CN101755996A - Casein phosphopept continuous production process by adopting immobilized enzyme hydrolysis crude cheese and ultrafiltration membrane separation method - Google Patents
Casein phosphopept continuous production process by adopting immobilized enzyme hydrolysis crude cheese and ultrafiltration membrane separation method Download PDFInfo
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Abstract
The invention relates to a casein phosphopept continuous production process by adopting an immobilized enzyme hydrolysis crude cheese and ultrafiltration membrane separation method, comprising the following steps: a. extraction of tryptase; b. preparation of tryptase immobilized reaction column; c. crude cheese hydrolysis; d. separation and purification of the casein phosphopept (CPP) with an ultrafiltration membrane; e. column chromatography of CPP ultrafiltrate. The process has rational design, strong operability, short production process, high concentration of intermediate products and low production energy consumption; the main separation and purification processes are carried out at normal temperature, the evaporation capacity is less in the drying process, the energy consumption in the production can be greatly reduced, and the cost is saved; the product is obtained by vacuum concentration and spray drying, the main ingredient of the product is micromolecule bioactive polypeptides, the polypeptide with 2000-6000 of molecular weight accounts for more than 45 percent of the polypeptides.
Description
Technical field
The present invention relates to the food product processing technology.
Background technology
CPP (Casein Phosphopeptides, CPP) being is raw material with the milk casein, through the hydrolysis of single or compound protease, again to the hydrolysate separation and purification, and then the natural physiological active peptide that contains phosphoserine bunch that obtains after the drying.
The core position of CPP is-SerP-SerP-SerP-Glu-Glu-, has the activity that very strong short calcium and other mineral element absorb, can combine with divalence such as calcium, iron, zinc and some trivalent mineral ion, not only can be used as the carrier of calcium, also can be used as the carrier of iron, manganese, zinc, selenium, so CPP has been acknowledged as application functional food additives more widely, it can promote the infant and teen-agely grow up healthy and sound and improve health-care effect to the elderly.
People such as Gu Kunrui are at Shaanxi Tech Univ's journal, No.4, and VOI.2l, Aug, 2003 have introduced immobilizing trypsinase and have been used to prepare the experimental study of CPP (CPP).Experiment is with the carrier of shitosan as immobilizing trypsinase, and method is: casein → casein hydrolyzate → filtration → immobilized enzyme hydrolysis → separation → concentrate → drying → CPP product.The characteristics of this method are: can repeatedly use after the immobilization of enzyme, save cost; Enzyme does not enter finished product, has promoted the local flavor of product.Its shortcoming is: with the shitosan is carrier, and carrier costs an arm and a leg, and the expense height is difficult for suitability for industrialized production.2006-12-8, University Of Tianjin has carried out " appraisal of scientific and technological achievements " to " efficiently continuous preparation technology's exploitation of the short calcium absorption factor CPP " project of carrying out, and applied for " the integrated technology of producing active casein polypeptide continuously of enzymolysis and membrane filtration " patent of invention, application number is 200310107554.3.Common problems such as this method is at ubiquitous reaction conversion ratio in the preparation enzymolysis process at CPP adopted intermittence and the target polypeptides productive rate is low, unstable product quality, enzyme consumption are big, technology cost height, propose the continuous new process for producing of enzyme membrane coupling one-step method, designed and installed the multistage enzyme membrane coupled reactor expansion test system that a cover is combined by the milipore filter spare of 20L enzymatic vessel and PSPP; Determined intermittently to prepare the process route of CPP, and optimized operating parameter with continuous enzymolysis; Finished 20L jar intermittent hydrolysis and 40L/hr long-time continuous, expansion runin stable, that efficiently produce CPP is sent out, the enzyme consumption reduces by 3 times than conventional method, reactor production capacity improves 1 times; And ion-exchange resin purification technology carried out design and optimization; Products at different levels detect proof through authoritative institution: the external calcium ability effect of holding is obvious, and every index all reaches country's health care (function) food universal standard.But, this method weak point be hydrolytic process in a reactor, easily cause substrate repeatedly hydrolysis produce little peptide, for later separation is brought difficulty, and influence finished product local flavor and quality.
Summary of the invention
" song draws " be the Tibetan area herdsman with milk spontaneous fermentation after, go out protein through thermal precipitation, make the yak coarse cheese then after the air dry, be commonly called as " song draws ".Its main component is a protein, and it is Tibetan area nomad's important foodstuffs source.
The present invention with the Tibetan area abundant low cost " song draws "---coarse cheese is a raw material, in conjunction with the advantage of prior art, its objective is provides a kind of immobilized enzyme hydrolysis crude cheese and milipore filter separation method to produce CPP continuously.This method is with immobilized enzyme hydrolysis crude cheese, and hydrolyzate is carried out effective separation and Extraction to obtain highly purified CPP product, promotes product special flavour, improves the continuity of production technology, reduces energy consumption and product cost.
The objective of the invention is to be achieved through the following technical solutions:
A kind of immobilized enzyme hydrolysis crude cheese and milipore filter separation method are produced CPP continuously, and its step is divided into:
A. extraction of tryptase method
I. after the fresh bovine pancreas being picked out fat, connective tissue, immerse ammonium sulfate, stir, press filtration obtains lixiviate filtrate;
Ii. in lixiviate filtrate, add solid ammonium sulfate, stir, leave standstill, go precipitation, obtain supernatant after the filtration;
Iii. below the supernatant adjust pH to 3.0, the ammonium sulfate concentrations in the maintenance solution staticly settles after stirring at 3.0～3.5mol/L, abandons supernatant, and sediment is the trypsinogen crude product;
Iv. the trypsinogen crude product adds ethanol, CaCl
2, dissolve, leave standstill activation, add ethanol again, stir, leave standstill, precipitate, promptly get trypsase after the filtration.
This technology can obtain hydrolysis " song draws "---the needed trypsase raw material of coarse cheese.
B. prepare tryptase immobilized reaction column
The rare resin anion (R.A.) of alkalescent benzene second adds 2.5% glutaraldehyde, 10 ℃～20 ℃ following cross-linking reactions 10～12 hours, adds 1～4% trypsase again, 10 ℃～15 ℃ following immobilizations 20 hours, and the diameter of packing into: highly=1: 10 stainless steel reaction post;
C. hydrolysis crude cheese
Coarse cheese with 10～15 times of water 55～60 ℃ of swellings 1 hour, NaOH adjustment pH value with 1mol/L is no more than 8.0, after the dissolving, after filtration, the protein solution that separates after the degreasing is reverse by the immobilised enzymes reaction column with the speed of 10L/min under 40 ℃～60 ℃, keep with immobilised enzymes circulation time of contact be 3 hours～5 hours; Then hydrolyzate is cooled to room temperature through heat exchange posts, precipitation is removed in press filtration, via obtaining the CPP hydrolyzate after the diatomite filter clarification;
D. milipore filter separates purification CPP (CPP)
The CPP hydrolyzate is through ultrafiltration membrance filter equipment twice separation, its technological parameter: diffusion barrier for the first time, the molecular weight upper limit≤50000 of damming, film initial pressure 2Bar, termination pressure 10Bar, circular flow 200L/hr, 2～5 hours mean circulation time (MCT)s; Milipore filter for the second time, diffusion barrier lower molecular weight limits≤1000 of damming, film initial pressure 4Bar, termination pressure 15Bar, circular flow 150L/hr, 3～6 hours mean circulation time (MCT)s;
E. the ultrafiltrate with CPP carries out column chromatography
Hydrolyzate after the separation carries out the chromatography absorption with 20～40 purpose granular activated carbon posts; flow velocity is 20L/hr; pressure is 1.2Bar～5Bar; then hydrolyzate is concentrated through cryogenic vacuum, 30 ℃～40 ℃ of vacuum 40mmHg～60mmHg, temperature are to solid content 30%～40%; spray-drying again; pressure 6Bar, 60 ℃～70 ℃ of temperature promptly obtain CPP (CPP) product.
The beneficial effect of advantage of the present invention and generation is:
1, compared with the prior art, protein liquid of the present invention carries out continuously circulating water through reverse injection immobilised enzymes reaction column and separates, hydrolytic process can be finished by the reaction column of 2～3 series connection, after each substrate hydrolysis is finished and is entered down the step operation, the enzyme reaction post can carry out the hydrolytic process of next time again, and immobilised enzymes repeats the hydrolysis number of times and is generally 8～10 times.Utilize tryptase immobilized reaction column, improve the utilization rate of enzyme, the saving enzyme dosage lacks 5～8 than the primary enzymolysis method accompanies, and has reduced the immobilization cost of enzyme.Course of reaction does not need heating to make enzyme deactivation, and enzyme and enzyme self decomposes the accessory substance that brings and also do not enter finished product.In addition, adopt the direct protease that extracts to carry out immobilization, as carrier, alternative expensive shitosan has reduced production cost, is easy to suitability for industrialized production with the rare resin anion (R.A.) of alkalescent benzene second in utilization processing back.
2, improve the continuity of explained hereafter: after removing hydrolysis technology in the production process and producing precipitation and utilize press filtration and diatomite filtration clarification, subsequent technique is the liquid phase flow operation all before obtaining finished product, is easy to control.Do not need in the course of reaction to add substrate, enzyme and other materials, each process is continued operation, and per step flow-control realizes to distribute each processing step equipment amount and flow velocity, to guarantee the equilibrium of product flow in the whole technology.
3, product purity height, the separative efficiency height, desalination is effective.This method with the rare resin anion (R.A.) of alkalescent benzene second (the resin model comprises 301～400) as preparing carriers immobilizing trypsinase reaction column, with coarse cheese (or " song draws ") is raw material, protein after the dissolving is behind the immobilised enzymes continuous hydrolysis, hold back via the two times of ultrafiltration film, one time activated carbon chromatography separates, can effectively remove the non-phosphopeptide in the product, salt, moisture content and big molecular polypeptide, use activated carbon chromatography absorption to remove little peptide simultaneously, product concentrates through vacuum, spray-drying, local flavor is greatly improved, CPP content can reach 45% in the product, has the body of promotion calcium, iron, the effect that zinc absorbs and storage is stayed, product quality is higher than domestic like product standard.
4, reasonable, the strong operability of technological design of the present invention, production process is short, the concentration of intermediate products height, energy consumption is low: the main separation and purification process all adopts normal temperature to handle, and the dry run evaporation capacity is few, has greatly reduced the energy consumption in producing, and has saved cost.
Description of drawings
Fig. 1 is a process chart of the present invention.
The specific embodiment
Below in conjunction with accompanying drawing the present invention is further described again:
Embodiment 1:
A kind of immobilized enzyme hydrolysis crude cheese and milipore filter separation method are produced CPP continuously, and its step is divided into:
A. extraction of tryptase is got fresh bovine pancreas 10kg, manually pick out fat, connective tissue, rubbing immersion immediately through meat grinder is equipped with in the 50L interlayer retort of 0 ℃ of 1L～4 ℃ of 0.1mol/L ammonium sulfates, after stirring, become slurries, the 0.1mol/L ammonium sulfate that adds 2 times of volumes of slurries, extracted 20 hours 0 ℃～4 ℃ interlayer retort, slowly stir every half an hour.Through the air pressure filter press filtration, collect filtrate, residue repeats lixiviate once with the 0.1mol/L ammonium sulfate of 1 times of slurry volume again, merges filtrate twice, discards residue.
Filtrate in the 50L interlayer retort adds the 0.2kg/L solid ammonium sulfate down to concentration 1.5mol/L at 0 ℃～4 ℃, leaves standstill after the stirring 8 hours, filters, goes precipitation.In filtrate, continue to add the 0.27kg/L solid ammonium sulfate, left standstill after the stirring 12 hours, obtain supernatant after the press filtration, discard residue to concentration 3.5mol/L.
Supernatant is with below the 2mol/L sulfuric acid 0.1L adjust pH to 3.0, and the ammonium sulfate concentrations in the maintenance solution staticly settled after stirring 10 hours at 3.5mol/L.Must precipitate after the press filtration supernatant discarded.This precipitation is the trypsinogen crude product.
With 25% ethanol of above-mentioned gained trypsinogen crude product (weight in wet base) adding 3 times (weight ratios), add the CaCl of trypsase crude product material quantity 2% simultaneously
2, making its dissolving and transferring the pH value of solution value is between the 5-6, and 0 ℃～4 ℃ leave standstill activation 12 hours, add ethanol then its concentration is reached more than 70%, fully stir, and 0 ℃～4 ℃ left standstill abundant precipitation 3 hours, promptly got trypsase after the filtration.Yield is about 3.0%.
B. tryptase immobilized reaction column preparation
Pack in 5L interlayer retort the rare resin anion (R.A.) 1kg of alkalescent benzene second after the activated processing stirs and slowly drips 2.5% heavy glutaraldehyde of 1/5 resin down, fully stirs cross-linking reactions 10 hours down at 10 ℃～20 ℃, filters, and glutaraldehyde is removed in washing.Be made into about 1.5% trypsin solution in the 0.2mol/L acetate buffer solution with 0.04kg trypsase (weight in wet base) adding 1L pH6.5～7.0 in the 2L container, with reacted resin 10～15 ℃ of following immobilizations 20 hours, diameter and the aspect ratio of packing into then is in 1: 10 the reaction column, every post immobilised enzymes amount of packing into is 0.2kg, and with the not immobilized enzyme of 0.2mol/L acetate buffer solution flush away of pH6.5～7.0, measure enzyme activity, standby.
The definition of resolvase vigor: under reaction condition, the filtrate light absorption increment behind the per minute enzyme hydrolysis tyrosine is a trypsase unit of activity (U/mg) with the enzyme amount of 1 μ mol tyrosine when 275nm place absorbance is suitable.
The vigor definition of immobilised enzymes: under same reaction conditions, the vigor of immobilised enzymes is the relative vigor of immobilised enzymes with the ratio of resolvase vigor.
Relative vigor (%)=(vigor of immobilised enzymes/resolvase vigor) * 100%.
The trypsase vigour-testing method: with the casein is that substrate is measured.See two the 625th page of " pancreatin " hurdles of Pharmacopoeia of the People's Republic of China version in 2005 for details.
This technology is to obtain the needed immobilised enzymes of hydrolytic process.The immobilised enzymes vigor is about about 75% of resolvase vigor.
C. the coarse cheese hydrolysis generates CPP
In 10L interlayer retort,, slowly add about the sodium hydroxide solution 50mL of 1mol/L under stirring, adjust pH8.0 water-soluble the expanding 1 hour of 0.5kg coarse cheese with 10 times, 55～60 ℃.After treating protein dissolution, separate degreasing through filter filtration, centrifuge.The protein solution that obtains is pressed into the immobilised enzymes reaction column by compression pump with the speed of 0.01L/min under 40 ℃～60 ℃, keeping liquid and immobilised enzymes circulation time of contact is 3 hours, and proteolysis can be by 2 to 3 immobilised enzymes reaction columns series connection realizations.Then hydrolyzate is cooled to room temperature through heat exchange posts, precipitation is removed in press filtration, via obtaining about CPP hydrolyzate 4L after the diatomite filter clarification.
D.CPP separates purification
The CPP hydrolyzate is through ultrafiltration membrance filter equipment twice separation, its technological parameter: diffusion barrier molecular weight≤50000 of damming for the first time, and film initial pressure 2Bar, termination pressure 10Bar, circular flow 200L/hr, the mean circulation time (MCT) is more than 2 hours.Milipore filter diffusion barrier molecular weight≤1000 of damming for the second time, film initial pressure 4Bar, termination pressure 15Bar, circular flow 1.50L/hr, the mean circulation time (MCT) is more than 3 hours.
Carry out the chromatography absorption with 20～40 purpose granular activated carbon posts that material quantity 0.5% (weight ratio) is housed about the hydrolyzate 3.5L after the separation, then that hydrolyzate is concentrated and dry through cryogenic vacuum, promptly obtain the CPP product.
E. product analysis testing result
The CPP yield that aforementioned production method is waited until is between 35%～45%, and molecular weight accounts for 45% at the polypeptide of 2000～6000 scopes in the product, CPP content 30%～35% in the product, and the product bitter taste is heavier.Production cost is 120～150 yuan/kg.
Product standard meets the product standard of determining with following detection method.
The main detection method of CPP (CPP) product:
1. protein measuring: use micro-Kjeldahl, see GB5424-85.
2. Zhi Fang mensuration: use the Soxhlet extraction process, see GB 5009.6-85.
3. the mensuration of moisture, ash content: use gravimetric method, see GB5424-85.
4. the mensuration of phosphoeptide: measure characteristic absorption peak among the CPP with high pressure liquid chromatography and column chromatography, compare with the total absorption peak area with CPP absworption peak area, and measure phosphorus content with molybdenum blue colorimetric method, again with CPP in 2.82 the comparing of average phosphorus content.(in May, 2005, front page was the 201st page for " functional food determination of bioactive constituent ", Chemical Industry Press).
5. sanitary index and microbiological indicator are measured and are as the criterion with state food and additive sanitary standard.Main standard has: GB/T 5009.11-2003 " mensuration of total arsenic and inorganic arsenic in the food ", GB/T5009.12-2003 " assay method of lead in the food ", GB/T 4789.2-2003 " microbiological test of food hygiene total plate count mensuration ", GB/T 5009.46-2003 " analytical method of breast and dairy products sanitary standard ", GB 2760-2007 " food additives use sanitary standard ".
6. CPP (CPP) product standard sees Table 1-3
Table 1: organoleptic indicator
Table 2: physical and chemical index
| Moisture (%) | ????≤10 |
| Ash content (%)) | ????≤6 |
| Holoprotein is (in kjeldahl determination, %) | ????≥80 |
| Phosphoeptide (%) | ????≥45 |
| PH (10% solution) | ????6.5±1 |
Table 3: health and microbiological indicator
| Total number of bacteria (individual/g) | ????≤5000 |
| Pathogenic bacteria | Must not detect |
| Arsenic | ????≤0.5ppm |
| Plumbous | ????≤0.5ppm |
Product is packed with the Lined with Kraft Paper bag, and loading amount is three kinds of 1Kg, 5Kg and 10Kg.Be stored in the dry environment, guard against damp.
Embodiment 2:
A. extraction of tryptase
Get fresh bovine pancreas 100kg, manually pick out fat, connective tissue, rub in the 500L interlayer retort that immerses the 0.1mol/L ammonium sulfate that 0 ℃～4 ℃ of 10L are housed immediately through meat grinder, after stirring, become slurries, the 0.1mol/L ammonium sulfate that adds 3 times of volumes of slurries extracted 30 hours 0 ℃～4 ℃ interlayer retort, slowly stirred every half an hour.Through the air pressure filter press filtration, collect filtrate, residue repeats lixiviate once with the 0.1mol/L ammonium sulfate of 1 times of slurry volume again, merges filtrate twice, discards residue.
Filtrate in the 500L interlayer retort adds the 0.2kg/L solid ammonium sulfate down to concentration 1.5mol/L at 0 ℃～4 ℃, leaves standstill after the stirring 10 hours, filters, goes precipitation.In filtrate, continue to add the 0.27kg/L solid ammonium sulfate, left standstill after the stirring 14 hours, obtain supernatant after the press filtration, discard residue to concentration 3.5mol/L.
Supernatant is with below the 2mol/L sulfuric acid 1.0L/100L adjust pH to 3.0, and the ammonium sulfate concentrations in the maintenance solution staticly settled after stirring 12 hours at 3.5mol/L.Must precipitate after the press filtration supernatant discarded.This precipitation is the trypsinogen crude product.
With 25% ethanol of above-mentioned gained trypsinogen crude product (weight in wet base) adding 5 times (weight ratios), add the CaCl of trypsase crude product material quantity 2% simultaneously
2, making its dissolving and transferring the pH value of solution value is between the 5-6, and 0 ℃～4 ℃ leave standstill activation 14 hours, add ethanol then its concentration is reached more than 70%, fully stir, and 0 ℃～4 ℃ left standstill abundant precipitation 4 hours, promptly got trypsase after the filtration.Yield is about 5.0%.
B. tryptase immobilized reaction column preparation
The rare resin anion (R.A.) 100kg of alkalescent benzene second after the activated processing packs in 500L interlayer retort, stir and slowly drip 2.5% heavy glutaraldehyde of 1/5 resin down, fully stirred cross-linking reaction 12 hours down at 10 ℃～20 ℃, be made into about 1.5% trypsin solution in the 0.2mol/L acetate buffer solution with 4kg trypsase (weight in wet base) adding 100L pH6.5～7.0 in the 200L basin, with 10～15 ℃ of following immobilizations 20 hours in the interlayer retort of reacted resin, diameter and the aspect ratio of packing into then is 1: 10 stainless steel reaction post, every post immobilised enzymes amount of packing into is 20kg, and with the not immobilized enzyme of 0.2mol/L acetate buffer solution flush away of pH6.5～7.0, measure enzyme activity, standby.
Enzyme activity definition and assay method are with embodiment 1.
This technology is to obtain the needed immobilised enzymes of hydrolytic process.The immobilised enzymes vigor is about more than 80% of resolvase vigor.
C. the coarse cheese hydrolysis generates CPP
In 1000L interlayer retort,, stir the sodium hydroxide solution 5L/100L that slowly adds 1mol/L down, adjust pH8.0 water-soluble the expanding 1 hour of 50kg coarse cheese with 15 times 55～60 ℃.After treating protein dissolution, separate degreasing through duplex strainer filtration, disk plate centrifuge.The protein solution that obtains is pressed into the immobilised enzymes reaction column by compression pump with the speed of 10L/min under 40 ℃～60 ℃, keeping liquid and immobilised enzymes circulation time of contact is 5 hours, can be according to hydrolysis effect by 3 immobilised enzymes reaction columns series connection realization proteolysis during hydrolysis.Then hydrolyzate is cooled to room temperature through heat exchange posts, precipitation is removed in press filtration, via obtaining about CPP hydrolyzate 700L after the diatomite filter clarification.
D.CPP separates purification
The CPP hydrolyzate is through ultrafiltration membrance filter equipment twice separation, its technological parameter: the diffusion barrier molecular weight≤500C0 that dams for the first time, and film initial pressure 2Bar, termination pressure 10Bar, circular flow 200L/hr, the mean circulation time (MCT) is more than 2 hours.Milipore filter diffusion barrier molecular weight≤1000 of damming for the second time, film initial pressure 4Bar, termination pressure 15Bar, circular flow 150L/hr, the mean circulation time (MCT) is more than 4 hours.
Carry out the chromatography absorption with 20～40 purpose granular activated carbon posts that material quantity 6% (weight ratio) is housed about the hydrolyzate 650L after the separation, then hydrolyzate is concentrated and spray-drying through cryogenic vacuum, promptly obtain the CPP product.
E. product analysis testing result
Product standard and detection method are with embodiment 1.
The CPP yield that present embodiment obtains is between 25%～35%, and molecular weight accounts for 45% at the polypeptide of 3000～6000 scopes in the product, CPP content 35%～45% in the product, and product has lighter bitter taste.Production cost is 180～200 yuan/kg.
The variation of process conditions of the present invention is to the influence of production process and product
1, the trypsase of Ti Quing temperature in immobilized reactant rises to more than 25 ℃ by 15 ℃～20 ℃, the immobilised enzymes vigor then reduces greatly, have only 50%～55% of resolvase vigor generally speaking, hydrolytic process will become not exclusively, finally have influence on product yield and drop to below 25%.
2, Rong Xie protein solution will if forward flow can cause reaction column to stop up owing to produce tiny precipitation in reaction, make technical process time lengthening with the reverse mode immobilised enzymes reaction column of flowing through, and when serious technology can not be carried out.
3, activated carbon chromatography absorption is in order to remove molecular weight less than 1000 free little peptide, these little peptides are the main components that have bitter taste in the product, in implementation process, do not remove bitter peptides if do not adopt activated carbon chromatography to carry out absorption, though can reduce the part producing cost, but bitter taste increases the weight of in the product, and product special flavour reduces.
4, milipore filter molecular cut off size has direct relation with product quality and yield, is limited to 1000 under the retaining molecular weight, on be limited to 50000.The big I of molecular cut off is divided into 1000～10000,1000～20000,1000～30000,1000～40000,1000～50,000 5 kinds of scopes, its product yield from 25% to 45% raises successively, and the CPP content in the product is from 45% to 30% reduction successively also.The penetrating amount of film influences process time and production cost, needs to keep the penetrating amount of film to reach more than 80% of factory-said value in the production process, otherwise needs film is carried out the milipore filter that CIP cleans or more renews.
5, product yield and production cost and raw material coarse cheese (" song draws ") quality has bigger relation, generally requires in the raw material protein content more than 70%, if protein content is lower than 65%, then the CPP yield can be lower than below 25%.
Claims (1)
1. immobilized enzyme hydrolysis crude cheese and milipore filter separation method are produced CPP continuously, and its step is divided into:
A. extraction of tryptase method
I. after the fresh bovine pancreas being picked out fat, connective tissue, immerse ammonium sulfate, stir, press filtration obtains lixiviate filtrate;
Ii. in lixiviate filtrate, add solid ammonium sulfate, stir, leave standstill, go precipitation, obtain supernatant after the filtration;
Iii. below the supernatant adjust pH to 3.0, the ammonium sulfate concentrations in the maintenance solution staticly settles after stirring at 3.0～3.5mol/L, abandons supernatant, and sediment is the trypsinogen crude product;
Iv. the trypsinogen crude product adds ethanol, CaCl
2, dissolve, leave standstill activation, add ethanol again, stir, leave standstill, precipitate, promptly get trypsase after the filtration;
B. prepare tryptase immobilized reaction column
The rare resin anion (R.A.) of alkalescent benzene second adds 2.5% glutaraldehyde, 10 ℃～20 ℃ following cross-linking reactions 10～12 hours, adds 1～4% trypsase again, 10～15 ℃ of following immobilizations 20 hours, and the diameter of packing into: highly=1: 10 stainless steel reaction post;
C. hydrolysis crude cheese
Coarse cheese with 10～15 times of water 55～60 ℃ of swellings 1 hour, NaOH adjustment pH value with 1mol/L is no more than 8.0, after the dissolving, after filtration, the protein solution that separates after the degreasing is reverse by the immobilised enzymes reaction column with the speed of 10L/min under 40 ℃～60 ℃, protein solution keep with immobilised enzymes circulation time of contact be 3～5 hours; Then hydrolyzate is cooled to room temperature through heat exchange posts, precipitation is removed in press filtration, via obtaining the CPP hydrolyzate after the diatomite filter clarification;
D. milipore filter separates purification CPP (CPP)
The CPP hydrolyzate is through ultrafiltration membrance filter equipment twice separation, its technological parameter: the diffusion barrier molecular weight upper limit≤50000 of damming for the first time, film initial pressure 2Bar, termination pressure 10Bar, circular flow 200L/h r, 2～5 hours mean circulation time (MCT)s; Milipore filter diffusion barrier lower molecular weight limits≤1000 of damming for the second time, film initial pressure 4Bar, termination pressure 15Bar, circular flow 150L/hr, 3～6 hours mean circulation time (MCT)s;
E. the ultrafiltrate with CPP carries out column chromatography
Hydrolyzate after the separation carries out the chromatography absorption with 20～40 purpose granular activated carbon posts; flow velocity is 20L/hr; pressure is 1.2Bar～1.5Bar; then hydrolyzate is concentrated through cryogenic vacuum, 30 ℃～40 ℃ of vacuum 40mmHg～60mmHg, temperature are to solid content 30%～40%; spray-drying again; pressure is 6Ba r, and temperature is 60 ℃～70 ℃, promptly obtains CPP (CPP) product.
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| CN104046673A (en) * | 2013-03-14 | 2014-09-17 | 中国食品发酵工业研究院 | Industrial manufacturing method and use of CPPs-containing hypoallergenic casein peptide whole-powder |
| CN105755084A (en) * | 2016-04-28 | 2016-07-13 | 李健 | Method for preparing CPPs through enzymolysis of cow milk casein |
| CN110922465A (en) * | 2019-10-12 | 2020-03-27 | 广州医科大学 | Method for preparing casein calcium peptide |
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| CN1903052B (en) * | 2005-07-26 | 2010-04-28 | 天津科技大学 | Method for prepairng whey powder containing casein phosphopeptide, anti-angiotonin converzyme peptide and oligo-galactose |
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|---|---|---|---|---|
| CN102849860A (en) * | 2011-07-02 | 2013-01-02 | 甘南州科瑞乳品开发有限公司 | Cyclic utilization method of wastewater from casein phosphoeptide production |
| CN102849860B (en) * | 2011-07-02 | 2013-09-04 | 甘南州科瑞乳品开发有限公司 | Cyclic utilization method of wastewater from casein phosphoeptide production |
| CN104046673A (en) * | 2013-03-14 | 2014-09-17 | 中国食品发酵工业研究院 | Industrial manufacturing method and use of CPPs-containing hypoallergenic casein peptide whole-powder |
| CN104046673B (en) * | 2013-03-14 | 2020-07-21 | 中国食品发酵工业研究院 | Industrial manufacturing method and application of low-sensitization casein peptide whole powder containing CPPs |
| CN105755084A (en) * | 2016-04-28 | 2016-07-13 | 李健 | Method for preparing CPPs through enzymolysis of cow milk casein |
| CN110922465A (en) * | 2019-10-12 | 2020-03-27 | 广州医科大学 | Method for preparing casein calcium peptide |
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| CN101755996B (en) | 2012-11-28 |
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