CN107604028A - The method that heating combined simulation digestion prepares collagen antioxidant polypeptide liquid - Google Patents
The method that heating combined simulation digestion prepares collagen antioxidant polypeptide liquid Download PDFInfo
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- CN107604028A CN107604028A CN201710679277.5A CN201710679277A CN107604028A CN 107604028 A CN107604028 A CN 107604028A CN 201710679277 A CN201710679277 A CN 201710679277A CN 107604028 A CN107604028 A CN 107604028A
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Abstract
The invention discloses the method that heating combined simulation digestion prepares collagen antioxidant polypeptide liquid, belong to foods processing technique and albumen utilizes technical field.The preparation technology used is using pepsin-solubilized collagen as raw material, high temperature pre-processes collagen, use in-vitro simulated gastro-intestinal digestion technology, by the peristole frequency containing simulation and amplitude, the bionical dynamic mouse peptic digest system for secreting glandular secretion peptic digest liquid, and simulation intestinal digestion afterwards, utilize pepsin trypsase, two step enzymatic isolation methods.The enzymatic hydrolyzation of albumen is drastically increased, improves raw material availability.The present invention uses dynamic models simulations gastro-intestinal digestion process, and under 37 DEG C of environment, closer to human body simulation digestive environments, process is more efficient, compared to artificial synthesized anti-oxidation peptide method, fermentation method safety and reliability.
Description
Technical field
The invention belongs to foods processing technique and albumen to utilize technical field, and in particular to is combined using heat pre-treatment imitative
Lively state mouse peptic digest system and simulation intestinal digestion prepare antioxidation polypeptide liquid.
Technical background
Collagen (collagen) is also referred to as collagen, be it is a kind of be widely present in it is non-aqueous in vertebra class animal connective tissue
Soluble fiber shape contains glycoprotein.Collagen is boiomacromolecule, the protein containing a small amount of galactolipin and glucose.
Its sterling is white, is the most abundant protein of mammal in-vivo content, the frame structure of composition extracellular matrix (ECM).
It is not very easily to digest and assimilate that collagen, which directly takes in human body, therefore collagen egg is commonly used in food processing field
White high-temperature denatured product gelatin is used as additive of candy, ice cream stabilizer etc., and antioxygen caused by collagen enzymolysis
It is even more to have substantial amounts of use in health food industry, beauty industry etc. to change peptide.
Anti-oxidation peptide is a kind of peptide with bioactivity, is the intermediate product of protein hydrolysis.Antioxidation polypeptide is main
Including the antioxidation polypeptide naturally occurred, such as glutathione;Antioxidation polypeptide caused by protein degradation, such as enzymatic hydrolysis of soybean egg
White enzymolysis product;Artificial synthesized antioxidation polypeptide, etc..The antioxidation polypeptide for wherein digesting formation meets the nutrition of human body
Demand, it is easy to industrialized production, and it is simple to operate, efficiently.More use alkali proteases of the polypeptide of enzymolysis protein generation at present,
The single enzyme solution such as papain.Such as:In Chinese patent CN106317170A, using natural purple perilla seed as raw material, utilize
Alkali protease extracts acid precipitation method, and anti-oxidation peptide is obtained by purifying.As a result easily cause enzymolysis incomplete, formation it is more
Peptide molecular weight distribution is more complicated, therefore brings and isolate and purify cumbersome, the problems such as cost is higher.
Stomach and intestine are the main places of human consumption's food, and stomach mixes food and stomachial secretion liquid by wriggling, and makes food
Grain grinding diminishes, and part biological chemical reaction occurs under one's belt, afterwards into intestines in the presence of intestinal digestion liquid it is most of
All digest so that food is more beneficial for human consumption's absorption.Therefore the present invention first uses heat pre-treatment technology, then passes through
Bionical dynamic mouse peptic digest system carries out simulation peptic digest, adds simulation intestinal digestion afterwards to prepare antioxidation polypeptide liquid.
The content of the invention
The present invention is entered using the pepsin-solubilized collagen of heat pre-treatment as raw material using bionical dynamic mouse peptic digest system
Row simulation peptic digest, simulation intestinal digestion is added afterwards to prepare antioxidation polypeptide liquid.
The method that the bionical dynamic mouse peptic digest of collagen and simulation intestinal digestion of heat pre-treatment prepare anti-oxidation peptide, is pressed
Carried out according to following steps:
(1) pepsin-solubilized collagen is dissolved into 0.02g/mL solution with distilled water, and concussion makes its dissolving complete, high-temperature water
Bath pretreatment, obtains raw material, hereinafter referred to as PSCH。
(2) bionical dynamic mouse peptic digest system is built:Simulate the gastric juice is prepared, and weighs 10.42g pepsins, 0.02g carbonic acid
Hydrogen sodium, 0.44g sodium chloride, 0.08g gastrons (mucin), stirring and dissolving is in distilled water, 50mL volumetric flask constant volumes, uses
1M HCl adjustment pH is 1.63 ± 0.01.In experiment, syringe pump selects mode of operation as perfusion, groundwater increment 3.4mL, flow
0.85mL/h, infusion time 4h.After 37 DEG C of preheating 10min of digestive system device, mouse stomach model the inside first adds before filling sample
0.6mL simulate the gastric juices remain to simulate mouse stomach gastric juice, then add sample and carry out simulation peptic digest.
(3) intestinal digestion system is simulated:Simulating intestinal digestion liquid, (preparation of simulation intestinal digestion liquid, weighs 0.68g biphosphates
Potassium, it is 6.8 with 1M sodium hydroxides regulation pH, with distilled water constant volume to 100mL, adds 1g trypsase.) before use 37 DEG C plus
Hot 10min.The peptic digest stage adds 4mL simulation intestinal digestion liquid after terminating, 6 h of lasting stirring digestion at 37 DEG C of water-bath.
(4) above-mentioned digestive juice removes high molecular weight protein, gained supernatant in 100 DEG C of enzyme deactivation 10min, 5000 × g centrifugation 10min
Liquid is anti-oxidation peptide liquid.
0.04g pepsin-solubilized collagens (extracting in laboratory) wherein are weighed in step (1), are dissolved with 2mL distilled water, it is molten
Liquid heats 10min, hereinafter referred to as PSC in 100 DEG C of water-bathsH。
High temperature enzyme deactivation is wherein used in step (4), centrifuges to remove high molecular weight protein and undissolved impurity, obtains antioxygen
Change peptide liquid.
Advantage of the invention is that:
(1) present invention uses heat pre-treatment collagen, and collagen can be made to be easier to be digested absorption.
(2) present invention using bionical dynamic mouse peptic digest system and device carries out simulation peptic digest, during simulation mouse stomach
Wriggling, three compression circulations of each minute, and the compression amplitude of fixed stomach make digestion more thorough.
(3) present invention using bionical dynamic mouse peptic digest system and device carries out simulation peptic digest, during in syringe pump
In the presence of simulate gland with necessarily secrete speed add simulate the gastric juice, peptic digest terminate after introduce intestinal digestion process, two steps
Enzymatic isolation method so that the albumen in enzymolysis liquid is less.
(4) digestive system of present invention simulation animal, process is dynamic model digestion, in food security aspect, than other
Antioxidation polypeptide production, such as the product more safe and reasonable obtained by fermentation method and artificial synthesized method.
Brief description of the drawings
Fig. 1 is the preparation flow figure of anti-oxidation peptide liquid.
Fig. 2 is standard molecular weight albumen and polypeptide reference substance chromatogram (A);Enzyme after bionical dynamic mouse peptic digest system digestion
Solve the relative molecular mass distribution figure (B) of liquid.
Fig. 3 heats collagen sample total amino acid test chromatogram.
Fig. 4 is bionical dynamic mouse stomach simulation digestive system material object installation diagram, wherein (1) motor;(2) FREQUENCY CONTROL;(3)
Axle;(4) gusset;(5) spring;(6) pressing plate;(7) external bionic-mouse stomach;(8) supporting plate.
Fig. 5 is simulation mouse stomach schematic diagram.
Embodiment
Bionical dynamic mouse peptic digest system and device (Fig. 4) buys gained by laboratory, and structure of a soft silica gel by being made up
Mouse stomach model, incubation chamber, external electric compression set and simulate the gastric juice secretion device four part composition.Simulate mouse stomach (Fig. 5)
Making be that stomach is taken out in Mice Body, will be translated into inside it outside, then pour model with specific paraffin, cut after reparation
The mouth of pipe for secreting gastric juice and emptying food is opened and flowed out, makes mouse stomach model with silica gel afterwards.Simulate the size of mouse stomach for 40 ×
30 × 21 mm, internal maximum water storage volume are 9mL, and relatively thin glandular stomach thickness is 0.65 ± 0.11mm, and glandular stomach thickness is 1.22
± 0.15mm.Duodenum and esophagus load onto check valve in the pharynx of the junction of stomach and pylorus, and the check valve of pharynx makes gas can be with
Into to ensure that the air pressure in digestion process stomach is constant, the check valve at pylorus can only empty digest, prevent its backflow
Enter in stomach.
External electric compression set is adjusted to close to 3 circulations i.e. per minute of true mouse peristole contraction frequency by motor.
By adjusting the angle (position of the fulcrum of spring) of gusset and the screw position of compression plate, thus it is possible to vary the initial bit of compression plate
Height i.e. amplitude is put and vertically moves, to ensure that solid particle is effectively broken, amplitude should be located at 4mm.
The present invention carries out bionical dynamic mouse peptic digest to the collagen of heat pre-treatment and simulation intestinal digestion prepares antioxygen
Change peptide, and the formation process of anti-oxidation peptide is monitored from the changes of contents of amino with process middle reaches, utilize high performance liquid chromatography
Determine the relative molecular weight distribution that bionical dynamic mouse stomach terminates rear digestive juice.Implementing procedure is as shown in Figure 1.
(1) soluble Amino assay
Appropriate digestive juice is taken in digestion process per 0.5h, 100 DEG C of water-bath 10min carry out destroy the enzyme treatment, 5000 × g centrifugations
10min removes high molecular weight protein.Take 125 μ L digestive juices centrifuged supernatants and phosphate buffer (pH8.2) and 2,4,6- trinitro-s
Benzene sulfonic acid solution concussion is well mixed, and 50 DEG C of water-bath lucifuges react 30min, with sodium sulfite solution terminating reaction, room temperature decentralization
After putting 15min, it is seen that light spectrophotometer method determines light absorption value under 420nm wavelength, and identical test method is surveyed with L-Leu
Determine free amino group content, standard curve (y=14.429x+0.0062, coefficient R are drawn with this2=0.9925), blank group
Enzymolysis liquid is replaced to determine with distilled water.
(2) polypeptide molecular weight is distributed
The chromatograph of Agilent 1260, chromatographic column:TSKgel-G2000SWXL(300mm × 7.8mm, Tosoh);Mobile phase:
Ultra-pure water prepares 0.1mol/L Na2SO4+ 0.1mol/L phosphate buffers (pH6.7) are well mixed, ultrasound degassing;Test column temperature
30℃;UV-detector wavelength:220nm;Detect flow velocity:0.5mL/min;The sample suitable μ L of multiple 0.22 of flowing phase dilution
Water film filtering ability loading, sample size is 10 μ L.
(3) total amino acid determines
The good collagen solution of 1mL heat pre-treatments is weighed in ampoule bottle, adds 5mL 6mol/L HCl, concussion is mixed
It is even.Alcohol blast burner seals, and 110 DEG C of oil baths hydrolyze 24h.Funnel filtering hydrolyzate (deionized water residue in washing bottle repeatedly
Matter), clarified solution constant volume in 50mL volumetric flasks.The sample liquid after 1mL constant volumes is taken in 1.5mL centrifuge tube, 60 DEG C of centrifugations are dense
Contracting repeats to be evaporated with deionized water again, and then plus 1mL amino acid analysis dilutions, oscillator mix, and syringe extracts, warp
Cross loading in 0.22 μm of water system filter membrane injection amino acid analysis bottle and carry out amino acid analysis measure.
Example 1:
Weighing the lyophilized pepsin-solubilized collagens of 0.04g, (collagen prepares gained by laboratory, and chicken feet skin is in 4 DEG C of bars
Under part, foreigh protein removing, fat and color are removed using 20% (w/v) NaCl (0.05mol/L Tris-HCl, pH7.5) solution
The impurity such as element.Pretreated chicken feet skin is carried with the 0.5M acetic acid of the precooling containing 0.1% (w/v) pepsin (2500U/mg)
Take, centrifuge, supernatant saltout gained precipitation vacuum freeze drying be pepsin-solubilized collagen.), 2mL distilled water is added, it is molten
Liquid heats 10min in 100 DEG C of water-baths.In experiment in-vitro simulated stomach and intestine are carried out with the collagen solution after heat pre-treatment
Digestion.Bionical dynamic mouse peptic digest system sets external electric compression set to be adjusted to receive close to true mouse peristole by motor
Contracting frequency is 3 circulations per minute, and amplitude is located at 3mm, and at 37 DEG C, syringe pump selects mode of operation, and to irrigate, groundwater increment is
3.4mL, flow 0.85mL/h, infusion time 4h.After 37 DEG C of preheating 10min of digestive system device, before dress sample in mouse stomach model
Face first adds 0.6mL simulate the gastric juices to simulate mouse stomach gastric juice residual, then adds 2mL samples and carries out simulating peptic digest 4h.Take out
Mixture, without enzyme deactivation, 37 DEG C of preheating 10min simulation intestinal digestion liquid is added, in 37 DEG C of thermostat water baths, is stirred, digestion
6h, at regular time and quantity sampling detect digestion process.The simulation stomach and intestine for the collagen that digestion can obtain heat pre-treatment after terminating disappear
Change anti-oxidation peptide liquid.The free amine group content of digestion process is determined, determines to simulate peptic digest and terminate free amine group content and is
0.015 mmol/L, it is 0.0201mmol/L that intestinal digestion, which terminates rear content,.Relative molecular mass accounts for 200-5000Da's
86.86%.
Example 2:
The lyophilized pepsin-solubilized collagens of 0.02g (laboratory is made by oneself, with embodiment 1) are weighed, add 2mL distilled water, it is molten
Liquid heats 10min in 100 DEG C of water-baths.In experiment in-vitro simulated stomach and intestine are carried out with the collagen solution after heat pre-treatment
Digestion.Bionical dynamic mouse peptic digest system sets external electric compression set to be adjusted to receive close to true mouse peristole by motor
Contracting frequency is 3 circulations per minute, and amplitude is located at 4mm, and at 37 DEG C, syringe pump selects mode of operation, and to irrigate, groundwater increment is
3.4mL, flow 0.85mL/h, infusion time 4h.After 37 DEG C of preheating 10min of digestive system device, before dress sample in mouse stomach model
Face first adds 0.6mL simulate the gastric juices to simulate mouse stomach gastric juice residual, then adds 2mL samples and carries out simulating peptic digest 4h.Take out
Mixture, without enzyme deactivation, 37 DEG C of preheating 10min simulation intestinal digestion liquid is added, in 37 DEG C of thermostat water baths, is stirred, digestion
6h, at regular time and quantity sampling detect digestion process.The simulation stomach and intestine for the collagen that digestion can obtain heat pre-treatment after terminating disappear
Change anti-oxidation peptide liquid.
The free amine group content of digestion process is determined, determines to simulate peptic digest and terminate free amine group content and is
0.0096mmol/L, it is 0.014mmol/L that intestinal digestion, which terminates rear content,.Molecular weight distribution such as Fig. 2, relative molecular mass exist
200-5000Da's accounts for 87.83%.The total amino acid composition (residue number/1000 residue) of heat pre-treatment collagen is surveyed
Determine result such as table 1.
Table 1 heats the amino acid composition (residue number/1000 residue) of collagen solution
Claims (5)
1. the method that the bionical dynamic analog peptic digest of heat pre-treatment collagen and simulation intestinal digestion prepare anti-oxidation peptide, its
It is characterised by following the steps below:
(1)Pepsin-solubilized collagen is dissolved with distilled water, high temperature bath processing, obtains raw material;
(2)Build bionical dynamic mouse peptic digest system:Syringe pump selection mode of operation is to irrigate in experiment, groundwater increment 3.4
ML, the mL/h of flow 0.85, infusion time are 4 h;
After 37 DEG C of 10 min of preheating of digestive system device, mouse stomach model the inside first adds 0.6 mL simulate the gastric juices to simulate before filling sample
Mouse stomach gastric juice remains, and then adds 2 mL samples and carries out simulation peptic digest;
(3)Simulate intestinal digestion system:37 DEG C of 10 min of heating before simulation intestinal digestion liquid use;
The peptic digest stage adds 4 mL simulation intestinal digestion liquid after terminating, 6 h of lasting stirring digestion at 37 DEG C of water-bath;
(4)Above-mentioned digestive juice centrifuges 10 min removing high molecular weight proteins, gained supernatant in 100 DEG C of enzyme deactivations 10min, 5000 × g
As anti-oxidation peptide liquid.
2. the bionical dynamic mouse stomach of the heat pre-treatment collagen according to described by claim 1 and simulation intestinal digestion prepare anti-
The method for aoxidizing peptide liquid, it is characterised in that step(1)Weigh 0.04 g pepsin-solubilized collagens(Extract in laboratory), with 2 mL
Distilled water dissolves, and solution heats 10 min, hereinafter referred to as PSC in 100 DEG C of water-bathsH。
3. the bionical dynamic mouse stomach of the heat pre-treatment collagen according to described by claim 1 and simulation intestinal digestion prepare anti-
The method for aoxidizing peptide liquid, it is characterised in that step(2)Middle simulation peptic digest liquid is formulated as weighing 10.42 g pepsins,
0.02 g sodium acid carbonates, 0.44 g sodium chloride, 0.08 g gastrons(mucin), in distilled water, 50 mL hold stirring and dissolving
Measuring bottle constant volume, it is 1.63 ± 0.01 with 1M HCl adjustment pH.
4. the bionical dynamic mouse stomach of the heat pre-treatment collagen according to described by claim 1 and simulation intestinal digestion prepare anti-
The method for aoxidizing peptide liquid, it is characterised in that step(3)The preparation of middle simulation intestinal digestion liquid:0.68 g potassium dihydrogen phosphates are weighed, with 1
M sodium hydroxides regulation pH is 6.8, with distilled water constant volume to 100 mL, adds 1 g trypsase.
5. the bionical dynamic mouse stomach of the heat pre-treatment collagen according to described by claim 1 and simulation intestinal digestion prepare anti-
The method for aoxidizing peptide liquid, it is characterised in that step(4)Using high temperature enzyme deactivation, then by centrifugation removing high molecular weight protein and not
Dissolved impurity, obtain antioxidation polypeptide liquid.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN110713534A (en) * | 2019-11-29 | 2020-01-21 | 福建农林大学 | Collagen peptide with photoaging improvement effect and preparation method thereof |
CN114155770A (en) * | 2021-11-18 | 2022-03-08 | 浙江工商大学 | Method for simulating old people to digest semi-solid food based on semi-dynamic digestion system |
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CN103882083A (en) * | 2014-02-26 | 2014-06-25 | 江苏大学 | Method for preparing antioxidant collagen peptide |
CN104263786A (en) * | 2014-09-09 | 2015-01-07 | 江苏大学 | Method for preparing rapeseed dreg protein antioxidative peptide solution by gastrointestinal simulated digestion |
CN104263789A (en) * | 2014-09-29 | 2015-01-07 | 江苏大学 | Method for preparing navodon septentrionalis skin anti-oxidative peptide liquid through in vitro simulation of gastrointestinal digestion |
CN105702146A (en) * | 2015-12-31 | 2016-06-22 | 南通东概念新材料有限公司 | Bionic dynamic mouse stomach-duodenum digestive system simulation device and simulation experiment method |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN103882083A (en) * | 2014-02-26 | 2014-06-25 | 江苏大学 | Method for preparing antioxidant collagen peptide |
CN104263786A (en) * | 2014-09-09 | 2015-01-07 | 江苏大学 | Method for preparing rapeseed dreg protein antioxidative peptide solution by gastrointestinal simulated digestion |
CN104263789A (en) * | 2014-09-29 | 2015-01-07 | 江苏大学 | Method for preparing navodon septentrionalis skin anti-oxidative peptide liquid through in vitro simulation of gastrointestinal digestion |
CN105702146A (en) * | 2015-12-31 | 2016-06-22 | 南通东概念新材料有限公司 | Bionic dynamic mouse stomach-duodenum digestive system simulation device and simulation experiment method |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN110713534A (en) * | 2019-11-29 | 2020-01-21 | 福建农林大学 | Collagen peptide with photoaging improvement effect and preparation method thereof |
CN114155770A (en) * | 2021-11-18 | 2022-03-08 | 浙江工商大学 | Method for simulating old people to digest semi-solid food based on semi-dynamic digestion system |
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