Optimization process for refining pig blood chlorhematin by using glacial acetic acid method

Abstract

The invention discloses an optimization process for refining pig blood chlorhematin by using a glacial acetic acid method, which comprises the following process flows: pretreating fresh pig blood to obtain pig blood red blood cell powder; preparing a pig blood chlorhematin crude product by adopting an optimized glacial acetic acid process; washing the crude product by adopting a hydrochloric acid solution washing method to obtain a pig blood chlorhematin refined product; neutral washing; and performing vacuum drying to constant weight to obtain a heme product. The pig blood chlorhematin refined by the glacial acetic acid extraction combined hydrochloric acid solution washing method provided by the invention has the advantages of high yield, high purity, great reduction of the concentration of glacial acetic acid, low energy consumption, environmental friendliness and reduction of the production cost of enterprises, and is suitable for large-scale industrial implementation.

Description

Technical Field

[0001] The invention relates to an optimized process for preparing hemin, in particular to an optimized process for preparing hemin from pig blood by using a glacial acetic acid method. Background Art

[0002] Anemia is the most common nutritional deficiency disease in the world and one of the most concerned public health issues. More than 95% of anemia is iron deficiency anemia. At present, the iron in blood tonic drugs is mainly non-heme iron with ferrous sulfate, ferrous fumarate, ferrous lactate, polysaccharide iron, etc. as the main components. However, studies have shown that the absorption rate of heme iron in the small intestine is as high as 25% to 30%, while non-heme iron is only 3% to 8%. In addition, heme iron can be directly absorbed by intestinal mucosal cells without causing damage, and can be used in iron-supplementing functional foods, nutritional enhancers and other products; heme is also a precursor of porphyrin drugs for the treatment of malignant tumors and sodium protoporphyrin diacid for the treatment of hepatitis.

[0003] Pig blood is an industrial byproduct that can produce a large amount of high nutritional value substances. A variety of biochemical products can be extracted from pig blood, such as hemoglobin, thrombin, albumin, superoxide dismutase (SOD) and other products. The hemoglobin content in pig blood is relatively high, accounting for about 5% of the whole blood. In other words, theoretically, the hemoglobin content in every 100mL of pig blood is as high as 40mg or more.

[0004] In recent years, a variety of methods for extracting hemin have been proposed through research, and the main methods currently include glacial acetic acid method, sodium carboxymethyl cellulose method, acid acetone method and enzymatic hydrolysis method, etc. These methods have their own advantages and disadvantages.

[0005] Among them, the glacial acetic acid method is simple to produce and has a faster speed. Most hemin production companies use the glacial acetic acid method. However, the use of high-concentration glacial acetic acid not only causes waste of resources, but also brings tremendous pressure to environmental protection. On the other hand, the use of high-concentration glacial acetic acid will also cause strong corrosion to steel structure production equipment.

[0006] The acid acetone method is an organic solvent method for extracting hemin. Although this method can extract hemin with high purity and yield, acetone is used in the extraction process, which is costly, highly volatile, flammable and explosive, difficult to recycle, and has high physiological toxicity. It cannot be used to directly produce hemin for food addition.

[0007] The process of extracting hemin with sodium carboxymethyl cellulose is simple and has low production cost. The extracted hemin is stable and can be stored for a long time. It can also be directly pressed into tablets to make medicine. However, the purity and yield of hemin are low. Although it is convenient to use, it cannot maximize economic benefits and is difficult to promote.

[0008] The principle of enzymatic hydrolysis is to hydrolyze hemoglobin to separate heme and protein, thereby extracting hemin. Enzymatic hydrolysis does not use organic solvents in extraction, consumes less energy, is environmentally friendly, and has high economic and social benefits. However, due to the different enzymatic hydrolysis processes, heme polypeptide complexes are easily formed, so the extraction effect is not satisfactory.

[0009] In summary, the current hemin extraction method mainly has the following problems:

[0010] 1) Cost issues, mainly reflected in the cost of raw materials and production cycle;

[0011] 2) Environmental issues: organic solvent residues in the production process pose safety and health risks. Summary of the invention

[0012] In order to solve the shortcomings of the above technology, the present invention provides an optimized process for refining pig blood hemin using glacial acetic acid method.

[0013] In order to solve the above technical problems, the technical solution adopted by the present invention is: an optimized process for refining pig blood hemin using glacial acetic acid method, including the following process flow:

[0014] S1. Pretreatment: taking fresh pig blood for pretreatment to obtain pig blood red blood cell powder;

[0015] S2. Crude product preparation: using pig blood red blood cell powder as raw material, adopting optimized glacial acetic acid process to prepare pig blood hemin crude product;

[0016] S3, preparation of refined product: washing the crude pig blood hemin obtained in S2 with a hydrochloric acid solution washing method to obtain a refined pig blood hemin;

[0017] S4. Neutral washing: Use 0.9% saline to wash the refined pig blood hemin to neutrality:

[0018] S5. Vacuum drying: under vacuum conditions at 50°C, the pig blood hemoglobin refined product after neutral washing is dried to constant weight to obtain the hemoglobin product.

[0019] Preferably, in S1, the pretreatment method includes preparation of fresh anticoagulated pig blood and ultrasonic disruption treatment.

[0020] Preferably, the preparation process of fresh anticoagulated pig blood is as follows: take fresh pig blood, add 0.5% anticoagulant trisodium citrate solution to the fresh pig blood, stir evenly, and then obtain fresh anticoagulated pig blood.

[0021] Preferably, fresh anticoagulated pig blood is used as raw material, and the red blood cell-rich precipitate is collected by centrifugation. The red blood cell precipitate is washed three times with 0.9% saline, then ultrasonically crushed with deionized water, centrifuged again to obtain a precipitate, and vacuum dried to obtain red blood cell powder.

[0022] Preferably, in S3, the optimal conditions of the optimized glacial acetic acid process used for the preparation of the crude product are: glacial acetic acid concentration of 75%, reaction temperature of 105°C, reaction time of 1 hour, and sodium chloride addition amount of 1.5%.

[0023] Preferably, the specific process of preparing the crude product of pig blood hemin by the glacial acetic acid method is as follows: glacial acetic acid with a concentration of 75% is mixed with pig blood red blood cell powder in a mass ratio of 4:1, and then sodium chloride with a mass ratio of 1.5% of the pig blood red blood cell powder is added, and the mixture is heated to 105° C. while being stirred evenly at a slow speed, and the reaction time is 1 hour to obtain a crude product solution of pig blood hemin;

[0024] After the crude pig blood hemin solution is cooled to room temperature, the precipitate is collected by centrifugation to obtain the crude pig blood hemin.

[0025] Preferably, in S4, the crude pig blood hemin is purified by washing the crude pig blood hemin three times with a pure hydrochloric acid solution with a pH of 1-3, and collecting the precipitate by centrifugation to obtain a refined pig blood hemin.

[0026] Preferably, the supernatant after centrifugation of the crude pig blood hemin solution is recovered with glacial acetic acid.

[0027] Preferably, the method for recovering glacial acetic acid is: adding saturated calcium chloride to the supernatant to be recovered.

[0028] Preferably, the glacial acetic acid recovery method is: adding the supernatant to be recovered into a distillation flask for distillation recovery treatment, and connecting a distillation column at the gas outlet of the distillation flask.

[0029] The present invention discloses an optimized process for refining pig blood hemin using a glacial acetic acid method, which has the following technical advantages:

[0030] 1) Compared with the traditional method, the concentration of glacial acetic acid used is reduced and the recovery rate of glacial acetic acid is ultimately improved, thus avoiding waste of resources and huge pressure on the environment; and avoiding the use of high-concentration glacial acetic acid can also reduce the strong corrosion caused to steel structure production equipment.

[0031] 2) The present invention takes pig blood hemin as the research object, adopts glacial acetic acid extraction combined with hydrochloric acid solution washing, and adopts orthogonal test to optimize the refining process, thereby improving the purity and recovery rate of the finished product, and is suitable for promotion to industrial implementation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the process of the present invention. DETAILED DESCRIPTION

[0033] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0034] An optimized process for refining pig blood hemin using glacial acetic acid method, such as Figure 1 As shown, the process includes the following:

[0035] S1. Pretreatment: taking fresh pig blood for pretreatment to obtain pig blood red blood cell powder;

[0036] S2. Crude product preparation: using pig blood red blood cell powder as raw material, adopting optimized glacial acetic acid process to prepare pig blood hemin crude product;

[0037] S3, preparation of refined product: washing the crude pig blood hemin obtained in S2 with a hydrochloric acid solution washing method to obtain a refined pig blood hemin;

[0038] S4. Neutral washing: Use 0.9% saline to wash the refined pig blood hemin to neutrality:

[0039] S5. Vacuum drying: under vacuum conditions at 50°C, the pig blood hemoglobin refined product after neutral washing is dried to constant weight to obtain the hemoglobin product.

[0040] The pretreatment method is mainly divided into two steps: one is to prepare fresh anticoagulated pig blood, and the other is ultrasonic disruption to fully release red blood cells.

[0041] Take fresh pig blood, add 0.5% anticoagulant trisodium citrate solution to the fresh pig blood, stir evenly, and then obtain fresh anticoagulated pig blood;

[0042] Centrifuge at 4500 r / min, pour out the supernatant (serum), collect the precipitate rich in red blood cells, wash the red blood cell precipitate three times with 0.9% saline, add deionized water and ultrasonically break it to release the red blood cells, then centrifuge at 4500 r / min to obtain the precipitate, and vacuum dry the precipitate to obtain red blood cell powder.

[0043] The optimal conditions for the optimized glacial acetic acid process were found through orthogonal experiments. The optimal conditions are: glacial acetic acid concentration of 75%, reaction temperature of 105℃, reaction time of 1 hour, and sodium chloride addition of 1.5%.

[0044] The specific process of preparing crude porcine blood hemin by the glacial acetic acid method is as follows: glacial acetic acid with a concentration of 75% is mixed with porcine blood red blood cell powder in a mass ratio of 4:1, and then sodium chloride with a mass percentage of 1.5% of the porcine blood red blood cell powder is added, and the mixture is heated to 105°C while being stirred slowly and evenly, and the reaction time is 1 hour to obtain a crude porcine blood hemin solution.

[0045] After the crude pig blood hemin solution is cooled to room temperature, it is centrifuged at a speed of 4500 r / min, and the precipitate is collected to obtain the crude pig blood hemin.

[0046] Next, the crude pig blood hemin is refined and prepared by washing it three times with a pure hydrochloric acid solution with a pH of 1-3, and collecting the precipitate by centrifugation to obtain a refined pig blood hemin.

[0047] Since the refined pig blood hemin product contains hydrochloric acid solution, the hydrochloric acid solution is washed away with 0.9% saline until the solution is neutral, and then the precipitate is collected by centrifugation again.

[0048] Finally, the pig blood hemoglobin refined product after neutral washing is dried to constant weight under vacuum conditions at 50°C to obtain the hemoglobin product.

[0049] Preferably, the supernatant after centrifugation of the crude pig blood hemin solution can be recovered with glacial acetic acid.

[0050] Preferably, the glacial acetic acid recovery method is: adding saturated calcium chloride to the supernatant to be recovered, or adding the supernatant to be recovered to a distillation flask for distillation recovery treatment, and connecting a distillation column at the gas outlet of the distillation flask to achieve reflux to increase the recovery rate.

[0051] Example

[0052] In order to explore the high yield, high purity, low energy consumption, environmentally friendly refining process of pig blood hemin and realize industrialization, orthogonal experimental design was used to explore the effects of the four factors of acetic acid addition, reaction temperature, reaction time, and sodium chloride addition on the yield, purity and yield of hemin products, and determine the optimal process parameters. The pilot test results with 50 kg of pig blood red blood cell powder as raw material showed that the optimal process conditions of the acetic acid method were: acetic acid concentration of 75%, reaction temperature of 105℃, reaction time of 1 hour, and sodium chloride addition of 1.5%; the crude product of pig blood hemin was prepared by the acetic acid method under the above process conditions, and the yield of the crude product of pig blood hemin was 49%, which was 11% higher than that of the enzyme method.

[0053] In the crude product refining experiment, various methods were tested, including alkali dissolution and acid precipitation, enzymatic decomposition, tannic acid precipitation, high temperature heating, acetic acid washing, strong acid hydrolysis, and DMI refining. The results showed that the refining method after glacial acetic acid extraction and hydrochloric acid solution washing can increase the purity of hemin to more than 95%, with low cost and a final yield of up to 49%, which is an ideal method for large-scale production. Finally, the glacial acetic acid method after process optimization was used to prepare pig blood hemin, and in accordance with the "National Food Safety Standard Food Nutrition Fortifier Hemin Chloride" (GB1903.522021 National Standard), the pilot production of the product was completed, and the product was used in blood tonic medicines or functional foods.

[0054] The specific processes of process optimization are:

[0055] 1. Pretreatment of pig blood

[0056] Take fresh pig blood, add 0.5% anticoagulant trisodium citrate solution to the fresh pig blood, stir evenly to obtain fresh anticoagulated pig blood, centrifuge at 4500r / min, pour out the supernatant (serum), and the supernatant can be spray-dried to prepare plasma protein powder.

[0057] Red blood cells are enriched in the precipitate. The collected red blood cell precipitate is washed three times with physiological saline, then ultrasonically broken with deionized water, and then centrifuged at 4500r / min to obtain a precipitate. The precipitate is dried into red blood cell powder and the filtrate is used for later use.

[0058] 2. Preparation of crude porcine blood hemin: In this example, orthogonal experiments were used to explore the optimal process conditions of the glacial acetic acid method.

[0059] The heme extraction rate was calculated using the following formula:

[0060]

[0061] Where: X0, X t The heme content in the product and the heme content in the raw materials respectively (converted from iron content to heme content / g).

[0062] The following formula is used to calculate the hemoglobin purity rate:

[0063]

[0064] Where: X1 and X2 are the iron content in the product and the iron content in pure heme, respectively. Iron content in the product (measured by ICP-MS / %); Iron content in pure heme (13.77% based on the theoretical value of heme).

[0065] With 50 kg of porcine red blood cell powder as raw material, the effects of acetic acid addition, reaction temperature, reaction time and sodium chloride addition on the preparation of hemin were investigated, and the optimal preparation conditions were obtained through response surface analysis.

[0066] An orthogonal experimental design was conducted for four factors: acetic acid concentration, reaction temperature, reaction time, and sodium chloride addition. Each factor was arranged to have three levels of repeated experiments. The experimental level settings are shown in Table 1. The orthogonal table L9(3 4 ) was tested to determine the optimal process parameters for preparing crude porcine blood hemin using the glacial acetic acid method based on the yield and purity of the crude porcine blood hemin.

[0067] Table 1 Orthogonal test factors and levels

[0068]

[0069] According to the orthogonal table L9(3 4 ) factor level combination, and an orthogonal test was conducted. The specific experimental arrangement and related results are shown in Table 2. The actual recovery rate and purity of pig blood hemin are jointly affected by acetic acid concentration, reaction temperature, reaction time and sodium chloride addition. It can be seen from Table 2 that the yield of pig blood hemin increases with the increase of acetic acid concentration. When the acetic acid concentration is 75%, the yield of hemin is significantly improved compared with 50% and 67%. In terms of reaction temperature, as the reaction temperature gradually increases, the yield of hemin is also significantly improved, and 105°C is the optimal reaction temperature. In terms of the reaction time of the pilot test, the optimal reaction time is 1 hour. Under the condition of equal other factors, the yield of hemin under a reaction time of 1 hour is the highest. Finally, when the amount of sodium chloride added is 1.5% of the mass of pig blood red blood cell powder, the yield of hemin is the highest, reaching 49.4%.

[0070] Table 2 Orthogonal experimental arrangement table and recovery correlation index table of pig blood hemin purification by glacial acetic acid method

[0071]

[0072] In summary, the pilot experiment with 50 kg of pig blood red blood cell powder as raw material showed that the optimal process conditions for preparing pig blood hemin by glacial acetic acid method were: glacial acetic acid concentration of 75%, reaction temperature of 105 ° C, reaction time of 1 hour, and sodium chloride addition of 1.5%. To further verify the results of the orthogonal experiment, three parallel experiments were carried out according to the optimal process conditions obtained in the experiment. The results are shown in Table 3. Under the optimal process conditions, the yield and purity of pig blood hemin were relatively high, with an average yield of 48.9%, and the repeatability of the experiment was also good.

[0073] Table 3 Orthogonal experimental arrangement table and recovery correlation index table of pig blood hemin purification by glacial acetic acid method

[0074]

[0075] 3. Refining of crude pig blood hemin

[0076] The refining of hemin is of great significance for the refining of crude hemin, and is one of the remedial methods after the product is unqualified. The crude hemin from pig blood prepared by the glacial acetic acid method with the highest yield (product No. 7 in Table 2) was tested with various methods such as alkali dissolution and acid precipitation, enzymatic decomposition, acid precipitation, high temperature heating, acetic acid washing, strong acid hydrolysis, and DMI refining. As shown in Table 4, DMI refining and hydrochloric acid solution washing can significantly improve the purity of hemin. Among them, DMI refining can improve the crude product to a very high purity (close to 100%), but its cost is high and the yield is low (the final yield is 32%). Hydrochloric acid solution washing can improve the purity of the finished product to more than 95%, with low cost and a final yield of 45%. Therefore, it is determined that hydrochloric acid washing is an ideal method suitable for the refining of crude hemin from pig blood.

[0077] Table 4 Information table of different purification methods for crude hemin

[0078]

[0079] 4. Verification and quality testing of the optimal purification process of porcine hemin

[0080] For further verification, three pilot process verification tests were carried out, with 50 kg of pig blood macrocell powder used in each test. Specifically, the crude hemin product was prepared according to the glacial acetic acid concentration of 75%, the reaction temperature of 105°C, the reaction time of 1 hour, and the amount of sodium chloride added of 1.5% of the pig blood red blood cell powder; the hydrochloric acid washing method was used to further refine and purify the hemin.

[0081] As shown in Table 5, the results of three repeated experiments show that after the process flow is optimized, the yield of refined porcine blood hemin is about 42.6% and the purity is above 97%. Therefore, the optimized process of refining porcine blood hemin using glacial acetic acid method explored in this embodiment is an ideal method suitable for large-scale production.

[0082] Table 5 Test results of the final process flow of pig blood hemin

[0083]

[0084] The three samples of fine pig blood hemin were tested for quality. All three samples met the company's internal control standards in terms of state, moisture, purity, total colony count, mold, yeast and E. coli colony count.

[0085] 5. Acetic acid recovery

[0086] The acetic acid recovery experiment was conducted by exploring methods such as vacuum distillation, room temperature distillation, low temperature crystallization using saturated calcium chloride, distillation after adding saturated calcium chloride, and segmented atmospheric distillation. Through the experimental results, two efficient and low-cost acetic acid recovery and purification methods (adding saturated calcium chloride and connecting a distillation column) were obtained, which can increase the acetic acid from 50% after extraction to more than 75%, and even more than 90%, while the loss rate of acetic acid each time can be controlled within 10%, as shown in Table 6.

[0087] Table 6 Comparison of different acetic acid recovery and purification methods

[0088]

[0089] Therefore, the pig blood hemin refined by the glacial acetic acid extraction combined with hydrochloric acid solution washing method provided in the present invention has high yield and high purity, greatly reduces the concentration of glacial acetic acid, has low energy consumption and is environmentally friendly, reduces the production cost of the enterprise, and is suitable for large-scale industrial implementation.

[0090] The above implementation modes are not limitations of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the scope of the technical solution of the present invention also belong to the protection scope of the present invention.

Claims

1. An optimized process for refining pig blood hemin using glacial acetic acid method, characterized in that: Including the following process: S1. Pretreatment: taking fresh pig blood for pretreatment to obtain pig blood red blood cell powder; S2. Crude product preparation: using pig blood red blood cell powder as raw material, adopting optimized glacial acetic acid process to prepare pig blood hemin crude product; S3, preparation of refined product: washing the crude pig blood hemin obtained in S2 with a hydrochloric acid solution washing method to obtain a refined pig blood hemin; S4. Neutral washing: Use 0.9% saline to wash the refined pig blood hemin to neutrality: S5. Vacuum drying: under vacuum conditions at 50°C, the pig blood hemoglobin refined product after neutral washing is dried to constant weight to obtain the hemoglobin product.

2. The optimized process for refining pig blood hemin by glacial acetic acid method according to claim 1, characterized in that: In S1, the pretreatment method includes preparation of fresh anticoagulated pig blood and ultrasonic disruption treatment.

3. The optimized process for refining pig blood hemin using glacial acetic acid according to claim 2, characterized in that: The preparation process of fresh anticoagulated pig blood is as follows: take fresh pig blood, add 0.5% anticoagulant trisodium citrate solution into the fresh pig blood, stir evenly, and then obtain fresh anticoagulated pig blood.

4. The optimized process for refining pig blood hemin using glacial acetic acid according to claim 3 is characterized in that: Fresh anticoagulated pig blood was used as the raw material, and the red blood cell-rich precipitate was collected by centrifugation. The red blood cell precipitate was washed three times with 0.9% saline, and then ultrasonically crushed with deionized water. The precipitate was centrifuged again, and the precipitate was vacuum dried to obtain red blood cell powder.

5. The optimized process for refining pig blood hemin using glacial acetic acid according to any one of claims 1 to 4, characterized in that: In S3, the best conditions of the optimized glacial acetic acid process used in the preparation of the crude product are: glacial acetic acid concentration of 75%, reaction temperature of 105° C., reaction time of 1 hour, and sodium chloride addition amount of 1.5%.

6. The optimized process for refining porcine hemin using glacial acetic acid according to claim 5, characterized in that: The specific process of preparing crude porcine blood hemin by glacial acetic acid method is as follows: glacial acetic acid with a concentration of 75% is mixed with porcine blood red blood cell powder in a mass ratio of 4:1, and then sodium chloride with a mass percentage of 1.5% of porcine blood red blood cell powder is added, and the mixture is heated to 105° C. while being stirred evenly at a slow speed, and the reaction time is 1 hour to obtain a crude porcine blood hemin solution; After the crude pig blood hemin solution is cooled to room temperature, the precipitate is collected by centrifugation to obtain the crude pig blood hemin.

7. The optimized process for refining pig blood hemin using glacial acetic acid according to claim 5, characterized in that: In S4, the refining process of the crude pig blood hemin is as follows: the crude pig blood hemin is washed 3 times with a pure hydrochloric acid solution with a pH of 1-3, and the precipitate is collected by centrifugation to obtain a refined pig blood hemin.

8. The optimized process for refining pig blood hemin using glacial acetic acid according to claim 6, characterized in that: The supernatant of the crude pig blood hemin solution after centrifugation is recovered with glacial acetic acid.

9. The optimized process for refining pig blood hemin using glacial acetic acid according to claim 8, characterized in that: The glacial acetic acid recovery method comprises: adding saturated calcium chloride to the supernatant to be recovered.

10. The optimized process for refining pig blood hemin using glacial acetic acid according to claim 8, characterized in that: The glacial acetic acid recovery method comprises the following steps: adding the supernatant to be recovered into a distillation flask for distillation recovery treatment, and connecting a distillation column at the gas outlet of the distillation flask.

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