CN103484516B - A kind of preparation method of two step enzymolysis process production high-quality hyperglobulinemia peptides - Google Patents
A kind of preparation method of two step enzymolysis process production high-quality hyperglobulinemia peptides Download PDFInfo
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a kind of preparation method of two step enzymolysis process production high-quality hyperglobulinemia peptides, comprising: fresh pig blood adds appropriate Trisodium Citrate anti-freezing, be separated with industrial tubular-bowl centrifuge high speed centrifugation, obtain fresh red blood corpuscle; Fresh red blood corpuscle is added appropriate water, regulates pH, add appropriate aspartic protease S1 enzymolysis, centrifugal simultaneously, removing centrifugation part obtains primary enzymolysis destainer; Primary enzymolysis destainer adds appropriate neutral protease S2 enzymolysis, and secondary enzymolysis liquid is warming up to 55 ~ 60 DEG C of insulation 3h after adding the harmless noresidue chemical decolorization agent of seldom amount can obtain secondary enzymolysis destainer; Secondary enzymolysis destainer adopts spraying dry after falling liquid film or ultrafiltration and concentration, just can obtain lurid enzymolysis protein powder.The object of the present invention is to provide that a kind of technique is simple, the preparation method of the high two step enzymolysis process production high-quality hyperglobulinemia peptides of production efficiency.
Description
Technical field
The present invention relates to hyperglobulinemia peptide field, particularly relate to a kind of preparation method of two step enzymolysis process production high-quality hyperglobulinemia peptides.
Background technology
Pig blood is a kind of byproduct of food industry be worth containing better nutritivity, protein content is up to 18.0%, wherein containing 20 kinds of natural amino acids, 8 kinds of essential amino acids content account for 44.3%, and aminoacids content is balanced, especially Methionin, tryptophane are high, are the protein resources of a kind of high-quality and cheapness.Porcine haemoglobin major part is oxyphorase (Hemoglobin), is present in red blood corpuscle, is made up of protoheme and globin, account for the 8O% of gross protein.PINPROL directly eats palatability difference and is difficult to digest and assimilate, in addition, a large amount of hemochrome is discharged in enzymolysis process, ferrous iron in hemochrome can be oxidized to ferric iron rapidly, enzymolysis solution is made to present Vandyke brown, affect purity and the quality of product, trace adds the Oxidation of Fat and Oils that will add in instant food, causes the deterioration of food color and local flavor.At present owing to not being applicable to business-like ripe decolouring technology, PINPROL is mainly processed to blood meal and uses as animal-feed, seriously constrains the exploitation of PINPROL.Therefore, decolouring is one of gordian technique of effectively utilizing of PINPROL.The pig blood hyperglobulinemia that can take on a new look by decolouring is difficult to received color, obtains the hyperglobulinemia peptide class product of the food grade of high-quality simultaneously, significantly promotes the added value of oxyphorase.
In recent decades, be separated the method that protoheme prepares high-quality protein product in order to study from oxyphorase, the scientific worker of countries in the world has done various trial, has made significant headway.On the whole, be to be divided into the method such as solvent decoloring method, sorbent material decoloring method, oxidizing decoloring method, acidifying oxidative decoloration method, enzymolysis absorption.Initial is adopt organic solvent method mostly, and from oxyphorase, remove protoheme as the people such as Tybor (1975) describe with the acetone soln of acidifying, the method causes organic solvent residual, and albumen is difficult to recycle, and environmental pollution is larger.People's (nineteen fifty-three) mistake carboxymethyl cellulose absorption protohemes such as the people such as Sato (1981) and Auto remove the heme moiety in haemoglobin molecule, and aforesaid method is all the method for somewhat expensive.Therefore, their successes are commercially restricted greatly.Some scholars, as Bingold(1949), Oord and Wesdorp (1979), and people's discovery hydrogen peroxide oxidation such as Mitsyk and Osadchaya (1970) is the method that oxyphorase very effectively removes protoheme.Danish Patent Application N0.5505/86 describes the method with mechanical treatment blood cell, to prepare the oxyphorase of decolouring, wherein add acid with adjust pH to 1 ~ 2, and add the oxygenant of 1-5%, particularly hydrogen peroxide, in reaction process and the carbohydrate derivative had containing dienol group prevents the oxidation of sulfydryl.Then the oxyphorase of by-product recovery decolouring is removed.European patent application, publication No.148114 discloses a kind of method preparing the oxyphorase of decolouring, wherein in pH=3.5 ~ 4, and processes whole blood when having decomposition of protein enzyme, to make globin sex change, thus heme moiety is made to be more vulnerable to the effect of oxygenant.Afterwards on this basis, Dane Yue Genweisima Byrd is gloomy proposed acidifying oxidative decoloration method, and achieved the patent (patent No.: CN88106521.8 in 1989 in China; Proprietary term: the preparation method being substantially free of the blood protein of protoheme).Add acid to reduce pH value to hemocyte suspension in the method, to make the red blood corpuscle cracking be present in suspension, and discharge oxyphorase, and heme moiety is separated from most oxyphorase.D/d protoheme gathers and forms precipitation, and protoheme precipitation is centrifuged together with cell debris and other solid matter to be removed, and then protoheme oxidizer treatment a small amount of in hemoglobin solutions is degraded to make it.This method does not need to use expensive chemical for avoiding the oxygenizement of protein portion, thus the oxyphorase of decolouring is prepared in more economical mode, the decoloring method of PINPROL is made to have striden forward major step, commercially there is certain use value, but this method is very high to the requirement of centrifugal force, below the 13000g oxyphorase rate of recovery is on the low side, decolouring is thorough not, and oxyphorase be still macromolecular, and palatability is poor and be difficult to the problems such as digested absorption.The problem of digested absorption is difficult in order to solve oxyphorase macromole, researchist afterwards, as (Houher, Synowiecki, ockerman) adopts the oxyphorase in enzymic hydrolysis extraction erythrocyte, enzymolysis product adopts activated carbon adsorption Apocytochrome porcine hemoglobin enzymolysis matter, but the oxyphorase matter rate of recovery is only 60 ~ 70%, enzymolysis product has heavier bitter taste, and protoheme is difficult to recycle.
China scientific worker has also carried out long-term research to PINPROL decolouring technology, has report to adopt the method such as ethanol and acetone, hydrogen peroxide, sodium alginate, carboxymethyl cellulose/sodium, carboxymethyl starch, protease hydrolyzed to decolour to PINPROL.Such as, Yang Wei and Peng Zhenrong (1996) report thermoase enzymolysis oxyphorase decolorizing with activated carbon prepares oxyphorase powder.Yang Yanjun (1997) once report carboxymethyl starch decolours to PINPROL enzymolysis solution.Chu Jie etc. (1999) adopt Sumizyme MP and neutral protease enzymatic hydrolysis pig blood, and decolorizing with activated carbon prepares PINPROL enzymolysis powder.Two kinds of enzyme composite hydrolysis are adopted, carboxymethyl starch decolouring preparation hydrolysis oxyphorase in (2007) such as Mei Juan Chu Jie.Guo is kind, and wide wait (2007) adopt compound protease (PancreatinandProtamex) enzymolysis, charcoal absorption decolouring preparation PINPROL.Sun Qian etc. (2009) adopt stomach en-enzymolysis, Hollow Fiber Ultrafiltration preparation decolouring PINPROL enzymolysis product.But these researchs are still in the laboratory study stage substantially, there is decolouring insufficient, oxyphorase loss is excessive; dissolvent residual, purity is not high, and product is poorly soluble; be difficult to the problem of the aspects such as large-scale production, distance business-like production technology can also have very large distance.In addition, the patent of China's existing preparation decolouring oxyphorase has " preparing edible heme (publication number: CN93108676.0) with stomach en-or gastric mucosa of animal hydrolysis of animal blood ", and this technique is more complicated, and decolorizing effect is poor; " a kind of method (publication number: CN1094618A) extracting protoheme and protein powder from poultry blood ", mainly with organic solvent extraction protoheme, there is organic pollution problem in this technique; " preparation method's (publication number: CN200710031826.4) of Apocytochrome porcine hemoglobin enzymolysis matter porcine hemoglobin zymolyte and heme peptide ", this process characteristic and advantage are: adopt compound protease to carry out efficient controlled hydrolysis to oxyphorase; Isoelectric point precipitation is adopted to realize the high efficiency separation of protease hydrolysis products and heme peptide; Product is without advantages such as bad flavors.But need are high-pressure homogeneous or supersound process carries out haemolysis process to erythrocyte, and need repeatedly repeatedly centrifugal, technique more complicated, repeatedly adjust ph saliferous is more, decolouring is problem not thoroughly.
In recent years, abroad make great progress the decolouring enzymolysis technical study of PINPROL, existing business-like production technology, quality product there occurs qualitative leap.Exploitation PINPROL deep process technology, expands its Application Areas, improves developing direction and trend that value-added content of product becomes world pig blood comprehensive development and utilization.
Summary of the invention
In order to solve the problems of the technologies described above, the object of the present invention is to provide that a kind of technique is simple, the preparation method of the high two step enzymolysis process production high-quality hyperglobulinemia peptides of production efficiency.
Complete skill scheme of the present invention is, a kind of preparation method of two step enzymolysis process production high-quality hyperglobulinemia peptides, comprises the following steps:
(1), erythrocytic preparation: fresh pig blood adds appropriate Trisodium Citrate anti-freezing, is separated, obtains fresh red blood corpuscle with industrial tubular-bowl centrifuge high speed centrifugation;
(2), erythrocytic primary enzymolysis decolouring: fresh red blood corpuscle is added appropriate water, regulates pH, add appropriate aspartic protease S1 enzymolysis, centrifugal simultaneously, removing centrifugation part obtains primary enzymolysis destainer;
(3), secondary enzymolysis decolouring: primary enzymolysis destainer adds appropriate neutral protease S2 enzymolysis, and secondary enzymolysis liquid is warming up to 55 ~ 60 DEG C of insulation 3h after adding the harmless noresidue chemical decolorization agent of seldom amount can obtain secondary enzymolysis destainer;
(4), the concentrated and spraying dry of secondary enzymolysis liquid: secondary enzymolysis destainer adopts spraying dry after falling liquid film or ultrafiltration and concentration, just can obtain lurid enzymolysis protein powder.
Erythrocytic primary enzymolysis decolouring: fresh red blood corpuscle is added appropriate water, regulates pH to 2.0 ~ 3.0.
Add appropriate aspartic protease S1, constant temperature enzymolysis 8h under 37 ~ 40 DEG C of conditions, with the centrifugal 5min of 5000g, removing centrifugation part obtains primary enzymolysis destainer.
Appropriate neutral protease S2 is added, constant temperature enzymolysis 8h under 40 DEG C of conditions at described primary enzymolysis destainer.
Therefore the present invention has following beneficial effect compared with present technology:
The preparation method of the present invention two step enzymolysis process production high-quality hyperglobulinemia peptide, desolventing technology removes the protoheme in PINPROL substantially, and the removing blood smell, makes hemoglobin product have good appearance luster, odorless; Make oxyphorase carry out controllable appropriateness degraded, be transformed into the oligopeptides of absorption easy to digest, little peptide, amino acids material, product has good water-soluble; After PINPROL decolouring enzymolysis, there is higher product recovery rate; Technology is applicable to the industrial production feature of automatization, serialization, mass-producing, and production cost will commercially produced within the scope of acceptable; The simpler less energy consumption of technical process of the present invention; mild condition; the requirement of serialization, large-scale production can be met; it is that auxiliary method carries out PINPROL decolouring that the present invention adopts comparatively unique centrifugation to be main, chemical decolorization; decolorizing efficiency is high; good decolorizing effect; overcome drawback and the deficiency of single decoloration method in the past; another technical characteristic comparatively given prominence to of the present invention is that not only PINPROL decolouring is more thorough; and oxyphorase degradable is abundant; the rate of recovery is high, decolouring enzymolysis protein powder good water solubility.
Accompanying drawing explanation
Accompanying drawing described herein is used to provide a further understanding of the present invention, forms a application's part, does not form inappropriate limitation of the present invention, in the accompanying drawings:
Fig. 1 is process schematic of the present invention.
Embodiment
Describe the present invention in detail below in conjunction with accompanying drawing and specific embodiment, be used for explaining the present invention in this illustrative examples of the present invention and explanation, but not as a limitation of the invention.
Embodiment 1:
The preparation method of a kind of two step enzymolysis process production high-quality hyperglobulinemia peptides of the present embodiment, comprises the following steps:
1, erythrocytic preparation: fresh pig blood adds appropriate Trisodium Citrate anti-freezing, is separated with industrial tubular-bowl centrifuge high speed centrifugation, obtains fresh red blood corpuscle.
2, erythrocytic primary enzymolysis decolouring: fresh red blood corpuscle is added appropriate water, regulate pH to 2.0 ~ 3.0, add appropriate aspartic protease S1, constant temperature enzymolysis 8h under 37 ~ 40 DEG C of conditions simultaneously, with the centrifugal 5min of 5000g, removing centrifugation part obtains primary enzymolysis destainer.
3, secondary enzymolysis decolouring: primary enzymolysis destainer adds appropriate neutral protease S2, constant temperature enzymolysis 8h under 40 DEG C of conditions, secondary enzymolysis liquid is warming up to 55 ~ 60 DEG C of insulation 3h after adding the harmless noresidue chemical decolorization agent of seldom amount can obtain secondary enzymolysis destainer.
4, the concentrated and spraying dry of secondary enzymolysis liquid: secondary enzymolysis destainer adopts spraying dry after falling liquid film or ultrafiltration and concentration, just can obtain lurid enzymolysis protein powder.
Fresh pig blood is separated the plasma proteins (otherwise processed) obtaining 60% through supercentrifuge, the hyperglobulinemia of about 40% amount of liquid enters enzymatic vessel, adds water and reconciles pH value 2-3, adds aspartic protease 40 DEG C of enzymolysis 8 hours, obtains primary enzymolysis liquid.Oxyphorase is mainly carried out Partial digestion by the effect of primary enzymolysis, makes protoheme be separated with oxyphorase simultaneously.Primary enzymolysis liquid obtains through centrifugation the primary enzymolysis destainer accounting for amount of liquid 85%, primary enzymolysis destainer is sorrel in test tube, and then add neutral protease 40 DEG C of enzymolysis 8 hours, add oxidizing decolouring and obtain secondary enzymolysis destainer, secondary enzymolysis destainer is faint yellow in test tube.Secondary enzymolysis destainer water content about 95%, enters cryogenic vacuum concentration tank below 50 DEG C, is concentrated to the hyperglobulinemia hydrolysis powder product that 85% rear spraying dry obtains creamy white.
The know-why of research
The bleaching principle of 1 oxyphorase: in red blood corpuscle, the overwhelming majority is red corpuscle, in addition containing being less than other hemocyte, hemocyte can discharge oxyphorase by rapid cleavage in acid condition, under the effect of acidic protein, oxyphorase is by Partial digestion, fully be dissolved in aqueous phase, because protoheme is insoluble in water in acid condition, with this understanding protoheme very easy from oxyphorase separate with cracking after hemocyte fragment form large particle, separate from the aqueous solution after centrifugal, desolventing technology can realize the separation of most of protoheme for the first time.Destainer is more conducive to being separated of a small amount of protoheme and oxyphorase after secondary enzymolysis, now can realize the Hemoglobin Peptide after to enzymolysis with the nontoxic residue-free chemical decolorization agent of seldom measuring again to decolour fully, substantially protoheme can be removed clean.
The deodorizing degradation principles of 2 oxyphorases: the bad smell of oxyphorase is caused by the existence of protoheme and other unknown taste compound, these materials fully can be removed, can eliminate the bad smell of oxyphorase after secondary enzymolysis and decolouring.Secondary enzymolysis can make oxyphorase more fully degrade, and destroys the tetramer structure that oxyphorase is original, obtains the enzymolysis product being mainly oligopeptides, group material.
Two step enzymolysis decoloring method hyperglobulinemia hydrolysis powder product the key technical indexes
Indicators of overall performance compares with domestic and international modern technique
1, the comparison of decolorizing effect: the decolouring of this technology hemoglobin product is comparatively thorough, and product appearance is light yellow, substantially not containing protoheme.The decoloring method of this technology and the gloomy acidifying oxidative decoloration method (patent No.: CN88106521.8) of Dane Yue Genweisima Byrd have similarity, but decolorizing effect is better, before Yue Genfa oxidative decoloration, protein liquid is chocolate, before this technology oxidative decoloration, protein liquid is sorrel, is faint yellow afterwards.The decoloring method of this technology and the enzymolysis isoelectric precipitation decoloring method (patent No.: CN200710031826.4) of Cui Chun also have similarity, and both collapse due to massive hemorrhage effects are substantially identical, but this technology decolorization process is simpler.
2, the comparison of decoloration process technology: the decolouring of this technology is the organic assembling of enzymolysis, acidifying isoelectric precipitation and oxidative decoloration, combines the advantage of multiple decoloring method, and decoloration process is simple, can realization flowization operation.About the acid precipitation step of root needs the centrifugal force 10min of more than 13000g, oxidative decoloration step needs 48h, centrifugal of this technology needs the centrifugal force 3min of more than 5000g, and oxidative decoloration need only 3h, greatly reduces the requirement of centrifugal force and shortens the oxidative decoloration time.The enzymolysis isoelectric precipitation collapse due to massive hemorrhage method of Cui Chun need to enzymolysis solution carry out 3 times centrifugal, 2 washing of precipitate process, operation more complicated; large-scale production applicability is poor; and carry out 3 adjustment pH in treating processes, more acid & alkali liquid need be added, more salinity can be brought into.General enzymolysis oxyphorase all needs to carry out high temperature (more than 90 DEG C) inactivation treatment to enzyme, a large amount of liquid heat energy consumption is large, remarkable increase production cost, this combine with technique 60 DEG C of oxidative decolorations and destainer spray drying step carry out inactivation treatment to twice enzymolysis enzyme, remarkable reduction energy consumption, cost-saving.
3, the comparison of product performance: the PINPROL enzymolysis powder that this technology is produced, the rate of recovery of protein can reach more than 90%, protein content is more than 95%, ash content is lower than 5%, the content of protoheme is lower than 0.1%, the peptide matters of molecular weight within the scope of 5000KDa accounts for more than 80%, and the nitrogen soluble index (NSI) of hydrolyzed solution in trichoroacetic acid(TCA) (TCA) is greater than 80%.The rate of recovery of protein and the content of small-molecular peptides higher, other index is suitable with the modern technique reported both at home and abroad.
Therefore, the preparation method of the present invention two step enzymolysis process production high-quality hyperglobulinemia peptide, desolventing technology removes the protoheme in PINPROL substantially, and the removing blood smell, makes hemoglobin product have good appearance luster, odorless; Make oxyphorase carry out controllable appropriateness degraded, be transformed into the oligopeptides of absorption easy to digest, little peptide, amino acids material, product has good water-soluble; After PINPROL decolouring enzymolysis, there is higher product recovery rate; Technology is applicable to the industrial production feature of automatization, serialization, mass-producing, and production cost will commercially produced within the scope of acceptable; The simpler less energy consumption of technical process of the present invention; mild condition; the requirement of serialization, large-scale production can be met; it is that auxiliary method carries out PINPROL decolouring that the present invention adopts comparatively unique centrifugation to be main, chemical decolorization; decolorizing efficiency is high; good decolorizing effect; overcome drawback and the deficiency of single decoloration method in the past; another technical characteristic comparatively given prominence to of the present invention is that not only PINPROL decolouring is more thorough; and oxyphorase degradable is abundant; the rate of recovery is high, decolouring enzymolysis protein powder good water solubility.
Above the technical scheme that the embodiment of the present invention provides is described in detail, apply specific case herein to set forth the principle of the embodiment of the present invention and embodiment, the explanation of above embodiment is only applicable to the principle helping to understand the embodiment of the present invention; Meanwhile, for one of ordinary skill in the art, according to the embodiment of the present invention, embodiment and range of application all will change, and in sum, this description should not be construed as limitation of the present invention.
Claims (1)
1. a preparation method for two step enzymolysis process production high-quality hyperglobulinemia peptides, is characterized in that, comprise the following steps:
(1), erythrocytic preparation: fresh pig blood adds appropriate Trisodium Citrate anti-freezing, is separated, obtains fresh red blood corpuscle with industrial tubular-bowl centrifuge high speed centrifugation;
(2), erythrocytic primary enzymolysis decolouring: fresh red blood corpuscle is added appropriate water, regulates pH, add appropriate aspartic protease S1 enzymolysis, centrifugal simultaneously, removing centrifugation part obtains primary enzymolysis destainer;
(3), secondary enzymolysis decolouring: primary enzymolysis destainer adds appropriate neutral protease S2 enzymolysis, and secondary enzymolysis liquid is warming up to 55 ~ 60 DEG C of insulation 3h after adding the harmless noresidue chemical decolorization agent of seldom amount can obtain secondary enzymolysis destainer;
(4), the concentrated and spraying dry of secondary enzymolysis liquid: secondary enzymolysis destainer adopts spraying dry after falling liquid film or ultrafiltration and concentration, just can obtain lurid enzymolysis protein powder;
Erythrocytic primary enzymolysis decolouring: fresh red blood corpuscle is added appropriate water, regulates pH to 2.0 ~ 3.0;
Add appropriate aspartic protease S1, constant temperature enzymolysis 8h under 37 ~ 40 DEG C of conditions, with the centrifugal 5min of 5000g, removing centrifugation part obtains primary enzymolysis destainer;
Appropriate neutral protease S2 is added, constant temperature enzymolysis 8h under 40 DEG C of conditions at described primary enzymolysis destainer.
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| CN1858228A (en) * | 2006-03-28 | 2006-11-08 | 华南理工大学 | Process for preparing low molecular peptide and amino acid by biological enzymolysis of pig blood haematoglobin |
| CN101748181A (en) * | 2010-01-15 | 2010-06-23 | 白求恩医科大学制药厂 | Method for preparing high-ferric content ferroheme polypeptide composite with pepsin hydrolysis method |
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| CN1858228A (en) * | 2006-03-28 | 2006-11-08 | 华南理工大学 | Process for preparing low molecular peptide and amino acid by biological enzymolysis of pig blood haematoglobin |
| CN101748181A (en) * | 2010-01-15 | 2010-06-23 | 白求恩医科大学制药厂 | Method for preparing high-ferric content ferroheme polypeptide composite with pepsin hydrolysis method |
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| 复合酶水解猪血工艺条件优化研究;李斌;《粮油食品科技》;20090630;第 17 卷(第 6 期);51-53 * |
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