CN1232649C - Process of continuous production of casein bioactive peptide by enzymolysis and filtering membrane concentration - Google Patents
Process of continuous production of casein bioactive peptide by enzymolysis and filtering membrane concentration Download PDFInfo
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- CN1232649C CN1232649C CN 200310107554 CN200310107554A CN1232649C CN 1232649 C CN1232649 C CN 1232649C CN 200310107554 CN200310107554 CN 200310107554 CN 200310107554 A CN200310107554 A CN 200310107554A CN 1232649 C CN1232649 C CN 1232649C
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Abstract
The present invention provides a technology for continuously preparing casein biological activity polypeptide by enzymolysis and filtering membrane concentration. In the technology, casein serves as a raw material, and the multifunctional biological activity polypeptide is made by the function of at least one kind of proteinase in a multi-stage enzyme membrane reactor which is combined by an enzymolysis tank and ultrafiltration nanofiltration membranes with different catch molecular weight. The technology combines pentagonal working procedures of enzymatic hydrolysis reaction, catalyst recovery reuse, separation and purification of products, concentration and desalting and elimination of bitterness peptide into a whole. Compared with the prior art, the conversion rate of the enzymolysis reaction is increased from 50% to 80%, and the unit time yield of the activity polypeptide is increased from 30 g/h to 50 g/h; the protein yield is enhanced from 50% to 70%, the proteinase utilization rate is enhanced by 1.5 times, and the technology cost is reduced by 1 time. The composition of the made activity polypeptide product tends uniformly, and the product is safe and nontoxic. The technology has wide application prospects in the field of food products, cosmetics, health products, medicine, etc.
Description
Technical field
The present invention relates to a kind ofly, belong to the technology of producing active polypeptide with the casein for the substrate enzymically hydrolyse enzyme digestion reaction and the integrated novel process of producing the Casein in Milk polypeptide continuously of membrane separation technique.
Background technology
Casein is the highest protein of Ruzhong content, is the complex body of several phosphor proteins, and main component is α
S1, α
S2, β, κ four peptide species chains.Casein is a hydrolytic action through single enzyme or prozyme, can make over one hundred kind of biologically active polypeptides of nearly 10 classes through separation and purification again.Complicated enzymolysis product, multifarious sequential structure makes such material have different physiological roles (table 1) such as immunomodulatory, free radical scavenging, mineral carrier, anticancer antibacterial, thrombolysis step-down, ataranesthesia, is to have the class biologically active substance that exploitation is worth.
Table 1 Casein in Milk biologically active polypeptides example
| Aminoacid sequence | Title | Physiological function |
| ThrThr-Met-Pro-Leu-Trp | α s1-Casokinin-6 α s1-junket kassinin kinin-6 | Immunomodulatory |
| Tyr-Gln-Gln-Pro-Val-Leu-Gly-Pro-Val-Arg | β-Casokinin-6 β-junket kassinin kinin-10 | Immunomodulatory |
| Phe-Phe-Val-Ala-Pro | α s1-ACEI-5 angiotensin converting enzyme inhibitor | Hypotensive |
| Ala-Val-Pro-Tyr-Pro-Gln-Arg | β-ACEI-7 angiotensin converting enzyme inhibitor | Hypotensive |
| Met-Ala-Ile-Pro-Pro-Lys-Asn-Asn-Gln-Asp-Lys | κ-Casoplatelin-11 casein platelet factor | Thrombolysis, anticoagulation |
| Asp-Ile-Gly-SerP-Glu-SerP-Thr-Glu-Asp-Gln-Ala-Met-Glu-Asp- Ile-Lys-Gln-Met-Glu-Ala-Glu-SerP-Ile-SerP-SerP-SerP-Glu-Glu- Ile-Val-Pro-Asn-SerP-Val-Glu-Gln-Lys | α s1-CPP-37 α s1-phosphopeptide caseinate-37 | Mineral carrier |
| Arg-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-Val -Glu-SerP-Leu-SerP-SerP-SerP-Glu-Glu-Ser-Ile-Thr-Arg | β-CPP-25 beta-casein phospho-peptide-25 | Mineral carrier |
| Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln- Glu-Val-Leu-Asn-Glu-Asn-Leu-Leu-Arg-Phe | α s1-Isracidin-23 casein is intended cidin | Antibiotic |
| Lys-Lys-Ile-Ser-Gln-Arg-Tyr-Gln-Lys-Phe-Ala-Leu-Pro- Gln-Tyr-Leu-Ths-Thr-Val-Tyr-Gln-His-Gln-Lys-Ala-Met -Lys-Pro-Trp-Ile-Gln-Pro-Lys-Thr-Lys-Val-Ile-Pro-Tyr | α s2-Casocidin-39 casein cidin | Antibiotic |
| Tyr-Pro-Ple-Pro-Gly-Pro-Ile | β-Casomorphin-7 β-junket deltorphin delta-7 | The opioid activity ataranesthesia |
| Ser-Thr-Tyr-Pro-Ser-Tyr | κ-Casoxin-5 κ-junket is newly plain | The opioid activity ataranesthesia |
At present, the production technique of casein biologically active polypeptides extensively adopts intermittently enzymolysis, promptly in common hydrolytic decomposition pot, carry out batchwise operation, after enzyme digestion reaction finishes, make proteolytic enzyme sex change inactivation by regulator solution pH value, and make the centrifugation of unhydrolysed casein post precipitation, again through multistep purification procedures such as solvent selectivity precipitation or ion exchange resin treatment, can make the target polypeptides product, there are the following problems in the technological process: (1) is owing to enzymolysis product can't in time separate from reaction system, cause product to suppress phenomenon easily, enzyme digestion reaction transformation efficiency and target polypeptides productive rate are all lower; (2) hydrolysising condition has certain difference, unstable product quality between the differential responses batch; (3) separated product and from reaction mixture enzyme reclaimed and use all very difficulty again from reaction system, so the enzyme consumption is big, technology cost height can not satisfy commercial requirement fully.
Summary of the invention
The object of the present invention is to provide a kind of with enzyme digestion reaction and the integrated novel process of producing active casein polypeptide continuously of membrane filtration technology.The biologically active polypeptides productive rate height that adopts this technology to make, cost is low, and has multiple physiological function, and safety non-toxic, can be used as healthcare products and eats.
For achieving the above object, the present invention adopts following technical proposals to be realized: in the multistage enzyme mebrane reactor that ultrafiltration, nanofiltration membrane by enzymatic vessel and PSPP combine, implement enzymolysis and the integrated technology of producing the casein biologically active polypeptides continuously of membrane filtration.Described enzyme digestion reaction is with whole casein, α
S1, α
S2, at least a in β, the κ-casein monomer, caseinate be substrate, under the condition of 25～50 ℃ of suitable temperature of reaction of proteolytic enzyme and pH value 4～10, a kind of or composite katalysis in pancreatin, trypsinase, stomach en-, neutral protease, Sumizyme MP, papoid, bromeline, carboxypeptidase and realize enzymically hydrolyse reaction, in the above-mentioned technology: the initial substrate mass concentration is 2～4%, and the ratio of initial enzyme-to-substrate mass concentration is 1: 20～1: 40; Constant by dripping NaOH solution maintenance system pH; By heating in water bath maintenance system homo(io)thermism.It is characterized in that: in the reaction that in enzymatic vessel, is hydrolyzed, be in 6000～10000 the ultra-fine filter continuously by at the bottom of the jar enzymolysis solution being introduced average molecular weight cut-off, the ultra-filtration membrane trapped fluid is back to enzymatic vessel and continues the circulation degraded, it is in 1000～2000 the nanofiltration device that the ultra-filtration membrane penetrating fluid is introduced average molecular weight cut-off continuously, the nanofiltration membrane penetrating fluid dumps, collect nanofiltration membrane and hold back concentrated solution, 150～200 ℃ of inlet temperatures, 70～85 ℃ of temperature outs, input speed 1.0～1.5L/h, under the condition of working pressure 0.1～1MPa, the spray-dried molecular weight that makes is 1000～10000 multi-functional Casein in Milk biologically active polypeptides, comprising: phosphopeptide caseinate, blood pressure lowering peptide, immune-active peptides, angiotensin-convertion enzyme inhibitor, opioid peptide, the thrombolysis peptide, bacteriostatic peptide, the free radical scavenging peptide.
Above-mentioned film separation unit comprises that one-level at least is used to hold back that the ultra-filtration membrane of casein substrate and proteolytic enzyme and one-level are used for that polypeptide solution concentrates, desalination and remove the nanofiltration membrane of bitter peptides: ultra-filtration membrane working pressure 0.02～0.05M Pa, circular flow 20～40L/h, reactant mean residence time 3～4h; Nanofiltration membrane working pressure 0.1～0.5M Pa.
Need constantly to add substrate solution in enzymatic vessel in the above-mentioned technology and proteolytic enzyme is constant with maintenance system: adding mass concentration to the enzymatic vessel Continuous Flow is 1～2% substrate solution, and flow acceleration is consistent with the ultra-filtration membrane permeation flux; Added proteolytic enzyme to enzymatic vessel in per 6～8 hours in batches, and made tank body endoproteinase virtual mass concentration keep 0.1～0.15%.
The present invention integrates enzymically hydrolyse reaction, catalyst recovery and removes five with, product separation and purification and concentrating and desalinating, bitter peptides again and go on foot operations, have following advantage compared to existing technology: (1) adopts membrane technique in time hydrolysate to be separated from reaction member, mobile chemical equilibrium, the elimination product suppresses, and makes the active polypeptide yield in unit time increase to 50g/h by 30g/h; (2) with the ultrafiltration and the nanofiltration membrane combined treatment enzymolysis solution of PSPP, can realize effective regulation and control, make that the target polypeptides molecular weight distribution is concentrated, composition trend homogeneous, stay in grade the enzymolysis product molecular weight distribution; (3) hold back the polymer substance that reacts in the system with ultra-filtration membrane, casein substrate effective concentration is raise, the enzyme digestion reaction transformation efficiency increases to 80% by 50%, and can realize the recirculation use of proteolytic enzyme, and the enzyme utilization ratio improves 1.5 times, and raw materials cost reduces by 1 times; (4) with nanofiltration membrane treatment ultra-filtration membrane penetrating fluid, can one the step realize efficient desalination (ratio of desalinization reaches 65～95%), permeablely concentrate (dehydration rate reaches 50～70%) and remove the assorted peptide of the small molecules that causes bitter taste, make product special flavour better, effect is higher; (5) enrichment enriched product in reactant flow makes the protein total recovery be increased to 70% by 50%, has simplified the downstream separation purification step simultaneously, and the technology cost obviously reduces; (6) strengthen reaction process, prolong effective reaction time significantly, make enzymolysis process be suitable for the requirement of large-scale continuous production; (7) normal pressure and temperature operation, gentleness is easily controlled, energy efficient, and do not have poisonous and harmful reagent contamination environment, belong to friendly process.
The prepared active casein polypeptide of the present invention with multiple biological function, safety non-toxic, have no side effect, be easily absorbed by the body, physics and chemistry and hygiology detect and reach country's health care (function) food universal standard, can be widely used in a plurality of fields such as food, healthcare products, makeup, also can be developed as active polypeptide class medicine through being further purified.
Description of drawings
Enzymolysis and the integrated process flow diagram of producing active casein polypeptide continuously of membrane filtration that accompanying drawing 1 proposes for the present invention.
Embodiment
For example the present invention is done below and describe in further detail.
Embodiment 1
Take by weighing casein 800g, add 20L water and place container for storing liquid, low-grade fever also fully stirs, the NaOH solution 16mL that slowly adds 1g/mL, be made into pH=8.5, concentration is 4% casein substrate solution, pump in the hydrolytic decomposition pot, the adjusting mixing speed is 80rmp, start water bath with thermostatic control, treat that homo(io)thermism is after 45 ℃, open the ultrafiltration circulation device, the average molecular weight cut-off of ultra-filtration membrane is 10000, make the circular flow of substrate solution with 30L/h, normal pressure stable circulation 15min, in hydrolytic decomposition pot, add 20 gram Sumizyme MPs, the reaction of beginning continuous enzymolysis is also clocked, regulate the ultra-filtration membrane working pressure behind the 10min and reach 0.05MPa, casein, macromolecular substance such as proteolytic enzyme and high-molecular polypeptide is held back by ultra-filtration membrane and is returned reaction member and continue the circulation degraded, and form penetrating fluid by film spare less than the polypeptide of the average molecular weight cut-off of ultra-filtration membrane, constant in the reaction process by the NaOH solution maintenance system pH=8.5 that drips 4mol/L, constant by 45 ℃ of heating in water bath maintenance system temperature, add with identical numerical value Continuous Flow according to survey ultra-filtration membrane permeation flux that 1.5% casein substrate solution is kept reaction volume and effectively concentration of substrate is constant, added 5g Sumizyme MP maintenance system enzyme activity every 6 hours constant, when ultra-filtration membrane per-meate side surge tank is collected the 5L transudate, open the nanofiltration circulation device to finish the polypeptide solution dehydration simultaneously, desalination and remove bitter peptides, the average molecular weight cut-off of nanofiltration membrane is 1000, working pressure 0.2MPa, when reaching 50%, thickening finishes nanofiltration, this moment, ratio of desalinization reached 70%, collect the nanofiltration membrane trapped fluid, 180 ℃ of inlet temperatures, 75 ℃ of temperature outs, input speed 1.5L/h, under the condition of working pressure 0.5MPa, the spray-dried molecular weight that makes is 1000～10000 multi-functional active casein polypeptide product, as calculated: protein yield＞70%, active polypeptide unit time generating rate is 50g/h.
Embodiment 2
Adopting average molecular weight cut-off is that 6000 ultra-filtration membrane and average molecular weight cut-off are 2000 nanofiltration membrane and 20L enzymatic vessel composition successive reaction separation system, to the 20L mass concentration is to add the 20g pancreatin in 2% the casein substrate solution, the maintenance system temperature is 40 ℃ in the reaction process, pH=8.0 is constant, Continuous Flow adds that 1% casein substrate solution is kept reaction volume and effectively concentration of substrate is constant, added 4g pancreatin maintenance system enzyme activity every 4 hours constant, other enzyme digestion reactions and membrane sepn operation steps and processing parameter are with embodiment 1, make active polypeptide product based on phosphopeptide caseinate, product is through the HPSEC check and analysis, molecular weight mainly concentrates in the spectrum peak of 3000～5000 correspondences, N/P ratio＜10.
Claims (3)
1. enzymolysis and the integrated technology of producing the casein biologically active polypeptides continuously of membrane filtration, this technology is specifically implemented in the multistage enzyme mebrane reactor that ultrafiltration, nanofiltration membrane by enzymatic vessel and PSPP combine; Described enzyme digestion reaction is with whole casein, α
S1, α
S2β, κ-casein monomer, at least a in the caseinate is substrate, under the condition of 25～50 ℃ of suitable temperature of reaction of proteolytic enzyme and pH value 4～10, through pancreatin, trypsinase, stomach en-, neutral protease, Sumizyme MP, papoid, bromeline, a kind of or composite katalysis in the carboxypeptidase and realize enzymically hydrolyse reaction, in the above-mentioned technology: the initial substrate mass concentration is 2～4%, the ratio of initial enzyme-to-substrate mass concentration is 1: 20～1: 40, constant by dripping NaOH solution maintenance system pH, by heating in water bath maintenance system homo(io)thermism, it is characterized in that: in the reaction that in enzymatic vessel, is hydrolyzed, be in 6000～10000 the ultra-fine filter continuously by at the bottom of the jar enzymolysis solution being introduced average molecular weight cut-off, the ultra-filtration membrane trapped fluid is back to enzymatic vessel and continues the circulation degraded, it is in 1000～2000 the nanofiltration device that the ultra-filtration membrane penetrating fluid is introduced average molecular weight cut-off continuously, the nanofiltration membrane penetrating fluid dumps, collect nanofiltration membrane and hold back concentrated solution, 150～200 ℃ of inlet temperatures, 70～85 ℃ of temperature outs, input speed 1.0～1.5L/h, under the condition of working pressure 0.1～1MPa, the spray-dried molecular weight that makes is 1000～10000 multi-functional Casein in Milk biologically active polypeptides, comprising: phosphopeptide caseinate, blood pressure lowering peptide, immune-active peptides, angiotensin-convertion enzyme inhibitor, opioid peptide, the thrombolysis peptide, bacteriostatic peptide, the free radical scavenging peptide.
2. the integrated technology of producing the casein biologically active polypeptides continuously of enzymolysis as claimed in claim 1 and membrane filtration, it is characterized in that: film separation unit comprises that one-level at least is used to hold back that the ultra-filtration membrane of casein substrate and proteolytic enzyme and one-level are used for that polypeptide solution concentrates, desalination and remove the nanofiltration membrane of bitter peptides, ultra-filtration membrane working pressure 0.02～0.05MPa, circular flow 20～40L/h, reactant mean residence time 3～4h; Nanofiltration membrane working pressure 0.1～0.5MPa.
3. the integrated technology of producing the casein biologically active polypeptides continuously of enzymolysis as claimed in claim 1 and membrane filtration, it is characterized in that: need constantly in enzymatic vessel, to add substrate solution in the reaction process and proteolytic enzyme is constant with maintenance system, adding mass concentration to the enzymatic vessel Continuous Flow is 1～2% substrate solution, and flow acceleration is consistent with the ultra-filtration membrane permeation flux; Added proteolytic enzyme to enzymatic vessel in per 6～8 hours in batches, and made tank body endoproteinase virtual mass concentration keep 0.1～0.15%.
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| CN103315128A (en) * | 2013-05-29 | 2013-09-25 | 中国农业科学院农产品加工研究所 | Casein powder and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103315128A (en) * | 2013-05-29 | 2013-09-25 | 中国农业科学院农产品加工研究所 | Casein powder and preparation method thereof |
| CN103315128B (en) * | 2013-05-29 | 2014-12-17 | 中国农业科学院农产品加工研究所 | Casein powder and preparation method thereof |
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