Swim bladder hydrolysate capable of improving male reproductive function as well as preparation method and application of swim bladder hydrolysate

Abstract

The invention relates to a swimming bladder hydrolysate capable of improving a male reproductive function as well as a preparation method and application thereof, and belongs to the technical field of biology. The invention provides a swimming bladder hydrolysate capable of improving a male reproductive function. The swimming bladder hydrolysate is obtained by performing enzymolysis on sturgeon swimming bladders through trypsin. The total protein content of the swimming bladder hydrolysate is 80%-90%; the amino acid composition of the swimming bladder hydrolysate comprises glycine, proline, glutamic acid, asparaginic acid, arginine, alanine, serine, lysine, threonine, phenylalanine, leucine, isoleucine, valine, histidine, tyrosine and methionine. Researches show that the swim bladder hydrolysate has very strong antioxidant activity, and the swim bladder hydrolysate can effectively relieve matt hair, unstable spirit, reduced exploration behavior, weight loss and male reproductive function damage of model mice caused by exposure of heavy metal cadmium, and has great application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to a swim bladder hydrolysate capable of improving male reproductive function and a preparation method and application thereof, and belongs to the technical field of biotechnology. BACKGROUND

[0002] The reproductive system is a general term for organs that reproduce offspring and continue the species. The organs of the reproductive system are different for men and women, but they are all composed of gonads, reproductive tracts, and accessory organs according to their functions. The male reproductive system includes testes, epididymides, seminal vesicles, prostate glands, and vas deferens, etc. Among them, the main purpose of the testes is to produce sperm and secrete testosterone. The epididymis is the site where sperm obtain energy and are stored. The seminal vesicles and the prostate glands are accessory glands of men, which mainly function to produce seminal vesicle fluid and prostatic fluid. The vas deferens is a duct connecting the epididymis and the seminal vesicles, which functions to transport sperm. Ensuring male reproductive health is one of the foundations of procreation.

[0003] However, investigations have found that the problem of male infertility is becoming more and more serious in social problems, and it is urgent to solve the problem of male reproductive health. At the present stage, clinical treatment relies on the use of hormone drugs such as testosterone preparations, anti-estrogens, and gonadotropins to solve the problem of male reproductive health. However, hormone drugs have obvious side effects, and therefore, it is urgent to find an effective and safer drug capable of improving male reproductive function to overcome the defects of existing hormone drugs.

[0004] Swim bladder of sturgeon is a dual-purpose medicinal and edible material with high protein, low heat and low fat, and is known for its high content of collagen. According to Records of Materia Medica, swim bladder of sturgeon has the functions of tonifying liver and kidney, nourishing blood and stopping bleeding, and dissipating blood stasis and swelling, and is a natural food material with potential health care effects. In recent years, it has been found that the enzymatic technology can effectively improve the active ingredients of swim bladder, so that it has a variety of biological functions. However, there is no report on the use of swim bladder hydrolysate to improve male reproductive function. SUMMARY

[0005] To solve the above problems, the present application provides a swim bladder hydrolysate capable of improving male reproductive function, which is obtained by trypsin enzymolysis of swim bladder of sturgeon. The total protein (total nitrogen) content of the swim bladder hydrolysate is 80% to 90%. The amino acid composition of the swim bladder hydrolysate includes glycine, proline, glutamic acid, aspartic acid, arginine, alanine, serine, lysine, threonine, phenylalanine, leucine, isoleucine, valine, histidine, tyrosine, and methionine.

[0006] In one embodiment of the present invention, in the fish maw hydrolysate, the components with a molecular weight less than 1000Da account for 65% to 75%, the components with a molecular weight between 1000Da and 2000Da account for 25% to 28%, the components with a molecular weight between 2000Da and 3000Da account for 1.5% to 2.5%, and the components with a molecular weight greater than 3000Da account for 0.5% to 1.5%.

[0007] In one embodiment of the present invention, the fish maw hydrolysate has a glycine content of 23% to 26%, a proline content of 11% to 14%, a glutamic acid content of 11% to 14%, an aspartic acid content of 5% to 8%, an arginine content of 7% to 10%, an alanine content of 10% to 13%, a serine content of 3% to 6%, a lysine content of 1% to 4%, a threonine content of 1% to 4%, a phenylalanine content of 1% to 4%, a leucine content of 1% to 4%, an isoleucine content of 0.5% to 3%, a valine content of 0.5% to 3%, a histidine content of 0.1% to 2%, a tyrosine content of 0.1% to 2%, and a methionine content of 0.5% to 2%.

[0008] In one embodiment of the present invention, the improvement of male reproductive function includes restoring male testicular index, alleviating male sperm abnormalities, reducing male reproductive organ damage, improving male reproductive hormone imbalance, improving male reproductive organ oxidative stress levels and / or regulating the distribution of bacterial flora related to reproductive function in the male intestine.

[0009] In one embodiment of the present invention, the alleviating male sperm abnormality includes increasing sperm count, enhancing sperm motility and / or reducing sperm deformity rate;

[0010] The said alleviating male reproductive organ damage includes improving the shedding of spermatogenic cells in male testicular tissue, alleviating the atrophy of the lumen of male testicular tissue and / or restoring the morphological structure of male testicular tissue;

[0011] The improvement of male reproductive hormone imbalance includes restoring the testosterone level in male serum, restoring the inhibin B level in male serum and / or restoring the luteinizing hormone level in male serum;

[0012] The improving the oxidative stress level of male reproductive organs includes reducing the malondialdehyde level in male testicular tissue, increasing the activity of superoxide dismutase in male testicular tissue and / or increasing the activity of catalase in male testicular tissue;

[0013] The improvement and regulation of the distribution of bacterial flora related to reproductive function in the male intestine includes increasing the abundance of Bacteroidetes in the male intestine and / or reducing the abundance of Firmicutes in the male intestine.

[0014] In one embodiment of the present invention, the sturgeon comprises a hybrid sturgeon.

[0015] In one embodiment of the present invention, the enzymatic hydrolysis is performed at a pH of 7.6 to 8.8, a temperature of 35° C. to 45° C., and a time of 3 to 4.5 hours.

[0016] In one embodiment of the present invention, the method for preparing the fish maw hydrolysate comprises the following steps:

[0017] Pretreatment step: soaking the sturgeon swim bladder in an organic reagent for a first time, then soaking it in an alkaline reagent for a second time, and then washing it until the pH is neutral to obtain a pretreated sturgeon swim bladder;

[0018] Premixing step: mixing the pretreated sturgeon maw and the dilution reagent and stirring to obtain a sturgeon maw mixed solution;

[0019] Enzymatic hydrolysis step: adding trypsin to the sturgeon swim bladder mixture and performing enzymatic hydrolysis to obtain an enzymatic hydrolysis reaction liquid; first performing enzyme inactivation treatment on the enzymatic hydrolysis reaction liquid, and then performing separation and purification to obtain a swim bladder hydrolyzate.

[0020] In one embodiment of the present invention, the organic reagent includes n-butanol solution and / or ethanol solution; the alkaline reagent includes sodium hydroxide solution; and the diluent includes water, physiological saline and / or buffer solution.

[0021] In one embodiment of the present invention, the organic reagent is a n-butanol solution with a concentration of 9.0% to 10.0% (v / v); and the alkaline reagent is a sodium hydroxide solution with a concentration of 3.8 g / L to 4.2 g / L.

[0022] In one embodiment of the present invention, the temperature of the first soaking is 4°C to 8°C, and the time is 24 hours to 28 hours; the temperature of the second soaking is 4°C to 8°C, and the time is 24 hours to 28 hours.

[0023] In one embodiment of the present invention, the ratio of the pretreated sturgeon swim bladder to the dilution reagent is (0.8-1.5) g: (8-15) mL.

[0024] In one embodiment of the present invention, the stirring speed is 100 r / min to 300 r / min, and the stirring time is not less than 30 minutes.

[0025] In one embodiment of the present invention, the amount of trypsin added to the sturgeon swim bladder mixture is 100 U / mL to 105 U / mL.

[0026] In one embodiment of the present invention, the separation and purification includes: centrifuging the enzymatic hydrolysis reaction liquid and filtering to obtain the filtrate; dialyzing and freeze-drying the filtrate in sequence to obtain a lyophilized powder; redissolving the lyophilized powder in an alcohol-soluble reagent, first shaking it, and then collecting the supernatant; vacuum concentrating the supernatant, centrifuging, dialysis and freeze-drying it in sequence to obtain an alcohol-soluble fish maw hydrolysate.

[0027] In one embodiment of the present invention, the alcohol-soluble reagent comprises an ethanol solution.

[0028] In one embodiment of the present invention, the alcohol-soluble reagent is an ethanol solution with a concentration of 75-85% (v / v).

[0029] In one embodiment of the present invention, the ratio of the lyophilized powder to the alcohol-soluble reagent is (0.9-1) g: (95-100) mL.

[0030] The present invention also provides a method for preparing the fish bladder hydrolyzate, which comprises: enzymatically hydrolyzing the sturgeon maw using trypsin to obtain the fish bladder hydrolyzate.

[0031] In one embodiment of the present invention, the sturgeon comprises a hybrid sturgeon.

[0032] In one embodiment of the present invention, the enzymatic hydrolysis is performed at a pH of 7.6 to 8.8, a temperature of 35° C. to 45° C., and a time of 3 to 4.5 hours.

[0033] In one embodiment of the present invention, the method for preparing the fish maw hydrolysate comprises the following steps:

[0034] Pretreatment step: soaking the sturgeon swim bladder in an organic reagent for a first time, then soaking it in an alkaline reagent for a second time, and then washing it until the pH is neutral to obtain a pretreated sturgeon swim bladder;

[0035] Premixing step: mixing the pretreated sturgeon maw and the dilution reagent and stirring to obtain a sturgeon maw mixed solution;

[0036] Enzymatic hydrolysis step: adding trypsin to the sturgeon swim bladder mixture and performing enzymatic hydrolysis to obtain an enzymatic hydrolysis reaction liquid; first performing enzyme inactivation treatment on the enzymatic hydrolysis reaction liquid, and then performing separation and purification to obtain a swim bladder hydrolyzate.

[0037] In one embodiment of the present invention, the organic reagent includes n-butanol solution and / or ethanol solution; the alkaline reagent includes sodium hydroxide solution; and the diluent includes water, physiological saline and / or buffer solution.

[0038] In one embodiment of the present invention, the organic reagent is a n-butanol solution with a concentration of 9.0% to 10.0% (v / v); and the alkaline reagent is a sodium hydroxide solution with a concentration of 3.8 g / L to 4.2 g / L.

[0039] In one embodiment of the present invention, the temperature of the first soaking is 4°C to 8°C, and the time is 24 hours to 28 hours; the temperature of the second soaking is 4°C to 8°C, and the time is 24 hours to 28 hours.

[0040] In one embodiment of the present invention, the ratio of the pretreated sturgeon swim bladder to the dilution reagent is (0.8-1.5) g: (8-15) mL.

[0041] In one embodiment of the present invention, the stirring speed is 100 r / min to 300 r / min, and the stirring time is not less than 30 minutes.

[0042] In one embodiment of the present invention, the amount of trypsin added to the sturgeon swim bladder mixture is 100 U / mL to 105 U / mL.

[0043] In one embodiment of the present invention, the separation and purification includes: centrifuging the enzymatic hydrolysis reaction liquid and filtering to obtain the filtrate; dialyzing and freeze-drying the filtrate in sequence to obtain a lyophilized powder; redissolving the lyophilized powder in an alcohol-soluble reagent, first shaking it, and then collecting the supernatant; vacuum concentrating the supernatant, centrifuging, dialysis and freeze-drying it in sequence to obtain an alcohol-soluble fish maw hydrolysate.

[0044] In one embodiment of the present invention, the alcohol-soluble reagent comprises an ethanol solution.

[0045] In one embodiment of the present invention, the alcohol-soluble reagent is an ethanol solution with a concentration of 75-85% (v / v).

[0046] In one embodiment of the present invention, the ratio of the lyophilized powder to the alcohol-soluble reagent is (0.9-1) g: (95-100) mL.

[0047] The present invention also provides the use of the above-mentioned fish maw hydrolyzate in preparing a product, wherein the product has any of the following functions:

[0048] (a) Improve male reproductive function;

[0049] (b) Improve the damage caused by heavy metal cadmium exposure;

[0050] (c) antioxidant;

[0051] and / or, (d) regulating intestinal flora.

[0052] In one embodiment of the present invention, the improvement of male reproductive function includes restoring male testicular index, alleviating male sperm abnormalities, reducing male reproductive organ damage, improving male reproductive hormone imbalance, improving male reproductive organ oxidative stress levels and / or regulating the distribution of male intestinal flora related to reproductive function;

[0053] The improvement of damage caused by heavy metal cadmium exposure includes improving dull hair caused by heavy metal cadmium exposure, improving mental instability caused by heavy metal cadmium exposure, improving reduced exploratory behavior caused by heavy metal cadmium exposure, improving weight loss caused by heavy metal cadmium exposure, improving impaired male reproductive function caused by heavy metal cadmium exposure and / or improving intestinal flora imbalance caused by heavy metal cadmium exposure;

[0054] Said anti-oxidation includes scavenging free radicals;

[0055] The regulating intestinal flora includes increasing the relative abundance of Mucispirillum in the intestine, increasing the relative abundance of Bacteroides_H in the intestine, reducing the relative abundance of Cryptobacteroides in the intestine, reducing the relative abundance of Dubosiella in the intestine, reducing the relative abundance of Mailhella in the intestine and / or reducing the relative abundance of Lactobacillus in the intestine.

[0056] In one embodiment of the present invention, the improvement of male reproductive function damage caused by heavy metal cadmium exposure includes restoring male testicular index, alleviating male sperm abnormalities, reducing male reproductive organ damage, improving male reproductive hormone imbalance, improving male reproductive organ oxidative stress levels and / or regulating the distribution of bacterial flora related to reproductive function in the male intestine.

[0057] In one embodiment of the present invention, the improvement of intestinal flora imbalance caused by exposure to heavy metal cadmium includes increasing the relative abundance of Mucispirillum in the intestine, increasing the relative abundance of Bacteroides_H in the intestine, reducing the relative abundance of Cryptobacteroides in the intestine, reducing the relative abundance of Dubosiella in the intestine, reducing the relative abundance of Mailhella in the intestine and / or reducing the relative abundance of Lactobacillus in the intestine.

[0058] In one embodiment of the present invention, the alleviating male sperm abnormality includes increasing sperm count, enhancing sperm motility and / or reducing sperm deformity rate;

[0059] The said alleviating male reproductive organ damage includes improving the shedding of spermatogenic cells in male testicular tissue, alleviating the atrophy of the lumen of male testicular tissue and / or restoring the morphological structure of male testicular tissue;

[0060] the improving male reproductive function comprises recovering testis index of male, relieving abnormality of sperm of male, reducing damage of male reproductive organ, improving male reproductive hormone disorder, improving oxidative stress level of male reproductive organ and / or regulating distribution of flora related to reproductive function in intestinal tract of male;

[0061] the improving oxidative stress level of male reproductive organ comprises reducing malondialdehyde level in testis tissue of male, increasing activity of superoxide dismutase in testis tissue of male and / or increasing activity of catalase in testis tissue of male;

[0062] the improving regulating distribution of flora related to reproductive function in intestinal tract of male comprises increasing abundance of Bacteroidota in intestinal tract of male and / or reducing abundance of Firmicute in intestinal tract of male.

[0063] In an embodiment of the present application, the product comprises a pharmaceutical product and / or a food product; the food product comprises a health food.

[0064] In an embodiment of the present application, the product is a pharmaceutical product; the components of the pharmaceutical product comprise the above-mentioned swim bladder hydrolysate and a pharmaceutical excipient.

[0065] In an embodiment of the present application, the pharmaceutical excipient comprises water, glycerol, vitamin C, ε-polylysine and / or tea polyphenol.

[0066] The present application also provides a product, the components of the product comprising the above-mentioned swim bladder hydrolysate; the product has any one of the following functions:

[0067] (a) improving male reproductive function;

[0068] (b) improving damage caused by heavy metal cadmium exposure;

[0069] (c) antioxidation;

[0070] and / or, (d) regulating intestinal flora.

[0071] In an embodiment of the present application, the improving male reproductive function comprises recovering testis index of male, relieving abnormality of sperm of male, reducing damage of male reproductive organ, improving male reproductive hormone disorder, improving oxidative stress level of male reproductive organ and / or regulating distribution of flora related to reproductive function in intestinal tract of male;

[0072] the improving damage caused by heavy metal cadmium exposure comprises improving hair luster loss caused by heavy metal cadmium exposure, improving mental instability caused by heavy metal cadmium exposure, improving reduction of exploration behavior caused by heavy metal cadmium exposure, improving weight loss caused by heavy metal cadmium exposure, improving damage of male reproductive function caused by heavy metal cadmium exposure and / or improving intestinal flora disorder caused by heavy metal cadmium exposure;

[0073] Said anti-oxidation includes scavenging free radicals;

[0074] The regulating intestinal flora includes increasing the relative abundance of Mucispirillum in the intestine, increasing the relative abundance of Bacteroides_H in the intestine, reducing the relative abundance of Cryptobacteroides in the intestine, reducing the relative abundance of Dubosiella in the intestine, reducing the relative abundance of Mailhella in the intestine and / or reducing the relative abundance of Lactobacillus in the intestine.

[0075] In one embodiment of the present invention, the improvement of male reproductive function damage caused by heavy metal cadmium exposure includes restoring male testicular index, alleviating male sperm abnormalities, reducing male reproductive organ damage, improving male reproductive hormone imbalance, improving male reproductive organ oxidative stress levels and / or regulating the distribution of bacterial flora related to reproductive function in the male intestine.

[0076] In one embodiment of the present invention, the improvement of intestinal flora imbalance caused by exposure to heavy metal cadmium includes increasing the relative abundance of Mucispirillum in the intestine, increasing the relative abundance of Bacteroides_H in the intestine, reducing the relative abundance of Cryptobacteroides in the intestine, reducing the relative abundance of Dubosiella in the intestine, reducing the relative abundance of Mailhella in the intestine and / or reducing the relative abundance of Lactobacillus in the intestine.

[0077] In one embodiment of the present invention, the alleviating male sperm abnormality includes increasing sperm count, enhancing sperm motility and / or reducing sperm deformity rate;

[0078] The said alleviating male reproductive organ damage includes improving the shedding of spermatogenic cells in male testicular tissue, alleviating the atrophy of the lumen of male testicular tissue and / or restoring the morphological structure of male testicular tissue;

[0079] The improvement of male reproductive hormone imbalance includes restoring the testosterone level in male serum, restoring the inhibin B level in male serum and / or restoring the luteinizing hormone level in male serum;

[0080] The improving the oxidative stress level of male reproductive organs includes reducing the malondialdehyde level in male testicular tissue, increasing the activity of superoxide dismutase in male testicular tissue and / or increasing the activity of catalase in male testicular tissue;

[0081] The improvement and regulation of the distribution of bacterial flora related to reproductive function in the male intestine includes increasing the abundance of Bacteroidetes in the male intestine and / or reducing the abundance of Firmicutes in the male intestine.

[0082] In one embodiment of the present invention, the product includes medicine and / or food; the food includes health food.

[0083] In one embodiment of the present invention, the product is a medicine; the ingredients of the medicine include the above-mentioned fish maw hydrolysate and pharmaceutical excipients.

[0084] In one embodiment of the present invention, the pharmaceutical excipients include water, glycerol, vitamin C, ε-polylysine and / or tea polyphenols.

[0085] The technical solution of the present invention has the following advantages:

[0086] The present invention provides a fish bladder hydrolyzate that can improve male reproductive function. The fish bladder hydrolyzate is obtained by enzymatic hydrolysis of sturgeon maw with trypsin; the total protein content of the fish bladder hydrolyzate is 80% to 90%; and the amino acid composition of the fish bladder hydrolyzate includes glycine, proline, glutamic acid, aspartic acid, arginine, alanine, serine, lysine, threonine, phenylalanine, leucine, isoleucine, valine, histidine, tyrosine, and methionine. Research shows that:

[0087] (1) The fish maw hydrolysate has strong antioxidant activity (including scavenging ABTS free radicals and reducing iron ions);

[0088] (2) The fish maw hydrolyzate can effectively alleviate the dull hair, mental instability, reduced exploratory behavior, weight loss, impaired male reproductive function (including restoring testicular index, alleviating sperm abnormalities, reducing reproductive organ damage, improving reproductive hormone imbalance, improving reproductive organ oxidative stress levels and regulating the distribution of intestinal flora related to reproductive function) and intestinal flora imbalance (including increasing the relative abundance of Mucispirillum and Bacteroides_H, and reducing the relative abundance of Cryptobacteroides, Dubosiella, Mailhella and Lactobacillus) caused by heavy metal cadmium exposure in model mice.

[0089] Therefore, the fish maw hydrolyzate has great application prospects in the preparation of drugs that can improve male reproductive function, improve damage caused by heavy metal cadmium exposure, resist oxidation, and / or regulate intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 : Validation results of antioxidant activity of sturgeon swim bladder hydrolysate SSBH-ES. Figure 1 In the figure, A is the ABTS free radical scavenging ability of sturgeon bladder hydrolysate SSBH-ES, and B is the iron ion reducing ability of sturgeon bladder hydrolysate SSBH-ES.

[0091] Figure 2 : Effects of sturgeon swim bladder hydrolysate SSBH-ES on body weight and organ indices in mice. Figure 2 In the figure, A shows the effect of fish bladder hydrolysate SSBH-ES on the body weight of mice, B shows the effect of fish bladder hydrolysate SSBH-ES on the testicular index of mice, and C shows the effect of fish bladder hydrolysate SSBH-ES on the epididymal index of mice.

[0092] Figure 3 : Effects of sturgeon swim bladder hydrolysate SSBH-ES on sperm parameters in mice. Figure 3 In the figure, A shows the effect of fish bladder hydrolysate SSBH-ES on the number of mouse epididymal sperm, B shows the effect of fish bladder hydrolysate SSBH-ES on the activity of mouse epididymal sperm, C shows the effect of fish bladder hydrolysate SSBH-ES on the sperm morphology of mouse epididymis, and D shows the observation picture of mouse epididymal sperm morphology staining.

[0093] Figure 4 : Pathological observation of the effects of sturgeon swim bladder hydrolysate SSBH-ES on mouse testicular structure.

[0094] Figure 5 : Effects of sturgeon swim bladder hydrolysate SSBH-ES on serum hormones and testicular oxidative stress in mice. Figure 5 In the figure, A shows the effect of fish maw hydrolysate SSBH-ES on the serum testosterone T level of mice, B shows the effect on the serum luteinizing hormone LH level of mice, and C shows the effect on the serum inhibin B INH-B level of mice.

[0095] Figure 6 : Effects of fish maw hydrolysate SSBH-ES on intestinal flora diversity in mice. Figure 6 In the figure, A represents the effect on the Chao1 index of the intestinal flora of mice, B represents the effect on the Shannon index of the intestinal flora of mice, C represents the effect on the Simpson index of the intestinal flora of mice, and D represents the effect on the Pielou_e index of the intestinal flora of mice.

[0096] Figure 7 : Effects of fish maw hydrolysate SSBH-ES on the phylum level composition of intestinal flora in mice.

[0097] Figure 8 : Effects of fish bladder hydrolysate SSBH-ES on the abundance of major intestinal flora in mice. Figure 8 In the figure, A shows the effect on the abundance of Bacteroidota in the mouse intestine, B shows the effect on the abundance of Firmicute in the mouse intestine, and C shows the effect on the F / B ratio in the mouse intestine.

[0098] Figure 9: Effects of fish maw hydrolysate SSBH-ES on species composition of intestinal flora in mice. DETAILED DESCRIPTION

[0099] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0100] If the specific experimental steps or conditions are not specified in the following examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be carried out. If the manufacturer of the reagents or instruments used is not specified, they are all conventional reagent products that can be obtained commercially. The trypsin involved in the following examples was purchased from Beijing Solebow Technology Co., Ltd., the hydroxyproline kit was purchased from Beijing Solebow Technology Co., Ltd., the CD-1 mouse was purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., the rapid sperm staining kit was purchased from Nanjing Jiancheng Bioengineering Research Institute, the ELISA kit was purchased from Wuhan Yilai Ruite Biotechnology Co., Ltd., the lipid peroxidation detection kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., and the DNA extraction kit was purchased from Illumina, USA. The blender involved in the following examples was purchased from Dalong Xingchuang Experimental Instrument (Beijing) Co., Ltd., model OS20-S; the dialysis bag involved in the following examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd., model SP131060.

[0101] Example 1: A sturgeon maw hydrolyzate and its preparation method

[0102] This embodiment provides a sturgeon maw hydrolysate, and the preparation method of the sturgeon maw hydrolysate comprises the following steps:

[0103] Pretreatment step: adding a sturgeon swim bladder (obtained from a hybrid sturgeon, strain Sturgeon Dragon Fish No. 1) to a 10% (v / v) n-butanol solution (the ratio of sturgeon swim bladder to n-butanol solution is 1 g: 10 mL), and soaking at 4°C for 24 hours (during the soaking process, fresh n-butanol solution is replaced every 12 hours); after the soaking, removing the sturgeon swim bladder and adding it to a 0.1M sodium hydroxide solution (the ratio of sturgeon swim bladder to sodium hydroxide solution is 1 g: 10 mL), and soaking at room temperature for 24 hours (during the soaking process, fresh sodium hydroxide solution is replaced every 8 hours); after the soaking, removing the sturgeon swim bladder and washing the sturgeon swim bladder with distilled water until the pH is neutral, thereby obtaining a pretreated sturgeon swim bladder;

[0104] Premixing step: The pretreated sturgeon maw and ultrapure water were mixed at a ratio of 1 g:10 mL, and stirred using a blender (at 250 rpm for 45 minutes) to obtain a sturgeon maw mixture (after treatment with n-butanol and sodium hydroxide, the sturgeon maw has a loose tissue structure. At this time, mixing it with ultrapure water in a certain proportion and stirring it using a bladed blender can form a relatively uniform sturgeon maw mixture);

[0105] Enzymatic hydrolysis step: After adding 100 U / mL of trypsin to the sturgeon swim bladder mixture, first use a pH regulator (hydrochloric acid or sodium hydroxide) to adjust the pH to 7.8, and then enzymatically hydrolyze at 37°C for 4 hours to obtain an enzymatic hydrolysis reaction solution; the enzymatic hydrolysis reaction solution is first placed in a 95°C water bath to inactivate the enzyme for 15 minutes, and then centrifuged (centrifuged at 5000 rpm for 20 minutes and then the supernatant is taken) and filtered to obtain the filtrate; the filtrate is successively vacuum concentrated, dialyzed (dialysis bag with ultrapure water as the dialysate for 7 2 hours) and vacuum freeze-dried to obtain a lyophilized powder; the lyophilized powder was first redissolved in an 80% (v / v) ethanol solution and then shaken (shake for 1 hour), and the supernatant was collected; the supernatant was sequentially vacuum concentrated, centrifuged (centrifuged at 10,000 rpm for 20 minutes, and the supernatant was collected), dialyzed (dialysis using ultrapure water as the dialysate using a dialysis bag for 72 hours, with a molecular weight cut-off of 100 Da), and vacuum freeze-dried to obtain an alcohol-soluble sturgeon swim bladder hydrolysate SSBH-ES.

[0106] Experimental Example 1: Component Analysis of Sturgeon Maw Hydrolysate

[0107] This experimental example provides a component analysis experiment of sturgeon swim bladder hydrolysate. The experimental process is as follows:

[0108] Experiment 1: Molecular weight determination

[0109] The molecular weight of sturgeon swim bladder hydrolysate (SSBH-ES) was determined using an Agilent 1260 high-performance liquid chromatograph equipped with a TSK gel G2000 SWXL (7.8 mm × 300 mm) column. The mobile phase consisted of acetonitrile / water / trifluoroacetic acid (TFA) (45:55:0.1, v / v / v). The sample preparation concentration was 1.0 mg / mL, the flow rate was set at 0.5 mL / min, the column temperature was set at 25°C, the UV detection wavelength was 220 nm, and the injection volume was 10 μL. A calibration curve was constructed using cytochrome C (12384 Da), aprotinin (6512 Da), bacillidase (1072 Da), tyrosine-tyrosine-arginine, and tyrosine-tyrosine-tyrosine as standards. The molecular weight results are shown in Table 1. The results showed that low molecular weight peptides accounted for a high proportion in sturgeon swim bladder hydrolysate SSBH-ES, of which components exceeding 3000Da accounted for only 0.94%, while components with molecular weight less than 3000Da accounted for 99.06%, and were mainly concentrated in 500Da-2000Da.

[0110] Table 1 Molecular weight distribution of sturgeon swim bladder hydrolysate SSBH-ES

[0111]

[0112]

[0113] Experiment 2: Amino Acid Composition Analysis

[0114] 20 mg of sturgeon swim bladder hydrolysate SSBH-ES was weighed into a hydrolysis tube, 10 mL of 6 M hydrochloric acid and 1 g of phenol were added, and the mixture was hydrolyzed in a 110°C oven for 24 hours to obtain a hydrolyzate. After the hydrolyzate was cooled to 20°C, it was quantified to 50 mL with ultrapure water to obtain a test sample. 1 mL of the test sample was placed in a clean Petri dish, evaporated to dryness, and then dissolved in 3 mL of ultrapure water, and evaporated to dryness twice. After the evaporation operation was completed, 1 mL of pH 2.2 citrate-hydrochloric acid buffer was added to the evaporated product for re-dissolution, and the mixture was shaken to obtain a reconstituted solution. The reconstituted solution was filtered through a 0.22 μm filter membrane to obtain a filtrate. The amino acid components in the filtrate were analyzed using a SYKAM-S-433D amino acid analyzer. The amino acid composition of sturgeon swim bladder hydrolysate SSBH-ES was tested as shown in Table 2. A total of 16 amino acids were detected, of which glycine (Gly) had the highest content, followed by proline (Pro), glutamic acid (Glu), aspartic acid (Asp), arginine (Arg), alanine (Ala), serine (Ser), lysine (Lys), threonine (Thr), phenylalanine (Phe), leucine (Leu), isoleucine (Ile), valine (Val), histidine (His), tyrosine (Tyr), and methionine (Met). Phenylalanine (Phe) and tyrosine (Tyr) are aromatic amino acids that can promote the antioxidant activity of proteins. The amino acid composition results also meet the amino acid composition unit Glycine-XY.

[0115] Table 2 Amino acid composition of sturgeon swim bladder hydrolysate SSBH-ES

[0116]

[0117]

[0118] Experimental Example 2: Antioxidant Activity Verification Experiment of Sturgeon Maw Hydrolysate

[0119] This experimental example provides a validation experiment on the antioxidant activity of sturgeon swim bladder hydrolysate. The experimental process is as follows:

[0120] Experiment 1: ABTS free radical scavenging ability

[0121] Sturgeon swim bladder hydrolysate SSBH-ES was diluted to a protein concentration of 0.5, 1, 2, 3, 4, 5, and 10 mg / mL using 80% (v / v) ethanol aqueous solution to obtain a series of samples with different concentrations; ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) was prepared into a 7 mM ABTS solution using a pH 7.4 phosphate buffer; a series of samples with different concentrations were mixed with the ABTS solution at a volume ratio of 1:20 and incubated at room temperature for 10 minutes to obtain incubation products; the absorbance of the incubation products was measured at 734 nm, and the absorbance was calculated according to the formula Calculate the ABTS free radical scavenging rate. The calculation results are shown in Figure 1 A; In the formula, A c As the control group (ethanol and ABTS + absorbance of the free radical working solution after mixing), A s is the absorbance of the sample solution, A b Does not contain ABTS + The experimental results showed that the ABTS free radical scavenging rate increased significantly with the increase of the concentration of sturgeon swim bladder hydrolysate. When the concentration reached 4 mg / mL, the scavenging rate reached 52.10±2.97%, showing strong antioxidant activity.

[0122] Experiment 2: Iron Ion Reducing Ability

[0123] Sturgeon swim bladder hydrolysate SSBH-ES was diluted with 80% (v / v) ethanol aqueous solution to a protein concentration of 0.5, 1, 2, 3, 4, 5, and 10 mg / mL to obtain a series of samples with different concentrations. The samples with different concentrations were mixed with FRAP reagent (acetate buffer: TPTZ: FeCl3 = 10:1:1, v / v / v) at a volume ratio of 1:1 and incubated at room temperature for 30 minutes to obtain incubation products. The absorbance of the incubation products was measured at 593 nm. The measurement results are shown in Figure 2. Figure 1 B. Experimental results showed that in the iron ion reducing capacity experiment, after different concentrations of sturgeon maw hydrolysate reacted with the FRAP reagent, the absorbance at a wavelength of 593 nm gradually increased, indicating enhanced reducing capacity. This result further demonstrates the antioxidant potential of sturgeon maw hydrolysate.

[0124] Experimental Example 3: Effect of Sturgeon Maw Hydrolysate on Male Reproductive Function

[0125] This experimental example provides an experiment on the effect of sturgeon swim bladder hydrolyzate on male reproductive function. The experimental process is as follows:

[0126] 1. Experimental process

[0127] Five-week-old healthy male CD-1 mice were housed under a 12-h light-dark cycle at constant temperature and humidity for 7 days. Mice were randomly divided into five groups according to a random number table: normal control (NC), cadmium-induced (Cd) group, low-dose SSBH-ES group (LD), medium-dose SSBH-ES group (MD), and high-dose SSBH-ES group (HD), with seven mice in each group. All mice had free access to food and water. The NC and model groups received oral administration of 0.01 mL / g bw normal saline daily. The LD, MD, and HD groups received SSBH-ES (sturgeon swim bladder hydrolyzate) at doses of 100, 200, and 400 mg / kg / day, respectively (using saline as the solvent, equivalent to that in the NC group). Two hours after oral administration, the NC group received saline, while the other groups received 5 mg / kg bw CdCl2 (using saline as the solvent, equivalent to that in the NC group). Each group had free access to food and water for 35 consecutive days, with oral administration doses adjusted based on daily body weight. On day 36, all mice were sacrificed, blood was collected, and the testicles and epididymis were removed.

[0128] During the experiment, the behavior and characteristics of the mice were observed and the weight of the mice was recorded. Figure 2 A. After the experiment, the mice were sacrificed and their feces were collected and stored frozen at -80°C for subsequent DNA extraction. After the mice were sacrificed, their blood was collected and their testicles and epididymis tissues were removed. The blood was centrifuged to separate the serum and stored frozen. The testicles and epididymis were weighed and the testicular and epididymal indices were calculated (organ weight / body weight × 100%). The calculation results are shown in Figure 2 B~ Figure 2 C.

[0129] The right epididymis was collected, minced, and placed in 1 mL of physiological saline preheated to 37°C to promote sperm release to obtain a mixture; the mixture was incubated at 37°C for 15 minutes to ensure optimal sperm motility, and then the sperm count and total active sperm were recorded using a Neubauer hemocytometer (the results were expressed as sperm counts per milliliter). After the sperm died naturally, the sperm deformity rate and morphology were analyzed using a rapid sperm staining kit. During the analysis, the sperm suspension was smeared, naturally dried, fixed, and stained, and the number of normal sperm, head defects, midpiece defects, and tail defects was recorded (at least 200 sperm were analyzed). The analysis results are shown in [ 15 ]. Figure 3 A~ Figure 3 D.

[0130] After the testicular tissue was removed, it was washed with physiological saline to remove surface blood stains, and then placed in 4% (w / v, g / 100 mL) paraformaldehyde solution for fixation for 24 hours. After fixation, the tissue was dehydrated, transparent, waxed, paraffin-embedded, and sectioned. After sectioning, the testicular tissue sections were routinely stained with hematoxylin-eosin (H&E). After staining, the morphological changes of the tissue were observed using an optical microscope, and pathological scores were performed. The pathological results are shown in Figure 4 .

[0131] ELISA kits were used to determine the levels of testosterone (T), luteinizing hormone (LH), and inhibin B (INH-B) in serum. Figure 5 A~ Figure 5 C. The detection steps using the ELISA kit include: adding the serum sample to the ELISA plate, incubating and washing the plate, adding the substrate for color development, and measuring the absorbance (450 nm); calculating the hormone concentration using the standard curve to evaluate the effect of sturgeon swim bladder hydrolysate on serum hormone levels.

[0132] Mouse testicular tissue was obtained and mixed with PBS buffer precooled to 4°C at a ratio of 1 mg:9 mL. The mixture was then homogenized using a tissue grinder to obtain a homogenate. A lipid peroxidation detection kit was used to detect the MDA level, SOD activity, and CAT activity of the homogenate to obtain oxidative stress indicators such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in the mouse testicular tissue, thereby evaluating the degree of oxidative damage in the mouse testicular tissue. The results are shown in Figure 5 D~ Figure 5 F.

[0133] Total genomic DNA was extracted from intestinal fecal samples using a DNA extraction kit. The extracted DNA was amplified from the V3-V4 hypervariable region of the 16S rRNA gene and then subjected to high-throughput sequencing. The resulting sequences were used to analyze the composition of the microbiota in the samples. α-diversity was measured from multiple perspectives using Chao 1, Shannon, Simpson, and Pieloué methods to assess the species richness and diversity of the intestinal flora. The evaluation results are shown in Figure 6 In order to study the effect of cadmium chloride exposure on the microbial community structure, a statistical analysis of the community composition was conducted at the phylum level. The results are shown in Figures 7 and 8 To further investigate the bacterial types that influence the changes in microbial composition, the distribution of species abundance in different samples was visualized using a genus-level species composition heat map to further compare the differences in species composition between groups. The comparison results are shown in Figure 9 .

[0134] 2. Experimental results

[0135] During the experiment, mice in the NC group exhibited active behavior and shiny fur throughout the study period. In contrast, mice in the Cd group showed signs of dull fur, mental instability, and reduced exploratory behavior after five days of exposure. However, improvements in these signs were observed after intervention with different doses of SSBH-ES. Figure 2 Results from A showed that the Cd group showed a trend of weight loss, indicating deterioration in health. However, supplementation with SSBH-ES restored some of the body weight. This suggests that sturgeon swim bladder hydrolyzate (SSBH-ES) can effectively mitigate the damage caused by cadmium exposure in mice.

[0136] Figure 2 B~ Figure 2 Results from the C study showed that the testicular index of mice exposed to cadmium was significantly lower than that of the control group. However, administration of sturgeon bladder hydrolyzate (SSBH-ES) significantly restored the testicular index, mitigating the damage caused by cadmium. However, no significant differences were observed between the different dose groups. In contrast, the epididymal index of mice did not change significantly across all groups. These results suggest that sturgeon bladder hydrolyzate (SSBH-ES) can effectively mitigate the reproductive damage caused by cadmium exposure in mice.

[0137] Figure 3 A~ Figure 3 Results from the D experiment showed that the sperm deformity rate in the NC group was slightly lower than in the other experimental groups, but the difference was not significant. Cadmium chloride treatment significantly reduced sperm count and motility, while sturgeon bladder hydrolyzate (SSBH-ES) effectively increased sperm count and motility in a dose-dependent manner, thereby mitigating the damage caused by cadmium. These results indicate that sturgeon bladder hydrolyzate (SSBH-ES) can effectively mitigate the reproductive damage caused by cadmium exposure in mice.

[0138] Figure 4 The results showed that the seminiferous tubules in the NC group were structurally intact, with tightly arranged spermatogenic cells and no obvious abnormalities. In contrast, the seminiferous tubules in the Cd group showed atrophy, disorganization and shedding of spermatogenic epithelial cells, and a decrease in the number of newly formed sperm in some tubular lumens. After continuous administration of sturgeon bladder hydrolyzate SSBH-ES, spermatogenic cell shedding improved, tubular lumen atrophy was alleviated, and morphological structure was relatively restored. However, compared with the other two groups, the recovery effect of the low-dose group was less obvious. These results indicate that sturgeon bladder hydrolyzate SSBH-ES can effectively reduce the structural damage of reproductive organs caused by cadmium exposure and protect their reproductive function.

[0139] Figure 5 A~ Figure 5Results from the C study showed that compared with the NC group, serum T and INH-B levels decreased in mice treated with Cd, while LH levels increased significantly, indicating that cadmium exposure disrupted reproductive hormone function in male mice. However, in mice treated with sturgeon bladder hydrolysate (SSBH-ES), serum T and INH-B levels rebounded, and LH levels returned to normal. These results indicate that SSBH-ES can effectively ameliorate cadmium-induced reproductive hormone imbalances and promote the recovery of hormone levels, further supporting its potential for improving reproductive function.

[0140] Oxidative stress is one of the factors leading to organ damage, which has also been confirmed in the cadmium stimulation model. Figure 5 D~ Figure 5 Results from F showed that compared with normal controls, malondialdehyde (MDA) levels in mice exposed to Cd were significantly increased, while superoxide dismutase (SOD) and catalase (CAT) activities were significantly decreased. These changes were significantly reversed in the SSBH-ES treatment group, and different doses of sturgeon bladder hydrolysate effectively improved MDA levels and SOD and CAT activities. These results suggest that SSBH-ES, a sturgeon bladder hydrolysate, exerts a beneficial effect on cadmium-induced testicular oxidative stress by activating antioxidant enzymes.

[0141] Figure 6 The results showed that compared with the normal group, the Shannon index, Simpson index and Pielou_e index decreased significantly after cadmium chloride treatment, indicating that bacterial diversity and uniformity decreased, while the bacterial diversity and uniformity of mice increased after supplementation with sturgeon bladder hydrolysate SSBH-ES. Figures 7 and 8 In the study, the top ten species in relative abundance at the phylum level were analyzed. The results showed that compared with the NC group, the abundance of Bacteroidota in the Cd group was significantly reduced, the abundance of Firmicute was significantly increased, and the F / B ratio was significantly increased. After treatment with sturgeon bladder hydrolysate SSBH-ES, the intestinal flora composition of mice improved, and the abundance of Bacteroidetes and Firmicute was restored. The Bacteroidetes has been shown to be beneficial microorganisms for glucose metabolism and can improve the energy supply of sperm cells by improving glycolysis. It can be seen that sturgeon bladder hydrolysate SSBH-ES may promote the proliferation of beneficial intestinal flora and improve the glycolysis of supporting cells to regulate spermatogenesis. Figure 9Results showed that compared with the NC group, the relative abundance of Cryptobacteroides, Dubosiella, Mailhella, and Lactobacillus significantly increased in the Cd group, while the relative abundance of Mucispirillum and Bacteroides_H significantly decreased. This suggests that Cd treatment disrupted the intestinal microbiota of mice, a shift reversed by sturgeon bladder hydrolysate intervention, bringing the microbiota closer to that of the NC group. These changes were associated with the restoration of intestinal health, further supporting the potential role of sturgeon bladder hydrolysate (SSBH-ES) in regulating intestinal microbiota balance. Taken together, these results suggest that sturgeon bladder hydrolysate can effectively reverse cadmium-induced intestinal microbial imbalance. This reversal not only helps restore a normal intestinal microecological environment but also may improve overall host health. The application of sturgeon bladder hydrolysate demonstrates its potential in regulating the intestinal microbiome, providing a new approach to preventing and controlling the health effects of environmental pollution. Therefore, sturgeon bladder hydrolysate is not only important for improving intestinal health but also offers a potential solution for optimizing male reproductive function.

[0142] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A fish maw hydrolyzate capable of improving male reproductive function, characterized in that: The fish maw hydrolysate is obtained by enzymatic hydrolysis of sturgeon maw with trypsin; the total protein content of the fish maw hydrolysate is 80% to 90%; and the amino acid composition of the fish maw hydrolysate includes glycine, proline, glutamic acid, aspartic acid, arginine, alanine, serine, lysine, threonine, phenylalanine, leucine, isoleucine, valine, histidine, tyrosine and methionine.

2. The fish maw hydrolyzate according to claim 1, wherein In the fish maw hydrolyzate, the components with a molecular weight less than 1000Da account for 65% to 75%, the components with a molecular weight between 1000Da and 2000Da account for 25% to 28%, the components with a molecular weight between 2000Da and 3000Da account for 1.5% to 2.5%, and the components with a molecular weight greater than 3000Da account for 0.5% to 1.5%.

3. The fish maw hydrolyzate according to claim 1 or 2, wherein The fish maw hydrolyzate has a glycine content of 23% to 26%, a proline content of 11% to 14%, a glutamic acid content of 11% to 14%, an aspartic acid content of 5% to 8%, an arginine content of 7% to 10%, an alanine content of 10% to 13%, a serine content of 3% to 6%, a lysine content of 1% to 4%, a threonine content of 1% to 4%, a phenylalanine content of 1% to 4%, a leucine content of 1% to 4%, an isoleucine content of 0.5% to 3%, a valine content of 0.5% to 3%, a histidine content of 0.1% to 2%, a tyrosine content of 0.1% to 2%, and a methionine content of 0.5% to 2%.

4. The fish maw hydrolyzate according to any one of claims 1 to 3, wherein The improvement of male reproductive function includes restoring male testicular index, alleviating male sperm abnormalities, reducing male reproductive organ damage, improving male reproductive hormone imbalance, improving male reproductive organ oxidative stress levels and / or regulating the distribution of bacterial flora related to reproductive function in the male intestine.

5. The fish maw hydrolyzate according to any one of claims 1 to 4, characterized in that The enzymatic hydrolysis has a pH of 7.6 to 8.8, a temperature of 35° C. to 45° C., and a time of 3 to 4.5 hours.

6. The fish maw hydrolyzate according to any one of claims 1 to 5, characterized in that The preparation method of the fish maw hydrolysate comprises the following steps: Pretreatment step: soaking the sturgeon swim bladder in an organic reagent for a first time, then soaking it in an alkaline reagent for a second time, and then washing it until the pH is neutral to obtain a pretreated sturgeon swim bladder; Premixing step: mixing the pretreated sturgeon maw and the dilution reagent and stirring to obtain a sturgeon maw mixed solution; Enzymatic hydrolysis step: adding trypsin to the sturgeon swim bladder mixture and performing enzymatic hydrolysis to obtain an enzymatic hydrolysis reaction liquid; first performing enzyme inactivation treatment on the enzymatic hydrolysis reaction liquid, and then performing separation and purification to obtain a swim bladder hydrolyzate.

7. A method for preparing the fish maw hydrolysate according to any one of claims 1 to 6, characterized in that: The method comprises: enzymatically hydrolyzing the sturgeon swim bladder using trypsin to obtain swim bladder hydrolysate.

8. Use of the fish maw hydrolyzate according to any one of claims 1 to 6 in preparing products, characterized in that: The product has any of the following functions: (a) Improve male reproductive function; (b) Improve the damage caused by heavy metal cadmium exposure; (c) antioxidant; and / or, (d) regulating intestinal flora.

9. The use according to claim 8, characterized in that The improvement of male reproductive function includes restoring male testicular index, alleviating male sperm abnormalities, reducing male reproductive organ damage, improving male reproductive hormone imbalance, improving male reproductive organ oxidative stress levels and / or regulating the distribution of male intestinal flora related to reproductive function; The improvement of damage caused by heavy metal cadmium exposure includes improving dull hair caused by heavy metal cadmium exposure, improving mental instability caused by heavy metal cadmium exposure, improving reduced exploratory behavior caused by heavy metal cadmium exposure, improving weight loss caused by heavy metal cadmium exposure, improving impaired male reproductive function caused by heavy metal cadmium exposure and / or improving intestinal flora imbalance caused by heavy metal cadmium exposure; Said anti-oxidation includes scavenging free radicals; The regulating intestinal flora includes increasing the relative abundance of Mucispirillum in the intestine, increasing the relative abundance of Bacteroides_H in the intestine, reducing the relative abundance of Cryptobacteroides in the intestine, reducing the relative abundance of Dubosiella in the intestine, reducing the relative abundance of Mailhella in the intestine and / or reducing the relative abundance of Lactobacillus in the intestine.

10. A product, characterized in that The ingredients of the product include the fish maw hydrolyzate according to any one of claims 1 to 6; the product has any of the following functions: (a) Improve male reproductive function; (b) Improve the damage caused by heavy metal cadmium exposure; (c) antioxidant; and / or, (d) regulating intestinal flora.

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