CN103484516A - Preparation method of high quality blood cell protein peptide by using two-step enzymolysis method - Google Patents
Preparation method of high quality blood cell protein peptide by using two-step enzymolysis method Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a preparation method of high quality blood cell protein peptide by using a two-step enzymolysis method, and the preparation method comprises adding an appropriate amount of sodium citrate in fresh pig blood for anticoagulation, using an industrial tubular centrifuge for high-speed centrifugal separation to obtain fresh red blood cells; adding an appropriate amount of water in the fresh red blood cells, regulating pH, adding an appropriate amount of acidic protease S1 for enzymolysis, centrifuging, and removing the centrifugal sedimentation part to obtain a first enzymolysis decolored solution; adding an appropriate amount of neutral protease S2 in the first enzymolysis decolored solution for enzymolysis, adding a very small amount of a harmless non-residual chemical decoloring agent into a secondary enzymatic hydrolysate, heating up to 55-60 DEG C and keeping warm for 3 h to obtain a secondary enzymolysis decolored solution; and performing spray drying of the secondary enzymolysis decolored solution after film falling or ultrafiltration concentration to obtain light yellow enzymolysis protein powder. The invention aims at providing the simple-process high-production-efficiency preparation method of the high quality blood cell protein peptide by using the two-step enzymolysis method.
Description
Technical field
The present invention relates to hyperglobulinemia peptide field, relate in particular to the preparation method that a kind of two step enzymolysis processs are produced high-quality hyperglobulinemia peptide.
Background technology
Pig blood is a kind of byproduct of food industry that better nutritivity is worth that contains, protein content is up to 18.0%, wherein contain 20 kinds of natural amino acids, 8 kinds of essential amino acids content account for 44.3%, and aminoacids content equilibrium, especially Methionin, tryptophane are high, are a kind of high-quality and cheap protein resource.The porcine haemoglobin major part is oxyphorase (Hemoglobin), is present in red blood corpuscle, protoheme and globin, consists of, and accounts for the 8O% of gross protein.PINPROL directly edible palatability is poor and be difficult to digest and assimilate, in addition, discharge a large amount of hemochrome in enzymolysis process, ferrous iron in hemochrome can be oxidized to rapidly ferric iron, make enzymolysis solution present Vandyke brown, affect purity and the quality of product, indium addition will add the Oxidation of Fat and Oils in instant food, causes the bad change of food color and local flavor.Owing to not being applicable to business-like ripe decolouring technology, PINPROL mainly is processed to blood meal and uses as animal-feed, has seriously restricted the exploitation of PINPROL at present.Therefore, decolouring is one of effective gordian technique of 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 high-quality food grade simultaneously, significantly promotes the added value of oxyphorase.
In recent decades, in order to study, separate the method that protoheme prepares high-quality protein product from oxyphorase, the scientific worker of countries in the world has done various trials, has made significant headway.On the whole, be to be divided into the methods such as solvent decoloring method, sorbent material decoloring method, oxygenant oxidative decoloration method, acidifying oxidative decoloration method, enzymolysis absorption.Be to adopt organic solvent method at first mostly, as the people such as Tybor (1975) have narrated with the acetone soln of acidifying, remove protoheme from oxyphorase, the method causes organic solvent residual, and albumen is difficult to recycle, and environmental pollution is larger.People's (nineteen fifty-three) such as the people such as Sato (1981) and Auto cross with carboxymethyl cellulose absorption protoheme and remove the heme moiety in haemoglobin molecule, and aforesaid method is all the method for somewhat expensive.Therefore, their successes commercially are restricted greatly.Some scholars, as Bingold(1949), Oord and Wesdorp (1979), and the people such as Mitsyk and Osadchaya (1970) find that with hydrogen peroxide oxidation be the method that oxyphorase is very effectively removed protoheme.Danish Patent Application N0.5505/86 has narrated the method with the mechanical treatment blood cell, oxyphorase with the preparation decolouring, wherein add acid with adjust pH to 1 ~ 2, and add the oxygenant of 1-5 %, hydrogen peroxide particularly, in reaction process and have an oxidation that prevents sulfydryl containing the carbohydrate derivative of dienol group.Then remove the oxyphorase of by-product recovery decolouring.European patent application, communique No.148114 has disclosed a kind of method for preparing the oxyphorase of decolouring, wherein in pH=3.5 ~ 4, and has in the situation of decomposition of protein enzyme and processes whole blood, in order to make the globin sex change, thereby make heme moiety be more vulnerable to the effect of oxygenant.Afterwards on this basis, the gloomy acidifying oxidative decoloration method that proposed of Dane Yue Genweisima Byrd, and obtained the patent (patent No.: CN88106521.8 in 1989 in China; Proprietary term: the preparation method who is substantially free of the blood protein of protoheme).In the method, to hemocyte suspension, add acid to reduce the pH value, so that be present in the red blood corpuscle cracking in suspension, and discharge oxyphorase, and heme moiety is separated from most oxyphorase.D/d protoheme gathers and forms precipitation, and the protoheme precipitation is centrifuged and removes together with cell debris and other solid matter, then uses protoheme a small amount of in hemoglobin solutions oxidizer treatment so that its degraded.This method does not need to use expensive chemical for avoiding the oxygenizement of protein portion, thereby prepare the oxyphorase of decolouring in more economical mode, make the decoloring method of PINPROL stride forward major step, certain use value is commercially arranged, but this method is very high to the requirement of centrifugal force, the following oxyphorase rate of recovery of 13000g is on the low side, the decolouring is thorough not, and oxyphorase is still the macromole aggregate, and palatability is poor and be difficult to the problem such as digested absorption.The problem that is difficult to digested absorption in order to solve the oxyphorase macromole, researchist afterwards, as adopting enzymic hydrolysis, (Houher, Synowiecki, ockerman) extract the oxyphorase in erythrocyte, enzymolysis product adopts the 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 the PINPROL decolouring technology, has the methods such as report employing ethanol and acetone, hydrogen peroxide, sodium alginate, carboxymethyl cellulose/sodium, carboxymethyl starch, protease hydrolyzed to be decoloured to PINPROL.For example, Yang Wei and Peng Zhenrong (1996) report prepares the oxyphorase powder with high temperature protease hydrolyzed oxyphorase decolorizing with activated carbon.Yang Yanjun (1997) once reported by carboxymethyl starch the PINPROL enzymolysis solution was decoloured.Chu Jie etc. (1999) adopt Sumizyme MP and neutral protease enzymatic hydrolysis pig blood, and decolorizing with activated carbon prepares PINPROL enzymolysis powder.Adopt two kinds of enzyme composite hydrolysis in (2007) such as the beautiful Chu Jie of U.S., carboxymethyl starch decolouring preparation hydrolysis oxyphorase.Guo is kind, and wide wait (2007) adopt compound protease (Pancreatin and Protamex) enzymolysis, charcoal absorption decolouring preparation PINPROL.Sun Qian etc. (2009) adopt the stomach en-enzymolysis, Hollow Fiber Ultrafiltration preparation decolouring PINPROL enzymolysis product.But these researchs basically or, in the laboratory study stage, the existence decolouring is insufficient, and the oxyphorase loss is excessive; dissolvent residual, purity is not high, and product is poorly soluble; the problem that is difficult to the aspects such as large-scale production, distance can business-like production technology also have very large distance.In addition, the patent of China's existing preparation decolouring oxyphorase have " with stomach en-or gastric mucosa of animal hydrolysis of animal blood prepare edible heme (publication number: CN93108676.0) ", this technique is more complicated, and decolorizing effect is poor; " a kind of method of extracting protoheme and protein powder from poultry blood (publication number: CN1094618A) ",, mainly with the organic solvent extraction protoheme, there is organic pollution problem in this technique; " preparation method of Apocytochrome porcine hemoglobin enzymolysis matter porcine hemoglobin zymolyte and heme peptide (publication number: CN200710031826.4) ", this process characteristic and advantage are: adopt compound protease to carry out efficient controlled hydrolysis to oxyphorase; Adopt isoelectric point precipitation to realize the high efficiency separation of protease hydrolysis products and heme peptide; Product is without advantages such as bad local flavors.But need are high-pressure homogeneous or supersound process is carried out the haemolysis processing to erythrocyte, need be repeatedly repeatedly centrifugal, the technique more complicated, repeatedly regulate pH value saliferous more, and decolouring is problem not thoroughly.
In recent years, abroad the decolouring enzymolysis technical study of PINPROL made great progress, existing business-like production technology, qualitative leap has occurred in quality product.Exploitation PINPROL deep process technology, expand 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 the preparation method that a kind of technique is simple, production efficiency Senior Two step enzymolysis process is produced high-quality hyperglobulinemia peptide.
Complete skill scheme of the present invention is that the preparation method that a kind of two step enzymolysis processs are produced high-quality hyperglobulinemia peptide comprises the following steps:
(1), erythrocytic preparation: fresh pig blood adds appropriate Trisodium Citrate anti-freezing, with industrial tubular-bowl centrifuge high speed centrifugation, separates, and obtains fresh red blood corpuscle;
(2), erythrocytic primary enzymolysis decolouring: fresh red blood corpuscle is added to appropriate water, regulate pH, add appropriate aspartic protease S1 enzymolysis, centrifugal simultaneously, remove centrifugation and partly obtain the primary enzymolysis destainer;
(3), secondary enzymolysis decolouring: the primary enzymolysis destainer adds appropriate neutral protease S2 enzymolysis, and secondary enzymolysis liquid adds after the harmless noresidue chemical decolorization agent of amount seldom and is warming up to 55 ~ 60 ℃ of insulation 3h and can obtains the secondary enzymolysis destainer;
(4), the concentrated and spraying drying of secondary enzymolysis liquid: spraying drying after secondary enzymolysis destainer employing falling liquid film or ultrafiltration and concentration just can obtain lurid enzymolysis protein powder.
Erythrocytic primary enzymolysis decolouring: fresh red blood corpuscle is added to appropriate water, regulate pH to 2.0 ~ 3.0.
Add appropriate aspartic protease S1, constant temperature enzymolysis 8h under 37 ~ 40 ℃ of conditions, with the centrifugal 5min of 5000g, remove centrifugation and partly obtain the primary enzymolysis destainer.
Add appropriate neutral protease S2 at described primary enzymolysis destainer, constant temperature enzymolysis 8h under 40 ℃ of conditions.
Therefore the present invention has compared following beneficial effect with present technology:
The preparation method that the present invention's two step enzymolysis processs are produced high-quality hyperglobulinemia peptide, the basic protoheme of removing in PINPROL is processed in decolouring, removes the blood smell, makes the oxyphorase 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 characteristics of automatization, serialization, mass-producing, and production cost will be in commercially producing the acceptable scope; The simpler less energy consumption of technical process of the present invention; mild condition; can meet the requirement of serialization, large-scale production; it is that master, chemical decolorization are that auxiliary method is carried out the PINPROL decolouring that the present invention adopts comparatively unique centrifugation; decolorizing efficiency is high; good decolorizing effect; drawback and the deficiency of single decoloration method have in the past been overcome; another comparatively outstanding technical characterictic of the present invention is that not only the PINPROL decolouring is more thorough; and the oxyphorase degradable is abundant; the rate of recovery is high, decolouring enzymolysis protein powder good water solubility.
The accompanying drawing explanation
Accompanying drawing described herein is used to provide a further understanding of the present invention, forms the application's a part, does not form inappropriate limitation of the present invention, in the accompanying drawings:
Fig. 1 is process schematic diagram of the present invention;
Fig. 2 is two step enzymolysis decoloring method hyperglobulinemia hydrolysis powder product the key technical indexes.
Embodiment
Describe the present invention in detail below in conjunction with accompanying drawing and specific embodiment, in this illustrative examples of the present invention and explanation, be used for explaining the present invention, but not as a limitation of the invention.
embodiment 1:
The preparation method that a kind of two step enzymolysis processs of the present embodiment are produced high-quality hyperglobulinemia peptide, comprise the following steps:
1, erythrocytic preparation: fresh pig blood adds appropriate Trisodium Citrate anti-freezing, with industrial tubular-bowl centrifuge high speed centrifugation, separates, and obtains fresh red blood corpuscle.
2, erythrocytic primary enzymolysis decolouring: fresh red blood corpuscle is added to appropriate water, regulate pH to 2.0 ~ 3.0, add appropriate aspartic protease S1, constant temperature enzymolysis 8h under 37 ~ 40 ℃ of conditions simultaneously, with the centrifugal 5min of 5000g, remove centrifugation and partly obtain the primary enzymolysis destainer.
3, secondary enzymolysis decolouring: the primary enzymolysis destainer adds appropriate neutral protease S2, constant temperature enzymolysis 8h under 40 ℃ of conditions, secondary enzymolysis liquid adds seldom after the harmless noresidue chemical decolorization agent of amount and is warming up to 55 ~ 60 ℃ of insulation 3h and can obtains the secondary enzymolysis destainer.
4, the concentrated and spraying drying of secondary enzymolysis liquid: spraying drying after secondary enzymolysis destainer employing falling liquid film or ultrafiltration and concentration just can obtain lurid enzymolysis protein powder.
Fresh pig blood separates the plasma proteins (otherwise processed) obtain 60% through supercentrifuge, approximately the hyperglobulinemia of 40% amount of liquid enters enzymatic vessel, adds water and reconciles pH value 2-3, adds aspartic protease 40 ℃ of enzymolysis 8 hours, obtains primary enzymolysis liquid.The effect of primary enzymolysis is mainly that oxyphorase is carried out to Partial digestion, makes protoheme separate with oxyphorase simultaneously.Primary enzymolysis liquid obtains accounting for the primary enzymolysis destainer of amount of liquid 85% through centrifugation, the primary enzymolysis destainer is sorrel in test tube, and then add neutral protease 40 ℃ of enzymolysis 8 hours, add the oxygenant oxidative decoloration to obtain the secondary enzymolysis destainer, it is faint yellow that the secondary enzymolysis destainer is in test tube.Secondary enzymolysis destainer water content approximately 95%, enter the cryogenic vacuum concentration tank and be concentrated to 85% rear spraying drying below 50 ℃ and obtain the hyperglobulinemia hydrolysis powder product of creamy white.
The know-why of research
The bleaching principle of 1 oxyphorase: in red blood corpuscle, the overwhelming majority is red corpuscle, contain in addition and be less than other hemocyte, hemocyte under acidic conditions fast cracking discharge oxyphorase, under the effect of acidic protein, oxyphorase is by Partial digestion, fully be dissolved in water, because protoheme is insoluble in water under acidic conditions, with this understanding protoheme very easily from oxyphorase separate with cracking after the hemocyte fragment form large particle, after centrifugal, from the aqueous solution, separate, the separation that can realize most of protoheme is processed in decolouring for the first time.Destainer more is conducive to separating of a small amount of protoheme and oxyphorase after secondary enzymolysis, now can realize the Hemoglobin Peptide after enzymolysis is decoloured fully with the nontoxic residue-free chemical decolorization agent of seldom measuring again, basically protoheme can be removed clean.
The deodorizing degradation principles of 2 oxyphorases: the stench flavor of oxyphorase is to cause due to the existence of protoheme and other unknown taste compound, through secondary enzymolysis with after decolouring, these materials fully can be removed, and can eliminate the stench flavor of oxyphorase.Secondary enzymolysis can make oxyphorase more fully degrade, and destroys the original tetramer structure of oxyphorase, obtains being mainly the enzymolysis product of oligopeptides, group material.
Two step enzymolysis decoloring method hyperglobulinemia hydrolysis powder product the key technical indexes (as shown in Figure 2)
Indicators of overall performance and the comparison of modern technique both at home and abroad
1, the comparison of decolorizing effect: the decolouring of present technique oxyphorase product is more thorough, and it is light yellow that product appearance is, substantially not containing protoheme.The acidifying oxidative decoloration method (patent No.: CN88106521.8) similarity is arranged that the decoloring method of present technique and Dane Yue Genweisima Byrd are gloomy, but decolorizing effect is better, before the Yue Genfa oxidative decoloration, protein liquid is chocolate, before the present technique oxidative decoloration, protein liquid is sorrel, is faint yellow afterwards.(patent No.: CN200710031826.4) similarity is also arranged, both collapse due to massive hemorrhage effects are basic identical, but present technique decolouring step is simpler for the enzymolysis isoelectric precipitation decoloring method of the decoloring method of present technique and Cui Chun.
2, the comparison of decoloration process technology: the present technique decolouring is the organic assembling of enzymolysis, acidifying isoelectric precipitation and oxidative decoloration, combines the advantage of multiple decoloring method, and decoloration process is simple, but the realization flow operation.Approximately the acid precipitation step of root needs the above centrifugal 10min of centrifugal force of 13000g, the oxidative decoloration step needs 48h, centrifugal of present technique needs the above centrifugal 3min of centrifugal force of 5000g, and oxidative decoloration need only 3h, greatly reduces the requirement of centrifugal force and has shortened 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 washings of precipitate are processed, the operation more complicated; the large-scale production applicability is poor; and carry out 3 times in treating processes and adjust pH, need add more acid & alkali liquid, can bring more salinity into.General enzymolysis oxyphorase all needs enzyme is carried out to high temperature (more than 90 ℃) inactivation treatment, a large amount of liquid heat energy consumptions are large, significantly increase production cost, present technique is carried out inactivation treatment to twice enzymolysis with enzyme in conjunction with 60 ℃ of oxidative decolorations and destainer spraying drying step, significantly reduce energy consumption, cost-saving.
3, the comparison of product performance: the PINPROL enzymolysis powder that present technique 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 in the 5000KDa scope 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 are higher, and other index is suitable with the modern technique of reporting both at home and abroad.
Therefore, the preparation method that the present invention's two step enzymolysis processs are produced high-quality hyperglobulinemia peptide, the basic protoheme of removing in PINPROL is processed in decolouring, removes the blood smell, makes the oxyphorase 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 characteristics of automatization, serialization, mass-producing, and production cost will be in commercially producing the acceptable scope; The simpler less energy consumption of technical process of the present invention; mild condition; can meet the requirement of serialization, large-scale production; it is that master, chemical decolorization are that auxiliary method is carried out the PINPROL decolouring that the present invention adopts comparatively unique centrifugation; decolorizing efficiency is high; good decolorizing effect; drawback and the deficiency of single decoloration method have in the past been overcome; another comparatively outstanding technical characterictic of the present invention is that not only the PINPROL decolouring is more thorough; and the oxyphorase degradable is abundant; the rate of recovery is high, decolouring enzymolysis protein powder good water solubility.
The above technical scheme that the embodiment of the present invention is provided is described in detail, applied specific case herein principle and the embodiment of the embodiment of the present invention are set forth, the explanation of above embodiment is only applicable to help to understand the principle of the embodiment of the present invention; , for one of ordinary skill in the art, according to the embodiment of the present invention, on embodiment and range of application, all will change, in sum, this description should not be construed as limitation of the present invention simultaneously.
Claims (4)
1. the preparation method that the step enzymolysis process is produced high-quality hyperglobulinemia peptide, is characterized in that, comprises the following steps:
(1), erythrocytic preparation: fresh pig blood adds appropriate Trisodium Citrate anti-freezing, with industrial tubular-bowl centrifuge high speed centrifugation, separates, and obtains fresh red blood corpuscle;
(2), erythrocytic primary enzymolysis decolouring: fresh red blood corpuscle is added to appropriate water, regulate pH, add appropriate aspartic protease S1 enzymolysis, centrifugal simultaneously, remove centrifugation and partly obtain the primary enzymolysis destainer;
(3), secondary enzymolysis decolouring: the primary enzymolysis destainer adds appropriate neutral protease S2 enzymolysis, and secondary enzymolysis liquid adds after the harmless noresidue chemical decolorization agent of amount seldom and is warming up to 55 ~ 60 ℃ of insulation 3h and can obtains the secondary enzymolysis destainer;
(4), the concentrated and spraying drying of secondary enzymolysis liquid: spraying drying after secondary enzymolysis destainer employing falling liquid film or ultrafiltration and concentration just can obtain lurid enzymolysis protein powder.
2. the preparation method that a kind of two step enzymolysis processs according to claim 1 are produced high-quality hyperglobulinemia peptide, is characterized in that, erythrocytic primary enzymolysis decolouring: fresh red blood corpuscle is added to appropriate water, regulate pH to 2.0 ~ 3.0.
3. the preparation method that a kind of two step enzymolysis processs according to claim 1 and 2 are produced high-quality hyperglobulinemia peptide, it is characterized in that, add appropriate aspartic protease S1, constant temperature enzymolysis 8h under 37 ~ 40 ℃ of conditions, with the centrifugal 5min of 5000g, remove centrifugation and partly obtain the primary enzymolysis destainer.
4. the preparation method that a kind of two step enzymolysis processs according to claim 3 are produced high-quality hyperglobulinemia peptide, is characterized in that, at described primary enzymolysis destainer, adds appropriate neutral protease S2, constant temperature enzymolysis 8h under 40 ℃ of conditions.
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| CN117886826A (en) * | 2023-12-13 | 2024-04-16 | 甘肃养泰和生物科技有限公司 | Method for extracting methemoglobin by heating and pressurizing |
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