CN112999897A - Method and process for extracting heme from blood meal by using reverse osmosis membrane - Google Patents
Method and process for extracting heme from blood meal by using reverse osmosis membrane Download PDFInfo
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- CN112999897A CN112999897A CN202110299597.4A CN202110299597A CN112999897A CN 112999897 A CN112999897 A CN 112999897A CN 202110299597 A CN202110299597 A CN 202110299597A CN 112999897 A CN112999897 A CN 112999897A
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- 239000012528 membrane Substances 0.000 title claims abstract description 109
- 238000001223 reverse osmosis Methods 0.000 title claims abstract description 76
- 150000003278 haem Chemical class 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 42
- 239000005996 Blood meal Substances 0.000 title description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 99
- 239000000243 solution Substances 0.000 claims abstract description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 37
- 229920001661 Chitosan Polymers 0.000 claims abstract description 35
- 239000008280 blood Substances 0.000 claims abstract description 29
- 210000004369 blood Anatomy 0.000 claims abstract description 29
- 239000000843 powder Substances 0.000 claims abstract description 25
- 239000004952 Polyamide Substances 0.000 claims abstract description 24
- 229920002647 polyamide Polymers 0.000 claims abstract description 24
- 239000002244 precipitate Substances 0.000 claims abstract description 22
- 239000000706 filtrate Substances 0.000 claims abstract description 19
- 239000002131 composite material Substances 0.000 claims abstract description 17
- 230000002378 acidificating effect Effects 0.000 claims abstract description 16
- 238000001035 drying Methods 0.000 claims abstract description 15
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 239000011259 mixed solution Substances 0.000 claims abstract description 12
- 238000001914 filtration Methods 0.000 claims abstract description 10
- 238000005516 engineering process Methods 0.000 claims abstract description 8
- 238000000926 separation method Methods 0.000 claims abstract description 7
- 241001465754 Metazoa Species 0.000 claims abstract description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 5
- 239000008213 purified water Substances 0.000 claims abstract description 5
- 239000002994 raw material Substances 0.000 claims abstract description 5
- 229920001184 polypeptide Polymers 0.000 claims abstract description 4
- 239000012716 precipitator Substances 0.000 claims abstract description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 4
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000000460 chlorine Substances 0.000 claims abstract description 3
- 229910052801 chlorine Inorganic materials 0.000 claims abstract description 3
- 230000003647 oxidation Effects 0.000 claims abstract description 3
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 3
- 238000000967 suction filtration Methods 0.000 claims abstract description 3
- 229920000642 polymer Polymers 0.000 claims abstract 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 25
- 239000011521 glass Substances 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 18
- 238000002791 soaking Methods 0.000 claims description 14
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 7
- 239000003513 alkali Substances 0.000 claims description 7
- 239000008367 deionised water Substances 0.000 claims description 7
- 229910021641 deionized water Inorganic materials 0.000 claims description 7
- 238000007711 solidification Methods 0.000 claims description 7
- 230000008023 solidification Effects 0.000 claims description 7
- ZIBGPFATKBEMQZ-UHFFFAOYSA-N triethylene glycol Chemical compound OCCOCCOCCO ZIBGPFATKBEMQZ-UHFFFAOYSA-N 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 102000001554 Hemoglobins Human genes 0.000 claims description 4
- 108010054147 Hemoglobins Proteins 0.000 claims description 4
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 claims description 4
- WNLRTRBMVRJNCN-UHFFFAOYSA-N adipic acid Chemical compound OC(=O)CCCCC(O)=O WNLRTRBMVRJNCN-UHFFFAOYSA-N 0.000 claims description 4
- 239000003431 cross linking reagent Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 3
- UMHJEEQLYBKSAN-UHFFFAOYSA-N Adipaldehyde Chemical compound O=CCCCCC=O UMHJEEQLYBKSAN-UHFFFAOYSA-N 0.000 claims description 2
- 239000001361 adipic acid Substances 0.000 claims description 2
- 235000011037 adipic acid Nutrition 0.000 claims description 2
- 239000012043 crude product Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000001294 propane Substances 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims 3
- SVTBMSDMJJWYQN-UHFFFAOYSA-N 2-methylpentane-2,4-diol Chemical compound CC(O)CC(C)(C)O SVTBMSDMJJWYQN-UHFFFAOYSA-N 0.000 claims 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 2
- 241000272525 Anas platyrhynchos Species 0.000 claims 1
- 241000283690 Bos taurus Species 0.000 claims 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims 1
- 238000004140 cleaning Methods 0.000 claims 1
- 238000004090 dissolution Methods 0.000 claims 1
- OVARTBFNCCXQKS-UHFFFAOYSA-N propan-2-one;hydrate Chemical compound O.CC(C)=O OVARTBFNCCXQKS-UHFFFAOYSA-N 0.000 claims 1
- 238000005185 salting out Methods 0.000 claims 1
- 239000011780 sodium chloride Substances 0.000 claims 1
- 239000002253 acid Substances 0.000 abstract description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 24
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 18
- 239000007864 aqueous solution Substances 0.000 description 18
- 238000005406 washing Methods 0.000 description 14
- WZCQRUWWHSTZEM-UHFFFAOYSA-N 1,3-phenylenediamine Chemical compound NC1=CC=CC(N)=C1 WZCQRUWWHSTZEM-UHFFFAOYSA-N 0.000 description 12
- 229940018564 m-phenylenediamine Drugs 0.000 description 12
- 239000000047 product Substances 0.000 description 9
- 229960000583 acetic acid Drugs 0.000 description 8
- 238000012696 Interfacial polycondensation Methods 0.000 description 6
- 239000008346 aqueous phase Substances 0.000 description 6
- 239000002585 base Substances 0.000 description 6
- UWCPYKQBIPYOLX-UHFFFAOYSA-N benzene-1,3,5-tricarbonyl chloride Chemical compound ClC(=O)C1=CC(C(Cl)=O)=CC(C(Cl)=O)=C1 UWCPYKQBIPYOLX-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 229910052742 iron Inorganic materials 0.000 description 6
- 238000011068 loading method Methods 0.000 description 6
- 239000004745 nonwoven fabric Substances 0.000 description 6
- 238000000643 oven drying Methods 0.000 description 6
- 229920002492 poly(sulfone) Polymers 0.000 description 6
- 238000000108 ultra-filtration Methods 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- -1 acetone heme Chemical class 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 238000007865 diluting Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000007872 degassing Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 241000587240 Cynanchum Species 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 1
- 208000015710 Iron-Deficiency Anemia Diseases 0.000 description 1
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 description 1
- 229940025294 hemin Drugs 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- SUBFIBLJQMMKBK-UHFFFAOYSA-K iron(3+);trithiocyanate Chemical compound [Fe+3].[S-]C#N.[S-]C#N.[S-]C#N SUBFIBLJQMMKBK-UHFFFAOYSA-K 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- ZNNZYHKDIALBAK-UHFFFAOYSA-M potassium thiocyanate Chemical compound [K+].[S-]C#N ZNNZYHKDIALBAK-UHFFFAOYSA-M 0.000 description 1
- 229940116357 potassium thiocyanate Drugs 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000003307 slaughter Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/06—Organic material
- B01D71/08—Polysaccharides
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/02—Reverse osmosis; Hyperfiltration ; Nanofiltration
- B01D61/025—Reverse osmosis; Hyperfiltration
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0002—Organic membrane manufacture
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
- B01D69/12—Composite membranes; Ultra-thin membranes
- B01D69/125—In situ manufacturing by polymerisation, polycondensation, cross-linking or chemical reaction
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/06—Organic material
- B01D71/56—Polyamides, e.g. polyester-amides
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- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Water Supply & Treatment (AREA)
- Nanotechnology (AREA)
- Manufacturing & Machinery (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
Abstract
The invention discloses a method for extracting heme from blood powder by using reverse osmosis technology, which comprises the steps of using animal blood powder as a raw material, particularly pig blood powder, dissolving the raw material by using purified water, adding a protein precipitator into a mixed solution, collecting precipitates after filtering, adding acidic acetone into the precipitates, performing suction filtration after dissolving, collecting filtrate, filtering the filtrate by using a reverse osmosis membrane to obtain concentrated heme, and drying to obtain high-purity heme. The method is characterized in that a protein precipitator is used for accelerating the separation of protein, and the use amount of acid acetone is reduced by half compared with the method for extracting heme by an acid acetone method; the reverse osmosis membrane used is a double-layer membrane of a polyamide composite reverse osmosis membrane and a high molecular polymer chitosan reverse osmosis membrane, the separation of polypeptide in the solution is enhanced through the action of chitosan, the purity of the obtained heme is improved, the removal rate of foreign matters in a system is improved through a double-layer membrane structure, and the chlorine resistance, oxidation resistance and pollution resistance of the membrane are improved.
Description
Technical Field
The invention relates to a method for producing heme, in particular to a method for preparing high-purity heme by utilizing a reverse osmosis membrane concentration technology.
Background art:
【1】 With the improvement of living standard of people, the demand of meat food is increasing day by day, the slaughter quantity of livestock and poultry is increasing, and simultaneously, a large amount of blood is generated, if the blood can not be utilized in time, the blood is not only waste of resources, but also causes pollution to the environment.
【2】 According to statistics, 60% of people in the society suffer from anemia, heme can be used as organic iron, can be well absorbed by human bodies, has no side effect, is a good iron-supplementing medicine, and has a good treatment effect on iron-deficiency anemia. The heme can be used as food additive, and can be modified to obtain pigment as edible pigment. The production of heme is therefore of increasing interest.
【3】 The existing methods for extracting heme comprise glacial acetic acid (Zhongguang: research on extracting heme by utilizing glacial acetic acid [ J ]. food science 2004, 20 (4): 90-95), acid acetone (Songyenarmy, etc.: new technology research on extracting and purifying heme [ J ] Guangzhou food industry science and technology, 2004, 20 (1): 6-11) and enzymolysis (Yanxinhong, summer water, enzymatic hydrolysis of hemoglobin to prepare heme peptide [ J ]. food and machinery, 2005(3): 314-:
firstly, a large amount of organic solvent is consumed and is difficult to recover, so that the production cost is increased and the environment is polluted;
secondly, the fresh blood is not easy to store and transport, and the production cost for preparing the heme by using the fresh blood as the raw material is higher. ③ the purity and the yield of the prepared heme are lower.
Disclosure of Invention
In order to solve the problems, the invention discloses a method for extracting heme from blood powder by using a reverse osmosis membrane, which specifically adopts the following technical scheme:
(1) adding 10-20 times of purified water into animal blood powder, and stirring for 15-30 to dissolve the blood powder completely.
(2) Slowly adding 0.5-2 times of protein precipitant into the blood powder solution, stirring for 0.5-1h, and precipitating completely, wherein the protein precipitant is acetone and water 1: 3-1: 5, and (c) a mixed solution.
(3) The solution was centrifuged at 3000r/min to collect the red precipitate. Acetone and water for precipitation 1: 3-1: 5, stirring and washing for 3-5 times, wherein the acetone aqueous solution used each time is 2-3 times of the precipitation volume, and after washing, performing suction filtration to collect precipitates.
(4) Adding acidic acetone solution with pH less than 3 and with precipitation mass of 3-5 times into the precipitate, stirring and extracting for 0.5-1h, vacuum filtering, collecting filtrate, washing the filtrate with acidic acetone for 2-3 times, and mixing the filtrates.
(5) Filtering the filtrate with reverse osmosis membrane concentrating device to obtain concentrated heme crude product, and oven drying to obtain high purity heme.
In a further embodiment of the present invention, the reverse osmosis membrane comprises two membranes, wherein the first membrane is a chitosan reverse osmosis membrane and the second membrane is a polyamide reverse osmosis membrane.
As a further scheme of the invention, the preparation process of the chitosan reverse osmosis membrane comprises the following steps:
(1) 2g to 10g of chitosan is put into a 100ml beaker, added with 3 percent to 6 percent aqueous acetone solution of acetic acid (acetone: water = 2: 4-8) and stirred fully for 0.5 to 1 hour.
(2) And adding 1-5g of triethylene glycol into the uniformly mixed solution, fixing the volume to 100mL, adding a cross-linking agent with the mass of 0.02 time of that of the chitosan, and stirring for 0.5-2h by using a stirrer. The cross-linking agent is one of glutaraldehyde, adipic dialdehyde, adipic acid and diepoxy propane.
(3) Pouring the mixed liquid on a clean glass plate, flattening the film by using a glass rod, placing the film in air for drying, and soaking the film into 2-5% diluted alkali solidification liquid of 278K for 0.5-2h after the fluidity is lost.
(4) Finally, the membrane was soaked with acetone until it was separated from the glass plate, washed clean with deionized water and stored in a moist environment.
As a further scheme of the invention, the preparation scheme of the polyamide reverse osmosis composite membrane comprises the following steps:
(1) preparing m-phenylenediamine aqueous solution with the concentration of 1.7-3.0%;
(2) soaking a polysulfone ultrafiltration membrane (with a film forming concentration of 16%) with PET non-woven fabric as a liner in the m-phenylenediamine aqueous solution in the step (1) for 1-5min to form an aqueous phase liquid layer on the surface of the porous base membrane to obtain a wet membrane;
(3) contacting the wet film with naphtha organic solution containing 0.02% -0.04% of trimesoyl chloride for interfacial polycondensation reaction for 1-5 min;
(4) drying in an oven at 110 ℃ for 2-10 minutes;
(5) and washing with clean water for 10 minutes to obtain the polyamide reverse osmosis membrane.
As a further scheme of the invention, the cross-linked chitosan reverse osmosis membrane and the polyamide composite reverse osmosis membrane are used by simultaneously loading into a reverse osmosis device, wherein the chitosan reverse osmosis membrane is a first layer, and the polyamide composite reverse osmosis membrane is a second layer.
The technical scheme provided by the invention has the beneficial effects that:
(1) the invention uses the animal blood powder as the raw material to extract the heme, and the dried blood powder has long shelf life, is convenient for production and utilization, and reduces the environmental pollution.
(2) The method uses the protein precipitator to accelerate the separation of the protein, thereby reducing the usage amount of the acidic acetone, reducing the usage amount of the acidic acetone by half compared with the method for preparing the heme by the acidic acetone method, and reducing the environmental pollution because the acetone used in the experimental process is filtered out by a reverse osmosis membrane and has high recoverability.
(3) The invention uses the cross-linked chitosan reverse osmosis membrane, and the hydroxyl and the amino contained in the molecular structure of the cross-linked chitosan can form hydrogen bond action with O and N atoms in polypeptide molecules, thereby enhancing the separation of the polypeptide in the solution and improving the purity of the obtained heme.
(4) The invention uses the double-layer membrane structure to greatly improve the removal rate of foreign matters in the solvent in reverse osmosis and improve the chlorine resistance, oxidation resistance and pollution resistance of the membrane. And the double-layer reverse osmosis membrane structure is suitable for solution purification of various systems.
Detailed description of the invention
The present invention will be further described below by way of specific examples.
The reverse osmosis concentration devices used in the following examples are hpml1812-2 reverse osmosis concentrators from Jilin sea technology, Inc. The operating parameters were: pressure: 80 PSI (temperature of 25 deg.C)
And the filtering flow rate is 3.5L/h.
Example 1:
preparation of acidic acetone heme solution:
(1) adding 15 times volume of purified water into 1kg of pig blood powder, and stirring for 30min to dissolve the blood powder completely.
(2) Slowly adding a 1.5-time volume of a mixed solution of acetone and water in a ratio of 1:5 into the blood powder solution, and fully stirring for 1h to fully precipitate the blood powder solution.
(3) The solution was centrifuged at 3000r/min to collect the red precipitate. And stirring and washing the precipitate for 3 times by using a mixed solution of acetone and water in a ratio of 1:5, wherein the acetone aqueous solution used in each time is 2 times of the volume of the precipitate, and filtering and collecting the precipitate after washing.
(4) Adding 2L of acidic acetone solution with pH of 3 into the precipitate, stirring and extracting for 0.5h, vacuum filtering, collecting filtrate, washing the filtrate with acidic acetone for 3 times, and mixing the filtrates to obtain acidic acetone heme solution.
Example 2:
the method comprises the following steps: preparing a cross-linked chitosan reverse osmosis membrane:
(1) 8g of chitosan was put in a 100ml beaker, and 3% acetic acid in acetone aqueous solution (acetone: water = 2: 5) was added thereto, followed by well stirring for 0.5 h.
(2) Adding 3g of triethylene glycol into the uniformly mixed solution, fixing the volume to 100mL, adding glutaraldehyde with the mass of 0.02 time of that of the chitosan, and stirring for 1 hour by using a stirrer.
(3) Pouring the mixed liquid on a clean glass plate, flattening the film by using a glass rod, placing the film in air for drying, and soaking the film in 3% diluted alkali solidification liquid of 278K for 2 hours after the fluidity is lost.
(4) Finally, the membrane was soaked with acetone until it was separated from the glass plate, washed clean with deionized water and stored in a moist environment.
Step two: preparing a polyamide composite reverse osmosis membrane:
(1) preparing m-phenylenediamine aqueous solution with the concentration of 2 percent;
(2) soaking a polysulfone ultrafiltration membrane (with a film forming concentration of 16%) with PET non-woven fabric as a liner in the m-phenylenediamine aqueous solution in the step (1) for 1min to form an aqueous phase liquid layer on the surface of the porous base membrane to prepare a wet membrane;
(3) contacting the wet film with naphtha organic solution containing 0.03% of trimesoyl chloride for interfacial polycondensation reaction for 5 min;
(4) oven drying at 110 deg.C for 10 min;
(5) and washing with clean water for 10 minutes to obtain the polyamide reverse osmosis membrane.
Step three: and (2) loading the cross-linked chitosan reverse osmosis membrane and the polyamide composite reverse osmosis membrane into a reverse osmosis concentration device, concentrating 100g of filtrate obtained in example 1 by using the reverse osmosis device, and drying to obtain 1.05g of finished heme product with the purity of 98.22% and the iron element content of 8.36%.
Example 3:
the method comprises the following steps: preparing a cross-linked chitosan reverse osmosis membrane:
(1) 6g of chitosan was put in a 100ml beaker, and added to a 4% aqueous acetone solution of acetic acid (acetone: water = 2: 8) and stirred well for 0.5 h.
(2) Adding 2g of triethylene glycol into the uniformly mixed solution, fixing the volume to 100mL, adding glutaraldehyde with the mass of 0.02 time of that of the chitosan, and stirring for 1 hour by using a stirrer.
(3) Pouring the mixed liquid on a clean glass plate, flattening the film by using a glass rod, placing the film in air for drying, and soaking the film in 3% diluted alkali solidification liquid of 278K for 2 hours after the fluidity is lost.
(4) Finally, the membrane was soaked with acetone until it was separated from the glass plate, washed clean with deionized water and stored in a moist environment.
Step two: preparing a polyamide composite reverse osmosis membrane:
(1) preparing a m-phenylenediamine aqueous solution with the concentration of 1.5 percent;
(2) soaking a polysulfone ultrafiltration membrane (with the film forming concentration of 16%) with PET non-woven fabric as a liner in the m-phenylenediamine aqueous solution in the step (1) for 3min to form an aqueous phase liquid layer on the surface of the porous base membrane to prepare a wet membrane;
(3) contacting the wet film with naphtha organic solution containing 0.04% of trimesoyl chloride to carry out interfacial polycondensation reaction for 3 min;
(4) oven drying at 110 deg.C for 10 min;
(5) and washing with clean water for 10 minutes to obtain the polyamide reverse osmosis membrane.
Step three: and (2) loading the cross-linked chitosan reverse osmosis membrane and the polyamide composite reverse osmosis membrane into a reverse osmosis concentration device, concentrating 100g of filtrate obtained in example 1 by using the reverse osmosis device, and drying to obtain 1.15g of finished heme product with the purity of 98.14% and the iron element content of 8.34%.
Example 4:
the method comprises the following steps: preparing a cross-linked chitosan reverse osmosis membrane:
(1) 10g of chitosan was put in a 100ml beaker, and 3% acetic acid in acetone aqueous solution (acetone: water = 2: 5) was added thereto, followed by well stirring for 0.5 h.
(2) Adding 4g of triethylene glycol into the uniformly mixed solution, fixing the volume to 100mL, adding glutaraldehyde with the mass of 0.02 time of that of the chitosan, and stirring for 1.5 hours by using a stirrer.
(3) Pouring the mixed liquid on a clean glass plate, flattening the film by using a glass rod, placing the film in air for drying, and soaking the film in 3% diluted alkali solidification liquid of 278K for 2 hours after the fluidity is lost.
(4) Finally, the membrane was soaked with acetone until it was separated from the glass plate, washed clean with deionized water and stored in a moist environment.
Step two: preparing a polyamide composite reverse osmosis membrane:
(1) preparing a 3% m-phenylenediamine aqueous solution;
(2) soaking a polysulfone ultrafiltration membrane (with the film forming concentration of 16%) with PET non-woven fabric as a liner in the m-phenylenediamine aqueous solution in the step (1) for 3min to form an aqueous phase liquid layer on the surface of the porous base membrane to prepare a wet membrane;
(3) contacting the wet film with naphtha organic solution containing 0.03% of trimesoyl chloride to carry out interfacial polycondensation reaction for 5 minutes;
(4) oven drying at 110 deg.C for 5 min;
(5) and washing with clean water for 10 minutes to obtain the polyamide reverse osmosis membrane.
Step three: and (2) loading the cross-linked chitosan reverse osmosis membrane and the polyamide composite reverse osmosis membrane into a reverse osmosis concentration device, concentrating 100g of filtrate obtained in example 1 by using the reverse osmosis device, and drying to obtain 1.27g of finished heme product with the purity of 97.93% and the iron element content of 8.28%.
Example 5:
the method comprises the following steps: preparing a cross-linked chitosan reverse osmosis membrane:
(1) in a 100ml beaker, 7.5g of chitosan was added to a 3% aqueous solution of acetic acid in acetone (acetone: water = 2: 4) and stirred well for 0.5 h.
(2) Adding 3.5g of triethylene glycol into the uniformly mixed solution, fixing the volume to 100mL, adding glutaraldehyde with the mass of 0.02 time of that of the chitosan, and stirring for 1.5 hours by using a stirrer.
(3) Pouring the mixed liquid on a clean glass plate, flattening the film by using a glass rod, placing the film in air for drying, and soaking the film in 3% diluted alkali solidification liquid of 278K for 2 hours after the fluidity is lost.
(4) Finally, the membrane was soaked with acetone until it was separated from the glass plate, washed clean with deionized water and stored in a moist environment.
Step two: preparing a polyamide composite reverse osmosis membrane:
(1) preparing m-phenylenediamine aqueous solution with the concentration of 2 percent;
(2) soaking a polysulfone ultrafiltration membrane (with the film forming concentration of 16%) with PET non-woven fabric as a liner in the m-phenylenediamine aqueous solution in the step (1) for 3min to form an aqueous phase liquid layer on the surface of the porous base membrane to prepare a wet membrane;
(3) contacting the wet film with naphtha organic solution containing 0.03% of trimesoyl chloride to carry out interfacial polycondensation reaction for 3 minutes;
(4) oven drying at 110 deg.C for 5 min;
(5) and washing with clean water for 10 minutes to obtain the polyamide reverse osmosis membrane.
Step three: and (2) loading the cross-linked chitosan reverse osmosis membrane and the polyamide composite reverse osmosis membrane into a reverse osmosis concentration device, concentrating 100g of filtrate obtained in example 1 by using the reverse osmosis device, and drying to obtain 1.35g of finished heme product with the purity of 98.31% and the iron element content of 8.38%.
Example 6:
the method comprises the following steps: preparing a cross-linked chitosan reverse osmosis membrane:
(1) 9g of chitosan was put in a 100ml beaker, and 3% acetic acid in acetone aqueous solution (acetone: water = 2: 5) was added thereto, followed by well stirring for 0.5 h.
(2) Adding 4.25g of triethylene glycol into the uniformly mixed solution, fixing the volume to 100mL, adding glutaraldehyde with the mass of 0.02 time of that of the chitosan, and stirring for 1.5 hours by using a stirrer.
(3) Pouring the mixed liquid on a clean glass plate, flattening the film by using a glass rod, placing the film in air for drying, and soaking the film in 278K 4% diluted alkali solidification liquid for 2 hours after the fluidity is lost.
(4) Finally, the membrane was soaked with acetone until it was separated from the glass plate, washed clean with deionized water and stored in a moist environment.
Step two: preparing a polyamide composite reverse osmosis membrane:
(1) preparing m-phenylenediamine aqueous solution with the concentration of 2.5 percent;
(2) soaking a polysulfone ultrafiltration membrane (with the film forming concentration of 16%) with PET non-woven fabric as a liner in the m-phenylenediamine aqueous solution in the step (1) for 3min to form an aqueous phase liquid layer on the surface of the porous base membrane to prepare a wet membrane;
(3) contacting the wet film with naphtha organic solution containing 0.03% of trimesoyl chloride to carry out interfacial polycondensation reaction for 4 minutes;
(4) oven drying at 110 deg.C for 5 min;
(6) and washing with clean water for 10 minutes to obtain the polyamide reverse osmosis membrane.
Step three: and (2) loading the cross-linked chitosan reverse osmosis membrane and the polyamide composite reverse osmosis membrane into a reverse osmosis concentration device, concentrating 100g of filtrate obtained in example 1 by using the reverse osmosis device, and drying to obtain 1.27g of finished heme product with purity of 97.96% and iron element content of 8.30%.
Example seven:
the acidic acetone method was used to extract hemoglobin from the solution (100 g) prepared in example 1 according to the literature (Songyun et al: New technology for extraction and purification of hemoglobin [ J ]. Guangzhou food industry science and technology, 2004, 20 (1): 6-11). The method comprises the following steps: and (3) taking 100ml of the filtrate in the example 1, adjusting the pH value to 6-7 by using 1.0mol/l NaOH solution, separating out a precipitate, and centrifuging for 10min at 3000r/min to obtain the precipitate. Dissolving the precipitate with 30ml of 0.1mol/l NaOH solution, adjusting the pH value to 5-6, centrifuging at 3000r/min for 10min, and finally washing the precipitate with distilled water to neutrality to obtain 0.87g of heme product with purity of 93.14% and iron content of 7.89%.
Example eight:
100g of the solution prepared in example 1 were worked up according to the seventh protocol of example, comprising the steps of: the pH of the filtrate in the example 1 is adjusted to 6-7 by using 1.0mol/l NaOH solution, precipitate is separated out, and the precipitate is separated by centrifuging at 3000r/min for 20 min. Dissolving the precipitate with 25ml of 0.2mol/l NaOH solution, adjusting the pH value to 6-7, centrifuging at 3000r/min for 20min, and finally washing the precipitate with distilled water to neutrality to obtain 0.86g of heme product, wherein the purity is 93.07%, and the content of iron element is 7.86%
The purity, yield and iron content of heme in each of the above examples were measured as follows:
(1) and (3) measuring the content of heme:
the scheme for measuring the content of the heme in the patent is measured by using a high performance liquid chromatography (Cynanchum, Xiandaqiang. HPLC for measuring the content of the hemin in Shuyue capsules. Xinjiang traditional Chinese medicine, 2005,23(6): 6-7), and the specific scheme is as follows:
selection of a mobile phase:
acetonitrile, methanol and water are respectively used as mobile phases, and the influence of the flow of the components on the separation effect of the heme is examined when the flow rate is 0.6mL/min and the column temperature is 30 ℃.
② drawing of HPLC standard curve
20.0mg of heme standard dried to constant weight at 105 ℃ is precisely weighed. Placing the mixture into a 100m1 volumetric flask, adding 0.1mol/L NaOH solution, diluting to the scale, and shaking up to obtain the product. Accurately measuring l.0, 2.0, 3.0, 4.0 and 5.0mL of heme standard solution, using 0.1mol/L NaOH solution to be fixed in a 100mL volumetric flask, and shaking up. Passing the prepared solution through a 0.22um microfiltration membrane, performing ultrasonic degassing, and performing machine detection.
Measuring the content of the heme sample:
taking a sample dried to constant weight at 105 ℃, and grinding into fine powder; an appropriate amount of the fine powder was weighed and placed in a 100mL volumetric flask. Dissolving with 0.1mol/L sodium hydroxide solution, diluting to scale, shaking, filtering, placing 10m1 filtrate in 100m1 volumetric flask, diluting with 0.1mol/L sodium hydroxide solution to scale, shaking, passing through membrane, degassing, and detecting on machine. Substituting the obtained value into a regression equation to calculate the content.
Purity = mass of heme x 100%/mass of product obtained
(2) Heme yield = (mass of produced heme/mass of blood powder used) × 100%
(3) Determination of iron ion content the determination was carried out using the method in the literature (evaluation of two determination methods of iron ion content in Yuan, Yuan. heme. Histra pharmaceutical industry, 2005,17(4): 63-65):
phenanthroline colorimetry. First, a standard curve is drawn. Then accurately weighing a proper amount of the sample in a 100mL beaker, adding 2mL of concentrated hydrochloric acid, and after completely dissolving, fixing the volume with distilled water. 10mL of the solution was added to a separatory funnel, and 1mL of 1:1 sulfuric acid solution and 2mL of 20% potassium thiocyanate solution were added and mixed. Then 30mL of anhydrous ether is added, ferric thiocyanate formed by ferric iron is extracted by shaking for a plurality of times until the ether layer is colorless, and the ether layer is removed. Transferring the residual water layer into a volumetric flask for constant volume. The absorbance is measured, and the ferrous content is calculated by a standard curve.
The heme yields, purities, iron contents of examples 2-8 were compared:
| item | Example 2 | Example 3 | Example 4 | Example 5 | Example 6 | Example 7 | Example 8 |
| Purity% | 98.22 | 98.14 | 97.93 | 98.31 | 97.96 | 93.14 | 93.07 |
| Yield% | 2.1 | 2.30 | 2.54 | 2.7 | 2.54 | 1.74 | 1.72 |
| Iron element content% | 8.36 | 8.34 | 8.28 | 8.38 | 8.30 | 7.89 | 7.86 |
Examples 2-6 use reverse osmosis membrane concentration to extract heme, and examples 7-8 use acid acetone method to extract heme, and it can be seen from the data that the method and process for extracting heme from blood powder using reverse osmosis membrane according to the present invention are significantly improved from aspects of heme yield, purity, and iron element content compared with the conventional process.
Claims (13)
1. The invention discloses a method for extracting heme from blood powder by using reverse osmosis technology, which comprises the steps of dissolving animal blood powder, particularly pig blood powder, by using purified water, adding a protein precipitator into a mixed solution, collecting precipitates after filtration, adding acidic acetone into the precipitates, performing suction filtration after dissolution, collecting filtrate, concentrating the filtrate by using a modified reverse osmosis membrane to obtain a concentrated heme crude product, and drying to obtain high-purity heme.
2. The method of claim 1, wherein the raw material is animal blood powder selected from one of pig blood powder, duck blood powder, and cattle blood powder.
3. The method of claim 1, wherein the amount of purified water is 10-20 times the mass of the dried blood.
4. The method according to claim 1, wherein the protein precipitant is selected from ethanol, methanol, acetone, dimethylformamide, dimethyl sulfoxide, acetonitrile, 2-methyl-2, 4-pentanediol, saturated ammonium sulfate, and 33% sodium chloride salting-out solution.
5. The process according to claims 1 to 4, wherein the protein precipitant used is acetone and water 1: 3-1: 5, and (c) a mixed solution.
6. A process according to claims 1 to 5, wherein the amount of the acetone/water mixture of the protein precipitant is 0.5 to 2 times the amount of the purified water-soluble blood powder solution.
7. The process according to claim 1, wherein the acidic acetone used has a pH of less than 3.
8. The method according to claims 1 to 7, characterized in that the amount of acidic acetone used is 3-5 times the amount of precipitate obtained by filtration with the addition of protein precipitant.
9. The method of claim 1, wherein the reverse osmosis membrane used comprises two membranes, the first membrane being a chitosan reverse osmosis membrane and the second membrane being a polyamide composite reverse osmosis membrane.
10. The method of claims 1 to 9, wherein the reverse osmosis membrane is prepared by placing 2g to 10g of chitosan in a 100ml beaker, adding 3% -6% acetic acid in acetone water solution (acetone: water = 2: 4-8), mixing well, adding 1-5g triethylene glycol, fixing the volume to 100mL scale, adding a certain amount of cross-linking agent, stirring with a stirrer for 1-2h, pouring the mixed liquid on a clean glass plate, and flattening the membrane by using a glass rod, drying the membrane in the air, soaking the membrane into 278K 2-5% diluted alkali solidification liquid for 0.5-2h after the fluidity is lost, soaking the membrane by using acetone until the membrane is separated from a glass plate, cleaning the membrane by using deionized water, storing the membrane in a humid environment, and putting the membrane into a reverse osmosis concentration device when the membrane is used.
11. The method as claimed in claims 1 to 9, wherein the cross-linking agent used by the chitosan reverse osmosis membrane is one of glutaraldehyde, adipic dialdehyde, adipic acid and diepoxy propane.
12. The method of claims 1-9 wherein the second membrane is a conventional polyamide reverse osmosis composite membrane.
13. The method as claimed in claim 1, wherein a protein precipitant is used to accelerate the separation of protein, and the amount of acidic acetone used is reduced by half compared with the acidic acetone method for extracting hemoglobin; the reverse osmosis membrane used is a double-layer membrane of a polyamide composite reverse osmosis membrane and a high molecular polymer chitosan reverse osmosis membrane, the separation of polypeptide in the solution is enhanced through the action of chitosan, the purity of the obtained heme is improved, the removal rate of foreign matters in the system is improved through a double-parameter membrane structure, and the chlorine resistance, oxidation resistance and pollution resistance of the membrane are improved.
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