CN101878849B - Polypeptide powder of crocodile and preparation method and application thereof - Google Patents
Polypeptide powder of crocodile and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a polypeptide powder of crocodile and a preparation method and application thereof, which relate to a crocodile product. The method comprises the following steps of: cleaning the crocodile meat, removing adipose tissues, cutting the crocodile meat into pieces; adding water in the crocodile meat pieces, boiling the crocodile meat pieces in a boiled water bath, homogenating to obtain homogenated liquid of crocodile meat; adding trypsin in the obtained homogenated liquid, carrying out water bath and enzymolysis to obtain an enzymolytic mixed liquid A; placing the enzymolytic mixed liquid A in the boiled water bath, inactivating enzyme to obtain an enzymolytic mixed liquid B; cooling the obtained enzymolytic mixed liquid B to room temperature and filtering to obtain the crocodile meat enzymolytic liquid; and drying the crocodile meat enzymolytic liquid to obtain the polypeptide powder of crocodile. The polypeptide powder of crocodile has a certain function of resisting fatigue and improving the exercise ability of the body and can be applied to the preparation of medicines for resisting antifatigue and improving body exercise ability.
Description
Technical field
The present invention relates to a kind of crocodile product, especially relate to a kind of polypeptide powder of crocodile and preparation method thereof and application.
Background technology
Albumen is as the nutrient of needed by human, and is very important to the life and health of human body.Epidemiology survey is the result show, if every day, the albumen intake was excessively low, just suffers from easily the cancer of the esophagus, cancer of the stomach, liver cancer etc.The expert of the World Health Organization and FAO (Food and Agriculture Organization of the United Nation) thinks the standard diet of a John Q. Public normal activity, and every day, the albumen intake can not be lower than 75g.Protein is divided into animal protein, vegetable protein, single cell protein and algae protein according to the source.These high molecular weight proteins replenish the albumen that needed by human body is wanted to a certain extent, but have the shortcomings such as nondigestible, absorption difference, non-acid resistance, and also there is certain anaphylaxis that causes in some albumen.
A large amount of scientific researches show, by selecting suitable protease, control certain hydrolysis degree, be hydrolyzed some protein and can obtain the large number of biological active peptides, these biologically active polypeptides not only have the advantages such as active height, absorption easy to digest and security height, and have a special physiological regulation function, such as reducing blood fat, delay senility, body beautification beauty treatment, anti-oxidant, anti-melancholy, antifatigue, improvement sleep, strengthening memory, suppress tumour etc.; Can also promote human body to the absorption of protein, vitamin and various healthy trace elements with household.Scientific research is found, active peptides in the body be link up between cell and the cell, the important chemical messenger between organ and the organ, by modes of action such as endocrine, paracrine, neuroendocrine and neurosecretions, transmit various special information, thus but the life processes such as growth regulation, growth, breeding, metabolism and behavior.
The development of biologically active polypeptide research field is very fast, be subjected to the great attention of various countries scientist and government, and along with modern protein engineering zymotechnic develops rapidly, enzymatic isolation method is produced biologically active polypeptide and has been replaced initial direct extraction method and synthetic method etc., becomes the focus of current polypeptide research field.By food proteins being carried out the difficult or processing of enzymolysis to obtain biologically active polypeptide, have the advantage of abundant raw material, with low cost, the uniqueness such as security good, be convenient to industrial production, application is good, be subject to the high praise of various countries' science.Active peptides is used and is summed up to get up to mainly contain medical and edible two broad aspect.Active peptides is as medicine, and its active function point concentrates on lacks on the polypeptide chain, not only can improve medicine virtual value and biologically active, and can reduce the bad reaction of non-targeted activity composition, improves its selectivity.The active peptides Applications in food mainly concentrates on the functional food.The functional polypeptide health food is at the early-stage in China, and added value is high, has the huge market space, higher economic and social benefit.
Crocodile belongs to the vertebrate Reptilia, and extensive growth is in South Asian nations such as Vietnam, Thailand, Malaysia, and as the existing unique offspring of archosaur, crocodile has the characteristics such as the bodily form is large, reproductive capacity strong, growth is fast, premunition is strong.The crocodile whole body is precious, and crocodile flesh of fish, bone, first and internal organ contain amino acid, unrighted acid, vitamin and the various trace element of abundant good protein and needed by human.
Modern medicine study finds that crocodile meat is not only delicious, and can fill blood, strengthen the bone, control asthma, and chronic cough, diabete (diabetes) are also had significant curative effect.
Publication number is for the application for a patent for invention of CN1465351 provides a kind of anticancer new product Crocodile immunization anti-cancer pill, be leather armour, meat bone, bile, blood etc. with the crocodile of artificial feeding behind processing and refining as main component.This product to the part leukaemia and existing shift in, patient with advanced cancer, receive better effects.Take this product without any toxicity or side effect.
Summary of the invention
The purpose of this invention is to provide a kind of polypeptide powder of crocodile and preparation method thereof and application.
The preparation method of polypeptide powder of crocodile of the present invention may further comprise the steps:
1) with behind the crocodile meat cleaning removal adipose tissue, is cut into fragment;
2) add entry in the crocodile fragment, after putting boiling water bath and boiling, homogenate gets crocodile meat homogenate;
3) in step 2) add trypsase in the crocodile meat homogenate of gained, water-bath, behind the enzymolysis the enzymolysis mixed liquor A;
4) with step 3) the enzymolysis mixed liquor A of gained places boiling water bath, makes enzyme deactivation, gets the enzymolysis mixed liquid B;
5) with step 4) the enzymolysis mixed liquid B of gained, be cooled to room temperature after, filter, get crocodile meat enzymolysis liquid;
6) with step 5) the crocodile meat enzymolysis liquid of gained is dry, and get product and be polypeptide powder of crocodile.
In step 1) in, the size of described fragment can be 1～2cm
3
In step 2) in, the described amount that adds entry, by volume, the crocodile fragment: water can be 1: (1～2.5); The time that described boiling water bath boils can be 20～40min.
In step 3) in, described tryptic addition by mass percentage, is 0.3%～0.5% of crocodile meat, and the temperature of described water-bath can be 40～45 ℃, and the time of described enzymolysis can be 4～6h.
In step 5) in, described filtration can be adopted 6～8 layers of filtered through gauze.
In step 6) in, described drying can adopt spray-drying.
Products obtained therefrom can be packed with capsule.
The prepared polypeptide powder of crocodile of the present invention contains protein, polypeptide, amino acid, sugar, fat, moisture, ash content and metallic element etc., is grouped into and can be found out by the one-tenth of polypeptide powder of crocodile, and polypeptide powder of crocodile belongs to low sugar, low fat, high-protein food.
Antioxidation in vitro measuring result through polypeptide powder of crocodile shows that polypeptide powder of crocodile has preferably scavenging action to various free radicals, and when its concentration was 6.0mg/ml, its clearance rate to DPPH can reach 44.2%; When its concentration is 3.43mg/ml, to the hydroxyl radical free radical clearance rate up to 95.5%; When its concentration is 3.5mg/ml, can reach 56.96% to the clearance rate of ultra-oxygen anion free radical.The feeding polypeptide powder of crocodile is 20 days continuously, the vertical rope climbing experiment of mouse and ice-water bath swimming with a load attached to the body experimental result show, rope climbing and swimming power that polypeptide powder of crocodile can prolong mouse preferably exhaust the time, and vertical rope climbing and the swimming with a load attached to the body power time of exhausting are the common counters of reflection animal movement degree of fatigue.This result shows that further polypeptide powder of crocodile has certain antifatigue, improves the locomitivity effect of body, can and improve in the body movement ability medicine and use in the preparation antifatigue.
Description of drawings
Fig. 1 is MALDI-TOF Mass Spectrometer Method polypeptide powder of crocodile range of molecular weight distributions.In Fig. 1, abscissa is the nucleocytoplasmic ratio m/z of each polypeptide ion in the polypeptide powder of crocodile, equals the relative molecular weight of this peptide section on the numerical value, and ordinate is polypeptide ion strength of flow (a.i) in the polypeptide powder of crocodile; Spectrum peak from left to right is respectively 684.30,1041.61, and 1107.50,1184.00,1238.59,1286.69.
Fig. 2 polypeptide powder of crocodile is to the scavenging action of DPPH.In Fig. 2, abscissa is the concentration C oncentration (mg/mL) of polypeptide powder of crocodile, and ordinate is that polypeptide powder of crocodile is to the removing percentage Clearup Rate (%) of DPPH.
Fig. 3 polypeptide powder of crocodile is to the scavenging action of hydroxy radical.In Fig. 3, abscissa is the concentration C oncentration (mg/mL) of polypeptide powder of crocodile, and ordinate is the removing percentage Clearup Rate (%) of the hydroxyl radical free radical of Fenton reaction generation, not add polypeptide powder of crocodile as the experiment contrast group.
Fig. 4 polypeptide powder of crocodile is to the scavenging action of ultra-oxygen anion free radical.In Fig. 4, abscissa is the concentration C oncentration (mg/mL) of polypeptide powder of crocodile, ordinate is the removing percentage Clearup Rate (%) of the ultra-oxygen anion free radical of pyrogallol autoxidation generation, not add polypeptide powder of crocodile as the experiment contrast group.
The specific embodiment
The present invention is further illustrated in connection with accompanying drawing for following examples.
The preparation of embodiment 1 polypeptide powder of crocodile
1) take by weighing 2～5kg crocodile meat, after adipose tissue was removed in cleaning, being cut into size was 1～2cm
3Fragment.
2) by volume, add 2: 5 distilled water, after putting boiling water bath and boiling 30min, homogenate in tissue mashing machine gets crocodile meat homogenate.
3) to step 2) in add the trypsase that accounts for crocodile meat quality 0.4% in the crocodile meat homogenate of gained, fully behind the mixing, in 40～45 ℃ of water-baths, enzymolysis 4～6h stirs once every 20min therebetween.
4) with step 3) in the enzymolysis mixed liquor of gained place boiling water bath 10～20min, make enzyme deactivation.
5) with step 4) in gained and enzymolysis mixed liquor, leave standstill naturally cool to room temperature after, with 6～8 layers of filtered through gauze, get crocodile meat enzymolysis liquid.
6) with step 5) middle gained enzymolysis liquid, carry out spray-drying, instrument is thrown condition setting and is: pressure 0.08～0.12MPa; Cold air blast velocity 0.6～1.0m
3/ min; 120～130 ℃ of inlet temperatures; 75～90 ℃ of outlet temperatures; Sample introduction speed 0.8～1.2L/h.Product is polypeptide powder of crocodile, is stored in the vial with cover.
7) pack with every 300mg with No. 2 capsules, namely get the polypeptide powder of crocodile finished product.
The composition measurement of embodiment 2 polypeptide powder of crocodile
Take by weighing a certain amount of polypeptide powder of crocodile, be mixed with the 200mg/mL mother liquor in the distilled water redissolution, adopt the Coomassie brilliant blue method to measure the protein concentration of mother liquor, take bovine serum albumin(BSA) as standard items; Adopt Folin-phenol method to measure the mother liquor content of peptides, take bovine serum albumin(BSA) as standard items; Adopt ninhydrin colorimetry to measure the concentration of mother liquor free amino acid, take glycine as standard items; Content adopts the total sugar concentration of the mother liquor of sulfuric acid-phynol method mensuration, with glucose as a standard product; Calculate albumen, polypeptide, free amino acid, total sugar content percentage.
The fat content percentage that adopts soxhlet extraction to measure.Adopt atmosphere pressure desiccation to measure moisture percentage.
Adopt the method for high temperature sintering to measure content of ashes percentage.Concrete steps are referring to " Food Inspection and analysis " (China Light Industry Press publishes, 1997).
The metal element content of embodiment 3 polypeptide powder of crocodile is measured
Take by weighing the 0.2g polypeptide powder of crocodile in the micro-wave digestion bottle, add the red fuming nitric acid (RFNA) of 5mL, the dense perchloric acid of 1mL places the micro-wave digestion difficulty to clear up, and clears up to be settled to 100mL with distilled water afterwards, adopts atomic absorption method, measures its metal element content.
The amino acid composition analysis of embodiment 4 polypeptide powder of crocodile
Take by weighing a certain amount of polypeptide powder of crocodile, after the little molecule of inorganic salts is removed in dialysis to redistilled water afterwards with the redistilled water dissolving, getting product solution adding final concentration is in the 6mol/L HCl solution, under 110 ℃ in sealed tube acid hydrolysis 24h, hydrolyzate pH is transferred to neutrality, and be diluted to suitable concn, measure various amino acid contents at Hitachi 835-50 automatic amino acid analyzer.
The acute toxicological experiment of embodiment 5 polypeptide powder of crocodile
By the acute toxicity test of GB GB1519.3-2003 food, take mouse as animal model.Choose a cleaning level KM mouse (provide credit number by anticancer research center laboratory animal room of Xiamen University: SCXK (Fujian) 2004-0001), 18～22g, male, each 10, mark, number and weigh.Take by weighing the 3g polypeptide powder of crocodile and be dissolved in the 4mL distilled water and shake up, the inspection product concentration that obtains is the 0.75g/mL polypeptide powder of crocodile.Mouse stomach 0.4mL/ is only after (that is: dosage is 15g/kg), Continuous Observation 7 days, and diet, drinking-water, the defecation of record mouse, urinate, the even in everyday situations such as action, carried out anatomic tissue and observed the organ lesion situation after weighing in the 7th day.
Embodiment 6 external removing free radical abilities
Take by weighing a certain amount of polypeptide powder of crocodile, be mixed with the 200mg/ml mother liquor in the distilled water redissolution.Carry out following experiment:
(1) free DPPH scavenging action is measured
1.9mL add the effector molecules (polypeptide powder of crocodile solution) of 1mL variable concentrations in the absolute ethyl alcohol, add 0.1mL 3mmol/L1, the bitter phenylhydrazine free radical (DPPH) of 1-diphenyl-2-, mixing immediately, room temperature reaction 30min surveys OD
517
Clearance rate is calculated as follows:
In the following formula, A
0Absorbance (control group) when not adding effector molecules; A
1Be the absorbance (application of sample group) behind the adding effector molecules.
(2) scavenging action of hydroxy radical is measured
Produce hydroxyl radical free radical with the Fenton reaction.0.5mL 9mmol/L FeSO
4In 1mL 9mmol/L salicylic acid-ethanolic solution, add 1mL variable concentrations effector molecules (polypeptide powder of crocodile solution), add at last 1mL 8.8mmol/LH
2O
2, mixing starts reaction immediately, room temperature reaction 1h, the light absorption value of survey 510nm.
The base clearance rate is calculated by following formula:
In the following formula, A
0Absorbance (control group) when not adding effector molecules; A
1Be the absorbance (application of sample group) behind the adding effector molecules.
(3) scavenging action of ultra-oxygen anion free radical is measured
Get the Tris hydrochloride buffer of 2.5mL pH8.4 concentration 20mmol/L, 1.0mL in 25 ℃ water-bath, be incubated 20min behind the distilled water mixing, be added in immediately the 0.5mmol/L pyrogallol 0.3mL of 25 ℃ of preheatings after the taking-up, pyrogallol autoxidation speed in the 1min was V after measured reaction started under 325nm
0It is the same that experimental group to be measured is got reagent, just adds respectively the crocodile active peptides mother liquor of a certain amount of variable concentrations before adding pyrogallol, and distilled water reduces respective volume, and sample suppresses the autoxidizable oxidation rate of pyrogallol in the rear 1min of measured reaction startup, is V
1
The base clearance rate is calculated by following formula:
The experiment of embodiment 7 mouse anti-reflecting fatigues
Select cleaning level KM mouse (to be provided by anticancer research center laboratory animal room of Xiamen University, credit number: SCXK (Fujian) 2004-0001), 18～22g, male, 40, being divided at random 4 groups is the high, medium and low dosage group of blank group and polypeptide powder of crocodile, 10 every group, mark, numbers and weighs.Successive administration 20 days.
The blank group: mouse stomach 0.9%NaCl 0.5mL/, once a day; High, medium and low each the dosage group concentration of polypeptide powder of crocodile is respectively: 0.01g/mL, 0.02g/mL, 0.06g/mL, the mouse stomach amount is 0.5mL/, once a day.Behind the last administration 12h, by only being placed on the cord of vertical hanging, make its muscle in tension or climb mouse, the record mouse since muscular fatigue from time that cord drops down.The 3rd stops experiment when falling, and the average time that accumulative total is 3 times is as the rope climbing time.Behind the last administration 24h, fasten the heavy counterweight of 5g at little tail at all, mouse is pursued only put into swimming cylinder went swimming, 0 ℃ of water temperature (frozen water mixes bath), depth of water 20cm.The record mouse is from the swimming beginning to the dead time.
The prepared polypeptide powder of crocodile of the present invention contains that protein, polypeptide and amino acid content are abundant to be respectively 1.55%, 72.75%, 2.02%, thorough enzymolysis; Sugar and fat content are lower, are respectively 1.24%, 1.89%, and its moisture, content of ashes are respectively 5.05%, 4.98% in addition, see Table 1.The prompting polypeptide powder of crocodile belongs to low sugar, low fat, high-protein food.The metal element content analysis is measured, metal element content to the human body beneficial is abundant, content is followed successively by K, Na, Ca, Mg, Fe, Zn, Cu from high to low, 31808.18,2623.64,1033.53,253.86,9.89,5.20,1.37mg/kg content is followed successively by:, and harmful heavy metal Ag, Pb equal size are significantly less than state food test stone (table 2).
Amino acid composition measuring result shows, amino acid A wide selection of colours and designs in this active peptides, essential amino acid (containing cystine, tyrosine) rich content, up to 30.11%, account for 39.08% of total amino acid content, and the ratio of branched-chain amino acid and aromatic amino acid is 3.63, is significantly higher than various fish, points out this active peptides to have certain liver protecting effect (table 3).
MALDI-TOF Mass Spectrometer Method, result show that the polypeptide powder of crocodile molecular weight distribution concentrates on this scope (Fig. 1) of 1000-1300Da.
Antioxidation in vitro measuring result through polypeptide powder of crocodile shows that polypeptide powder of crocodile has preferably scavenging action to various free radicals, and when its concentration was 6.0mg/ml, its clearance rate to DPPH can reach 44.2% (Fig. 2).
When its concentration is 3.43mg/ml, to the hydroxyl radical free radical clearance rate up to 95.5% (Fig. 3).
When its concentration is 3.5mg/ml, can reach 56.96% (Fig. 4) to the clearance rate of ultra-oxygen anion free radical.
The feeding polypeptide powder of crocodile is 20 days continuously, the vertical rope climbing experiment of mouse and ice-water bath swimming with a load attached to the body experiment, the result shows that rope climbing and swimming power that this product can prolong mouse preferably exhaust the time (table 4), and vertical rope climbing and the swimming with a load attached to the body power time of exhausting are the common counters of reflection animal movement degree of fatigue.This result shows that further polypeptide powder of crocodile has certain antifatigue, improves the locomitivity effect of body.
By the acute toxicity test of GB GB1519.3-2003 food, take Kunming kind small white mouse as animal model, the result shows that this active peptides belongs to nontoxic food, and safety can be reliable.Mouse stomach dosage is 15g/kg, and Continuous Observation 7 days was weighed on the 7th day.The result: 10 mouse all grow up healthy and sound, blunt, the tired volt of action, ataxia, exaggerated respiration, feed minimizing, irregular, the abnormal responses such as four limbs are weak and limp, stupor of few, the little stool of drinking times obviously, appear in the body weight increase.The various organs such as the heart of dissection discovery mouse, liver,spleen,kidney do not have obvious pathology.
The constituent analysis of table 1 polypeptide powder of crocodile
The amino acid composition analysis of table 2 polypeptide powder of crocodile
The metal element content analysis of table 3 polypeptide powder of crocodile
Table 4 polypeptide powder of crocodile mouse anti-reflecting fatigue analysis of experimental data
Claims (3)
1. the preparation method of polypeptide powder of crocodile is characterized in that may further comprise the steps:
1) with behind the crocodile meat cleaning removal adipose tissue, being cut into size is 1～2cm
3Fragment;
2) add entry in the crocodile fragment, after putting boiling water bath and boiling, homogenate gets crocodile meat homogenate, the described amount that adds entry, and by volume, the crocodile fragment: water is 1: 1～2.5, the time that described boiling water bath boils is 20～40min;
3) in step 2) add trypsase in the crocodile meat homogenate of gained, water-bath, behind the enzymolysis the enzymolysis mixed liquor A, described tryptic addition by mass percentage, is 0.3%～0.5% of crocodile meat; The temperature of described water-bath is 40～45 ℃, and the time of described enzymolysis is 4～6h;
4) with step 3) the enzymolysis mixed liquor A of gained places boiling water bath, makes enzyme deactivation, gets the enzymolysis mixed liquid B;
5) with step 4) the enzymolysis mixed liquid B of gained, be cooled to room temperature after, with 6～8 layers of filtered through gauze, get crocodile meat enzymolysis liquid;
6) with step 5) middle gained enzymolysis liquid, carry out spray-drying, the condition setting of instrument is: pressure 0.08～0.12MPa; Cold air blast velocity 0.6～1.0m
3/ min; 120～130 ℃ of inlet temperatures; 75～90 ℃ of outlet temperatures; Sample introduction speed 0.8～1.2L/h gets product and is polypeptide powder of crocodile.
2. the prepared polypeptide powder of crocodile of the preparation method of polypeptide powder of crocodile as claimed in claim 1.
3. the application of polypeptide powder of crocodile as claimed in claim 2 in preparation antifatigue and raising body movement ability medicine.
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| CN108813248A (en) * | 2018-06-20 | 2018-11-16 | 百生康（北京）健康产业科技有限公司 | A kind of crocodile drink and preparation method thereof |
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| CN109797184A (en) * | 2019-03-29 | 2019-05-24 | 集美大学 | A kind of crocodile peptide and its production method |
| CN114426998A (en) * | 2021-09-16 | 2022-05-03 | 海南三元星生物科技股份有限公司 | Crocodile peptide preparation method and prepared crocodile peptide |
| CN117069803B (en) * | 2023-08-18 | 2024-05-14 | 广东贞山鳄鱼养殖有限公司 | Crocodile soup additive and preparation method and application thereof |
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Effective date of registration: 20170911 Address after: Haicang District, Haicang District, Fujian, Xiamen Haicang Road, finance letter City office building, room 1903 Patentee after: Fujian Tuo dragon industry limited liability company Address before: Xiamen City, Fujian Province, 361005 South Siming Road No. 422 Co-patentee before: Tailand Sriracha Tiger Zoo Co., Ltd. Patentee before: Xiamen University |