CN101283805B - Zinc-polypeptides complex and its preparation method and application - Google Patents
Zinc-polypeptides complex and its preparation method and application Download PDFInfo
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Abstract
The invention provides a zinc-polypeptide composition, which is prepared from a zinc mineral and an animal material in a weight ratio of (1:1) to (1:5) by hydrolyzing the animal material by protease to obtain polypeptide, and complex-reacting with the zinc mineral. The composition not only contains zinc elements absorbable by human and animal bodies but contains polypeptide component, thus supplementing zinc and enhancing immunity. The composition can be used as a food, a nutritional product, a healthy product or an animal feed additive for supplementing zinc and improving immunity. The materials are selected from natural animal materials, which has low cost, such as low-value agricultural and fishery products or scraps or leftovers of agricultural and fishery products.
Description
Technical field
The present invention relates to chemical field, be specifically related to the metal polypeptides complexe.
Background technology
Zinc (Zn) is the necessary trace element of humans and animals, is called as biological element.At present, organic zinc mainly is the amino acid chelate thing, as the amino acid chelate thing of trace elements such as zinc, selenium or calcium.The amino-acid chelate of edible trace element not only can replenish the required trace element of human body or animal, can also replenish the required amino acid of human body or animal simultaneously.State knows that office discloses an application for a patent for invention " preparation method of amino-acid zinc and application thereof " (application number 94114854.8) on April 2nd, 1997, this application discloses a kind of preparation method of triglycine zinc huge legendary turtle compound, be about to the mixed in molar ratio that amino acid and zinc oxide were pressed 2: 1, in the aqueous solution, concentrate after reaction a period of time, use alcohol evolution reaction product at last.State knows that office discloses an application for a patent for invention " preparation method of amino acid chelate thing " (application number 200380105098.7) on February 11st, 2006, this application discloses a kind of method for preparing metal-amino acid huge legendary turtle compound, the metal carbonate and the acidic amino acid (as glutamic acid, aspartic acid etc.) that are about to mol ratio and are 1: 1～1: 4 react in 0～100 ℃ the aqueous solution, get the reactant supernatant at last and get final product through freeze drying.State know the office on February 22nd, 2006 Granted publication a patent of invention " a kind of preparation technology of organic selenium " (application number 200410016499.1), this patent disclosure a kind of preparation method of selenium methionine: methionine salt and selenic chloride were reacted in the aqueous solution or organic solution 0.5～10 hour, when reaction control pH2～9, temperature-10～100 ℃, at last after filtration, concentrate and dry, get final product.The used chelating agent of said method all is the amino acid of the single component of non-natural, make gained huge legendary turtle compound nutritional labeling single, in actual use, often need being used just of several amino acid trace element chelated things can embody effect preferably, application cost is higher relatively.And, because amino acid whose preparation is the normal chemical acid-hydrolysis method that adopts, can introduce chemical pollutant or chemical residue unavoidably, need in addition tight monitoring and detection.Along with micromolecule polypeptide can be by the confirmation of animal economy absorption approach, the exploitation of relevant polypeptide just becomes the new focus of nutrition or Food Science field.Because the chemical synthesis difficulty of pure product peptide is big, cost is high, and chemical synthesising peptide often the biologically active than preparation peptide from animals and plants is low, so the application by chemical synthesising peptide and micro-prepared in reaction metal complex also can be subjected to certain limitation.
Summary of the invention
The purpose of this invention is to provide a kind of Zinc-polypeptides complexe.
Another object of the present invention provides the preparation method and the application of this complex.
The present invention realizes that the technical scheme of above-mentioned purpose is:
A kind of Zinc-polypeptides complexe, this complex is that 1: 1～1: 5 animal material and inorganic zinc made by the following method by weight ratio: add the polypeptide that alkali protease or bacillus subtilis neutral proteinase hydrolysis obtain in animal material, carry out complex reaction with inorganic zinc again and make; Wherein, described inorganic zinc is zinc oxide, zinc chloride or zinc carbonate, preferred zinc oxide or zinc carbonate; Described animal material is leftover bits and pieces or the discarded object after low value fish and shellfish aquatic products, the fish and shellfish processing.
Zinc-polypeptides complexe of the present invention is prepared by following method:
(1) preliminary treatment of raw material: animal material is shredded or grinds;
(2) protease hydrolytic: get the raw material after step (1) is handled, the water that adds 3～5 times, stir, transfer pH to 6.5～9.5 with the PH conditioning agent, amount by every gram raw material 700～1000U adds alkali protease or bacillus subtilis neutral proteinase then, under 45～60 ℃, stirs or sways at last, water-bath 2～3 hours gets hydrolyzate; Described pH conditioning agent is sodium acid carbonate, NaOH or hydrochloric acid;
(3) enzyme-deactivating is handled: hydrolyzate is heated to 95～100 ℃, kept 10 minutes, be cooled to room temperature, and centrifugal, filter, get clear liquid;
(4) complex reaction:
1. with acetic acid or NaHCO
3PH to 4.0～7.0 of regulating step (3) gained clear liquid: when described clear liquid pH less than 4.0 the time, use NaHCO
3Or NaOH regulates, when described clear liquid pH greater than 7.0 the time, regulate with acetic acid;
2. add inorganic zinc solution, the pure water that adds 4～5 times of clear liquids then reacts 0.5～10h under 25 ℃ to 100 ℃ condition in reaction system, and the course of reaction discontinuous stirs; Wherein, adding the volume of 0.1g/ml inorganic zinc solution and the volume ratio of clear liquid is 1: 10;
3. after reaction finishes it is concentrated, pour into after the cooling and carry out sedimentation in 95%～98% ethanol, isolated by filtration, the sediment that separation is obtained washs with a small amount of distilled water or ethanol at last, drains 80～100 ℃ of dry down the getting final product in back; Directly carry out heated-air drying or spray-drying after perhaps concentrating; Wherein the method for Nong Suoing can be that vacuum concentrates, and also can be that heating concentrates under the normal pressure; Generally between 25～100 ℃, temperature is low more for the temperature that heating concentrates, and it is good more that the activity of gained complex keeps, but required time is long more.
In the inventive method,
After the raw material that grinds or shred is water-soluble, promptly in the step (2), before transferring pH to 6.5～9.5 with the PH conditioning agent, can also be in 55 ℃ of water-baths 30 minutes, in 100 ℃ of following heat treatments 20 minutes, make the protein denaturation in the raw material then, fully be hydrolyzed to polypeptide after helping adding protease;
When the used protease of step (2) was alkali protease, preferably the pH value with the mixture of raw material and water transferred to 9.0～9.5, and the bath temperature after enzyme-added is 50～60 ℃;
When the used protease of step (2) was bacillus subtilis neutral proteinase, preferably the pH value with the mixture of raw material and water transferred to 6.5～7.5, and the bath temperature after enzyme-added is 45～55 ℃;
The pH value of clear liquid preferably is adjusted to 4.5～5.5 described in the step (4); The amount that adds inorganic zinc is preferred: with step (1) raw material weight ratio be 1: 2～1: 3; Preferred 1～5 hour of reaction time, preferred 25 ℃～70 ℃ of reaction temperature.
Zinc-polypeptides complexe of the present invention not only contains can be by the zinc element of human body and animal body absorption, also contain abundant polypeptide composition, has the effect that strengthens body immunity, the nutriment, health products or the animal feed additive that can be used as zinc supplementation and improve immunity of organisms; Raw materials used is the natural animal raw material, and is waste material or leftover bits and pieces after low value farming fish product or the processing of agricultural fish product, cheap.
The method that complex of the present invention is used for preparing the drink that contains base furnish such as water, fruit juice, milk, sucrose, vitamin, mineral matter is: add 0.1～0.3% complex of the present invention in described base furnish.
The method that complex of the present invention is used for preparing beans (milk) powder food is: the complex of the present invention that adds 0.3-0.5% at soymilk or bean powder.
It is that the method for the solid food of raw material is with starch that complex of the present invention is used for preparing main: add 0.1～0.5% complex of the present invention at raw material.
It is that the using method of additive of the aquatic feeds of raw material is with the bean cake powder that complex of the present invention is used for preparing main: add 3～5% complex of the present invention at raw material.
It is that the using method of additive of the pig feed of raw material is that complex of the present invention is used for preparing mainly with corn flour, bean cake powder, flour, wheat bran, chaff cake etc.: add 1～3% complex of the present invention at raw material.
Complex of the present invention is as being that the using method of additive of the bird feed of raw material is with corn flour, bean cake powder, fish meal, the dish dregs of rice, red dog flour, rice bran etc. mainly: add 2～4% complex of the present invention in raw material.
To prove technique effect of the present invention by zoopery below.
One, experiment material: Zinc-polypeptides complexe, the preparation method sees embodiment 1.
Two, animal used as test and grouping
Body weight 19.85 ± 0.91g, the healthy male mice in kunming in mouse 9 weeks of age (providing by Traditional Chinese Medicine University Of Guangzhou's animal center) are provided for use, under peace and quiet, 22 ± 2 ℃ of conditions of room temperature, are fed, natural lighting, regularly ventilate every day.Adopt national standard rodent feed to raise by free diet, filling stomach physiological saline or Zinc-polypeptides complexe grouping: the 1st group (standard feed control group), the feeding standard feed is irritated stomach physiological saline; The 2nd group (low dose group, Zinc-polypeptides complexe concentration are 0.25mg/ml), the feeding standard feed is irritated the stomach Zinc-polypeptides complexe; The 3rd group (middle dosage group, Zinc-polypeptides complexe concentration are 0.5mg/ml), the feeding standard feed is irritated the stomach Zinc-polypeptides complexe; The 4th group (high dose group, Zinc-polypeptides complexe concentration are 1.0mg/ml), the feeding standard feed is irritated the stomach Zinc-polypeptides complexe.Feed 30d continuously, measure the phagocytic function and the indexs of correlation such as liver GSH-PX vigor, SOD vigor of mouse macrophage then.
Three, experimental technique and result
1, Zinc-polypeptides complexe of the present invention is to the influence of mouse macrophage vigor
Engulf chicken red blood cell experiment (half intracorporal method) by Turnover of Mouse Peritoneal Macrophages, measure the phagocytic percentage and the phagocytic index of macrophage, thus the phagocytic function of reflection macrophage, the resistance against diseases of embodiment body.Because macrophage can be engulfed chicken red blood cell, with macrophage with engulf chicken red blood cell incubation poststaining, the percentage of the macrophage of chicken red blood cell is engulfed in calculating under oily mirror, and the form of the interior chicken red blood cell of observation of cell, judges the phagocytic function and the digestive function of macrophage in view of the above.
The chicken erythrocyte suspension preparation:
The chicken broken end is got blood, mixes in 1: 9 ratio with physiological saline, places the conical flask of bead, fully shakes towards a direction, with defiber.With physiological saline washing 2～3 times, centrifugal (2000r/min 10min), removes supernatant, is made into the chicken erythrocyte suspension of 1% (v/v) with physiological saline.
Phagocytic function is measured:
(1) injects sterilization broth bouillon 1ml macrophage derivant in every mouse peritoneal;
(2) every mouse lumbar injection 1% chicken erythrocyte suspension 1mL behind the 24h.Interval 30min, mouse is put to death in the cervical vertebra dislocation;
(3) it is faced upward the position and be fixed on the mouse plate, abdominal skin is cut off in the center, injects physiological saline 2mL through the abdominal cavity, gently rubs belly 1min;
(4) sucking-off abdominal cavity washing lotion 1mL then, average mark drips smear on 2 slides, 37 ℃ of incubator incubations of dislocation 30min;
(5) incubate completely, rinsing in physiological saline is to remove not paster cell;
(6) dry, with the fixing 5min of 1: 1 acetone methanol solution;
(7) 1: 10 (v/v) Giemsa-phosphate buffer dyeing 30min use the distilled water rinsing again, dry;
(8) microscopically is observed the situation of macrophage phagocytic chicken red blood cell.
The oil mirror is the counting macrophage down, and every sheet is counted 100, is calculated as follows phagocytic percentage and phagocytic index.
The phagocytic percentage of table 2 various dose complex and phagocytic index (
N=7)
| Group | Phagocytic percentage (%) | Phagocytic index |
| Control group | 35.71±2.31 | 1.19±0.14 |
| Low dose group | 38.86±1.96 | 1.26±0.08 |
| Middle dosage group | 42.71±3.65 | 1.50±0.08 |
| High dose group | 42.57±3.33 | 1.40±0.09 |
As shown in table 2, phagocytic percentage, between the phagocytic percentage of the macrophage after the middle dosage group of complex of the present invention and high dose group are handled and the 1st group (control group) notable difference (P＜0.05) is arranged, utmost point significant difference (P＜0.01) is arranged between phagocytic index and the 1st group (control group).As seen, Zinc-polypeptides complexe has the phagocytic activity that stimulates macrophage, has antiviral, as to suppress tumour cell growth, strengthens the anti-infection ability of animal to pathogen, obviously reduces effects such as the infectious diseases incidence of disease.
2, Zinc-polypeptides complexe of the present invention is to the influence of mouse GSH-PX vigor and SOD vigor
The mensuration of GSH-PX vigor is measured according to kit (Nanjing is built up bio-engineering research and provided) specification.The mensuration of SOD vigor is measured according to kit (Nanjing is built up bio-engineering research and provided) specification.
Table 3 complex to the influence of mouse kidney liver GSH-Px and SOD vigor (
N=7)
| Group | The GSH-Px vigor | SOD vigor (U/mg prot) |
| The 1st group (control group) | 55.63±752 | 105.43±5.63 |
| The 2nd group (low dose group) | 99.15±7.13 | 139.59±8.03 |
| The 3rd group (middle dosage group) | 117.39±10.00 | 190.16±23.48 |
| The 4th group (high dose group) | 110.95±14.65 | 218.46±25.66 |
As shown in table 3, each dosage group of complex of the present invention is to all having utmost point significant difference (P＜0.01) between the influence of GSH-Px and SOD vigor and the 1st group (control group), as seen, Zinc-polypeptides complexe of the present invention has and improves body liver kidney GSH-PX vigor and SOD vigor, strengthens immunity of organisms, promotes effect such as animal health growth.
3, Zinc-polypeptides complexe of the present invention is removed the mensuration of effect to superoxide radical
Under alkali condition autoxidation can take place according to pyrogallol, generate coloured intermediate product and ultra-oxygen anion free radical (O
2-), ultra-oxygen anion free radical (O
2-) autoxidation there is the principle of catalytic action, adopt AAS to measure Zinc-polypeptides complexe, shown in seeing the following form to the superoxide radical clearance rate.
Table 4 complex is removed effect to superoxide radical
| Zinc-protein hydrolysate complex consumption (mg) | 0.6 | 1.2 | 1.8 | 2.4 | 3.0 |
| Clearance rate (%) | 55.51 | 69.78 | 78.78 | 80.69 | 80.82 |
As can be seen from Table 4, Zinc-polypeptides complexe has superoxide radical removes effect preferably, and along with the increase of Zinc-polypeptides complexe consumption, can be better to the removing effect of superoxide radical.
From above-mentioned experimental result as can be seen, complex of the present invention has the phagocytic activity that stimulates macrophage, has the effect that improves body liver kidney GSH-PX vigor and SOD vigor, superoxide radical had remove effect preferably, can strengthen the anti-infection ability of animal, strengthen immunity of organisms, promote the animal health growth pathogen.
Description of drawings
Fig. 1 is the scintigram of contrast protein hydrolysate polypeptide ultra-violet absorption spectrum.
Fig. 2 is the scintigram of complex ultra-violet absorption spectrum of the present invention.
Fig. 3 is the infrared spectrum comparison diagram of protein hydrolysate polypeptide and complex of the present invention, and wherein S1 is the infrared spectrum of protein hydrolysate polypeptide, and S2 is the infrared spectrum of Zn-polypeptides complexe.
The specific embodiment
Preparation example
Example 1
The preliminary treatment of animal material: take by weighing the Tilapia mossambica processing fent, chop up fast, avoid raw material to form gel with tissue mashing machine or cutmixer.
Proteolysis: get a certain amount of raw material, by material: water=1: 3 is soluble in water, stirs, and regulates pH to 9.0, adds alkali protease 700U/g raw material, sways 50 ℃ of waters bath with thermostatic control and carries out enzyme digestion reaction 2h in the shaking table, sways once every 10min.Then hydrolyzate is heated to 100 ℃, keeps 10min, be cooled to room temperature, filter to such an extent that clear liquid is standby.Used alkali protease is Alcalase 2.4L FG during hydrolysis, is provided by letter Bioisystech Co., Ltd of Novi.
The complex preparation: get clear liquid 100ml, regulate pH to 4.0, adding concentration is the liquor zinci chloridi 10ml of 0.1g/ml, electric furnace preheating 10min, remove by filter insoluble matter, add 400ml water again, carried out complex reaction 4 hours at 70 ℃ then, vacuum is concentrated into about 10ml then, pour into after the cooling and carry out sedimentation in 95% ethanol, isolated by filtration, precipitation is washed with a small amount of distilled water and ethanol, drain and be placed on drying in 80 ℃ of baking ovens, promptly get solid-state Zn-polypeptides complexe.
The evaluation of gained complex:
1, the infrared spectrum of complex of the present invention detects:
Protein hydrolysate polypeptide (PEP) and Zinc-polypeptides complexe (Zn-PEP) are respectively by determination of infrared spectroscopy, and contrast protein hydrolysate polypeptide and complex thereof are at 4000～500cm
-1Infrared spectrum (FTIR) absorption band in zone and ownership statistics thereof are as shown in table 1, and Fig. 3 is infrared spectrum (FTIR) figure.
The infrared spectrum frequency and the ownership of table 1 Zinc-polypeptides complexe
| 1046.05 | 1045.61 | C-N-C or CO stretching vibration (acid amides band V) |
| 929.70 | 929.05 | |
| 878.56 | ||
| (591.09 very weak) | 602.26 | |
| 531.80 |
As can be seen from Table 1, contrast protein hydrolysate polypeptide (PEP) all exists than big-difference aspect intensity and the absorption frequency with the IR spectrum of Zinc-polypeptides complexe of the present invention (Zn-PEP).PEP is at 3392.96cm
-1The absworption peak at place moves to 3325.67cm in Zn-PEP
-1, red shift 71.92cm
-1Behind the synthetic Zn-PEP of PEP, at 3080.61cm
-1The absworption peak at place disappears; These bands of a spectrum corresponding respectively NH symmetrical stretching vibration and the antisymmetric stretching vibration of PEP.Amino and carboxyl that peptide molecule is described have all participated in Zn
2+Coordination, and the oxygen that combines with zinc among the PEP is with also existing hydrogen bond between the NH.PEP is at 2970.89cm
-1The absworption peak at place moves to 2968.85cm in Zn-PEP
-1, red shift 2.04cm
-1, this is CH among the PEP
2, the stretching vibration of CH group, this illustrates Zn
2+Cause the part of PEP node configuration to change with the effect of PEP.Behind the synthetic Zn-PEP of PEP, at 2084.13cm
-1The absworption peak at place disappears; PEP is at 1662.11cm
-1The absworption peak at place moves to 1654.34cm in Zn-PE
-1, red shift 7.71cm
-1, this is COO
-Or the CO stretching vibration, illustrate that the oxygen of carboxyl has participated in Zn among the PEP
2+Coordination.PEP is at 1583.22cm
-1The absworption peak at place disappears, and further specifies the direct and Zn of oxygen on the carboxyl
2+Direct coordination is arranged.PEP is at 1452.59cm
-1, 1398.70cm
-1, 1319.41cm
-1, 1161.86cm
-1, 1115.31cm
-1, 1079.64cm
-1, 1049.05cm
-1Red shift Deng the absworption peak of locating takes place all shows PEP and Zn
2+Coordination is arranged, and generated new complex.PEP is at 1262.64cm
-1The absworption peak at place disappears, show amino on the residue and carboxyl oxygen all may with Zn
2++Direct Cheng Jian, and by hydrogen bond association.Zn-PEP is at 878.56cm
-1Near the place absworption peak is strengthened and at 602.26cm
-1The red shift of place's absworption peak might be Zn
2+Cause with the stretching vibration of N, the formed fit key of O.
By the spectrum elucidation result as can be known, PEP and Zn
2+Complexation reaction has taken place really, and has formed Zn
2+And the fit key between N, the O, having caused that the part of PEP molecular skeleton changes, mating reaction occurs on the N, O atom of amino residue and peptide bond.
2, the ultra-violet absorption spectrum of protein hydrolysate polypeptide and Zinc-polypeptides complexe
The ultra-violet absorption spectrum of contrast protein hydrolysate polypeptide is seen Fig. 1, and the ultra-violet absorption spectrum of Zinc-polypeptides complexe of the present invention is seen shown in Figure 2.As can be seen from Figure 1, the maximum absorption band of protein hydrolysate is at the 210nm place, and this is by carbonyl on the peptide bond (C=O) n → π
*Electron transition causes.Fig. 2 is as can be seen: the maximal ultraviolet absorption peak of complex is compared with the maximum absorption band 210nm place of protein hydrolysate polypeptide at the 216nm place, displacement 6nm.This is owing to behind N, the O formation fit key in zinc ion and the protein hydrolysate polypeptide, influenced carbonyl on the peptide bond (C=O) n → π
*Electron transition; Also can find out have a more weak new absworption peak to occur at the 272nm place from zinc-complex ultra-violet absorption spectrum scintigram, this is π → π in the part (N-C-O)
*Due to the electron transition; In addition, zinc-complex less than 200nm and greater than the zone of 300nm without any absworption peak.
Infrared spectrum and uv absorption spectra by Zinc-polypeptides complexe and protein hydrolysate polypeptide the analysis showed that bonding action has taken place for N, O in zinc ion and the protein hydrolysate polypeptide, have formed zinc-protein hydrolysate polypeptides complexe.
Example 2
The preliminary treatment of animal material: take by weighing the Tilapia mossambica processing fent, chop up fast, avoid raw material to form gel with tissue mashing machine or cutmixer.
Proteolysis: get a certain amount of raw material, by material: water=1: 5 is soluble in water, stirs, and regulates pH to 7.5, adds bacillus subtilis neutral proteinase 1000U/g raw material, sways 55 ℃ of waters bath with thermostatic control and carries out enzyme digestion reaction 3h in the shaking table, sways once every 10min.Then hydrolyzate is heated to 100 ℃, keeps 10min, be cooled to room temperature, centrifugal that clear liquid is standby.Bacillus subtilis neutral proteinase used during hydrolysis is provided by Nanning Pang Bo bioengineering Co., Ltd.
The complex preparation: get clear liquid 100ml, regulate pH to 4.0, adding concentration is the liquor zinci chloridi 10ml of 0.1g/ml, electric furnace preheating 3min, remove by filter insoluble matter, add 500ml water again, under 100 ℃, carried out complex reaction 5 hours then, vacuum is concentrated into about 10ml then, pour into after the cooling and carry out sedimentation in 95% ethanol, isolated by filtration, precipitation is washed with a small amount of distilled water and ethanol, drain and be placed on drying in 100 ℃ of baking ovens, promptly get solid Zn-polypeptides complexe.
Example 3
The preliminary treatment of animal material: take by weighing the Tilapia mossambica processing fent, chop up fast, avoid raw material to form gel with tissue mashing machine or cutmixer.
Proteolysis: get a certain amount of raw material, by material: water=1: 4 is soluble in water, stirs, and regulates pH to 9.0, adds alkali protease 8000U/g raw material, sways 55 ℃ of waters bath with thermostatic control and carries out enzyme digestion reaction 2.5h in the shaking table, sways once every 10min.Then hydrolyzate is heated to 100 ℃, keeps 10min, be cooled to room temperature, filter to such an extent that clear liquid is standby.Used alkali protease is Alcalase 2.4L FG during hydrolysis, is provided by letter Bioisystech Co., Ltd of Novi.
The complex preparation: get clear liquid 100ml, regulate pH to 5.0, adding concentration is the burnett's solution 10ml of 0.1g/ml, electric furnace preheating 7min, remove by filter insoluble matter, add 400ml water again, carried out complex reaction 3 hours at 70 ℃ then, vacuum is concentrated into about 10ml then, pour into after the cooling and carry out sedimentation in 95% ethanol, isolated by filtration, precipitation is washed with a small amount of distilled water and ethanol, drain and be placed on drying in 90 ℃ of baking ovens, promptly get solid Zn-polypeptides complexe.
Example 4
The preliminary treatment of animal material: take by weighing the Tilapia mossambica processing fent, chop up fast, avoid raw material to form gel with tissue mashing machine or cutmixer.
Proteolysis: get a certain amount of raw material, by material: water=1: 3 is soluble in water, stirs, and regulates pH to 7.0, adds bacillus subtilis neutral proteinase 1000U, sways 50 ℃ of waters bath with thermostatic control and carries out enzyme digestion reaction 3.0h in the shaking table, sways once every 10min.Then hydrolyzate is heated to 100 ℃, keeps 10min, be cooled to room temperature, centrifugal that clear liquid is standby.Bacillus subtilis neutral proteinase used during hydrolysis is provided by Nanning Pang Bo bioengineering Co., Ltd.
The complex preparation: get clear liquid 100ml, regulate pH to 5.0, adding concentration is the liquor zinci chloridi 10ml of 0.1g/ml, electric furnace preheating 3-10min, remove by filter insoluble matter, add 400ml water again, carried out complex reaction 4 hours at 70 ℃ then, vacuum is concentrated into about 10ml then, pour into after the cooling and carry out sedimentation in 95% ethanol, isolated by filtration, precipitation is washed with a small amount of distilled water and ethanol, drain and be placed on drying in 80 ℃ of baking ovens, promptly get solid Zn-polypeptides complexe.
Example 5
The preliminary treatment of animal material: take by weighing the Tilapia mossambica processing fent, chop up fast, avoid raw material to form gel with tissue mashing machine or cutmixer.
Proteolysis: get a certain amount of raw material, by material: water=1: 3 is soluble in water, stirs, and regulates pH to 9.0, adds alkali protease 1000U/g raw material, sways 55 ℃ of waters bath with thermostatic control and carries out enzyme digestion reaction 3.0h in the shaking table, sways once every 10min.Then hydrolyzate is heated to 100 ℃, keeps 10min, be cooled to room temperature, filter to such an extent that clear liquid is standby.Used alkali protease is Alcalase 2.4L FG during hydrolysis, is provided by letter Bioisystech Co., Ltd of Novi.
Complex preparation: get clear liquid 100ml, regulate pH to 5.0, adding concentration is the zinc carbonate solution 10ml of 0.1g/ml, electric furnace preheating 10min removes by filter insoluble matter, adds 400ml water again, carry out complex reaction 1h at 25 ℃ then, reaction is finished after concentrate down in 45 ℃, normal pressure, and last spray-drying promptly obtains solid-state Zinc-polypeptides complexe.
Example 6
The preliminary treatment of animal material: take by weighing the Tilapia mossambica processing fent, chop up fast, avoid raw material to form gel with tissue mashing machine or cutmixer.
Proteolysis: get a certain amount of raw material, by material: water=1: 5 is soluble in water, stirs, and regulates pH to 7.5, adds bacillus subtilis neutral proteinase 1000U/g raw material, sways 45 ℃ of waters bath with thermostatic control and carries out enzyme digestion reaction 2h in the shaking table, sways once every 10min.Then hydrolyzate is heated to 95 ℃, keeps 10min, be cooled to room temperature, filter to such an extent that clear liquid is standby.Bacillus subtilis neutral proteinase used during hydrolysis is provided by Nanning Pang Bo bioengineering Co., Ltd.
Complex preparation: get clear liquid 100ml, regulate pH to 5.0, adding concentration is the liquor zinci chloridi 10ml of 0.1g/ml, electric furnace preheating 10min removes by filter insoluble matter, adds 400ml water again, under 50 ℃, carry out complex reaction 0.5h then, it is concentrated down in 60 ℃, normal pressure that the back is finished in reaction, and spray-drying or heated-air drying promptly obtain the solid-state Zinc-polypeptides complexe of solubility.
Example 7
The preliminary treatment of animal material: take by weighing the Tilapia mossambica processing fent, chop up fast, avoid raw material to form gel with tissue mashing machine or cutmixer.
Proteolysis: get a certain amount of raw material, by material: water=1: 3 is soluble in water, stirs, and regulates pH to 9.0, adds alkali protease 1000U/g raw material, sways 60 ℃ of waters bath with thermostatic control and carries out enzyme digestion reaction 3.0h in the shaking table, sways once every 10min.Then hydrolyzate is heated to 100 ℃, keeps 10min, be cooled to room temperature, filter to such an extent that clear liquid is standby.Used alkali protease is Alcalase 2.4L FG during hydrolysis, is provided by letter Bioisystech Co., Ltd of Novi.
Complex preparation: get clear liquid 100ml, regulate pH to 7.0, adding concentration is the liquor zinci chloridi 10ml of 0.1g/ml, electric furnace preheating 10min removes by filter insoluble matter, adds 400ml water again, carry out complex reaction 10h at 70 ℃ then, reaction is finished after vacuum concentrates, and spray-drying promptly obtains the solid-state Zinc-polypeptides complexe of solubility.
Example 8
The preliminary treatment of animal material: take by weighing the Tilapia mossambica processing fent, chop up fast, avoid raw material to form gel with tissue mashing machine or cutmixer.
Proteolysis: get a certain amount of raw material, by material: water=1: 3 is soluble in water, stir, 55 ℃ of water-baths 30 minutes, in 100 ℃ of following heat treatments 20 minutes, pH to 7.0 was regulated in the cooling back then, added bacillus subtilis neutral proteinase 1000U/g raw material, sway 50 ℃ of waters bath with thermostatic control and to carry out enzyme digestion reaction 3.0h in the shaking table, sway once every 10min.Then hydrolyzate is heated to 100 ℃, keeps 10min, be cooled to room temperature, filter to such an extent that clear liquid is standby.Bacillus subtilis neutral proteinase used during hydrolysis is provided by Nanning Pang Bo bioengineering Co., Ltd.
Complex preparation: get clear liquid 100ml, regulate pH to 5.0, adding concentration is the liquor zinci chloridi 10ml of 0.1g/ml, electric furnace preheating 10min removes by filter insoluble matter, adds 400ml water again, carry out complex reaction 4h at 70 ℃ then, it is concentrated down in 100 ℃, normal pressure that the back is finished in reaction, and heated-air drying promptly gets Zinc-polypeptides complexe.
Application examples
Example 1
The application of Zinc-polypeptides complexe in drink
A kind of drink, mainly by base furnish such as water, fruit juice, milk, sucrose, vitamin, mineral matter and quite the Zinc-polypeptides complexe of the present invention of base furnish weight 0.2% form, its preparation method is: Zinc-polypeptides complexe of the present invention is disperseed to join in the base furnish, abundant mixing, according to the operation of drink processing technology, get final product then.
Example 2
The application of Zinc-polypeptides complexe in powder or granular food
A kind of bean powder food, mainly by base furnish such as whole soya-bean powder, rice meal, syrup, vegetable oil, white sugar, lecithin, mineral matter, vitamin and quite the Zinc-polypeptides complexe of the present invention of base furnish weight 0.4% form, its preparation method is: Zinc-polypeptides complexe of the present invention is disperseed to join in the base furnish, abundant mixing, according to the operation of bean powder processing technology, get final product then.
Example 3
The application of Zinc-polypeptides complexe in solid food
A kind of cake food, mainly by base furnish such as smart powder, white granulated sugar, edible oil, egg, milk powder, starch, essence, calcium lactate, carbonic hydroammonium, sodium bicarbonate, sodium hydrogensulfite and quite the Zinc-polypeptides complexe of the present invention of base furnish weight 0.3% form, its preparation method is: Zinc-polypeptides complexe of the present invention is disperseed to join in the base furnish, abundant mixing, according to the operation of cake processing technology, get final product then.
Example 4
Zinc-polypeptides complexe is as the application of feeding additive aquatic animal
A kind of aquaculture feed, mainly formed originally and the Zinc-polypeptides complexe of the present invention of suitable raw material weight 3% is formed by bean cake powder, fish meal, vitamin, mineral matter, binder etc., its preparation method is: Zinc-polypeptides complexe of the present invention is disperseed to join in the described raw material dry powder, abundant mixing, according to the preparation of production of fodder processing technology, get final product then.
Example 5
Zinc-polypeptides complexe is as the application of pig feed additive
A kind of pig feed, mainly by raw material such as corn flour, bean cake powder, flour, wheat bran, chaff cake, vitamin, mineral matter, amino acid and quite the Zinc-polypeptides complexe of the present invention of raw material weight 1-3% form, its preparation method is: Zinc-polypeptides complexe of the present invention is disperseed to join in the raw material dry powder, abundant mixing, according to the preparation of production of fodder processing technology, get final product then.
Example 6
Zinc-polypeptides complexe is as the application of bird feed additive
A kind of bird feed, mainly by raw material such as corn flour, bean cake powder, fish meal, the dish dregs of rice, red dog flour, rice bran, vitamin, calcium monohydrogen phosphate, amino acid and quite the Zinc-polypeptides complexe of the present invention of raw material weight 3% form, its preparation method is: Zinc-polypeptides complexe of the present invention is disperseed to join in the original dry powder, abundant mixing, according to the preparation of production of fodder processing technology, get final product then.
Claims (6)
1. the preparation method of a Zinc-polypeptides complexe, this method may further comprise the steps:
(1) preliminary treatment of raw material: animal material is shredded or grinds;
(2) protease hydrolytic: get the raw material after step (1) is handled, the water that adds 3～5 times, stir, transfer pH to 6.5～9.5 with the PH conditioning agent, amount by every gram raw material 700～1000U adds alkali protease or bacillus subtilis neutral proteinase then, under 45～60 ℃, stirs or sways at last, water-bath 2～3 hours gets hydrolyzate; Described pH conditioning agent is sodium acid carbonate, NaOH or hydrochloric acid;
(3) enzyme-deactivating is handled: hydrolyzate is heated to 95～100 ℃, kept 10 minutes, be cooled to room temperature, and centrifugal, filter, get clear liquid;
(4) complex reaction:
1. with acetic acid or NaHCO
3PH to 4.0～7.0 of regulating step (3) gained clear liquid: when described clear liquid pH less than 4.0 the time, use NaHCO
3Or NaOH regulates, when described clear liquid pH greater than 7.0 the time, regulate with acetic acid;
2. add inorganic zinc solution, the pure water that adds 4～5 times of clear liquids then reacts 0.5～10h under 25 ℃～100 ℃ condition in reaction system, and the course of reaction discontinuous stirs; Wherein, adding the volume of 0.1g/ml inorganic zinc solution and the volume ratio of clear liquid is 1: 10;
3. carry out after reaction finishes that vacuum concentrates or the normal pressure heating concentrates, pour into after the cooling and carry out sedimentation in 95%～98% ethanol, isolated by filtration, the sediment that separation is obtained is drained the back drying and is got final product with a small amount of distilled water or ethanol washing at last; Perhaps, concentrated heated-air drying or the spray-drying of directly carrying out later;
In the above-mentioned steps, the weight ratio of described animal material and inorganic zinc is 1: 1～1: 5; Described inorganic zinc is zinc oxide, zinc chloride or zinc carbonate; Described animal material is leftover bits and pieces or the discarded object after low value fish and shellfish aquatic products, the fish and shellfish processing.
2. the method for claim 1 is characterized in that in the step (2), the following processing of process earlier before transferring pH to 6.5～9.5 with the PH conditioning agent: in 55 ℃ of water-baths 30 minutes, follow 100 ℃ of following heat treatments 20 minutes.
3. method as claimed in claim 1 or 2 is characterized in that in the step (2), and when used protease was alkali protease, raw material adds behind the water mixing regulated pH value to 9.0～9.5 with sodium acid carbonate or NaOH, and the bath temperature after enzyme-added is 50～60 ℃.
4. method as claimed in claim 1 or 2 is characterized in that in the step (2), and when used protease was bacillus subtilis neutral proteinase, raw material added behind the water mixing with salt acid for adjusting pH value to 6.5～7.5, and the bath temperature after enzyme-added is 45～55 ℃.
5. method as claimed in claim 1 or 2 is characterized in that the time of complex reaction in the step (4) is 1～5 hour.
6. method as claimed in claim 1 or 2 is characterized in that the temperature of complex reaction in the step (4) is 25 ℃～70 ℃.
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| CN103931907A (en) * | 2013-01-21 | 2014-07-23 | 李宗禄 | Formula of polypeptide organic pig feed and preparation method of pig feed |
| CN104095242B (en) * | 2014-07-15 | 2016-04-13 | 广州大学 | A kind of composite zinc selenium polypeptide compound and preparation method thereof |
| CN106072662B (en) * | 2016-06-21 | 2019-11-19 | 常熟理工学院 | A kind of preparation method of Corbicula fluminea polypeptide chelate zinc |
| CN107411100A (en) * | 2017-05-04 | 2017-12-01 | 济南大学 | A kind of method for preparing high F value corn oligopeptide chelates of zinc |
| CN111253468B (en) * | 2020-01-22 | 2021-12-07 | 广州大学 | Zinc ion complex peptide and complex and application thereof |
| CN111693644B (en) * | 2020-07-19 | 2022-09-20 | 广州天科生物科技有限公司 | Method for detecting content of free zinc in amino acid zinc complex |
| CN112063674B (en) * | 2020-08-03 | 2022-07-05 | 广州大学 | Preparation method and application of zinc ion complex peptide based on charge property and hydrophobicity |
| CN114634882B (en) * | 2022-03-25 | 2023-06-20 | 广州大学 | Method for promoting yeast growth and fermentation |
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| CN1329465A (en) * | 1998-12-09 | 2002-01-02 | N.V.努特里西阿 | Baby food stimulating growth of the thymus |
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