CN113637720A - Preparation method of desalted cartilage oligopeptide powder - Google Patents
Preparation method of desalted cartilage oligopeptide powder Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract
The invention relates to a preparation method of desalted cartilage oligopeptide powder, which mainly comprises the following steps: the method comprises the steps of taking meat poultry trachea and cartilage as raw materials, carrying out cooking, enzymolysis and the like to obtain enzymolysis liquid, carrying out ultrafiltration separation on the interception side and the permeation side respectively to obtain chondroitin sulfate and oligopeptide liquid, and enabling the oligopeptide liquid to enter a nanofiltration membrane device for desalination and preconcentration treatment. Firstly, concentrating a raw material liquid by 3-5 times by adopting nanofiltration equipment, then carrying out continuous intermittent nanofiltration constant volume desalination, namely adding pure water to maintain the volume in a tank body to be constant, carrying out percolation, and suspending operation until the conductivity of a permeation solution is lower than 100 mu s/cm. And then pumping the nanofiltration membrane permeate into reverse osmosis membrane equipment, and recycling the water produced by reverse osmosis for the nanofiltration dialysis stage. And (3) carrying out vacuum evaporation concentration and spray drying on the nanofiltration membrane concentrated solution to obtain desalted oligopeptide powder, and recycling evaporated water obtained by evaporation concentration in the cooking stage. The oligopeptides prepared by the method are all active oligopeptides with the molecular weight lower than 2000 Da, the preparation method has better decoloring and odor removing effects, and the ash content of oligopeptide powder is lower than 2 percent.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of desalted cartilage oligopeptide powder.
Background
The protein is hydrolyzed by protease to form free amino acid which is finally absorbed by human body, and some recent researches show that the protein is not absorbed in the form of amino acid, and a large part of the protein is absorbed in the form of peptide, so that the oligopeptide has some special biological effects and nutritional values. In recent years, some peptides which can regulate the physiological function of human body, enhance and exert the physiological activity of human body have been discovered: antibacterial active peptides (antibacterial peptide D, nisin, and hevein selected from silkworm), anticancer polypeptides (such as tumor cell necrosis factor and cyclicpeptide), anti-AIDS peptides (such as GLQ protein), mineral absorption promoting peptides (CPPS), enzyme modulators (such as tryptic peptide), hormone peptides (such as growth hormone releasing factor (GRFS)), etc. In addition, oligopeptides also have thermal stability, water solubility, and fluidity, and thus, oligopeptides are widely added as functional factors to various foods.
The oligopeptide produced in the chondroitin sulfate production process belongs to a byproduct, and has a great economic value after refining, purifying and enriching. The oligopeptide separated from the animal cartilage tissue belongs to hydrolyzed protein, has small molecular weight and easy absorption, not only influences the growth of body tissues such as muscles, but also participates in aspects such as hormone production, immune function maintenance, transport of other nutrients and oxygen, generation of hemoglobin, blood coagulation and the like. It can be used as the main raw material of a microorganism culture medium and applied to the fields of fermentation industry, biochemical products, microorganisms and the like; can be used for animal feed to increase the protein content in the feed; can be used as culture medium for microorganism and animal cell, and ingredient for special cosmetic, and can improve disease resistance of plant in crops.
In the production process of chondroitin sulfate, animal cartilage and trachea are generally subjected to enzymolysis by protease, a certain amount of alkali is continuously added in the enzymolysis process to regulate acid groups generated by enzymolysis, the salt content in the solution is gradually increased by the method, and further, the ash content of an oligopeptide powder product is overhigh, and the ash content of the oligopeptide powder without desalination treatment is up to 6%. Ash content is one of important standards for measuring quality of polypeptide powder, and the ash content of high-quality polypeptide powder in domestic and foreign standards is generally required to be below 2%, so that the oligopeptide solution needs to be desalted.
The existing common desalination method comprises an ion exchange method, electrodialysis and nanofiltration, the ion exchange method has high purification efficiency but is complex to operate, Chinese patent CN 108623654A discloses a preparation method of corn active peptide, a large amount of acid and alkali are consumed in the resin regeneration and elution processes, environmental pollution is caused, and the ion exchange resin has no universal applicability; the electric field is used as a driving force for electrodialysis, and the anion and cation membranes are used for selectively permeating ions, and the Chinese patent CN 110407914A discloses an electrodialysis purification method of glutamine dipeptide, wherein the problems of membrane pollution, sewage treatment and energy consumption cause that the production cost is higher, which is a main limiting factor for the industrial application. The nanofiltration membrane technology has been widely researched in desalination application, the separation performance of the nanofiltration membrane technology is between reverse osmosis and ultrafiltration, the nanofiltration membrane technology is one of the membrane separation technologies which develop rapidly internationally in recent decades, and compared with the two technologies, the nanofiltration membrane can play a role in concentration and can also carry out desalination treatment. The aperture of the nanofiltration membrane is about a few nanometers, the cut-off molecular weight is usually 200-1000 Da, and some inorganic salt and small molecular organic matters are allowed to permeate the membrane under the driving of pressure, so that the separation of macromolecular organic matters and small molecular organic matters and the separation of organic matters and inorganic salts can be realized. Compared with ultrafiltration and reverse osmosis, the nanofiltration membrane has two unique separation characteristics: the retention rate of small molecular substances with the relative molecular mass of about hundreds of Da is higher; the nanofiltration membrane usually has charge property, and due to the influence of the southwest effect, the charge property and the ionic strength of materials have great influence on the separation efficiency of target substances; the nanofiltration membrane has high removal rate of monovalent ions, and the salt ions mainly existing in the oligopeptide liquid are the monovalent ions. Nanofiltration is used as an industrial technology, has the advantages of no phase change in the separation process, simple and convenient operation, low energy consumption, easy integration and amplification and the like, and has extremely wide application prospect. Chinese patent CN1392155A has reported that nanofiltration technology is applied to desalination and concentration of animal and plant oligopeptide solution, but the dialysis process needs to consume a large amount of pure water, and no effective measure is provided for membrane pollution regulation.
Disclosure of Invention
In order to solve the problems and improve the quality and the added value of the oligopeptide powder, the invention aims to provide a method for preparing the cartilage oligopeptide powder based on a membrane separation technology, which prepares the desalted oligopeptide powder by taking meat and poultry cartilage enzymolysis liquid as a raw material and combining ultrafiltration, nanofiltration and reverse osmosis membrane integrated processes. The method can not only reduce the ash content of the oligopeptide powder, but also play a role in decoloring and deodorizing, can save water through water recycling, and reduce sewage discharge. Meanwhile, in the membrane operation process, the flow velocity of the membrane surface is intermittently regulated and controlled to relieve concentration polarization and filter cake accumulation, and an intermittent pulse electric field is added, so that the membrane flux is favorably improved. The method is simple to operate, low in cost and easy to realize industrial application.
A method for preparing desalted cartilage oligopeptide powder comprises the following steps:
s1 mixing trachea and cartilage of meat and poultry according to a certain proportion, cleaning and decontaminating, removing pure water to immerse the raw materials, and steaming at high temperature to keep the boiling liquid boiling;
s2, cooling, adding trypsin for primary enzymolysis, adding NaOH to adjust pH, adding protease after primary enzymolysis, adjusting pH, and heating for inactivation;
s3, clarifying and standing the feed liquid, filtering the feed liquid by a safety filter screen, and feeding the feed liquid into ultrafiltration membrane equipment to obtain ultrafiltration membrane permeate and retentate;
s4, desalting and pre-concentrating the ultrafiltration permeate in a nanofiltration membrane device, and dialyzing by adding pure water after concentration to obtain nanofiltration concentrate and nanofiltration permeate;
s5, allowing the nanofiltration membrane permeate to enter reverse osmosis membrane equipment, and obtaining pure water at the permeate side for reuse in a nanofiltration dialysis stage;
and (3) feeding the nanofiltration membrane concentrated solution S6 into vacuum evaporation concentration equipment, performing spray drying to obtain desalted oligopeptide powder, and reusing the evaporated water in the cooking stage.
As a limitation, in the step S1, the cooking time in the step S2 is 4-8 hours, the used enzyme is protease, the enzymolysis process comprises primary enzymolysis and secondary enzymolysis, the time of the primary enzymolysis is 1-3 hours, and the time of the secondary enzymolysis is 4-8 hours;
in step S3, the concentration multiple of ultrafiltration is 7-10 times, then pure water is added for dialysis, and ultrafiltration is finished when the conductivity of dialysate is constant;
the ultrafiltration membrane component is a spiral wound membrane, a hollow fiber membrane or a plate-type membrane, is made of cellulose acetate, cellulose acetate esters, polyethylene, polysulfone or polyamide, and has a molecular weight cutoff of 2000-5000 Da;
as a further limitation, the operating temperature in the ultrafiltration process is 15-45 ℃, the operating pressure is 0.2-0.7 MPa, and the membrane surface flow rate is 1-5 m/s;
as a second limitation, the nanofiltration in step S4 is characterized by a nanofiltration operation mode of concentration-dialysis, the initial mass fraction of the polypeptide liquid of nanofiltration being 2-15%;
the nanofiltration is characterized in that raw material liquid is concentrated by 3-5 times, then pure water is continuously added for dialysis, the addition amount of the pure water is consistent with the flux of nanofiltration membrane permeate liquid until the conductivity of the nanofiltration permeate liquid is less than 100 mus/cm, and the polypeptide solution is concentrated to 10-50% (w/w).
The molecular weight cut-off of the nanofiltration membrane component involved in the nanofiltration process is 150-300 Da.
The nanofiltration membrane component is a spiral wound membrane, a plate-type membrane and a tubular membrane, and the nanofiltration membrane is made of polyamide, cellulose acetate, polyether sulfone, sulfonated polyether sulfone or polyvinyl alcohol.
As a further limitation, the operating temperature of the nanofiltration membrane in operation is 15-50 ℃, the operating pressure is 0.5-2.5 MPa, and the flow rate of the membrane surface is 1-5 m/s.
The reverse osmosis membrane component is a wide-flow-channel spiral wound membrane.
As a third limitation, in step S6, a double-effect tube-type falling film evaporator is used for vacuum evaporation and concentration, the single-effect evaporation temperature is 60-75 ℃, and the single-effect vacuum degree is 0.07-0.08 MPa; the double-effect evaporation temperature is 50-60 ℃, and the double-effect vacuum degree is 0.08-0.09 MPa.
The temperature of the air inlet of the spray dryer is 130-170 ℃, and the temperature of the air outlet is 65-85 ℃.
The invention has the following characteristics and advantages due to the adoption of the technical scheme: the invention belongs to the technology of deep processing of byproducts, improves the product value, has simple operation process, low energy consumption and less produced wastewater, and is suitable for large-scale industrial production; the prepared oligopeptide powder product has high quality, mainly contains oligopeptides with the molecular weight less than 2000 Da, is easy to absorb, has high nutritional value, is high-quality functional animal protein, and can be used as food additives, feeds and the like; the invention synchronizes the wastewater treatment and production through the membrane integration technology, can greatly save the water consumption through the water reuse, reduces the sewage discharge, and is green and environment-friendly.
Drawings
FIG. 1 is a diagram of a system for concentrating and desalting demineralized oligopeptide powder;
FIG. 2 is a graph showing the molecular weight distribution of oligopeptide powder;
FIG. 3 is a flow chart of oligopeptide powder preparation.
In fig. 1: 1. a raw material tank 2, a reaction tank 3 with a jacket, a pump 4, a circulating water bath 5, a nanofiltration membrane 6, a membrane inlet pressure gauge 7, a membrane outlet pressure gauge 8, trapped liquid 9, permeate liquid 10, a nanofiltration permeate receiving tank 11, a reverse osmosis membrane 12, a flowmeter 13, a thermometer 14 and a pure water receiving tank.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited to the scope of the examples.
Example 1
Accurately weighing 300g of pig cartilage and 600g of pig trachea, washing off surface dirt with clear water, adding clear water, continuously cooking for 5h, cooling to 52 ℃, adding 30g of pig pancreas, adding 2g of NaOH for multiple times to adjust the pH value to 8.50-9.00, and keeping the temperature at 52 ℃ for 2.5 h. Weighing 1.70g of alkaline protease for carrying out second enzymolysis, adding 2g of NaOH for multiple times to adjust the pH to 8.50-9.00, keeping the pH in the whole enzymolysis process in the interval all the time, keeping the temperature at 52 ℃, carrying out enzymolysis for 5h, then inactivating, filtering, entering ultrafiltration membrane equipment with the molecular weight cutoff of 5000Da to effectively separate chondroitin sulfate and oligopeptide solution, and entering the protein material liquid obtained by separation into ultrafiltration membrane equipment with the molecular weight cutoff of 150 plus 300DaAnd (3) polyamide nanofiltration membrane equipment, namely concentrating the oligopeptide solution to the minimum volume, adding deionized water for percolation, regulating the membrane surface flow rate to be 3 m/s, and regulating the membrane surface flow rate to be 5m/s after 3 h of percolation. The pulse voltage of the membrane module is controlled at 15V, the operation is interrupted for 7min, and the average membrane flux reaches 30 L.m-2·h-1When the permeate had a conductivity of 87. mu.s/cm, the filtration was stopped. Spray drying is carried out after desalting to obtain 61.89 g of oligopeptide powder product, and the ash content is measured to be 1.9%.
Example 2
Accurately weighing 350g of chicken cartilage and 650g of duck trachea, washing off surface dirt with clear water, adding clear water, continuously cooking for 4-6 h, cooling to 48 ℃, adding 28g of pig pancreas, continuously stirring at 120rpm, adding 2.5g of NaOH for multiple times to adjust the pH value to 8.50-9.00, and keeping the temperature at 48 ℃ for 2 h. Weighing 1.50g of alkaline protease for carrying out second enzymolysis, adjusting the pH to 8.50-9.00, keeping the pH in the whole enzymolysis process in the interval all the time, continuously stirring in the enzymolysis process to ensure full enzymolysis, keeping the process temperature at about 50 ℃ all the time, carrying out enzymolysis for 4 hours, then stopping stirring, feeding the inactivated and filtered feed liquid into ultrafiltration membrane equipment with the molecular weight cutoff of 5000Da to effectively separate chondroitin sulfate and oligopeptide solution, feeding the separated protein feed liquid into polyethersulfone nanofiltration membrane equipment with the molecular weight cutoff of 150 and 300Da, concentrating the oligopeptide solution to the minimum volume, adding deionized water for carrying out percolation, firstly regulating and controlling the membrane surface flow rate to be maintained at 3 m/s, and regulating the membrane surface flow rate to be 5.5 m/s after the percolation for 3.5 hours. The pulse voltage of the membrane module is controlled at 17V, the operation is interrupted for 7min, and the average membrane flux reaches 42.5 L.m-2·h-1When the permeate had a conductivity of 78. mu.s/cm, the filtration was stopped. Collecting the nanofiltration permeate, preparing pure water by reverse osmosis, recycling the pure water for the nanofiltration dialysis process, and performing spray drying to obtain 68.93 g of oligopeptide powder product, wherein the ash content is determined to be 1.67%.
Example 3
Accurately weighing 450g of duck cartilage and 550g of pig trachea, washing off surface dirt with clear water, adding clear water, continuously steaming for 3-5 h, cooling to 50 ℃, adding 30g of pig pancreas, adding 3 g of NaOH for multiple times to adjust pH to 8.50-9.00, keeping temperature at 50 DEG CLasting for 2 h. Weighing 1.85 g of alkaline protease for carrying out second enzymolysis, adjusting the pH to 8.50-9.00, keeping the pH in the whole enzymolysis process in the interval all the time, keeping the temperature at 50 ℃, carrying out enzymolysis for 4h, adding 17 g of hydrochloric acid and 3.00g of hydrogen peroxide for inactivation, filtering by using a double-layer 400-mesh filter screen, enabling the feed liquid to enter ultrafiltration membrane equipment with the molecular weight cutoff of 4000Da so as to effectively separate chondroitin sulfate and oligopeptide solution, enabling the separated protein feed liquid to enter cellulose acetate nanofiltration membrane equipment with the molecular weight cutoff of 200Da, concentrating the oligopeptide solution to the minimum volume, adding deionized water for carrying out diafiltration, firstly regulating and controlling the membrane surface flow rate to be maintained at 3 m/s, and regulating the membrane surface flow rate to be 6 m/s after carrying out diafiltration for 4 h. The pulse voltage of the membrane module is controlled at 16V, the operation is intermittent for 8 min, and the average membrane flux reaches 35.5 L.m-2·h-1When the permeate had a conductivity of 98. mu.s/cm, the filtration was stopped. Collecting the nanofiltration permeate, preparing pure water by adopting reverse osmosis, and recycling the pure water for the nanofiltration percolation process. And (4) evaporating and concentrating the desalted oligopeptide solution to 2-3 times of the initial volume, and stopping the operation. And then spray drying to obtain 65.30g of oligopeptide powder product, and determining that the ash content is 1.8%.
The oligopeptide powder sample obtained in the above embodiment is used for ion content determination and molecular weight distribution detection by atomic emission spectroscopy and time-of-flight mass spectroscopy, and the result is shown in the following chart.
Table 1 change in ash content and metal ion content of oligopeptide powder before and after desalting.
| Item | As received | Polyamide nanofiltration membrane | Polyethersulfone nanofiltration membrane | Cellulose acetate film |
| Ash content | 6.9% | 1.9 % | 1.67% | 1.8% |
| Na | 14000 | 3000 | 2100 | 2400 |
| K | 4200 | 1400 | 1700 | 1200 |
Claims (9)
1. The preparation method of the desalted cartilage oligopeptide powder is characterized by comprising the following steps: the method comprises the following steps of taking cartilage and air pipes from meat and poultry such as chicken, duck, pig, cattle and the like as raw materials, carrying out cooking, enzymolysis and the like to obtain chondroitin sulfate and oligopeptide mixed liquid, and separating by an ultrafiltration membrane component to obtain oligopeptide liquid and chondroitin sulfate solution; the oligopeptide liquid enters a nanofiltration membrane component, concentration is carried out firstly, then pure water is added for dialysis, the dialysate enters a reverse osmosis membrane component, and the prepared pure water is recycled in a nanofiltration dialysis stage; the nanofiltration membrane concentrated solution is subjected to vacuum evaporation concentration and spray drying to obtain oligopeptide powder, and the evaporated water is reused in the cooking stage.
2. The method according to claim 1, wherein the raw material solution is a mixture of chondroitin sulfate and oligopeptide, the cooking time is 4-8 h, the enzyme is protease, the enzymolysis process comprises primary enzymolysis and secondary enzymolysis, the primary enzymolysis time is 1-3 h, and the secondary enzymolysis time is 4-8 h.
3. The method of claim 1, wherein the concentration factor of ultrafiltration is 7-10 times, and then pure water is added for dialysis, and the ultrafiltration is terminated when the conductivity of the dialysate is constant; the ultrafiltration membrane component is a spiral wound membrane, a hollow fiber membrane or a plate-type membrane, is made of cellulose acetate, cellulose acetate ester, polyethylene, polysulfone or polyamide, and has a molecular weight cutoff of 2000-5000 Da; the operating temperature in the ultrafiltration is 15-45 deg.C, the operating pressure is 0.2-0.7 MPa, and the membrane surface flow rate is 1-5 m/s.
4. The method of claim 1, wherein the concentration-dialysis nanofiltration operation mode comprises concentrating the feed solution by 3-5 times, continuously adding pure water for dialysis, wherein the amount of the added pure water is consistent with the flux of the nanofiltration permeate, and concentrating the polypeptide solution to 10-50% (w/w) until the nanofiltration permeate conductivity is less than 100 μ s/cm; the initial mass fraction of the nanofiltration polypeptide liquid is 2-15%; the molecular weight cut-off of the nanofiltration membrane component involved in the nanofiltration process is 150-300 Da.
5. The method according to claim 1, wherein the nanofiltration membrane module is a spiral wound membrane, a plate-type membrane or a tubular membrane, and the nanofiltration membrane is made of polyamide, cellulose acetate, polyethersulfone, sulfonated polyethersulfone or polyvinyl alcohol.
6. The method according to claim 1, wherein the operating temperature of the nanofiltration membrane is 15-50 ℃, the operating pressure is 0.5-2.5 MPa, and the flow rate of the membrane surface is 1-5 m/s; the nanofiltration membrane component is added with an intermittent pulse electric field, the pulse voltage is maintained at 10-20V, and the operation is performed for 5-10 min intermittently.
7. The method of claim 1 wherein the reverse osmosis membrane module is a wide flow path spiral wound membrane.
8. The method as claimed in claim 1, wherein the vacuum evaporation concentration is performed by using a double-effect tube-type falling film evaporator, the one-effect evaporation temperature is 60-75 ℃, and the one-effect vacuum degree is 0.07-0.08 MPa; the double-effect evaporation temperature is 50-60 ℃, and the double-effect vacuum degree is 0.08-0.09 MPa.
9. The method as claimed in claim 1, wherein the temperature of the air inlet is 130-170 ℃ and the temperature of the air outlet is 65-85 ℃.
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| CN1392155A (en) * | 2002-07-23 | 2003-01-22 | 江南大学 | Desalting and concentrating of oligopeptide solution by nano filtering technology |
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