Water-soluble heme as well as preparation method and application thereof

Abstract

The invention belongs to the field of nano material preparation, and particularly relates to a preparation method of water-soluble heme, which adopts an anti-solvent precipitation method, accurately controls an anti-solvent precipitation process through an ultrasonic microreactor or a micromixer, and realizes continuous preparation of water-soluble heme with small size, high solid content and excellent stability. The particle size of the water-soluble heme obtained through the preparation method is 200 nm or below, the solid content in water can reach 200-500 mg / mL, and the water-soluble heme can be used as a health care product or a food additive.

Description

Technical Field

[0001] The invention belongs to the field of nano material preparation, and specifically relates to water-soluble hemoglobin and a preparation method and application thereof. Background Art

[0002] The lack of iron, an essential trace element for the human body, is a global problem. Iron in the diet is divided into heme and non-heme. Heme has a high iron absorption and bioavailability, and is easier to absorb than non-organic iron in plants or iron salts commonly used in food fortification. Traditional iron supplements such as ferrous sulfate, ferrous gluconate, and ferrous fumarate are all inorganic iron or plant iron. Although they have a good iron supplement effect, they have low utilization in the body (absorption rate is generally 5% to 8%), large side effects, and a special metallic rust taste, making them difficult to consume for a long time. Heme is a biological iron that is absorbed through heme transporters, which are mainly expressed in the brush boundary membranes of duodenal enterocytes and hepatocytes. Its iron absorption is more complete than that of non-heme iron, and its bioavailability is high (absorption rate reaches 15% to 25%).

[0003] The effective absorption rate of iron in heme is 25% on average, and can reach 40% when iron is deficient. In addition, the absorption of heme iron is also affected by food factors. Heme has long been widely used as an iron fortifier in countries such as Japan and the United States. The most common heme-fortified foods on the Japanese market are low-calorie candies and low-calorie biscuits. The heme fortifiers on the Chinese market are also mainly iron tablets, iron soft candies, and iron capsules. Heme iron tablets, biscuits, etc. are more convenient to carry, but the human body absorption rate of heme in a water-insoluble form is relatively low, while the absorption rate of heme iron in a dissolved state can be increased by 5 times. The smaller the particle size of heme, the higher the iron absorption rate. Since free heme is insoluble in water, most heme iron nutritional fortifiers on the market appear in solid form. Now researchers are exploring ways and methods to increase the iron content of plant foods from many aspects. Directly ingesting food in a solution state or ingesting solid food into the digestive tract and dissolving in the digestive juice has a significantly higher bioavailability than insoluble heme. At the same time, water-soluble hemoglobin can meet the addition needs of any kind of food.

[0004] The iron content of commercially available heme iron supplements ranges from 2.0 to 8.0%, and is often a complex of heme (also known as ferrous protoporphyrin) tightly bound to globin. During the preparation of this complex, peptides and proteins often directly flocculate and precipitate, and non-small molecule heme forms nanoparticles under the driving force of supersaturation. This makes it difficult for conventional preparation methods to control particle size and water solubility at high concentrations. The existing technology precipitates heme by repeatedly and finely controlling pH. The heme prepared by this method has a large particle size and is often a large aggregate, resulting in general stability. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing water-soluble hemoglobin, as well as the water-soluble hemoglobin obtained by the method and its application, so as to solve the problem that the existing preparation method is difficult to achieve precise control of small-particle hemoglobin, thereby affecting the quality and stability of water-soluble hemoglobin.

[0006] According to one aspect of the present invention, a method for preparing water-soluble hemoglobin is provided, comprising the following steps: (1) mixing hemoglobin with an alkaline aqueous solution to prepare an alkaline aqueous solution of hemoglobin with a concentration of 5 to 500 mg / mL; (2) mixing the alkaline aqueous solution of hemoglobin with the anti-solvent, and performing the mixing reaction in an ultrasonic microreactor or micromixer to form a suspension, and hemoglobin will precipitate after the reaction; (3) Solution replacement and impurity removal, pH adjustment: The suspension is dialyzed as a dialysis sample, the dialysis sample is placed in a dialysis bag, both ends are clamped with dialysis clamps, and an appropriate amount of dialysis fluid (pure water) is added to the dialysis device; the volume of the dialysis fluid should be 90-110 times the volume of the sample, and the dialysis fluid is replaced every 2-5 hours. The dialysis is continued for 1-2 days until the pH value is between 7.0-7.3 to ensure that excess impurities (such as inorganic salts, by-products, etc.) are removed; (4) After drying, water-soluble hemoglobin powder is obtained.

[0007] In some embodiments, when performing solution replacement in step (3), in addition to dialysis, tangential flow can also be used.

[0008] In some embodiments, the alkaline aqueous solution is an aqueous solution of amines (such as urea) or sodium hydroxide, and the concentration is 0.1-2.0 mol / L.

[0009] In some embodiments, the anti-solvent used to prepare water-soluble hemoglobin is an acid or an ester; specifically, the acid is at least one of hydrochloric acid, sulfuric acid, acetic acid, caprylic acid and capric acid, and the acid concentration is 0.1~1N; the ester is at least one of ethyl acetate, diisooctyl adipate, trioctyl acetyl citrate, caprylic and capric glyceride, caprylic acid ester, caprylic acid ester and caprylic acid ester.

[0010] In some embodiments, the conditions for the reaction of the antisolvent and the base to precipitate heme are: the flow ratio of the heme alkaline aqueous solution to the antisolvent is (1.0~10.0):(1.0~2.0), and the rapid mixing reaction is carried out at room temperature for 0.1~90 minutes. Specifically, when the antisolvent is an acid, the flow ratio of the heme alkaline aqueous solution to the antisolvent is 1:(1.0~2.0); when the antisolvent is an ester, the flow ratio of the heme aqueous solution to the antisolvent is (1.0~10.0):1.

[0011] In some embodiments, during drying, spray drying is specifically used to obtain water-soluble heme powder. During spray drying, ethyl acetate can also be removed.

[0012] In some embodiments, the reaction conditions in the ultrasonic microreactor are: ultrasonic power 10-1000 W, ultrasonic frequency 10-200 kHz, and residence time 1-300 s.

[0013] In some embodiments, the reaction temperature of step (2) is 25-90°C.

[0014] In some embodiments, the micro-mixing structure includes but is not limited to the structures disclosed in patents 202222930543.6, 202110770263.0, and 202121541511.6 applied for by the inventor team, as well as common micro-mixing structures such as T-type, Y-type, and spiral types.

[0015] In some embodiments, the desalination treatment method is any one of dialysis, centrifugation and tangential flow filtration.

[0016] The present invention provides a method for preparing water-soluble hemoglobin, which adopts an anti-solvent precipitation method, uses an ultrasonic microreactor or a micromixer, sets the flow rate ratio, the total flow rate and the ultrasonic power to adjust the mixing performance, so that the time required for sufficient mixing (mixing time) is less than the nucleation and precipitation time of hemoglobin, and accurately controls the anti-solvent precipitation process, thereby realizing the continuous preparation of water-soluble hemoglobin with small size, high solid content and excellent stability.

[0017] According to a second aspect of the present invention, there is provided water-soluble hemoglobin prepared by the above-mentioned method for preparing water-soluble hemoglobin, the particle size of which is below 200 nm and the suspended solid content in water can reach 200-500 mg / mL.

[0018] According to the third aspect of the present invention, there is provided the use of the water-soluble hemoglobin as a health product or a food additive. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is a schematic diagram of the preparation process of the water-soluble hemoglobin of the present invention. Figure 2 This is a comparison chart of the effects of different residence times on the particle size of water-soluble hemoglobin. Figure 3 This is a comparison chart of the effects of different ultrasonic powers on the particle size of water-soluble hemoglobin. Figure 4 This is a comparison chart of the effects of different heme solution concentrations on the particle size of water-soluble heme. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments. Unless otherwise specified, the following raw materials are commercially available. The purity of hemoglobin used in the following examples is 20%-25%.

[0021] Example 1 A method for preparing water-soluble hemoglobin, using an anti-solvent precipitation method, accurately controlling the anti-solvent precipitation process through an ultrasonic microreactor, comprising the following steps: S1: Weigh 3 g of hemoglobin, prepare a 30 mg / mL hemoglobin solution with 0.1 M NaOH solution, and let stand for later use; S2: Use a 50ml syringe to extract 50mL of heme solution, and use another syringe to extract 5mL of ethyl acetate (purity ≥ 99%); S3: Connect the syringe to the syringe pump, set the flow rate of the aqueous hemoglobin solution to 2.127 mL / min, the flow rate of the organic ethyl acetate to 0.213 mL / min, the corresponding residence time to 20 s, and the power of the ultrasonic microreactor to 40 W; S4: Turn on the syringe pump and ultrasonic power supply, wait for 5 minutes to start receiving samples; the reaction temperature is around 35°C; stop collecting after receiving 40-45mL of sample; S5: The suspension was placed in a 30000Da dialysis bag and dialyzed with pure water to remove inorganic salts. The pure water was replaced every 4 hours until the pH value of the dialyzed sample was 7-7.3 to ensure that the by-products were completely removed during the reaction; S6: Use a spray dryer to spray dry the dialyzed sample at a spray drying temperature of 120°C and a sample flow rate of 15 mL / min. After spray drying, water-soluble hemoglobin powder is obtained.

[0022] Example 2 A method for preparing water-soluble hemoglobin, using an antisolvent precipitation method, accurately controlling the antisolvent precipitation process through a micro mixer, comprising the following steps: S1: Weigh 3 g of hemoglobin, prepare a 30 mg / ml hemoglobin solution with 0.1 M NaOH solution, and let stand for later use; S2: Use a 50 ml syringe to draw 50 ml of heme solution, and use another syringe to draw 50 ml of 0.1 M anti-solvent HCl solution; S3: Connect the syringe to the syringe pump, set the flow rate of the heme NaOH solution to 1.17 ml / min, the flow rate of the HCl solution to 1.17 ml / min, and the residence time to 20 s; S4: Turn on the syringe pump and ultrasonic power supply, wait for 5 minutes to start receiving samples; stop collecting after receiving 40-45 ml of sample, and the heme solution and anti-solvent form a suspension; S5: The collected suspension was placed in a 10000Da dialysis bag and dialyzed with pure water to remove inorganic salts. The bag was changed every 30 minutes for 4 times, and then changed every 6 hours for 3 times. S6: Use a spray dryer to spray dry the dialyzed sample at a spray drying temperature of 120°C and a sample flow rate of 15 mL / min. After spray drying, water-soluble hemoglobin powder is obtained.

[0023] The heme powder prepared in Example 1-2 was measured for parameters such as particle size and PDI, and the measurement results are recorded in Table 1.

[0024] Specifically, the method for determining the particle size of water-soluble heme is as follows: ①Testing instrument: Nanoparticle size analyzer (Nanolink S901); ②Parameter setting: select 1.6 as the refractive index of the sample test; ③Sample preparation: Weigh 100 mg of water-soluble hemoglobin solid powder, prepare it into 20 mg / ml hemoglobin solution with pure water, take 50 ul, dilute it 200 times with pure water, take 1.5 ml of the diluted sample and inject it into a quartz cuvette, put the cuvette into the sample slot, close the slot door and start measuring.

[0025] Table 1 Parameters of heme powder prepared in Example 1-2 Experiment 1: Investigating the effect of residence time on the particle size of water-soluble hemoglobin Based on Example 1, only the residence time was changed. When the residence time was 10s, 20s, and 30s, water-soluble hemoglobin powders with different residence times were prepared. The particle sizes were measured. The results are as follows: Figure 2 As shown, the particle sizes of hemoglobin powders obtained within the residence time range of 10 to 40 s are all below 115 nm. When the residence time is 40 s, the particle size of water-soluble hemoglobin is the smallest, indicating that its particle size gradually decreases with the extension of the residence time.

[0026] Experiment 2: Investigating the effect of ultrasonic power on the particle size of water-soluble hemoglobin Based on Example 1, the residence time was set to 40 s, and the remaining parameters were changed only by the ultrasonic power. When the ultrasonic power was 20 W, 30 W, 40 W, and 50 W, water-soluble hemoglobin powders under different ultrasonic powers were prepared, and their particle sizes were measured. The results are as follows: Figure 3 As shown in the figure, the particle size of hemoglobin powder obtained when the ultrasonic power is in the range of 20~50W is all below 140nm, and the particle size of water-soluble hemoglobin is the smallest when the ultrasonic power is 50W, indicating that with the increase of ultrasonic power, its particle size gradually decreases.

[0027] Experiment 3: Investigating the effect of heme solution concentration on the particle size of water-soluble heme On the basis of Example 1, the residence time was set to 40s and the ultrasonic power was set to 50W. The remaining parameters were changed only in the heme solution (i.e., the heme alkaline aqueous solution). When the heme solution concentrations were 10mg / mL, 20mg / mL, 30mg / mL, 40mg / mL, and 50mg / mL, water-soluble heme powders were prepared under different ultrasonic powers, and their particle sizes were measured. The results are as follows: Figure 4 As shown, it shows that the particle size of hemoglobin powder obtained when the ultrasonic power is in the range of 20~50W is all below 105nm, and with the increase of the concentration of hemoglobin solution, its particle size gradually decreases.

[0028] Through the above experiments, the residence time, ultrasonic power and hemoglobin concentration were controlled separately to explore the influence of different factors on the particle size. It was found that when the residence time was 40s, the ultrasonic power was 50W, and the hemoglobin concentration was 50mg / ml, the hemoglobin particle size was the smallest, which was 79.9nm.

[0029] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing water-soluble hemoglobin, characterized in that: The steps include: (1) mixing hemoglobin with an alkaline aqueous solution to prepare an alkaline aqueous solution of hemoglobin with a concentration of 5 to 500 mg / mL; (2) mixing the alkaline aqueous solution of heme and the anti-solvent in an ultrasonic microreactor or a micromixer to form a suspension; the anti-solvent is an acid or an ester, and the flow ratio of the alkaline aqueous solution of heme to the anti-solvent is (1.0-10.0):(1.0-2.0); (3) using the suspension as a sample for solution replacement until the pH value of the suspension is between 7.0 and 7.3; (4) Drying the sample after the solution replacement to obtain water-soluble hemoglobin powder.

2. The method for preparing water-soluble hemoglobin according to claim 1, characterized in that: The alkaline aqueous solution in step (1) is an aqueous solution of amines or sodium hydroxide, and the concentration is 0.1-2.0 mol / L.

3. The method for preparing water-soluble hemoglobin according to claim 2, characterized in that: The acid in step (2) is at least one of hydrochloric acid, sulfuric acid, acetic acid, caprylic acid and capric acid, and the acid concentration is 0.1~1N.

4. The method for preparing water-soluble hemoglobin according to claim 2, characterized in that: The ester in step (2) is at least one of ethyl acetate, diisooctyl adipate, trioctyl acetylcitrate, caprylic acid glyceryl ester, caprylic acid neoester, octyl palmitate and isooctyl laurate.

5. The method for preparing water-soluble hemoglobin according to any one of claims 1 to 4, characterized in that: The reaction temperature of step (2) is carried out at 25-90°C.

6. The method for preparing water-soluble hemoglobin according to claim 5, characterized in that: When mixing in an ultrasonic microreactor or an ultrasonic micromixer, the ultrasonic power is 10-1000 W, the ultrasonic frequency is 10-200 kHz, and the residence time is 1-300 s.

7. The method for preparing water-soluble hemoglobin according to claim 5, characterized in that: When the solution replacement in step (3) is performed by dialysis, the specific operation is as follows: the suspension is placed in a 8000-50000Da dialysis bag and dialyzed with pure water until the pH value is 7-7.

3.

8. Water-soluble hemoglobin obtained by the method for preparing water-soluble hemoglobin according to any one of claims 1 to 7.

9. The water-soluble hemoglobin according to claim 8, characterized in that Its particle size is below 200nm, and the solid content in water can reach 200-500mg / mL.

10. Use of the water-soluble hemoglobin according to claim 8 or 9 as a health product or food additive.

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