CN116918976A - ADPP polypeptide with functions of promoting vital essence production and strengthening yang, composition and application thereof - Google Patents
ADPP polypeptide with functions of promoting vital essence production and strengthening yang, composition and application thereof Download PDFInfo
- Publication number
- CN116918976A CN116918976A CN202210361635.9A CN202210361635A CN116918976A CN 116918976 A CN116918976 A CN 116918976A CN 202210361635 A CN202210361635 A CN 202210361635A CN 116918976 A CN116918976 A CN 116918976A
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- China
- Prior art keywords
- adpp
- polypeptide
- temperature
- alkaline protease
- bromelain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Classifications
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/02—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from meat
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A23L33/15—Vitamins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
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Abstract
The application relates to the field of biology, in particular to an ADPP polypeptide with the functions of promoting vital essence production and strengthening yang, a composition and application thereof. The ADPP polypeptide is obtained by hydrolyzing giant salamander muscle with hydrolase selected from temperature-resistant alkaline protease and bromelain to obtain hydrolysate; the alkaline protease is temperature-resistant alkaline protease produced by bacillus pumilus or bacillus licheniformis temperature-resistant alkaline protease; the working temperature of the alkaline protease is 60-65 ℃. The ADPP protein peptide has the functions of producing sperm, activating sperm or strengthening yang.
Description
Technical Field
The application relates to the field of biology, in particular to an ADPP polypeptide with the functions of promoting vital essence production and strengthening yang, a composition and application thereof.
Background
Recent reports indicate that during the last 60 years, the semen quality of humans has decreased, which has led to a substantial increase in the rate of male infertility. The factors responsible for the decline in semen quality are often complex and the cause of the pathogenesis of many patients is ambiguous. With the development of the living standard, people are gradually paying attention to the connection between environmental problems and infertility. For the last two decades, many clinical, epidemiological and basic experimental efforts have demonstrated that environmental endocrine disruptors can reduce male fertility. Bisphenol A (BPA) is widely used in the production of polycarbonate and epoxy resin, and has weak estrogen and antiandrogen effects due to its structure similar to estrogen, thus being an important environmental endocrine disrupter. More and more studies have shown that BPA can cause different forms and degrees of damage to the reproductive system. Studies in male animal experiments have shown that BPA exposure in the perinatal period can affect reproductive ability and destroy the Sertoli cell-associated proteins that play an important role in the spermatogenesis process. In addition, there may be a certain link between the decrease in BPA content in urine and the increase in sperm DNA damage. In addition, BPA exposure reduces testosterone levels in serum, inhibits steroid synthases, causes hypermethylation of the sperm cell estrogen receptor promoter region, and these changes may ultimately lead to male reproductive dysfunction. Modern medicine is not completely known about the pathogenesis of male infertility, and no specific therapeutic drug exists clinically.
After 21 st century, a great deal of resources are required by human beings, so that the problems of land resource shortage, population expansion, environmental deterioration and the like are increasingly serious. In view of this, various countries in the world have made a step of arming fresh water organisms and amphibians, and climax of developing and utilizing aquatic and amphibian biological resources worldwide has been rapidly raised.
The Chinese giant salamander has delicious meat, unique flavor, nutrition and nourishing, is reputation of "live ginseng in water", and has very high scientific research, eating, medical, beautifying and ornamental values. In which 14 fatty acids having extremely strong antioxidant stress activation are detected in total in the muscle. After the 60 th century, in order to protect giant salamander resources, some domestic enterprises begin to artificially breed giant salamanders. At present, the giant salamander product is mainly used for being directly eaten as high-end dishes on a dining table, and if the deep processing development of giant salamander in the fields of food, medicine, cosmetology, health care and the like is further developed, the industry of giant salamanders will be facilitated to be mature.
In view of this, the present application has been made.
Disclosure of Invention
The application aims at providing an ADPP polypeptide with the functions of promoting vital essence production and strengthening yang.
It is a second object of the present application to provide a composition comprising an ADPP polypeptide.
It is a third object of the application to provide the use of ADPP polypeptides and compositions thereof.
In order to solve the technical problems of the application, the technical scheme adopted is as follows:
the application provides an ADPP polypeptide, which is obtained by hydrolyzing giant salamander muscle by hydrolase to obtain a hydrolysate and then separating; the hydrolase is selected from temperature-resistant alkaline protease and bromelain; the alkaline protease is temperature-resistant alkaline protease produced by bacillus pumilus or bacillus licheniformis temperature-resistant alkaline protease; preferably, the alkaline protease operates at a temperature of 60 to 65 ℃.
Optionally, giant salamander muscle: alkaline protease: the mass ratio of bromelain is 100:0.05 to 0.1:0.025 to 0.05.
Optionally, the preparation method of the ADPP polypeptide at least comprises the following steps:
s1, taking muscles of artificially cultured giant salamanders, adding water with the mass of 5-10 times, mincing to prepare minced meat, and performing rough filtration and homogenization to obtain a homogenized liquid;
s2, adding temperature-resistant alkaline protease into the homogenized solution, regulating the pH value to 7.5-8.0, carrying out enzymolysis for 20-40 minutes at the temperature of 55-65 ℃, and adding bromelain for enzymolysis for 0.5-1.5 h;
s3, centrifuging or filtering to remove undigested cell fragments and residues, ultrafiltering with an ultrafiltration membrane with a molecular weight cutoff of 10KD, and collecting filtrate to obtain the ADPP polypeptide.
Optionally, in S1, the meat emulsion is ground with a tissue pestle or refiner and homogenized with a homogenizer.
Optionally, the enzymolysis temperature of the temperature-resistant alkaline protease is 60-65 ℃ and the enzymolysis time is 30 minutes; the enzymolysis temperature of the bromelain is 55-65 ℃, preferably 60 ℃.
Optionally, the preparation method further comprises the step of drying the giant salamander hydrolysate; preferably, the low-temperature spray drying method is selected for drying, the inlet temperature is 100-120 ℃, and the outlet temperature is 50-70 ℃.
The application provides a composition containing the ADPP polypeptide, and the composition also contains at least one of vitamins, trace elements and traditional Chinese medicine extracts.
Optionally, the vitamins are at least one selected from vitamin B12 and vitamin E, the microelements are selected from zinc gluconate, and the Chinese medicinal extract is at least one selected from icariin extract, fructus Schisandrae extract and Corni fructus extract.
Optionally, the composition further comprises adjuvants selected from one or more of bulking agent, sweetener, and correctant.
The application provides application of the ADPP polypeptide or the composition in preparing foods or medicines for producing sperms, activating sperms or strengthening yang.
The application has at least the following beneficial effects:
the application has the surprising discovery in research that the polypeptide has the functions of producing sperm, activating sperm or strengthening yang. The ADPP is used as a basic raw material source to develop the 'aquatic Vigor' health care product with the functions of improving sexual desire and sexual function and improving sperm quantity, and an auxiliary reproduction service market is developed, so that the problem of ultralow fertility rate caused by advanced age and environmental pollution in China at present can be effectively solved from another dimension.
Drawings
FIG. 1 shows the result of SDS-polyacrylamide gel electrophoresis of ADPP polypeptides according to the application;
FIG. 2 is a graph showing the general morphology of testis and the results of tissue sections of testis in an example of the present application;
FIG. 3 is a graph showing the results of semen analysis in an embodiment of the present application.
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application.
Detailed Description
The embodiment of the application provides an ADPP polypeptide (ADPP, andrias davidianus protein and peptide production, giant salamander polypeptide), which is obtained by hydrolyzing giant salamander muscle by hydrolase to obtain a hydrolysate and then separating; the hydrolase is selected from temperature-resistant alkaline protease and bromelain; the alkaline protease is temperature-resistant alkaline protease produced by Bacillus pumilus (Bacillus Subtilis) or Bacillus licheniformis temperature-resistant alkaline protease. Namely, the two bacteria are gradually heated and domesticated from the optimal temperature of 37 ℃ to about 65 ℃ to obtain the bacillus pumilus or the bacillus licheniformis which are domesticated at high temperature, and the working temperature of alkaline protease secreted by the bacteria after being domesticated at high temperature is 60-65 ℃.
A large amount of large molecular proteins with complex structures exist in giant salamander muscles (see SDS-polyacrylamide gel electrophoresis), and the giant salamander muscles are required to be degraded into small molecular proteins, polypeptides and amino acids for better absorption and utilization. A large number of screening experiments show that the temperature-resistant alkaline protease is adopted for hydrolysis, and then bromelain is adopted, so that giant salamander muscle can be decomposed into ADPP polypeptides with specific molecular weight, and the effects of producing sperm, activating sperm and strengthening yang are achieved.
In order to further improve the bioactivity, yield and purity of the ADPP polypeptide, the embodiment of the application discovers that the temperature-resistant alkaline protease produced by bacillus pumilus (Bacillus Subtilis) or bacillus licheniformis after heat induction domestication is particularly suitable for decomposing giant salamander muscles after a large number of strains for secreting alkaline protease are screened, and the temperature is increased during enzymolysis, so that the growth of mixed bacteria can be inhibited, and the yield and purity of the polypeptide are improved.
The heat induction domestication temperature-resistant alkaline protease method specifically comprises the following steps: according to the main source of alkaline protease is microbial extraction, bacillus pumilus or bacillus licheniformis strain producing alkaline protease is transferred into conventional culture solution such as NA culture medium and beef extract peptone solution to be fermented and cultured at 37 ℃, after the strain grows up, the temperature is slowly raised to perform fermentation and culture, other fermentation and culture conditions are not changed, and the temperature is gradually raised to about 65 ℃, so that the temperature-resistant alkaline protease of fermentation and culture is separated and purified. The domestication method can remarkably improve the activity, stability (temperature resistance and alkali resistance), oxidation resistance and chelation resistance of the enzyme.
In a second step, the present example further hydrolyzes alkaline protease hydrolysates using bromelain. The embodiment of the application utilizes the characteristics of bromelain that bromelain preferentially hydrolyzes peptide chains on the carboxyl side of basic amino acid (such as arginine) or aromatic amino acid (such as phenylalanine and tyrosine) and alcohol soluble protein is antiallergic, and has the supplementing effect on domesticating and inducing the alkaline protease to degrade giant salamander muscle protein. The biological activity of the enzymolysis product can be further improved by the combined application of the two.
As a preferred implementation of the embodiment of the application, giant salamander muscle: alkaline protease: the mass ratio of bromelain is 1:0.05 to 0.1:0.025 to 0.05. If the amount of alkaline protease and bromelain is reduced, the enzyme degradation effect of macromolecular protein in muscle and the yield of polypeptide are affected, and the experimental purpose is not achieved. If the dosage of alkaline protease and bromelain is too large, giant salamander protease is digested into a large amount of amino acids and a small amount of small molecular polypeptides, and the yield of the polypeptides and the composition ratio of the polypeptides are affected. The ratio of peptide fragments in the enzymatically hydrolyzed polypeptide can also be affected if the ratio between alkaline protease and bromelain is altered.
As a preferred embodiment of the present application, the preparation method of the ADPP polypeptide specifically comprises at least the following steps:
s1, taking muscles of artificially cultured giant salamanders, adding water with the mass of 5-10 times, mincing to prepare minced meat, rough filtering to remove fascia and homogenizing to obtain a homogenized liquid;
s2, adding temperature-resistant alkaline protease into the homogenized solution, regulating the pH value to 7.5-8.0, carrying out enzymolysis for 20-40 minutes at the temperature of 55-65 ℃, and adding bromelain for enzymolysis for 0.5-1.5 h;
s3, centrifuging or filtering to remove cell fragments, ultrafiltering with an ultrafiltration membrane with a molecular weight cutoff of 10KD, and collecting filtrate to obtain giant salamander hydrolysate.
Specifically, in S1, the meat emulsion is crushed by a tissue crusher or refiner and homogenized by a homogenizer. When macromolecular proteins and polypeptides in giant salamander meat are subjected to enzymolysis, the macromolecular proteins and polypeptides are released from tissue cells firstly, and in the treatment process of the giant salamander muscle tissue, the released proteins are kept in the original natural state and the activity is not lost. The method for breaking the tissue cells is a plurality of methods including a mechanical method, a physical method, a chemical method, a biochemical method and the like; the chemical method and biochemical method are methods of disrupting cells using chemical reagents such as acids, bases, surfactants and organic solvents or lysing cells using enzymes, which are liable to cause protein denaturation or affect subsequent experiments, and are not suitable for the present application. The physical method mainly comprises a method for breaking tissue cells by various physical factors, such as repeated freeze-thawing method, flash-heat quenching method, ultrasonic treatment and the like, and is not suitable for breaking giant salamander muscle tissue cells due to difficult industrial production or protein denaturation. The method for breaking tissue cells by mechanical shear force mainly comprises a tissue masher, a homogenizer, a mortar, a grinding and squeezing device and the like, and the method is characterized in that the giant salamander muscle is treated by the tissue masher or the pulping device after the screening experiment.
In a further preferred embodiment, the cells are disrupted using a high pressure homogenizer (a biospecific high pressure cell disrupter); the working principle is as follows: the device comprises one or a plurality of reciprocating plunger pumps for pressurizing a cell chamber, materials enter a valve group with adjustable pressure under the action of the plungers, the pressure can reach 1800bar/27000psi, a sample is extruded into a narrow gap, when the materials pass through a limited gap (working area) with a specific width, the materials are extruded by a very high pressure (such as 1800 bar) in the gap, when the samples pass through the gap and bear a very low pressure (generally 1 bar), the materials which are instantaneously decompressed can generate a very high blasting force, can be ejected at a very high flow rate (1000-1500 m/s) and collide on an impact ring of one of the collision valve assemblies, and can generate a very strong impact force; the sample also generates certain shearing force among sample particles in the high-speed spraying process; by the blasting force, impact force and shear force produce three effects: cavitation effect, impact effect, shear effect. After the three effects are treated, the particle size of the material can be uniformly thinned to below 100nm, so that the effects of breaking bacteria or homogenizing and crushing liquid samples are very good. The crushing rate is more than 95 percent. Can be equipped with an in-situ cooler and an external cooler, and can be optionally equipped with a cooling coil at a temperature lower than 20 ℃ to ensure higher bioactivity.
Specifically, in S2, the enzymolysis temperature of the temperature-resistant alkaline protease is preferably 60-65 ℃, and under the temperature condition, other miscellaneous bacteria such as common escherichia coli and the like cannot be propagated, so that the pollution of the miscellaneous bacteria during enzymolysis is prevented or reduced, the purity of the polypeptide product can be improved, and the activity and the safety of the polypeptide product are ensured.
In a further preferred embodiment, the enzymatic hydrolysis time is 30 minutes; if the time is too short, the enzymolysis is insufficient, and if the time is too long, the enzyme can digest more polypeptide, which affects the enzymolysis of the next enzyme, and the composition of the polypeptide in the final product can be affected.
In a further preferred embodiment, the enzymolysis temperature of bromelain is 60 ℃, and the enzymolysis time is more preferably 1 hour; if the time is too short, the enzymolysis is insufficient, and if the time is too long, the proportion of peptide fragments of bromelain enzymolysis increases, which may affect the peptide yield and composition. Specifically, in S3, ultrafiltration is carried out by using an ultrafiltration membrane with the molecular weight cutoff of 10KD, and giant salamander proteins with the molecular weight of more than 10KD and added enzymes (the molecular weight of the enzymes is more than 10 KD) which are not subjected to enzymolysis digestion are removed, so that the quality of ADPP polypeptides is ensured; and collecting the filtrate.
In a further preferred embodiment, the method of preparation further comprises the step of drying the giant salamander hydrolysate; preferably, the low-temperature spray drying method is selected for drying, because the small molecular proteins and polypeptides are sensitive to temperature, when common vacuum drying and spray drying are used, the biological activity or structure of the small molecular proteins and polypeptides can be greatly damaged, the freeze drying time is long, the energy efficiency is low, and the dried materials are in a block shape and need to be crushed for the second time. The low-temperature spray dryer creatively combines the advantages of vacuum drying and spray drying, can rapidly dry materials (for a few seconds) under the low-temperature condition, and avoids denaturation and inactivation of small molecular proteins and polypeptides. Working principle of low-temperature spray dryer: air passes through the filter and the heater, enters the air distributor at the top of the dryer, and hot air uniformly enters the dryer in a spiral shape. The feed liquid is pumped to a centrifugal atomizer at the top of the dryer from a feed liquid tank through a filter, so that the feed liquid is sprayed into tiny mist droplets, the atomization is very uniform, the feed liquid is contacted with hot air in parallel flow, the moisture is rapidly vaporized, the moisture is instantaneously vaporized (for a few seconds), and the finished product is dried in a very short time. The dried solid particles are separated from the waste gas by a cyclone separator and then enter a collecting bottle. The exhaust gas is then directly discharged to the atmosphere or to an air filtration device. The low-temperature spray dryer is specially developed for heat-sensitive materials, and under the drying condition, substances which are easy to oxidize, volatilize and heat-sensitive can well maintain chemical structure and biological activity. The prepared ADPP polypeptide is dried by a low-temperature spray dryer, the inlet temperature is set at 100-120 ℃, and the outlet temperature is set at 50-70 ℃.
The embodiment of the application also relates to a composition containing the ADPP polypeptide, and the composition also contains at least one of vitamins, trace elements and traditional Chinese medicine extracts. The vitamin is at least one selected from vitamin B12 and vitamin E; the microelements are selected from zinc gluconate, and the Chinese medicinal extract is selected from at least one of icariin extract, fructus Schisandrae extract, and Corni fructus extract. The composition also contains adjuvants selected from one or more of bulking agent, sweetener and correctant.
The embodiment of the application also relates to application of the ADPP polypeptide and the composition in preparing foods or medicines for producing sperms, activating sperms or strengthening yang. The ADPP polypeptide has the functions of producing sperm, activating sperm or strengthening yang. The ADPP is used as a basic raw material source to develop the 'aquatic Vigor' health care product with the functions of improving sexual desire and sexual function and improving sperm quantity, and an auxiliary reproduction service market is developed, so that the problem of ultralow fertility rate caused by advanced age and environmental pollution in China at present can be effectively solved from another dimension.
The technical scheme of the embodiment of the application is further described by the following specific implementation manner:
instrument, reagent source:
high pressure homogenizer, AH-1500, ATS Industrial systems Co., ltd; autoclaving pot, tomy sx-700, tomy in Japan; culturing shaking table at constant temperature, TH2-100, shanghai-Heng science instruments Co., ltd; clean bench, SW-CJ-2FD, available from air technologies, inc. of Antai, suzhou; electrophoresis apparatus, DYY-6C, six instrument factories in Beijing; protein purification apparatus, AKTA prime Plus, GE healthcare group, usa; constant temperature incubator, 9H-360, beijing Zhongxing Wei industry instruments Co., ltd; 5L stainless steel microorganism fermenter, biostatcplus, sidoris Germany; high-speed organization masher, DS-1, on the sea sample model factory; ultrapure water instrument, direct-Pure Plu, acer Truncatum Bunge (Rephile) Biotechnology Co., ltd; a small Spray dryer, H-Spray Mini, beijing Hall biotechnology Co., ltd; high capacity refrigerated centrifuge, DL-6M, shanghai Lu Xiangyi centrifuge instruments limited; high-speed refrigerated centrifuge, DL-21M, shanghai Lu Xiangyi centrifuge instruments limited; ultrafilters, ZJMP-12-053 and OM-141 types, MILLIPORE, USA; microscopic imaging system, carl Zeiss, germany; full-automatic sperm analyzer, shanghai san Jose Western medical Co., ltd; the enzyme-labeled instrument, tecan, australia; inverted fluorescence microscope, OLYMPUS, japan; a low temperature high speed centrifuge, beckman, united states; a chemiluminescent imaging system, clinx; manual rotary microtomes, frozen microtomes, leica, germany.
BPA, purity >99.9%, sigma, usa; corn oil, sigma, usa; DMSO, sigma, usa; hematoxylin-eosin staining, beyotime; TUNEL staining kit, roche Applied Science, switzerland; testosterone ELISA kit, cloud-Clone; sodium citrate antigen retrieval solution, solabio; human anti-rabbit polyclonal antibody GAPDH, abclone; human anti-rabbit polyclonal antibody MTA2, abcam, USA; sheep anti-rabbit immunofluorescence secondary antibody kit, beyotime; anti-fluorescence quenching contains DAPI envelope tablet, dalian Meren; sheep anti-rabbit immunohistochemical secondary antibody, st John's Laboratory, USA; oil red O staining kit, league; mouse testis TM4 cell line; DMEM/F12 medium, gibco, USA; penicillin and streptomycin, mi mouse, china; fetal bovine serum, gibco, usa; other chemical reagents are domestic conventional reagents.
Example 1
ADPP polypeptide is prepared from giant salamander meat, giant salamander muscle is firstly prepared, artificially cultured giant salamander is slaughtered, muscle is taken, water is washed, 5-10 times of water is added for mincing, meat paste is prepared by a tissue masher or a paste grinder, fascia is roughly filtered, and then cells are crushed by a high-pressure homogenizer (a biological special high-pressure cell crusher) to release muscle protein and polypeptide. Repeating for 1-2 times if necessary to obtain homogeneous solution.
Adding high-temperature domesticated temperature-resistant alkaline protease enzymolysis protein into a homogeneous solution containing giant salamander muscle protein and polypeptide, wherein the mass ratio is as follows: giant salamander meat: alkaline protease = 1:0.05; regulating the pH to 7.5-8.0 (7.8), uniformly mixing, carrying out enzymolysis for 0.5h at the temperature of 60 ℃, and adding bromelain for continuous enzymolysis for 1h, wherein the mass ratio is as follows: giant salamander meat: bromelain=1: 0.025.
centrifuging (12000 rpm/min) or filtering to remove cell debris, ultrafiltering with ultrafiltration membrane with molecular weight cut-off of 10KD, and removing giant salamander protein and enzyme with molecular weight of more than 10KD without enzymolysis digestion to ensure the quality of ADPP polypeptide; and collecting the filtrate.
Spray drying the filtrate with low temperature spray drier at inlet temperature of 100-120 deg.c and outlet temperature of 50-70 deg.c. Light yellow peptide powder was collected.
SDS-electrophoresis results obtained with 12.5% of the ADPP polypeptide are shown in FIG. 1. Wherein:
note that: m: protein molecular weight standard;
a, giant salamander muscle protein sample;
b, carrying out enzymolysis on the giant salamander muscle protein sample by using alkaline protease;
c, carrying out double enzymolysis on a sample with incomplete protein of giant salamander muscle by using alkaline protease and bromelain, wherein the giant salamander muscle is prepared by the steps of: alkaline protease: the mass ratio of bromelain is 100:0.04:0.02;
and d, carrying out double enzymolysis on the giant salamander muscle protein sample by using alkaline protease and bromelain under the condition of the embodiment.
As can be seen from FIG. 1, the ADPP polypeptides obtained by the enzymolysis under the conditions of the present example have smaller molecular weight and more complete enzymolysis.
Experimental example 2
Modeling basis and verification purpose: bisphenol A (BPA) is widely used in the production of polycarbonate and epoxy resin, and has weak estrogen and antiandrogen effects due to its structure similar to estrogen, thus being an important environmental endocrine disrupter. More and more studies have shown that BPA can cause different forms and degrees of damage to the reproductive system. And (3) preparing a mouse sterile model by using BPA, verifying the protection effect of the SP on the reproductive disorder caused by BPA contamination, and reflecting the sperm generating, activating and strengthening yang of the SP.
The method comprises the following steps: 40C 57BL/6 male mice were divided into four groups, namely a control group, a BPA group, a BPA+ physiological saline (NACL) group and a BPA+ ADPP group. The BPA group was used to treat animals with 50mg/kg/d BPA, which was dissolved in corn oil and administered by intraperitoneal injection; the control group was only intraperitoneally injected with corn oil. The BPA+ADPP group was subjected to gastric lavage with the ADPP polypeptide of example 1 in an amount of 4g/kg/d in addition to the BPA treatment of 50mg/kg/d, and the polypeptide of example 1 was dissolved in physiological saline; the bpa+nacl group saline lavage. And (3) continuously molding for 28 days, measuring the weight of the mice weekly, measuring the volume of testis and the body-to-testicle ratio after molding, taking epididymal sperms, measuring the number of sperms and the sperm motility by using a CASA computer aided analysis system, and analyzing the histopathology of testis by HE staining. The results are shown in FIG. 2.
From figure 2 (a) analysis of testicle gross morphology, BPA treatment significantly reduced testicle volume, while supplementation with ADPP effectively reversed the gross morphology changes in testicle due to BPA, suggesting that ADPP has efficacy in combating BPA reproductive toxicity, promoting spermatogenesis.
As can be seen from fig. 2 (b), the tissue sections were stained with hematoxylin-eosin, and pathological examination showed that the seminiferous tubule lumen of the mouse testis was substantially disappeared after 28 days of BPA treatment, a large number of seminiferous cells were apoptotic, and the seminiferous epithelium was severely deformed; and the ADPP feeding group can be used for basically recovering the seminiferous tubule structure, so that the apoptosis of seminiferous cells is greatly reduced, and the lumen reappears in part of seminiferous tubules to prompt the effective recovery of spermatogenesis.
The results of Computer Aided Semen Analysis (CASA) are shown in figure 3.
As can be seen from fig. 3, the ADPP-fed group effectively increased sperm density, forward motility (PR), non-forward motility (NP), and non-motile (IM) sperm count due to BPA, i.e., SP significantly enhanced sperm density and sperm motility.
The result shows that the ADPP obtained by the embodiment of the application can effectively improve the spermatogenesis of the mice damaged by BPA treatment, effectively recover and obviously enhance the sperm density and sperm motility, has obvious protection effect of resisting BPA spermatogenesis damage, and is presumed to be because the hydrolysis method of the application obtains some polypeptides with stronger functions for sperm density and sperm motility. The composition and proportion of peptide fragments in the polypeptide are changed without adopting the product obtained by enzymolysis of the application, so that the efficacy of enhancing sperm density and sperm motility is changed or reduced.
EXAMPLE 3ADPP polypeptide compositions
The main raw materials are mixed according to the following parts by weight:
other auxiliary materials such as common filler, compound sweetener, flavoring powder, etc.
While the application has been described in terms of the preferred embodiment, it is not intended to limit the scope of the claims, and any person skilled in the art can make many variations and modifications without departing from the spirit of the application, so that the scope of the application shall be defined by the claims.
Claims (10)
1. An ADPP polypeptide, wherein the ADPP polypeptide is obtained by hydrolyzing giant salamander muscle with hydrolase to obtain a hydrolysate, and then separating;
the hydrolase is selected from the group consisting of thermostable alkaline protease and bromelain;
the alkaline protease is temperature-resistant alkaline protease produced by bacillus pumilus or bacillus licheniformis temperature-resistant alkaline protease; preferably, the working temperature of the alkaline protease is 60-65 ℃.
2. The ADPP polypeptide of claim 1, wherein the giant salamander muscle: alkaline protease: the mass ratio of bromelain is 100:0.05 to 0.1:0.025 to 0.05.
3. ADPP polypeptide according to claim 1 or 2, characterized in that the method for producing the ADPP polypeptide comprises at least the following steps:
s1, taking muscles of artificially cultured giant salamanders, adding water with the mass of 5-10 times, mincing to prepare minced meat, and performing rough filtration and homogenization to obtain a homogenized liquid;
s2, adding the temperature-resistant alkaline protease into the homogeneous solution, adjusting the pH value to 7.5-8.0, carrying out enzymolysis for 20-40 minutes at the temperature of 55-65 ℃, and adding bromelain for enzymolysis for 0.5-1.5 h;
s3, centrifuging or filtering to remove undigested cell fragments and residues, ultrafiltering with an ultrafiltration membrane with a molecular weight cutoff of 10KD, and collecting filtrate to obtain the ADPP polypeptide.
4. An ADPP polypeptide according to claim 3, wherein in S1, the meat emulsion is crushed using a tissue rammer or refiner and homogenized using a homogenizer.
5. An ADPP polypeptide according to claim 3, wherein the temperature resistant alkaline protease is enzymatically hydrolyzed at 60-65 ℃ for 30 minutes;
the enzymolysis temperature of the bromelain is 55-65 ℃, preferably 60 ℃.
6. The ADPP polypeptide of claim 3, wherein the method of preparing further comprises the step of drying the giant salamander hydrolysate;
preferably, the drying is carried out by adopting a low-temperature spray drying method, the inlet temperature is 100-120 ℃, and the outlet temperature is 50-70 ℃.
7. A composition comprising an ADPP polypeptide as set forth in any one of claims 1 to 6, wherein the composition further comprises at least one of vitamins, trace elements, and a herbal extract.
8. The composition according to claim 7, wherein the vitamin is at least one selected from vitamin B12, vitamin E; the microelements are selected from zinc gluconate, and the traditional Chinese medicine extract is selected from at least one of icariin extract, fructus Schisandrae extract and Corni fructus extract.
9. The composition according to claim 8, further comprising an auxiliary material selected from one or more of a filler, a sweetener, and a flavoring agent.
10. Use of an ADPP polypeptide according to any one of claims 1 to 6, or a composition according to any one of claims 7 to 9, for the preparation of a seminiferous, seminiferous or yang-invigorating food or pharmaceutical product.
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