CN111072771A - Co-production process for extracting SOD and heme from chicken blood - Google Patents

Co-production process for extracting SOD and heme from chicken blood Download PDF

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CN111072771A
CN111072771A CN201811224015.0A CN201811224015A CN111072771A CN 111072771 A CN111072771 A CN 111072771A CN 201811224015 A CN201811224015 A CN 201811224015A CN 111072771 A CN111072771 A CN 111072771A
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heme
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陈石良
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)

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Abstract

The invention discloses a co-production process for extracting SOD and heme from chicken blood, which takes the chicken blood as a raw material, and prepares SOD by hemolysis, thermal denaturation reaction, filter pressing, ultrafiltration concentration, purification and freeze drying of filtrate; the heme is prepared by extracting, separating and drying blood residues through acidic acetone. The invention utilizes modern biochemical separation technologies such as thermal denaturation, isoelectric precipitation, ultrafiltration, extraction and the like to prepare the co-production process of SOD and heme, realizes the comprehensive utilization and high-value development of chicken blood, has no waste, no pollution and short production period, is suitable for industrial production, and the activity of the obtained SOD lyophilized powder reaches more than 5000U/mg dry powder and the purity of the obtained heme reaches more than 85 percent.

Description

Co-production process for extracting SOD and heme from chicken blood
Technical Field
The invention relates to the technical field of chicken blood deep processing, in particular to a co-production process for extracting SOD and heme from chicken blood.
Background
The poultry blood is a kind of nutrient resource rich in animal protein, contains various nutrients and bioactive substances, such as protein, amino acid, various enzymes, mineral elements and the like, and has high potential nutritive value. As a world breeding country in China, the sources of poultry blood are extremely rich, according to preliminary statistics, the slaughter amount of chickens (including broiler chickens and hens) in China per year is nearly 90 hundred million at present, and according to the calculation of 100g of blood generated after each chicken is slaughtered, 900 million tons of blood can be collected per year only by taking the chicken blood, and in addition, hundreds of millions of blood volumes of slaughtered other kinds of poultry (including ducks, geese and the like) are quite huge protein resources. However, at present, most of poultry blood in the slaughtering industry of China is directly processed into common protein feed raw materials, a very small amount of poultry blood is directly used for eating, and the other part of poultry blood is still directly discharged as waste, so that precious protein resources are wasted, and environmental pollution is caused. Therefore, the comprehensive utilization and the fine and deep processing of the poultry blood are developed, so that the environment pollution can be eliminated, products with high nutritive value and high economic added value can be produced, and the method has important significance for improving the economic benefit and the social benefit of enterprises.
Disclosure of Invention
The invention aims to provide a co-production process for extracting SOD and heme from chicken blood, which is researched and developed based on the above situation.
In order to achieve the purpose, the invention is realized by adopting the following technical scheme: a co-production process for extracting SOD and heme from chicken blood comprises the following process steps:
(1) preparation of blood cell liquid: collecting fresh chicken blood, performing anticoagulation treatment, performing centrifugal separation, and collecting red blood cells to obtain blood cell liquid;
(2) hemolysis: adding deionized water with the volume 1 time of that of the hemocyte solution, and continuously stirring for hemolysis for 60min to obtain hemolysis solution;
(3) thermal denaturation reaction I: adjusting pH of the hemolysis solution to 5.2 with HCl, rapidly heating to 60 deg.C, maintaining the temperature for 60min, rapidly cooling to below 40 deg.C, press-filtering with plate-and-frame filter press, and collecting filtrate I and blood residue I respectively.
(4) And (3) thermal denaturation reaction II: adjusting the pH value of the filtrate I to 6.5 by using NaOH, rapidly heating to 70 ℃, preserving heat for 20min, rapidly cooling to below 40 ℃, and carrying out filter pressing by using a plate-and-frame filter press to respectively collect filtrate II and blood residue II.
(5) And (3) ultrafiltration concentration: and (3) carrying out ultrafiltration concentration on the collected filtrate II by an ultrafiltration membrane device with the molecular weight cutoff of 5000Da, concentrating to 1/10 of the original volume, and collecting crude SOD enzyme liquid.
(6) And (3) purification: heating the SOD crude enzyme solution to 45 ℃, adjusting the pH value to 9.0 by using NaOH, dropwise adding a protective agent lauroyl chloride accounting for 0.02-0.05% of the total mass of the SOD crude enzyme solution, keeping the temperature for reaction for 1 hour, quickly cooling to below 20 ℃, centrifuging at 6000r/min for 10min, collecting supernatant, adjusting the pH value of the supernatant to 6.5 by using HCl, centrifuging at 6000r/min for 10min, and collecting the supernatant to obtain the blue-green SOD enzyme solution;
(7) and (3) freeze drying: adding trehalose accounting for 2.0% of the weight of the SOD enzyme solution into the SOD enzyme solution, uniformly stirring and mixing, and freeze-drying to obtain light blue green SOD freeze-dried powder;
(8) extraction: adding acidic acetone with the volume 5 times of that of the blood residue I obtained in the step (3) and the blood residue II obtained in the step (4), stirring for 30min, centrifuging at 6000r/min for 10min, and collecting supernatant;
(9) separation: adjusting the pH value of the supernatant obtained in the step (8) to 5.0, adding 1% of sodium acetate, uniformly stirring, standing for 30min, centrifuging at 6000r/min for 10min, and collecting precipitate;
(10) and (3) drying: and (4) washing the precipitate obtained in the step (9) with deionized water and absolute ethyl alcohol, and drying in vacuum at 50-60 ℃ to obtain the heme.
The centrifugal separation in the step (1) is continuous separation by adopting a tubular centrifuge, and the centrifugal rotating speed is more than 16000 r/min.
The acidic acetone in the step (8) is 3% (v/v) acetone hydrochloride solution.
Through the implementation of the technical scheme, the invention has the following beneficial effects:
(1) the invention uses rich chicken blood as raw material, applies modern biotechnology and modern processing technology, and works out a co-production process for jointly extracting high-activity SOD and heme iron in the same production process flow, and the co-production process is not reported in similar patents.
(2) The SOD prepared by the process has high activity, the unit enzyme activity reaches more than 5000U/mg dry powder, and the purity of the prepared heme reaches more than 85 percent.
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
(1) Preparation of blood cell liquid: collecting 1000L of fresh chicken blood in a slaughtering chicken factory, performing anticoagulation treatment, and continuously separating with a tubular centrifuge to obtain 280L of blood cell liquid and 720L of blood serum liquid; spray drying the blood serum to obtain chicken plasma protein powder;
(2) hemolysis: pumping the blood corpuscle into a reaction tank, adding 280L of deionized water, and continuously stirring and hemolysis for 60min at the stirring speed of 150r/min to obtain 560L of hemolysis solution;
(3) thermal denaturation reaction I: adjusting the pH value of the hemolytic solution to 5.2 by using HCl under stirring, rapidly heating to 60 ℃, keeping the temperature for 60min, rapidly cooling to below 40 ℃, and performing filter pressing by using a plate-and-frame filter press to respectively obtain 325L of filtrate and 103 kg of blood residue I;
(4) and (3) thermal denaturation reaction II: pumping the filtrate I into a reaction tank by using a pump, adjusting the pH value to 6.5 by using NaOH, quickly heating to 70 ℃, keeping the temperature for 20min, quickly cooling to below 40 ℃, performing filter pressing by using a plate-and-frame filter press, and respectively collecting 24 kg of filtrate II 262L and blood residues II;
(5) and (3) ultrafiltration concentration: carrying out ultrafiltration concentration on the collected filtrate II by an ultrafiltration membrane device with the molecular weight cutoff of 5000Da, and concentrating to 1/10 of the original volume to obtain 26L of SOD crude enzyme liquid;
(6) and (3) purification: heating the SOD crude enzyme solution to 45 ℃, adjusting the pH value to 9.0 by using NaOH, dropwise adding a protective agent lauroyl chloride accounting for 0.02% of the total mass of the SOD crude enzyme solution, carrying out heat preservation reaction for 1 hour, then rapidly cooling to below 20 ℃, centrifuging at 6000r/min for 10min, collecting supernatant, adjusting the pH value of the supernatant to 6.5 by using HCl, centrifuging at 6000r/min for 10min, and collecting the supernatant to obtain the blue-green SOD enzyme solution;
(7) and (3) freeze drying: adding trehalose with the weight of 2.0% of the SOD enzyme solution into the SOD enzyme solution, stirring and mixing uniformly, and freeze-drying to obtain 22.5g of light blue green SOD freeze-dried powder with the enzyme activity of 5310U/mg by detection;
(8) extraction: adding acidic acetone with the volume 5 times of that of the blood residue I obtained in the step (3) and the blood residue II obtained in the step (4), stirring for 30min, centrifuging at 6000r/min for 10min, and collecting supernatant;
(9) separation: adjusting the pH value of the supernatant obtained in the step (8) to 5.0, adding 1% of sodium acetate, uniformly stirring, standing for 30min, centrifuging at 6000r/min for 10min, and collecting precipitate;
(10) and (3) drying: washing the precipitate obtained in the step (9) with deionized water and absolute ethyl alcohol, and drying at 50-60 ℃ in vacuum to obtain 3.56 kg of heme with the heme purity of 87.2% by detection.
Example 2
(1) Preparation of blood cell liquid: collecting 1000L of fresh chicken blood in a slaughtering chicken factory, performing anticoagulation treatment, and continuously separating with a tubular centrifuge to obtain 300L of blood cell liquid and 700L of plasma liquid; spray drying the blood serum to obtain chicken plasma protein powder;
(2) hemolysis: pumping the blood corpuscle liquid into a reaction tank, adding 300L of deionized water, and continuously stirring for hemolysis for 60min at the stirring speed of 150r/min to obtain 600L of hemolysis liquid;
(3) thermal denaturation reaction I: adjusting the pH value of the hemolytic solution to 5.2 by using HCl under stirring, rapidly heating to 60 ℃, keeping the temperature for 60min, rapidly cooling to below 40 ℃, and performing filter pressing by using a plate-and-frame filter press to respectively obtain filtrate I369L and blood residue I99 kg;
(4) and (3) thermal denaturation reaction II: pumping the filtrate I into a reaction tank by using a pump, adjusting the pH value to 6.5 by using NaOH, quickly heating to 70 ℃, keeping the temperature for 20min, quickly cooling to below 40 ℃, performing filter pressing by using a plate-and-frame filter press, and respectively collecting 26 kg of filtrate II 287L and blood residue II;
(5) and (3) ultrafiltration concentration: carrying out ultrafiltration concentration on the collected filtrate II by an ultrafiltration membrane device with the molecular weight cutoff of 5000Da, and concentrating to 1/10 of the original volume to obtain 29L of SOD crude enzyme liquid;
(6) and (3) purification: heating the SOD crude enzyme solution to 45 ℃, adjusting the pH value to 9.0 by using NaOH, dropwise adding a protective agent lauroyl chloride accounting for 0.02% of the total mass of the SOD crude enzyme solution, carrying out heat preservation reaction for 1 hour, then rapidly cooling to below 20 ℃, centrifuging at 6000r/min for 10min, collecting supernatant, adjusting the pH value of the supernatant to 6.5 by using HCl, centrifuging at 6000r/min for 10min, and collecting the supernatant to obtain the blue-green SOD enzyme solution;
(7) and (3) freeze drying: adding trehalose with the weight of 2.0% of the SOD enzyme solution into the SOD enzyme solution, stirring and mixing uniformly, and freeze-drying to obtain light blue green SOD freeze-dried powder with the weight of 25.8g, wherein the activity of the enzyme is 5160U/mg by detection;
(8) extraction: adding acidic acetone with the volume 5 times of that of the blood residue I obtained in the step (3) and the blood residue II obtained in the step (4), stirring for 30min, centrifuging at 6000r/min for 10min, and collecting supernatant;
(9) separation: adjusting the pH value of the supernatant obtained in the step (8) to 5.0, adding 1% of sodium acetate, uniformly stirring, standing for 30min, centrifuging at 6000r/min for 10min, and collecting precipitate;
(10) and (3) drying: washing the precipitate obtained in the step (9) with deionized water and absolute ethyl alcohol, and drying at 50-60 ℃ in vacuum to obtain 4.82 kg of heme with the heme purity of 85.8% by detection.
Example 3
(1) Preparation of blood cell liquid: collecting 1000L of fresh chicken blood in a slaughtering chicken factory, performing anticoagulation treatment, and continuously separating with a tubular centrifuge to obtain 265L of blood cell liquid and 735L of blood serum; spray drying the blood serum to obtain chicken plasma protein powder;
(2) hemolysis: pumping the blood corpuscle liquid into a reaction tank, adding 260L of deionized water, continuously stirring and hemolysis for 60min at the stirring speed of 150r/min to obtain 520L of hemolysis liquid;
(3) thermal denaturation reaction I: adjusting the pH value of the hemolytic solution to 5.2 by using HCl under stirring, rapidly heating to 60 ℃, keeping the temperature for 60min, rapidly cooling to below 40 ℃, and performing filter pressing by using a plate-and-frame filter press to respectively obtain 331L filtrate and 82 kg blood residue I;
(4) and (3) thermal denaturation reaction II: pumping the filtrate I into a reaction tank by using a pump, adjusting the pH value to 6.5 by using NaOH, quickly heating to 70 ℃, keeping the temperature for 20min, quickly cooling to below 40 ℃, performing filter pressing by using a plate-and-frame filter press, and respectively collecting 256L of filtrate II and 25 kg of blood residues II;
(5) and (3) ultrafiltration concentration: carrying out ultrafiltration concentration on the collected filtrate II by an ultrafiltration membrane device with the molecular weight cutoff of 5000Da, and concentrating to 1/10 of the original volume to obtain 26L of SOD crude enzyme liquid;
(6) and (3) purification: heating the SOD crude enzyme solution to 45 ℃, adjusting the pH value to 9.0 by using NaOH, dropwise adding a protective agent lauroyl chloride accounting for 0.02% of the total mass of the SOD crude enzyme solution, carrying out heat preservation reaction for 1 hour, then rapidly cooling to below 20 ℃, centrifuging at 6000r/min for 10min, collecting supernatant, adjusting the pH value of the supernatant to 6.5 by using HCl, centrifuging at 6000r/min for 10min, and collecting the supernatant to obtain the blue-green SOD enzyme solution;
(7) and (3) freeze drying: adding trehalose with the weight of 2.0% of the SOD enzyme solution into the SOD enzyme solution, stirring and mixing uniformly, and freeze-drying to obtain 19.2g of light blue green SOD freeze-dried powder with the enzyme activity of 5240U/mg by detection;
(8) extraction: adding acidic acetone with the volume 5 times of that of the blood residue I obtained in the step (3) and the blood residue II obtained in the step (4), stirring for 30min, centrifuging at 6000r/min for 10min, and collecting supernatant;
(9) separation: adjusting the pH value of the supernatant obtained in the step (8) to 5.0, adding 1% of sodium acetate, uniformly stirring, standing for 30min, centrifuging at 6000r/min for 10min, and collecting precipitate;
(10) and (3) drying: washing the precipitate obtained in the step (9) with deionized water and absolute ethyl alcohol, and drying at 50-60 ℃ in vacuum to obtain 4.25 kg of heme, wherein the purity of the heme is 86.6% by detection.

Claims (3)

1. A co-production process for extracting SOD and heme from chicken blood is characterized by comprising the following steps:
(1) preparation of blood cell liquid: collecting fresh chicken blood, performing anticoagulation treatment, performing centrifugal separation, and collecting red blood cells to obtain blood cell liquid;
(2) hemolysis: adding deionized water with the volume 1 time of that of the hemocyte solution, and continuously stirring for hemolysis for 60min to obtain hemolysis solution;
(3) thermal denaturation reaction I: adjusting the pH value of the hemolytic solution to 5.2 by using HCl, rapidly heating to 60 ℃, preserving heat for 60min, rapidly cooling to below 40 ℃, and performing filter pressing by using a plate-and-frame filter press to respectively collect filtrate I and blood residue I;
(4) and (3) thermal denaturation reaction II: adjusting the pH value of the filtrate I to 6.5 by using NaOH, rapidly heating to 70 ℃, preserving heat for 20min, rapidly cooling to below 40 ℃, and performing filter pressing by using a plate-and-frame filter press to respectively collect filtrate II and blood residue II;
(5) and (3) ultrafiltration concentration: ultrafiltering and concentrating the collected filtrate II by an ultrafiltration membrane device with the molecular weight cutoff of 5000Da to 1/10 of the original volume, and collecting crude SOD enzyme solution;
(6) and (3) purification: heating the SOD crude enzyme solution to 45 ℃, adjusting the pH value to 9.0 by using NaOH, dropwise adding a protective agent lauroyl chloride accounting for 0.02-0.05% of the total mass of the SOD crude enzyme solution, keeping the temperature for reaction for 1 hour, quickly cooling to below 20 ℃, centrifuging at 6000r/min for 10min, collecting supernatant, adjusting the pH value of the supernatant to 6.5 by using HCl, centrifuging at 6000r/min for 10min, and collecting the supernatant to obtain the blue-green SOD enzyme solution;
(7) and (3) freeze drying: adding trehalose accounting for 2.0% of the weight of the SOD enzyme solution into the SOD enzyme solution, uniformly stirring and mixing, and freeze-drying to obtain light blue green SOD freeze-dried powder;
(8) extraction: adding acidic acetone with the volume 5 times of that of the blood residue I obtained in the step (3) and the blood residue II obtained in the step (4), stirring for 30min, centrifuging at 6000r/min for 10min, and collecting supernatant;
(9) separation: adjusting the pH value of the supernatant obtained in the step (8) to 5.0, adding 1% of sodium acetate, uniformly stirring, standing for 30min, centrifuging at 6000r/min for 10min, and collecting precipitate;
(10) and (3) drying: and (4) washing the precipitate obtained in the step (9) with deionized water and absolute ethyl alcohol, and drying in vacuum at 50-60 ℃ to obtain the heme.
2. The co-production process of extracting SOD and heme from chicken blood as claimed in claim 1, wherein: and (2) the centrifugal separation in the step (1) is continuous separation by adopting a tubular centrifuge, and the centrifugal rotating speed is more than 16000 r/min.
3. The co-production process of extracting SOD and heme from chicken blood as claimed in claim 1, wherein: the acidic acetone in the step (8) is 3% (v/v) acetone hydrochloride solution.
CN201811224015.0A 2018-10-19 2018-10-19 Co-production process for extracting SOD and heme from chicken blood Pending CN111072771A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112841406A (en) * 2021-01-25 2021-05-28 窦靖鑫 Deep processing technology of black-bone chicken and black-bone chicken polypeptide protein powder

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112841406A (en) * 2021-01-25 2021-05-28 窦靖鑫 Deep processing technology of black-bone chicken and black-bone chicken polypeptide protein powder

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