CN104945503A - Method for preparing nitrosohemoglobin by using porcine blood - Google Patents
Method for preparing nitrosohemoglobin by using porcine blood Download PDFInfo
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- CN104945503A CN104945503A CN201410122785.XA CN201410122785A CN104945503A CN 104945503 A CN104945503 A CN 104945503A CN 201410122785 A CN201410122785 A CN 201410122785A CN 104945503 A CN104945503 A CN 104945503A
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Abstract
The invention provides a method for preparing nitrosohemoglobin by using porcine blood. The method comprises the following steps: (1) separating blood cells from fresh porcine blood; (2) extracting a hemoglobin crude extract from blood cells; (3) purifying the hemoglobin and measuring the purity of the hemoglobin, namely adding an antioxidant to the hemoglobin crude extract obtained in the step (2) for vacuum degassing, introducing N2 for protection, heating in a water bath at 60 DEG C for 30 minutes so that impurity proteins can be precipitated and then centrifuging to obtain a supernatant; adding 10mg/ml glycerol to the purified hemoglobin solution and then storing the purified hemoglobin solution in a refrigerator at 4 DEG C for later use; finally, measuring the content of the hemoglobin; (4) synthesizing the nitrosohemoglobin, namely adding NaOH to the purified hemoglobin, and meanwhile, adding sodium nitrite and vitamin C, regulating the pH to acidity, heating to the range of 50-70 DEG C and keeping for 25-45 minutes, and performing vacuum freeze-drying to generate a red powder, namely the nitrosohemoglobin. The method is simple in process, relatively low in equipment requirement and suitable for industrial production.
Description
Technical field
The present invention relates to a kind of production method of foodstuff additive, particularly a kind of method utilizing pig blood to prepare nitrosohaemoglobin.
Background technology
Dinitrosyl ferrohemochrome is considered to the material that can make butcher's meat color development usually.Meat produces red Nitrosyhemoglobin (HbNO) after nitrite (or nitrate) is pickled, more stable pigment can be transformed into because of globin sex change again under the effect of salt and heating---dinitrosyl ferrohemochrome is a kind of peach pigment.The consistence of nitrosohaemoglobin and dinitrosyl ferrohemochrome structure determines the Meat Additive that it will be a kind of very safety non-pollution.
Safe and reliable as the oxyphorase wide material sources of one of synthesis nitrosohaemoglobin raw material.According to data, pig blood accounts for 5% of pig live body gross weight, and butcher the 60%-70% that the rear blood that can collect accounts for total amount, in pig blood, protein content about 19%, is wherein mainly oxyphorase, accounts for 2/3 of whole blood total protein.Chinese since 1993, meat production amount occupy world-class position always.Therefore, be practicable with pig blood economically as Material synthesis nitrosohaemoglobin.And pig blood is nutritious, whole blood contains the protein of 17%-21%, and from amino acid composition, hematoglobin protein is a kind of high-quality protein, and especially lysine content is very high, and close to 9.0%, its essential amino acids content is also higher than human milk and shell egg.From the angle of complementary action of protein, because in corn protein, lysine content is low, methionine(Met) and dirty histidine content high, isoleucine content is suitable, and the high lysine content of blood becomes good com gluten protein complement and easily absorbs.Therefore, not only can not destroy the nutritive value of blood with pig blood synthesis nitrosohaemoglobin, and animal blood comprehensive resource utilization rate can be improved and improve added value of product.Therefore nitrosohaemoglobin will be a kind of multi-functional foodstuff additive, have development and application prospect widely.
Summary of the invention
Object of the present invention provides a kind of method utilizing pig blood to prepare nitrosohaemoglobin, and the method utilizes swelling, the centrifugal method combined separation and purification of hemoglobin from pig blood, then by oxyphorase and Sodium Nitrite Reactive Synthesis nitrous acid oxyphorase.The method technique is simple, required equipment requires lower, is applicable to suitability for industrialized production.
As above conceive, technical scheme of the present invention is: a kind of method utilizing pig blood to prepare nitrosohaemoglobin, is characterized in that: comprise the steps:
1. from fresh pig blood, hemocyte is isolated;
2. oxyphorase crude extract can only strangely be extracted from hemocyte;
3. purified hemoglobin, and measure its purity: step 2. in add antioxidant vacuum outgas in the oxyphorase crude extract that obtains, and pass into N
2protection, makes foreign protein precipitate in 60 DEG C of heating in water bath 30min, then centrifuging and taking supernatant liquor; Hemoglobin solutions after purifying adds 10mg/ml glycerine and is placed in 4 DEG C of refrigerators and saves backup; Last Measuring hemoglobin content again;
4. nitrosohaemoglobin is synthesized: in oxyphorase after purification, add NaOH, add Sodium Nitrite, vitamins C simultaneously, regulate pH to be acid, be heated to 50-70 DEG C and keep 25-45min, generate red meal after vacuum lyophilization and be nitrosohaemoglobin.
Above-mentioned steps concrete grammar is 2.: the distilled water adding oxyphorase volume 1.5 times, mixes rear induction stirring 30min; When solution by dark red transfer to scarlet time, by solution with the centrifugal 15min of 3000r/min, discard precipitation, obtain oxyphorase crude extract.
The method of above-mentioned steps 3. Measuring hemoglobin content is:
1. the preparation of AHD-575 reagent: accurately take Triton X-100 25.0g, adds 0.1mol/LNaOH solution and is settled to 1L, filters, saves backup under room temperature;
2. the making of typical curve: solution PINPROL standard substance being mixed with different concns, draws 0.1ml respectively and joins in 30mLAHD-575 reagent, standing and reacting 5min, under 575m wavelength, with AHD-575 reagent for blank, measure absorbancy; With the concentration of PINPROL standard substance (g/L) for X-coordinate, be ordinate zou with absorbancy, draw standard lines and the regression equation of the relation of hemoglobin solutions concentration X and absorbance Y;
3., in the beaker of cleaning, draw hemoglobin extract sample 0.1ml, measure absorbance A with the method for AHD-575
575, look into typical curve and try to achieve content of hemoglobin (g/L).
Above-mentioned steps is oxyphorase 4.: Sodium Nitrite: vitamins C is according to the proportions of mol ratio=1:1 ~ 3:4 ~ 8.
Above-mentioned steps is 4. middle regulates pH to be 5.0 ~ 6.0.
Above-mentioned steps 4. in add 0.5gNaOH.
The present invention has following advantage and positively effect:
1. the present invention utilizes swelling, the centrifugal method combined separation and purification of hemoglobin from pig blood, then by oxyphorase and Sodium Nitrite Reactive Synthesis nitrous acid oxyphorase.The method technique is simple, required equipment requires lower, is applicable to suitability for industrialized production.
2. the present invention adopts one of raw material pig blood, abundance and cost is low.
3. raw material pig blood of the present invention contains abundant nutritive value, carries the nutritive substance of multiple needed by human body in the nitroso-group albumen therefore synthesized.
4. the product nitrosohaemoglobin that prepared by the present invention is a kind of foodstuff additive of safety non-pollution.
Accompanying drawing illustrates:
Fig. 1 is oxyphorase typical curve figure;
Fig. 2 is nitro oxyphorase acetone extract UV scanning figure.
Embodiment:
Below in conjunction with embodiment, the present invention will be further described:
Embodiment 1: a kind of method utilizing pig blood to prepare nitrosohaemoglobin, comprises the steps:
1. the separation of hemocyte: get 1000mL fresh pig blood and add 1g sodium citrate anticoagulant fast, fully mix with pig blood along a direction stirring with glass stick, low temperature transports laboratory back.Filter after stirring 5min, the centrifugal 30min of 3000r/min, abandons upper serum; Add the physiology salt of 0.9%, stir, the centrifugal 10min of 3000r/min, abandon supernatant (repeating twice), collect red blood corpuscle and recording volume.
2. extract oxyphorase: the distilled water adding blood cell volume 1.5 times, mixes rear induction stirring 30min; When solution by dark red transfer to scarlet time, by solution with the centrifugal 15min of 3000r/min, discard precipitation, obtain oxyphorase crude extract.
3. oxyphorase purifying: appropriate antioxidant Vc will be added in oxyphorase crude extract, vacuum outgas, and pass into N2 protection, in 60 DEG C of heating in water bath 30min, foreign protein is precipitated, the centrifugal 15min of 3000r/min, gets supernatant liquor.Hemoglobin solutions after purifying adds 10mg/ml glycerine and is placed in 4 DEG C of refrigerators and saves backup.
Oxyphorase purity testing: solution PINPROL standard substance being mixed with different concns, draws 0.1ml respectively and joins in 30mLAHD-575 reagent, standing and reacting 5min, under 575m wavelength, with AHD-575 reagent for blank, measure absorbancy.With the concentration of PINPROL standard substance (g/L) for X-coordinate, be ordinate zou with absorbancy, draw standard lines and the regression equation of the relation of hemoglobin solutions concentration X and absorbance Y.In the beaker of cleaning, draw hemoglobin extract sample 0.1ml, measure absorbance A with the method for AHD-575
575, look into typical curve and try to achieve content of hemoglobin (g/L).
The compound method of described AHD-575 reagent is: accurately take Triton X-100 25.0g, adds 0.1mol/LNaOH solution and is settled to 1L, filters, saves backup under room temperature.
4. synthesize nitrosohaemoglobin: in the ratio of oxyphorase, Sodium Nitrite, vitamins C l:2:6 in molar ratio, pH is 5.5, be heated to 60 DEG C and keep 30min, generate red meal after vacuum lyophilization and be nitrosohaemoglobin.
Embodiment 2: a kind of method utilizing pig blood to prepare nitrosohaemoglobin, comprises the steps:
1. the separation of hemocyte: get 1000mL fresh pig blood and add 1g sodium citrate anticoagulant fast, fully mix with pig blood along a direction stirring with glass stick, low temperature transports laboratory back.Filter after stirring 5min, the centrifugal 30min of 3000r/min, abandons upper serum; Add the physiology salt of 0.9%, stir, the centrifugal 10min of 3000r/min, abandon supernatant (repeating twice), collect red blood corpuscle and recording volume.
2. extract oxyphorase: the distilled water adding blood cell volume 1.5 times, mixes rear induction stirring 30min; When solution by dark red transfer to scarlet time, by solution with the centrifugal 15min of 3000r/min, discard precipitation, obtain oxyphorase crude extract.
3. oxyphorase purifying: appropriate antioxidant Vc will be added in oxyphorase crude extract, vacuum outgas, and pass into N2 protection, in 60 DEG C of heating in water bath 30min, foreign protein is precipitated, the centrifugal 15min of 3000r/min, gets supernatant liquor.Hemoglobin solutions after purifying adds 10mg/ml glycerine and is placed in 4 DEG C of refrigerators and saves backup.
Oxyphorase purity testing: solution PINPROL standard substance being mixed with different concns, draws 0.1ml respectively and joins in 30mLAHD-575 reagent, standing and reacting 5min, under 575m wavelength, with AHD-575 reagent for blank, measure absorbancy.With the concentration of PINPROL standard substance (g/L) for X-coordinate, be ordinate zou with absorbancy, draw standard lines and the regression equation of the relation of hemoglobin solutions concentration X and absorbance Y.In the beaker of cleaning, draw hemoglobin extract sample 0.1ml, measure absorbance A with the method for AHD-575
575, look into typical curve and try to achieve content of hemoglobin (g/L).
The compound method of described AHD-575 reagent is: accurately take Triton X-100 25.0g, adds 0.1mol/LNaOH solution and is settled to 1L, filters, saves backup under room temperature.
4. synthesize nitrosohaemoglobin: in the ratio of oxyphorase, Sodium Nitrite, vitamins C l:3:4 in molar ratio, pH is 6.0, be heated to 60 DEG C and keep 30min, generate red meal after vacuum lyophilization and be nitrosohaemoglobin.
The measuring principle of described oxyphorase purity is: under alkaline environment, non-ionic surface-active substance in oxyphorase and derivative and reagent thereof is reacted, the alkaline high ferro non-ionic type table that rapid formation is stable and promoting agent mixture, this mixture has an absorption peak at wavelength 575nm place.The stable reagent, nontoxic of this method application, and the result of oxyphorase is had no significant effect.
Claims (6)
1. utilize pig blood to prepare a method for nitrosohaemoglobin, it is characterized in that: comprise the steps:
1. from fresh pig blood, hemocyte is isolated;
2. oxyphorase crude extract can only strangely be extracted from hemocyte;
3. purified hemoglobin, and measure its purity: step 2. in add antioxidant vacuum outgas in the oxyphorase crude extract that obtains, and pass into N
2protection, makes foreign protein precipitate in 60 DEG C of heating in water bath 30min, then centrifuging and taking supernatant liquor; Hemoglobin solutions after purifying adds 10mg/ml glycerine and is placed in 4 DEG C of refrigerators and saves backup; Last Measuring hemoglobin content again;
4. nitrosohaemoglobin is synthesized: in oxyphorase after purification, add NaOH, add Sodium Nitrite, vitamins C simultaneously, regulate pH to be acid, be heated to 50-70 DEG C and keep 25-45min, generate red meal after vacuum lyophilization and be nitrosohaemoglobin.
2. a kind of method utilizing pig blood to prepare nitrosohaemoglobin according to claim 1, is characterized in that: above-mentioned steps concrete grammar is 2.: the distilled water adding oxyphorase volume 1.5 times, mixes rear induction stirring 30min; When solution by dark red transfer to scarlet time, by solution with the centrifugal 15min of 3000r/min, discard precipitation, obtain oxyphorase crude extract.
3. a kind of method utilizing pig blood to prepare nitrosohaemoglobin according to claim 1, is characterized in that: the method for above-mentioned steps 3. Measuring hemoglobin content is:
1. the preparation of AHD-575 reagent: accurately take Triton X-100 25.0g, adds 0.1mol/LNaOH solution and is settled to 1L, filters, saves backup under room temperature;
2. the making of typical curve: solution PINPROL standard substance being mixed with different concns, draws 0.1ml respectively and joins in 30mLAHD-575 reagent, standing and reacting 5min, under 575m wavelength, with AHD-575 reagent for blank, measure absorbancy; With the concentration of PINPROL standard substance (g/L) for X-coordinate, be ordinate zou with absorbancy, draw standard lines and the regression equation of the relation of hemoglobin solutions concentration X and absorbance Y;
3., in the beaker of cleaning, draw hemoglobin extract sample 0.1ml, measure absorbance A 575 with the method for AHD-575, look into typical curve and try to achieve content of hemoglobin (g/L).
4. a kind of method utilizing pig blood to prepare nitrosohaemoglobin according to claim 1, is characterized in that: above-mentioned steps is oxyphorase 4.: Sodium Nitrite: vitamins C is according to the proportions of mol ratio=1:1 ~ 3:4 ~ 8.
5. a kind of method utilizing pig blood to prepare nitrosohaemoglobin according to claim 1, is characterized in that: above-mentioned steps is 4. middle regulates pH to be 5.0 ~ 6.0.
6. a kind of method utilizing pig blood to prepare nitrosohaemoglobin according to claim 1, is characterized in that: above-mentioned steps 4. in add 0.5gNaOH.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420323A (en) * | 2015-12-23 | 2016-03-23 | 吉林大学 | Phytochelatin iron supplementation powder containing iron and preparation method of phytochelatin iron supplementation powder |
| CN106566278A (en) * | 2016-11-07 | 2017-04-19 | 中国农业科学院农产品加工研究所 | Hemoglobin coloring agent and preparation method thereof |
| CN107266563A (en) * | 2017-08-01 | 2017-10-20 | 四川沃文特生物技术有限公司 | A kind of preparation method of hemoglobin quality-control product |
-
2014
- 2014-03-28 CN CN201410122785.XA patent/CN104945503A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420323A (en) * | 2015-12-23 | 2016-03-23 | 吉林大学 | Phytochelatin iron supplementation powder containing iron and preparation method of phytochelatin iron supplementation powder |
| CN106566278A (en) * | 2016-11-07 | 2017-04-19 | 中国农业科学院农产品加工研究所 | Hemoglobin coloring agent and preparation method thereof |
| CN107266563A (en) * | 2017-08-01 | 2017-10-20 | 四川沃文特生物技术有限公司 | A kind of preparation method of hemoglobin quality-control product |
| CN107266563B (en) * | 2017-08-01 | 2020-08-11 | 四川沃文特生物技术有限公司 | Preparation method of hemoglobin control product |
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