 hours, and centrifuged at 3500r / min for 10 minutes. The supernatant was taken to determine the NO and testosterone content. After the blood collection, the mice were killed by dislocating the cervical vertebrae, and the cavernous tissue on both sides of the mouse penis was peeled off in an ice bath, and the cGMP and PDE5 content was detected. The NO content in serum was determined according to the instructions of the NO detection kit (Biyuntian Biotechnology Research Institute). The instructions for the testosterone ELISA kit for the determination of serum testosterone content (No.: PT872, Biyuntian). The cyclic guanosine monophosphate (cGMP) kit and the phosphodiesterase 5 (PDE5) kit were purchased from Beijing Andy Huatai Technology Co., Ltd.

[0069] 6. Statistical methods

[0070] The experimental data were expressed as mean ± standard deviation (x ± s), and statistical analysis was performed using SPSS 22.0 software.

[0071] 2. Results

[0072] like Figure 6 As shown, compared with the blank group, there was no statistical difference in the serum testosterone content of mice in the negative group, sea cucumber peptide group 1, sea cucumber peptide group 2, group 3, and group 4. Group 1 was significantly higher than the blank group, and group 2 was significantly higher than the blank group, indicating that the modified sea cucumber peptides provided by groups 1 and 2, respectively, can increase the serum testosterone content of mice. Groups 3 and 4 cannot increase the serum testosterone content of mice. In addition, there were statistical differences between group 5 and group 1, group 6 and group 2, and group 8 and group 4, indicating that the combination of modified sea cucumber peptides and yam powder to make an emulsion is beneficial to provide modified sea cucumber peptides to increase the serum testosterone content of mice.

[0073] like Figure 7As shown, compared with the blank group, there was no statistical difference in the serum NO content of mice in the negative group, sea cucumber peptide group 1, sea cucumber peptide group 2, group 3, and group 4. However, group 1 was significantly higher than the blank group, and group 2 was significantly higher than the blank group, indicating that the modified sea cucumber peptides provided by groups 1 and 2, respectively, can increase the serum NO content of mice. However, groups 3 and 4 cannot increase the serum NO content of mice. In addition, there were statistical differences between group 6 and group 2, group 7 and group 3, and group 8 and group 4, indicating that the combination of modified sea cucumber peptides and yam powder to make an emulsion is beneficial to provide the modified sea cucumber peptides to improve the serum NO content of mice.

[0074] like Figure 8 As shown, compared with the blank group, there was no statistical difference in the cGMP content of the mouse corpus cavernosum in the negative group, sea cucumber peptide group 1, sea cucumber peptide group 2, group 3, and group 4. Group 1 was significantly higher than the blank group, and group 2 was significantly higher than the blank group, indicating that the modified sea cucumber peptides provided by groups 1 and 2, respectively, can increase the cGMP content of the mouse corpus cavernosum. Groups 3 and 4 cannot increase the cGMP content of the mouse corpus cavernosum. In addition, there were statistical differences between group 6 and group 2, group 7 and group 3, and group 8 and group 4, indicating that the combination of modified sea cucumber peptides and yam powder to make an emulsion is beneficial to provide the modified sea cucumber peptide to improve the cGMP content of the mouse corpus cavernosum.

[0075] like Fig. 9 As shown, compared with the blank group, there was no statistical difference in the PDE5 content of the mouse corpus cavernosum in the negative group, sea cucumber peptide group 1, sea cucumber peptide group 2, group 3, and group 4. However, group 1 was significantly higher than the blank group, and group 2 was significantly higher than the blank group, indicating that the modified sea cucumber peptides provided by groups 1 and 2, respectively, can reduce the PDE5 content of the mouse corpus cavernosum, while groups 3 and 4 cannot reduce the PDE5 content of the mouse corpus cavernosum.

[0076] like Fig.10 As shown, compared with the blank group, there was no statistical difference in the mounting latency in the male mice mating experiment in the negative group, sea cucumber peptide group 1, sea cucumber peptide group 2, group 3, and group 4. However, group 1 was significantly higher than the blank group, and group 2 was significantly higher than the blank group, indicating that the modified sea cucumber peptides provided by group 1 and group 2, respectively, can reduce the mounting latency in the male mice mating experiment, while group 3 and group 4 cannot reduce the mounting latency in the male mice mating experiment.

[0077] The study showed that the modified sea cucumber peptide and its emulsion with yam powder increased the serum testosterone and NO content and the cavernous cGMP level of male mice, decreased the cavernous PDE5 level, and shortened the mouse mounting latency. The unmodified sea cucumber peptide did not have these biological activities.

[0078] Whether male sexual function is strong or weak is the result of the combined action of many factors inside and outside the body, such as testosterone secreted by interstitial cells of the testicles, which promotes the expression of nitric oxide synthase by regulating the expression of sexual function-related genes and then catalyzes the NO synthesized by L-arginine. For example, NO inhibits the production of PDE5 and promotes the synthesis of guanylate cyclase to reduce the hydrolysis of cGMP in the body, so that the penile arterioles and corpus cavernosum smooth muscles can be dilated, and blood is rapidly pumped into the cavernous sinus to cause erection. The above is a relatively clear pathway in the process of erection, and the determination of its key factor content can indirectly reflect the erectile ability. It can be seen that the modified sea cucumber peptides and emulsions prepared by the present invention can increase the serum testosterone and NO content and the corpus cavernosum cGMP level of male mice, reduce the corpus cavernosum PDE5 level, and shorten the mouse riding latency, indicating that these modified sea cucumber peptides and emulsions have obvious effects on improving the sexual function of male mice.

[0079] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a modified sea cucumber peptide, characterized in that: Includes steps: (1) adding the synthesized sea cucumber peptide to a hydrochloric acid solution, wherein the final concentration of the added sea cucumber peptide is 3 wt %, and stirring the solution; (2) heat treating the reaction solution obtained in step (1) for 10 min; (3) adjusting the pH value of the solution obtained in step (2) to neutral, dialyzing, and freeze-drying to obtain a primary modified sample; (4) dissolving the initial modified sample in an ammonia-ammonium chloride buffer solution at pH 8.0 to prepare an initial modified sample solution, adding sodium tripolyphosphate to the initial modified sample solution, and reacting by magnetic stirring; (5) The reaction solution obtained in step (4) is dialyzed and freeze-dried to obtain a modified sea cucumber peptide.

2. The preparation method according to claim 1, characterized in that: In step (1), the concentration of the hydrochloric acid solution is 0.05-0.15 mol / L, the final concentration of the sea cucumber peptide is 2.5-3.5 wt%, and the stirring treatment time is 1-3 h.

3. The preparation method according to claim 1, characterized in that: In step (2), the reaction solution obtained in step (1) is subjected to wet heat treatment for 8 to 15 minutes.

4. The preparation method according to claim 3, characterized in that: In step (2), a wet heat treatment at a temperature of 105 to 120°C is used.

5. The preparation method according to claim 1, characterized in that: In step (3), the pH is adjusted to neutral using 0.1 mol / L NaOH solution.

6. The preparation method according to claim 1, characterized in that: In step (4), the final concentration of sodium tripolyphosphate is 4 to 8 wt % and the reaction time is 1 to 2 h.

7. The preparation method according to claim 1, characterized in that: In step (3) and step (5), dialysis is performed using an SP131060 cellulose dialysis bag.

8. The modified sea cucumber peptide obtained by the preparation method according to any one of claims 1 to 7.

9. A method for preparing an emulsion, characterized in that: Includes steps: The modified sea cucumber peptide and yam polysaccharide prepared by the preparation method as claimed in any one of claims 1 to 7 are mixed by equal weight, dissolved in distilled water, the pH is adjusted to 7.0 with a PBS solution of pH 7.4, and stirred at a uniform speed for homogenization at 28000r / min for 3min.

10. Use of the modified sea cucumber peptide prepared by the preparation method according to any one of claims 1 to 7 or the emulsion prepared by the preparation method according to claim 9 in the preparation of products for improving sexual function.

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