CN101664543A - Hemokinin-iron oral liquid - Google Patents

Hemokinin-iron oral liquid Download PDF

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CN101664543A
CN101664543A CN200910171107A CN200910171107A CN101664543A CN 101664543 A CN101664543 A CN 101664543A CN 200910171107 A CN200910171107 A CN 200910171107A CN 200910171107 A CN200910171107 A CN 200910171107A CN 101664543 A CN101664543 A CN 101664543A
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iron
juice
heme
bivalent
peptide
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CN101664543B (en
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张滨
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Zhang Bin
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Abstract

The invention provides a divalent hemokinin-iron oral liquid, the preparation method thereof comprises: separating hematid out of blood of livestock or poultry, adding doubleness distilled water, combining ultrasonication to break wall, releasing hemoglobin, adjusting pH value of hemoglobin solution, adding protease, placing in a vacuum dry chamber, leading in pulse current for acting for 4 hours,carrying out heating enzyme killing treatment at 95 DEG C for 30 minutes, obtaining divalent hemokinin-iron with purity more than 90% by metal-chelating affinity chromatography and electromagnetic separation, then adding tomato juice, red date juice, liquorice juice, angelica sinensis juice, astragalus mongholicus juice, juice of dried rhizome of rehmannia, stevioside, glycoprotein, carrageenan and algae ester sodium to blend so as to obtain the hemokinin-iron oral liquid. The oral liquid beverage product prepared by the invention has small dose, good mouth feel and high portability, can be directly absorbed by intestinal mucosal cells, cannot directly generate any digestion characteristic irritation symptom, and the absorptivity is high.

Description

Heme peptide-iron oral liquid
Technical Field
The invention relates to a biological product and a preparation method thereof, and particularly provides a preparation method of a bivalent heme peptide-iron and a separation and purification method thereof. The application fields are iron-supplementing capsules, iron-supplementing medicinal granules, iron-supplementing oral liquid or iron-supplementing beverage.
Background
Iron is an indispensable nutrient in most organisms, and is about 4-5 g in adults, which accounts for one ten-thousandth of the body weight, with a very low proportion, and most of the iron in the human body exists intracellularly in the form of hemoglobin (76%) and ferritin (23%). But its role is very important. Iron is a basic raw material for generating hemoglobin, myoglobin and iron-containing enzymes by organisms, is a necessary trace element for human bodies, participates in the transportation of oxygen and carbon dioxide in the bodies, and is a component of tissue respiratory enzymes such as cytochrome oxidase and peroxidase. The importance of iron metabolism has therefore far exceeded the range of red blood cells and haemoglobins.
If the iron deficiency or iron absorption disorder occurs for a long time in the diet, Iron Deficiency Anemia (IDA) can be caused, the metabolism of red blood cells is influenced, the immunologic function of the human body is reduced, and the growth and the intelligence development of the teenagers and children are seriously influenced. According to statistics, 95.65% of nutritional anemia in China is caused by iron deficiency anemia, and the iron deficiency anemia is about 20% -70% in children under 7 years old. At present, the traditional iron supplement agents such as ferrous sulfate, ferrous fumarate and the like are inorganic iron, have low in-vivo utilization rate, about 5-8% of absorption rate, have large side effect, often cause symptoms such as nausea, abdominal distension, dyspepsia, diarrhea, constipation and the like, and have special fishy smell. Iron in the peptide iron chelate is easier to utilize and absorb than ferrous sulfate.
Iron deficiency anemia is a ubiquitous nutritional deficiency disease worldwide, particularly in developing countries. At present, various oral iron preparations in the world are nearly hundreds of kinds, such as ferrous sulfate, ferrous gluconate, ferrous citrate and the like. For IDA, iron therapy is mostly adopted at home and abroad, and although the products have certain curative effect, the following problems exist: (1) the bioavailability is low: if the ferrous sulfate which is popular for more than 200 years is difficult to be absorbed by human body, the absorption rate is about 1.6 percent, and the absorption rate of other inorganic iron agents and organic iron agents is between 3 percent and 5 percent. (2) The side effect is great: these iron products all destroy the balance of metal ions in the human body, cause gastrointestinal adverse reactions, and require to be taken after meals in order to reduce the adverse side effects of gastrointestinal discomfort, and at this time, the absorption rate of the dietary phosphoric acid and phytic acid is further reduced because of the inhibition of iron absorption. (3) Poor mouthfeel, difficult persistence: the products have certain rust taste and poor mouthfeel, and the treatment course of IDA (IDA) is usually as long as several months (2-6 months), so the IDA is more difficult to persist.
The above problems of iron supplement agents are also the main reason why IDA has not been improved fundamentally in the global area for a long time.
The iron supplement agent has three types: i.e., inorganic, such as ferrous sulfate; organic phases, such as ferrous gluconate; biological states such as the heme peptide iron. For the benefit of the three types of iron agents to the human body, the sequence is: biological iron > organic iron > inorganic iron.
The absorption test of scientists on iron agents proves that the absorption rate of heme iron peptide in small intestine can be from 23-27%, while the absorption rate of non-heme iron peptide (iron contained in vegetable food such as vegetables, fruits, beans) is only 3-6%. The so-called bivalent heme iron peptide (Hemepeptide-iron) is biological iron, can be directly absorbed by small intestinal mucosa cells, does not directly produce any stimulation symptom with digestion characteristics, and has the absorption rate as high as 91 percent. The research shows that bivalent iron is more beneficial to the absorption of human body than trivalent iron, organic iron has less irritation to intestines and stomach than inorganic iron and is easy to absorb, and heme iron is easier to absorb than non-heme iron.
Therefore, the bivalent heme peptide iron is the biological iron agent which is most easily absorbed by human bodies, is the best utilization state of the biological iron agent, and has the highest bioavailability.
The livestock and poultry blood resources are very rich, China is a big meat producing country, the livestock and poultry yield in China is continuously increased after 20 years of innovation, and the total meat yield is increased by 11% every year. The development is slow in the aspect of deep processing of the bone blood of the livestock and poultry slaughtering by-products, the utilization rate is less than 10%, except that part of the bone blood is utilized in the primary processing forms of blood bean curd, blood meal, bone meal and the like, a certain part of the bone blood is discharged or discarded in the form of sewage, so that a large amount of valuable nutrient resources are lost, and serious environmental pollution is caused. Some processed products belong to feed grade, food grade products need to be imported from foreign countries, and the production technology of plasma protein powder and blood cell protein powder has a certain gap with the foreign countries. For example, the denaturation rate of immunoglobulin in plasma protein powder is high, the erythrocyte wall breaking technology is not effectively solved, the digestion and absorption of the erythrocyte protein powder are poor, and the like. Some key technologies in the comprehensive utilization of livestock and poultry bone blood are weak, and the comprehensive utilization rate is low.
The bivalent heme peptide-iron is prepared with animal blood as main material and through anticoagulation, hemoglobin degradation and other technological processes. In the case of pig, the pig blood accounts for about 5.0-6.0% of the weight of the pig, and the protein content in the blood is up to 17-20%, wherein 2% of the blood is the immunoglobulin with physiological activity. The pig blood protein contains 8 essential amino acids required by human body, and its composition is balanced, so that it is a high-quality protein. Active substances such as immunoglobulin, transfer factor and the like in blood have remarkable effects on passive immune function and the like, the iron content in pig blood reaches 2.1-2.2 g/kg, and the pig blood is natural organic iron (iron porphyrin), the iron porphyrin is an organic form of iron, is beneficial to absorption by a human body, has no adverse reaction, and can effectively supplement the iron in the human body. Is suitable for increasing iron demand of human body (such as infants and pregnant women), insufficient intake of meat and liver and various iron-deficiency anemia, and is a good natural blood-enriching agent.
The application of heme iron peptide in food and medicine is rapidly developed, and the preparation method is more, such as glacial acetic acid method. Base-organic solvent method, ion-exchange cellulose method, etc. However, they all have certain limitations and defects, which affect the yield and quality of the product. Chinese patent CN1094618A discloses a method for extracting heme and protein powder from livestock blood, which comprises preparing qualified livestock blood into blood powder, extracting heme from the blood powder with mixed solvent mainly containing pyridine under anhydrous condition, washing the extracted blood powder with ethanol to obtain protein powder, and evaporating the extractive solution under reduced pressure to obtain heme. The process is complicated, the yield is not high due to certain loss of effective components, a large amount of chemical reagents are consumed, the cost is high, and the extracted heme is extremely unstable. In addition, the mixed solvent mainly containing pyridine is used for extracting the heme, so that the heme has certain toxicity and peculiar smell, pollutes the environment and is harmful to health. And thus its application is greatly limited. Chinese patent CN1362525 discloses a method for preparing small peptide heme by ultrafiltration and enzymatic hydrolysis, which, although less chemical reagents are introduced, is too cumbersome in the whole process (step 12), and the heme loss is too much during ultrafiltration, and the cost of the added enzyme preparation is too high, thus still not satisfying the application requirements.
Therefore, there is a need for a cost-effective method for separating and purifying iron heme peptides that avoids the excessive use of chemical reagents, is simple in process, and can utilize waste blood sources of livestock.
Disclosure of Invention
The principle of the invention is that the livestock and poultry blood which is not developed in large scale at present is used as a raw material, a high-frequency pulse electric field is combined under the condition of vacuum environment, the hemoglobin is degraded by protease, and the bivalent heme iron peptide is separated by methods such as metal chelating affinity chromatography or electromagnetic separation, the purity of the bivalent heme iron peptide reaches more than 90 percent, and the application fields of the bivalent heme iron peptide are iron-supplementing capsules, iron-supplementing medicinal granules, iron-supplementing oral liquid or iron-supplementing beverage.
Accordingly, a first object of the present invention is to provide a method for preparing a bivalent heme peptide iron, comprising:
(1) separating red blood cells from the blood of livestock and poultry, and adding an anticoagulant for treatment;
(2) dispersing red blood cells, and centrifugally collecting red blood cell sediments;
(3) breaking the wall of the red blood cells by ultrasonic waves, releasing hemoglobin, and adjusting the pH according to the used degrading enzyme;
(4) adding a degrading enzyme solution, and performing high-frequency pulse treatment in a vacuum chamber to ensure that the enzyme fully degrades hemoglobin and releases bivalent heme peptide iron;
(5) after inactivating the enzyme at high temperature, centrifuging and collecting supernatant;
(6) and purifying and collecting the bivalent heme peptide iron by metal chelating affinity chromatography or electromagnetic separation of the supernatant.
Wherein, in one embodiment, the high-frequency pulse treatment is performed by applying a pulse frequency of 200kHz for 4 hours in the step 4. In a specific embodiment, the electric field parameter of the high-frequency pulse in step 4 is T1-500.0us、T2500.0us、Δ1.000ms、f1.000KHz、V11.88v、V2120mv and h20.3, the pulse wave type is bidirectional square wave, the electrode spacing is 1.2cm, and the pulse frequency is 200 kHz. In another embodiment, in step 4, the vacuum degree in the vacuum chamber is reduced to 0.01MPa within 1 minute, the power is turned on to make the temperature in the vacuum chamber constant at 37 deg.CAnd (4) keeping for 4 hours, and degrading the hemoglobin by using protease under the conditions of vacuum and high-frequency pulse electric field.
In another embodiment, the flow rate of the hemoglobin degradation liquid passing through the electromagnetic separator in step 6 is 1-2 cm/min, reflux treatment is performed for 3-4 times, then deionized distilled water is washed for 5min, and the magnetism is removed, so that the released column inner wall substance is bivalent heme peptide iron. In a specific embodiment, the central magnetic field of the electromagnet is 1.2T, a glass column with the diameter of 5mm and the length of 200mm is arranged at the center of the magnetic field, 130m stainless steel wires with the length of 5um and the diameter of 300 alloy compositions are wound at the periphery of the glass column to form the magnetic glass column, the current of a direct current power supply for electromagnetic separation is not less than 20A, and the voltage is not less than 50V. In another specific embodiment, the hemoglobin degradation liquid uniformly flows through the center of the electromagnet from top to bottom along the inner wall of the tube at a flow rate of 40-120 ml/h, and the angle between the cross section of the glass column tube and the center of the electromagnet is 15 degrees. The power is turned on and the current reaches 20A. When the volume of the effluent reaches 20ml, the power supply is cut off, the electromagnetism is relieved, and the substances in the tube are washed by cell separation buffer solution, namely the bivalent heme peptide iron.
Also in the previous embodiment, 6000U/mL trypsin was added in step 4; in step 5, the enzyme is extinguished at high temperature by maintaining the vacuum degree at 0.01MPa, raising the temperature of the vacuum chamber to 95 ℃ for 10-30 minutes.
In one embodiment, step 1 is performed using a mixture of 0.2% trisodium citrate and 0.15% sodium citrate as an anticoagulant, added to the blood at 5% by weight of the blood; adding 2% of D-glucose, 0.85% of NaCl, 0.05% of citric acid and 0.08% of sodium citrate according to the weight of 10% of blood in the step 2 to prepare a cell dispersion, standing for 5 minutes, centrifuging at the rotating speed of 5000r/min for 30 minutes, discarding the supernatant, and collecting the precipitate. In one embodiment, trypsin is used to adjust the pH to 8.0 and neutral protease is used to adjust the pH to 7.0 in step 3 to prevent oxidation of the iron ions in the hemoglobin.
The second purpose of the invention is to provide a preparation method of the bivalent heme peptide iron blood-enriching beverage, which comprises the following steps:
(1) purifying and collecting the bivalent heme peptide iron according to any scheme;
(2) adding tomato juice, fructus Jujubae juice, Glycyrrhrizae radix juice, radix Angelicae sinensis juice, radix astragali juice, radix rehmanniae juice, steviosin, aspartame, carrageenan, and sodium alginate, blending, adjusting to 1L with ultra-pure water, and adjusting pH to 4.0 with citric acid, dipotassium hydrogen phosphate or potassium dihydrogen phosphate;
(3) heating the mixed solution to 65 ℃, vacuum degassing under the vacuum degree of 0.18MPa, and then homogenizing twice under the vacuum degree of 30MPa by using a homogenizer;
(4) sterilizing at high temperature, and packaging to obtain the bivalent heme peptide iron blood-replenishing beverage.
The third purpose of the invention is to provide the bivalent heme peptide iron blood-enriching beverage prepared by the method.
In one embodiment, the beverage composition prepared comprises: 5g/L of bivalent heme peptide-iron, 38mL/L of tomato juice, 28mL/L of red date juice, 8mL/L of liquorice juice, 6.5mL/L of angelica sinensis juice, 4.0mL/L of astragalus juice, 2.7mL/L of radix rehmanniae juice, 1.5g/L of stevioside, 1.0g/L of aspartame, 0.8g/L of carrageenan and 0.5g/L of sodium alginate, wherein the volume of the extract is adjusted to 1L by using ultrapure water, and the pH value of the extract is adjusted to 4.0 by using citric acid, dipotassium hydrogen phosphate or potassium dihydrogen phosphate; or,
5g/L of heme peptide-iron, 100mL/L of tomato juice, 160mL/L of red date juice, 150mL/L of liquorice juice, 45mL/L of honey, 30mL/L of astragalus membranaceus, 120mL/L of codonopsis pilosula, 40mL/L, CMCNa 0.3g/L of medlar, 0.2g/L of sodium alginate, 0.1g/L of vitamin C and 0.035mg/L of amaranth, and the pH value is adjusted to 4.0 by 0.85g/L of citric acid; or
5g/L of heme peptide-iron, 200ml/L of tomato juice, 140ml/L of red date juice, 50ml/L of liquorice juice, 25ml/L, CMCNa 0.3.3 g/L of honey, 0.2g/L of sodium alginate and 0.1g/L of Vc, and the pH is adjusted to 4.0 by using citric acid, dipotassium hydrogen phosphate or potassium dihydrogen phosphate.
Advantageous effects
1. The method has simple steps, and the used chemical reagents are all edible additives, so that the residual toxicity and peculiar smell of the chemical reagents are avoided;
2. the enzyme preparation is cheap in industry, and the cost is low;
3. the hemoglobin can be released to the maximum extent by treating the blood cells with ultrasonic waves;
4. the real-time automatic operation can be realized by electric field high-frequency pulse treatment, and the heme peptide iron component can be completely released after the degradation of the combined enzyme preparation;
5. by electromagnetic separation, loss of effective components caused by ultrafiltration or chromatographic separation is avoided, thereby obtaining higher yield.
6. The purity of the bivalent heme peptide-iron purified by the method can reach more than 90 percent, and the production requirement of heme peptide-iron functional food or beverage is completely met.
Detailed description of the preferred embodiments
Example 1: erythrocyte separation of livestock and poultry blood
(1) Blood collection
The qualified healthy livestock and poultry are eaten at no time and kept for 24 hours (only water is fed during the period), and then are transported to a slaughtering workshop, and the blood is rapidly collected in a sealable container (a non-toxic plastic bag, a non-toxic plastic bottle, stainless steel and other containers) by using a tubular vacuum blood collecting knife.
(2) Anticoagulation treatment
3-5 minutes before blood collection, a mixture of 0.2% trisodium citrate and 0.15% sodium citrate is used as an anticoagulant, 5% of the blood collection amount is added into a blood collection container, and the mixture and the blood are stirred for 3 minutes at a speed of 100 r/min.
(3) Erythrocyte isolation
Adding 2% D-glucose, 0.85% NaCl, 0.05% citric acid and 0.08% sodium citrate in an amount which is 10% of the blood volume into the anticoagulated blood to prepare a cell dispersion, standing for 5 minutes, centrifuging at the rotating speed of 5000r/min for 30 minutes, discarding the supernatant, collecting the precipitate, and repeating the operation twice.
Example 2: breaking cell wall of erythrocyte
(1) Breaking cell wall of erythrocyte
Adding distilled water according to 2 times of erythrocyte precipitate amount, placing in ultrasonic processor for 2 min, breaking erythrocyte wall, and releasing hemoglobin.
(2) Adjusting the pH of a hemoglobin solution
Adjusting the pH value of the hemoglobin solution to be in accordance with the pH value of the optimal degradation of the protease, if trypsin is used, adjusting the pH value to 8.0, if neutral protease is used, adjusting the pH value to 7.0, and adjusting the pH within 2 minutes, wherein the oxidation reaction of iron ions in the hemoglobin is mainly prevented.
Example 3: under the condition of vacuum high-frequency pulse environment, the protease degrades the hemoglobin
(1) Requirement for equipment for proteolysis of hemoglobin by vacuum high-frequency pulse
A vacuum chamber: the vacuum chamber can bear the vacuum degree below 0.01MPa and maintain the vacuum degree change within +/-0.2 Pa for 6 hours.
High-frequency pulse: the parameter of the pulse electric field is T1-500.0us、T2500.0us、Δ1.000ms、f1.000KHz、V11.88v、V2120mv and h20.3, the pulse wave type is bidirectional square wave, the electrode spacing is 1.2cm, and the pulse frequency is 200 kHz.
(2) Adding protease
Adding 6000U/mL protease solution into the solution subjected to wall breaking and hemoglobin solution pH adjustment, stirring for 2 minutes at a speed of 50r/min, sealing the container, and placing the container in a vacuum chamber.
(3) Inserting pulse electrode
Inserting the pulse electrode connected with the pulse signal generator into a closed container containing hemoglobin solution, wherein the distance between the two electrodes is 1.2cm, checking whether a vacuum pump, a pulse signal generator pipeline and a circuit are connected, closing a door of the vacuum chamber, and starting the vacuum pump to pump vacuum.
(4) Adjusting the temperature and degree of vacuum in the vacuum chamber
Reducing the vacuum degree in the vacuum chamber to 0.01MPa within 1 minute, switching on a power supply to keep the temperature in the vacuum chamber constant at 37 ℃, maintaining for 4 hours, and degrading the hemoglobin by using protease under the conditions of vacuum and a high-frequency pulse electric field environment.
Example 4: termination of protease Activity
(1) Enzyme deactivation
The power supply to the pulse generator was turned off and the temperature in the vacuum chamber was raised to 95 ℃ for 10 minutes. The vacuum in the vacuum chamber was maintained at 0.01MPa during the heating.
(2) Cooling down
And (3) closing the heating power supply of the vacuum chamber, closing the power supply of the vacuum pump when the temperature in the vacuum chamber reaches the room temperature, relieving the vacuum degree in the vacuum chamber, and taking out the closed container containing the hemoglobin degradation liquid.
(3) Centrifugation
Quickly pouring the hemoglobin degradation liquid into a sealable centrifugal tube, adding 3/4 with the height of the centrifugal tube, quickly sealing the centrifugal tube, putting the centrifugal tube into a centrifugal machine, and centrifuging for 15 minutes at 5000 r/min. Collecting supernatant, sealing in a sealed container, and storing at-18 deg.C if the time of standing is more than 4 hr.
Example 5: isolation of Iron (II) heme peptides
(1) Separation of iron (II) heme peptides by metal chelate affinity chromatography (IMAC)
Preparing an affinity chromatography column filler: weighing 30 g of agar powder, adding 0.04mol/L sodium acetate buffer solution with pH4.0 according to the proportion of 1g of agar powder to 50ml of buffer solution, stirring and extracting, filtering precipitates after extraction, and washing with water for 3 times. And extracting the agar powder by using 0.05mol/L sodium carbonate-sodium bicarbonate buffer solution with the pH of 9.0 according to the proportion, stirring the mixture for 6 hours, washing the mixture to be neutral, and draining the mixture to obtain the wet agar powder. 150ml of 1.6mol/L sodium hydroxide solution is added into the wet agar powder, 57ml of acetone and 28.5ml of epichlorohydrin are added into the wet agar powder at the temperature of 25 ℃ while stirring, and after reaction for 12 hours, the wet agar powder is washed by deionized water to be neutral. Then, 520ml of a 0.1mol/L sodium carbonate solution (containing 9% iminodiacetic acid) was added thereto, the pH was adjusted to 11 with 6mol/L sodium hydroxide, and the reaction was stirred at 25 ℃ for 12 hours. Taking 50g of reactants, adding 80ml of 50mmol/L sodium phosphate buffer solution (containing 5mg/ml copper chloride and 1mol/L sodium chloride) with pH of 6.0, stirring and reacting for 12 hours at 25 ℃, fully washing with deionized water and draining to obtain the affinity chromatography column filler.
Column assembling: preparing a chromatographic column with the diameter phi of 25cm and the length of 100cm, vertically fixing the chromatographic column on a titration test tube bracket, closing a knob, injecting 1/4 distilled water with the length of a tube, dissolving the prepared affinity chromatographic column filling material with 1 time of distilled water, slowly injecting the mixture into the chromatographic column at the flow rate of 2cm/min, and when the filling material is 3cm away from the chromatographic column opening, finishing filling, and balancing with distilled water for 30 minutes.
Chromatography: and (4) installing a column chromatography separation system, adjusting the ultraviolet detection absorption peak to 410nm, and automatically collecting the separation liquid.
(2) Electromagnetic separation of ferrous heme peptide iron
Electromagnetic separation equipment: the electromagnetic valve consists of an electromagnet made of superconductor material, a vacuum bottle filled with liquid helium and a plastic rubber tube surrounded by a stainless steel wire. Filling liquid helium into the vacuum bottle; the height of the electromagnet is generally designed to be 25cm, the inner diameter is 50mm, the outer diameter is 200mm, and the central magnetic field of the electromagnet is 1.0-1.5T; a stainless steel wire of 40um with a length of 5cm is wound outside the center of the plastic rubber tube, and the density is 150cm2In/cm, to enhance the electromagnetic intensity of the center.
A direct-current power supply: the current of the DC power supply is not less than 20A, and the voltage is not less than 50V.
Electromagnetic separation: the hemoglobin degradation liquid uniformly flows through the center of the electromagnet from top to bottom along the inner wall of the tube at a flow rate of 40-120 ml/h, and the angle between the cross section of the glass column tube and the center of the electromagnet is 15 degrees. The power is turned on and the current reaches 20A. When the volume of the effluent reaches 20ml, the power supply is cut off, the electromagnetism is relieved, and the substances in the tube are washed by cell separation buffer solution, namely the ferrous iron solution of the heme peptide.
Example 6: concentrating, drying and low-temperature preserving ferrous heme iron
The obtained bivalent heme iron peptide is utilized within 4 hours and needs to be cooled and stored; if used after 4 hours, the product is preserved or freeze-dried.
(1) Concentrating and drying
And (3) putting the bivalent heme peptide iron subjected to metal chelating affinity chromatography (IMAC) and electromagnetic separation into a vacuum freeze drying oven, and drying at-40 ℃ and the vacuum degree of 0.018mPa for 10 hours to obtain concentrated and dried bivalent heme peptide iron.
(2) Low temperature preservation
Vacuum packaging the concentrated and dried ferrous heme iron peptide, and freezing at-18 deg.C.
Example 7: determination of ferrous purity and molecular mass of bivalent heme peptide
(1) Determination of iron purity of heme peptide
The purity of the bivalent heme peptide iron is determined by three modes of high-efficiency gel chromatography, Native-PAGE electrophoresis and ultraviolet visible scanning.
The high-efficiency gel chromatography method comprises the following steps: chromatographic column selection of glucose gel-200 (Sephacry 1)TmS-200); mobile phase: ultra-pure water, flow rate: 0.5 ml/min; the detector temperature was 25 ℃. Filtering the enzymolysis product of pig hemoglobin with 0.5 μm microporous membrane, injecting 50 μ l into chromatograph, and measuring at 300nThere is only one distinct absorption peak at m.
Native-PAGE electrophoresis method: Native-PAGE (Native-PAGE) is to perform polyacrylamide gel electrophoresis on protein with activity maintained without adding denaturants such as SDS mercaptoethanol and the like, so that the natural shape and charge of biomacromolecules can be maintained in the electrophoresis process, and higher resolution can be obtained, and the Native-PAGE has important significance for identification of the biomacromolecules. The native polyacrylamide gel and the denatured SDS-PAGE electrophoresis are essentially identical in operation, except that the native polyacrylamide gel is formulated and the running buffer cannot contain denaturing agents such as SDS and the like, and only one distinct band is found in the native-PAGE protein gel electrophoresis image.
③ the ultraviolet visible scanning method: and scanning the sample in the range of 200-800nm by using an ultraviolet spectrophotometer to observe whether a characteristic absorption peak exists or not.
(2) Determination of the molecular mass of the iron of the heme peptide
Measuring the content of ferrous heme peptide
A three band scanning method is used. The absorbance values were measured at 525, 545, 565 and 575nm, as heme peptide (mmol/L) — (0.166R)1+0.086R2+0.088R3+0.099)×A525Wherein: r1＝A575/A525、R2＝A565/A525、R3＝A545/A525And calculating the content.
② measurement of ferrous heme peptide iron molecular mass
Adopting a matrix-assisted laser desorption tandem time-of-flight mass spectrometry method: after 1. mu.L of the sample was spotted on a MALDI target plate and allowed to dry naturally, 1. mu.L of a saturated matrix CHCA (α -cyano-4-hydroxycinnamic acid) solution was spotted and allowed to dry naturally at room temperature. Mass spectrometry was performed using an autoflex III smartpeak tandem time-of-flight mass spectrometer, using Smartbeam solid state laser with a wavelength of 335nm and an acceleration voltage of 20kV, and data were collected in positive ion mode. The Peptide Mass Fingerprint (PMF) mass scanning range of the sample is 3000-20000Da, and the iron molecular mass of the bivalent heme peptide is determined to be 7529.0968 Da.
Third, measuring iron ion in bivalent heme peptide iron
Adopting an atomic absorption spectrophotometer method: atomic absorption working parameters: wavelength 248.3nm, slit 0.2nm, lamp current 3mA, air-acetylene 1.5L/min, argon flow 6.5L/min. Weighing 5mg of heme peptide-iron, placing in a conical flask, adding 10.0mL of nitric acid and perchloric acid (4: 1), heating on an electric hot plate until no yellow smoke is emitted, taking down, cooling, adding 2.0mL of 5% hydrochloric acid, and continuously heating to remove acid until white smoke is emitted. Cooling, washing with 5% hydrochloric acid for several times, and diluting to 50mL volumetric flask to obtain the solution to be measured. Under the condition of the set working parameters of the atomic absorption spectrophotometer, the content of the iron in the heme peptide iron is measured to be 1013.500 ug/L.
(3) Yield and purity of pilot-scale produced heme peptide iron
The yield and purity of the ferrous heme peptide produced by pilot plant experiments are shown in the following table.
TABLE 1 yield of pilot plant production of heme peptide-iron
Example 8: bivalent heme peptide iron blood-enriching beverage
(1) Raw materials
The separated bivalent heme iron peptide, tomato, red date (dried), liquorice, a sweetening agent and a thickening agent.
(2) Taking juice
Extracting juice from tomatoes by a juicing method: soaking tomatoes which meet the processing requirements in a clean water pool for 30 minutes, cleaning with flowing clear liquid for 15 minutes, draining, and juicing in a spiral juicer.
Extracting red date (dried) by adopting a heat preservation extraction method to obtain juice: cleaning the dried red dates without diseases, insect pests and mildew for 3 minutes by using flowing clear water, and blowing and drying by using an air blower. Then placing the mixture into a far infrared electric oven with the surface fire of 150 ℃ and the bottom fire of 120 ℃ and baking for 15 minutes. Taking out, adding purified water 8 times of the weight of the red dates, and preserving the heat for 8 hours at the temperature of 85 ℃. Then, the juice is squeezed in a plate-type squeezer.
Extracting liquorice juice: selecting pulp-fruit type Glycyrrhrizae radix with yellowish meat and abundant powder, pulverizing, adding distilled water 15 times of Glycyrrhrizae radix weight, boiling at 100 deg.C for 30 min, and filtering to obtain Glycyrrhrizae radix juice.
(3) Blending
Three formulas are designed according to different sensory requirements of people in different regions on the product.
1L of the divalent heme peptide-iron blood replenishing oral liquid comprises the following formula I: 5g/L of bivalent heme peptide-iron, 38mL/L of tomato juice, 28mL/L of red date juice, 8mL/L of liquorice juice, 6.5mL/L of angelica sinensis juice, 4.0mL/L of astragalus juice, 2.7mL/L of radix rehmanniae juice, 1.5g/L of stevioside, 1.0g/L of aspartame, 0.8g/L of carrageenan and 0.5g/L of sodium alginate, wherein the volume of the extract is adjusted to 1L by using ultrapure water, and the pH value of the extract is adjusted to 4.0 by using citric acid, dipotassium hydrogen phosphate or potassium dihydrogen phosphate;
the second formula of the 1L of the bivalent heme peptide-iron blood replenishing oral liquid is as follows: 5g/L of heme peptide-iron, 100mL/L of tomato juice, 160mL/L of red date juice, 150mL/L of liquorice juice, 45mL/L of honey, 30mL/L of astragalus membranaceus, 120mL/L of codonopsis pilosula, 40mL/L, CMCNa 0.3g/L of medlar, 0.2g/L of sodium alginate, 0.1g/L of vitamin C and 0.035mg/L of amaranth, and the pH value is adjusted to 4.0 by 0.85g/L of citric acid;
the third formula of the 1L of the bivalent heme peptide-iron blood replenishing oral liquid is as follows: 5g/L of heme peptide-iron, 200ml/L of tomato juice, 140ml/L of red date juice, 50ml/L of liquorice juice, 25ml/L, CMCNa 0.3.3 g/L of honey, 0.2g/L of sodium alginate and 0.1g/L of Vc, and the pH is adjusted to 4.0 by using citric acid, dipotassium hydrogen phosphate or potassium dihydrogen phosphate.
(4) Homogenizing
Heating the mixed solution to 65 ℃, vacuum degassing for 15 minutes under the vacuum degree of 0.18MPa, and homogenizing twice under 30MPa by using a homogenizer.
(5) Sterilization
Heating at 100 deg.C for 20 min.
(6) Filling
The bottles were filled with 10mL glass bottles.
The industrial production is realized, and the full-automatic control production is carried out in the whole operation process under the aseptic condition.
The beverage product of the invention has small dosage, good taste, and convenient carrying, storage and taking. The finished product is sterilized, sealed and packaged, has stable quality, is not easy to deteriorate, has good mouthfeel and is easily accepted by people. Can be directly absorbed by small intestinal mucosa cells, can not directly produce any digestive characteristic irritation symptom, and has high absorption rate.

Claims (5)

1. A heme peptide-iron oral liquid preparation is prepared by the following steps:
(1) separating red blood cells from the blood of livestock and poultry, and adding an anticoagulant for treatment;
(2) dispersing red blood cells, and centrifugally collecting red blood cell sediments;
(3) breaking the wall of the red blood cells by ultrasonic waves, releasing hemoglobin, and adjusting the pH according to the used degrading enzyme to prevent iron ions in the hemoglobin from generating oxidation reaction. (ii) a
(4) Adding a degrading enzyme solution, and performing 200kHz high-frequency pulse treatment in a vacuum chamber to ensure that the enzyme fully degrades hemoglobin and release bivalent heme peptide iron;
(5) maintaining the vacuum degree at 0.01MPa, raising the temperature of the vacuum chamber to 95 ℃ for 10-30 minutes, inactivating the enzyme at high temperature, centrifuging and collecting supernatant;
(6) purifying and collecting the bivalent heme peptide iron by metal chelating affinity chromatography or electromagnetic separation of the supernatant;
(7) adding purified and collected ferrous heme iron into tomato juice, red date juice, licorice juice, angelica sinensis juice, astragalus juice, radix rehmanniae juice, stevioside, aspartame, carrageenan and sodium alginate for blending, adjusting the volume to 1L by using ultra-pure water, and adjusting the pH to 4.0 by using citric acid, dipotassium hydrogen phosphate or potassium dihydrogen phosphate;
(8) heating the mixed solution to 65 ℃, vacuum degassing under the vacuum degree of 0.18MPa, and then homogenizing twice under the vacuum degree of 30MPa by using a homogenizer;
(9) sterilizing at high temperature, and packaging to obtain the bivalent heme peptide iron blood-replenishing beverage.
2. The preparation of claim 1, wherein the hemoglobin degradation solution passing through the electromagnetic separator in step 6 has a flow rate of 1-2 cm/min, is treated with reflux for 3-4 times, is washed with deionized distilled water for 5min, and is demagnetized, and the released substances on the inner wall of the column are bivalent heme peptide iron.
3. The formulation of claim 1 or 2, consisting of: 5g/L of bivalent heme peptide-iron, 38mL/L of tomato juice, 28mL/L of red date juice, 8mL/L of liquorice juice, 6.5mL/L of angelica sinensis juice, 4.0mL/L of astragalus juice, 2.7mL/L of radix rehmanniae juice, 1.5g/L of stevioside, 1.0g/L of aspartame, 0.8g/L of carrageenan and 0.5g/L of sodium alginate, wherein the volume of the extract is adjusted to 1L by using ultrapure water, and the pH value of the extract is adjusted to 4.0 by using citric acid, dipotassium hydrogen phosphate or potassium dihydrogen phosphate.
4. The preparation of claim 1 or 2, wherein the heme peptide is iron 5g/L, tomato juice 100mL/L, red date juice 160mL/L, licorice juice 150mL/L, honey 45mL/L, astragalus root 30mL/L, codonopsis pilosula 120mL/L, wolfberry 40mL/L, CMCNa 0.3g/L, sodium alginate 0.2g/L, vitamin C0.1 g/L, amaranth 0.035mg/L, and the pH is adjusted to 4.0 with citric acid 0.85 g/L; or alternatively.
5. The preparation of claim 1 or 2, wherein the heme peptide is iron 5g/L, tomato juice 200ml/L, red date juice 140ml/L, licorice juice 50ml/L, honey 25ml/L, CMCNa 0.3g/L, sodium alginate 0.2g/L, Vc 0.1g/L, and pH is adjusted to 4.0 with citric acid, dipotassium hydrogen phosphate or potassium dihydrogen phosphate.
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