Method for improving ferrous chelating amount of ovotransferrin by using low-temperature plasma

Abstract

The invention relates to a method for increasing the ferrous chelating amount of ovotransferrin by using low-temperature plasma, which comprises the following steps of: preparing ovotransferrin by using fresh egg white as a raw material and adopting cation exchange chromatography; preparing a protein solution with a certain concentration, sequentially adding NaHCO3 and FeSO4 solutions, and adjusting the pH value of the mixed solution; under a certain temperature condition, low-temperature plasma with certain power is adopted for treatment, and the ovotransferrin ferrous chelate with the high chelation rate is prepared within a certain reaction time range. The low-temperature plasma treatment improves the ferrous chelating amount of ovotransferrin, not only conforms to the development trend of modern nutritional supplements, but also provides a new technical path for solving the global health problem of iron-deficiency anemia.

Description

Technical Field

[0001] The present invention relates to a method for increasing the ferrous chelation amount of ovotransferrin by low-temperature plasma. Background Art

[0002] According to the statistical data of the World Health Organization (WHO), about 24.8% of the global population is affected by anemia, and iron deficiency anemia (IDA) accounts for up to 50%. Nutritional iron deficiency anemia has become a focus issue in the field of international public health. At present, oral iron supplements are still the core strategy for preventing and treating iron deficiency anemia, but how to improve the bioavailability of iron and reduce side effects remains the research focus. Inorganic iron, organic acid iron, heme iron, protein chelated iron, etc. are common iron supplements on the market. Inorganic iron such as ferrous sulfate and ferrous fumarate is relatively common, with low price, but low absorption rate by the body and large gastrointestinal irritation; organic acid iron includes ferrous gluconate, ferrous succinate, and ferric citrate. Compared with inorganic iron, such products are relatively mild, but may still irritate the intestine.

[0003] As a new generation of highly efficient iron supplement, protein chelated iron has become the key research direction of current nutritional iron supplements due to its significant advantages such as high bioavailability, low gastrointestinal irritation, multi-functional nutritional synergistic effect, and excellent food compatibility. Such iron supplements are not only suitable for long-term and safe supplementation, but also can effectively overcome many defects of traditional iron agents, showing broad application prospects.

[0004] Among many protein carriers, ovotransferrin has attracted much attention due to its unique biological characteristics. As an important member of the transferrin family, ovotransferrin is derived from egg white, not only has natural nutritional value, but also shows excellent iron binding and release ability. These characteristics make it an ideal protein source for preparing high-quality protein chelated ferrous, and has important application value in the fields of food processing and nutritional fortification.

[0005] It is worth noting that the protein iron chelation amount is a key index for evaluating the quality of such iron supplements. A high chelation amount can not only ensure the stable binding and efficient absorption of iron elements, but also maximize the functional characteristics of proteins. Therefore, developing methods to increase the chelation amount of ovotransferrin ferrous chelate not only conforms to the development trend of modern nutritional supplements, but also provides a new technical path for solving the global health problem of iron deficiency anemia.

[0006] Low-temperature plasma is an emerging non-thermal processing technology that generates plasma rich in active particles by ionizing gas, and has functions such as efficient sterilization, enzyme inactivation, and degradation of pesticide residues, while maintaining food quality. Low-temperature plasma shows broad prospects in food processing, and its environmental protection and high-efficiency characteristics meet the requirements of the sustainable development of the future food industry, and is expected to become an important supplement or alternative technology to traditional thermal processing. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for improving the ferrous chelation amount of ovotransferrin by low-temperature plasma, and the prepared ferrous chelate of ovotransferrin has a relatively high chelation amount.

[0008] The technical solution adopted by the present invention is as follows:

[0009] A method for improving the ferrous chelation amount of ovotransferrin by low-temperature plasma, the method comprising the following steps:

[0010] (1) Pretreat fresh egg white to prepare a protein solution; purify and prepare a pure ovotransferrin solution by cation exchange chromatography; desalt the protein by dialysis and freeze-dry to obtain a protein freeze-dried powder;

[0011] (2) Prepare a protein solution with a certain concentration, and optimize single-factor conditions such as anion type, Fe 2+ concentration, protein concentration, chelation temperature, etc.;

[0012] (3) Based on the optimal single-factor conditions obtained in the above step (2), optimize single-factor conditions of response surface methods such as pH value, low-temperature plasma power, low-temperature plasma treatment time, etc.;

[0013] (4) Based on the optimization scheme in step (3), select three factors of pH, low-temperature plasma power, and treatment time for response surface optimization;

[0014] Further, the protein freeze-dried powder in step (1) is an ovotransferrin freeze-dried powder.

[0015] Further, the cation exchange column used for the cation exchange chromatography purification in step (1) is

[0016] CM-Sepharose Fast Flow.

[0017] Further, the buffer solution for slow flushing and elution used in the cation exchange chromatography in step (1) is HAc-NaAc buffer solution, and the flow rate is 1-3 mL / min.

[0018] Further, the detection method for chelation in step (2) is atomic absorption spectrometry.

[0019] Further, the anion types in step (2) are sodium salts such as NaHCO3 and NaCl.

[0020] Further, the Fe 2+ concentration in step (2) is 6.00 - 30.00 mM FeSO4.

[0021] Furthermore, the protein concentration in step (2) is 6.00 - 30.00 mg / mL.

[0022] Furthermore, the temperature of the chelation reaction in step (2) is 4°C - 45°C.

[0023] Furthermore, the optimized pH value in step (3) is pH 6.0 - 9.0.

[0024] Furthermore, the power of the low-temperature plasma in step (3) is 385 W - 550 W.

[0025] Furthermore, the treatment time of the low-temperature plasma in step (3) is 10 s - 40 s.

[0026] Furthermore, the response surface experimental design in step (4) is as follows:

[0027] Table 1 Response surface analysis and levels of the ferrous chelating ability of ovotransferrin

[0028]

[0029] Compared with the prior art, some inventions have the following beneficial effects:

[0030] This invention mainly uses low-temperature plasma treatment, conducts a chelation reaction between ovotransferrin and ferrous sulfate, and then optimizes the chelation process through single-factor and response surface methods to increase the ferrous chelation amount of the ovotransferrin-ferrous chelate, ensure the stable binding and efficient absorption of iron elements, and maximize the functional properties of proteins, providing a certain scientific basis for expanding the application of ovotransferrin in foods. Description of the Drawings

[0031] Figure 1 SDS-PAGE gel results of purified ovotransferrin, M: marker; 1: prepared ovotransferrin; 2: ovotransferrin standard

[0032] Figure 2 Effect of anion type on the ovotransferrin-ferrous chelation reaction

[0033] Figure 3 Effect of ferrous ion concentration on the ovotransferrin-ferrous chelation reaction

[0034] Figure 4 Effect of temperature on the ovotransferrin-ferrous chelation reaction

[0035] Figure 5 Effect of ovotransferrin concentration on the ovotransferrin-ferrous chelation reaction

[0036] Figure 6Effect of pH Value on the Chelation Reaction of Ovalbumin Transferrin with Ferrous Iron

[0037] Figure 7 Effect of Low Temperature Plasma Power on the Chelation Reaction of Ovalbumin Transferrin with Ferrous Iron

[0038] Figure 8 Effect of Low Temperature Plasma Treatment Time on the Chelation Reaction of Ovalbumin Transferrin with Ferrous Iron

[0039] Figure 9 Analysis of the Results of Orthogonal Experiments Specific Embodiments

[0040] The present invention will be described in detail below with reference to specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but not to limit the present invention.

[0041] Example 1 Preparation of Ovalbumin Transferrin

[0042] Take 80 mL of fresh egg white, add 160 mL of distilled water under the condition of 4 °C, and stir slowly for 40 min. Adjust the pH value to 6.0 with sodium acetate, continue to stir for 4 h under the condition of 4 °C, and centrifuge at 8000 rpm / min for 15 min to take the supernatant. Use a CM-Sepharose Fast Flow cation exchange column to separate and purify ovalbumin transferrin with a HAc-NaAc buffer solution of pH 6.0 and 50 mM. Place the eluate in a dialysis bag of 8000 - 14000 Da, dialyze to remove salts at 4 °C for 48 h, and change distilled water 6 times. After freeze-drying the protein solution in a freeze dryer for 48 h, a white powder, which is the ovalbumin transferrin sample, is obtained and stored at -20 °C for standby. As shown in the appendix Figure 1 The purity of the prepared ovalbumin transferrin is higher than 80%.

[0043] Example 2 Optimization of Single-Factor Conditions

[0044] (1) Optimization of anion type: Sequentially add 1 mL of ovalbumin transferrin solution with a concentration of 15 mg / mL, 0.5 mL of NaHCO3 (or NaCl) solution with a concentration of 150 mM, 1 mL of 0.01 M citric acid-sodium citrate buffer solution with pH 7.0, and finally 0.5 mL of FeSO4 solution with a concentration of 30 mM into a centrifuge tube, mix evenly, let stand for 30 min, and after the reaction is complete, perform dialysis. After dialysis, volume-fix the target solution to 5 mL, and use flame atomic absorption spectrophotometry to measure the iron content in the sample. The results are as shown in the appendix Figure 2 As shown, when the anion is selected as HCO3 - There is a relatively high chelation amount between ovalbumin transferrin and ferrous iron.

[0045] (2) Fe2+ Concentration optimization: Sequentially add 1 mL of ovotransferrin solution with a concentration of 15 mg / mL, 0.5 mL of NaHCO3 with a concentration of 150 mM, 1 mL of 0.01 M citric acid-sodium citrate buffer with pH 7.0 into a centrifuge tube. Finally, add 0.5 mL of FeSO4 solutions with concentrations of 6.00, 12.00, 18.00, 24.00, 30 mM respectively, so that the final concentration in the solution reaches 0.10×10 -3 ,0.20×10 -3 ,0.30×10 -3 ,0.40×10 -3 ,0.50×10 -3 mol / L. After mixing the solution evenly, let it stand for 30 min. After dialysis, volume-fix the target solution to 5 mL, and use atomic absorption spectrophotometry to determine the iron content in the sample. The results are as attached Figure 3 shown. When the chelation amount of ferrous ions increases with the increase of ferrous ion concentration, when the concentration reaches 0.5×10 -3 mol / L, the chelation amount is the highest.

[0046] (3) Optimization of protein treatment temperature: Add 1 mL of ovotransferrin solution with a concentration of 6.00 mg / mL, 0.5 mL of NaHCO3 with a concentration of 150 mM, 1 mL of 0.01 M citric acid-sodium citrate buffer with pH 7.0 into a centrifuge tube in sequence. Then add 0.5 mL of FeSO4 solution with a concentration of 30 mM, and treat it at 4℃, 25℃, 37℃, 45℃ for 30 min respectively. Mix evenly, and after dialysis, volume-fix it to 5 mL. The results are as attached Figure 4 shown. When the reaction temperature is 25℃, the chelation effect is the best.

[0047] (4) Optimization of ovotransferrin concentration: Add 1 mL of OVT with concentrations of 6.00 mg / mL, 9.00 mg / mL, 15.00 mg / mL, 30.00 mg / mL into centrifuge tubes respectively, so that the final concentration in the solution reaches 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL. Sequentially add 0.5 mL of NaHCO3 with a concentration of 150 mM, 1 mL of 0.01 M citric acid-sodium citrate buffer with pH 7.0, and finally add 0.5 mL of FeSO4 solution with a concentration of 30 mM. Mix evenly, let it stand for 30 min, and after dialysis, volume-fix the target solution to 5 mL, and use atomic absorption spectrophotometry to determine. The results are as attached Figure 5 shown. Ovotransferrin with a concentration of 6 mg / mL has the best chelation effect.

[0048] Example 3 Optimization of Chelation Process by Response Surface

[0049] (1) Chelation PH optimization: Based on the optimized single-factor results obtained above, that is, ovotransferrin with a volume of 1 mL and a concentration of 6 mg / mL, NaHCO3 with a volume of 0.5 mL and a concentration of 150 mM, and FeSO4 solution with a volume of 0.5 mL and a concentration of 30 mM, the effects of citric acid-sodium citrate buffers with different PH values of 6.0, 7.0, 8.0, and 9.0 on the chelation reaction were investigated respectively. After mixing evenly, it was transferred to a reaction dish. The low-temperature plasma power was set at 470 W, the treatment reaction time was 20 s, and after dialysis, it was made up to 8 mL. It was measured by atomic absorption spectrophotometry. The results are as follows Figure 6 As shown, when the pH value is 7, the chelation effect is the best.

[0050] (2) Optimization of low-temperature plasma treatment power: Ovotransferrin with a volume of 1 mL and a concentration of 6 mg / mL, NaHCO3 with a volume of 0.5 mL and a concentration of 150 mM, FeSO4 solution with a volume of 0.5 mL and a concentration of 30 mM, and the effect of citric acid-sodium citrate buffer with a pH value of 7.0 on the chelation reaction. After mixing evenly, it was transferred to a reaction dish. The effects of low-temperature plasma powers of 0 W, 385 W, 440 W, 495 W, and 550 W on the chelation amount were investigated respectively. The reaction time was 20 s, and after dialysis, it was made up to 8 mL. It was measured by atomic absorption spectrophotometry. The results are as follows Figure 7 As shown, when the power is 440, the effect is the best.

[0051] (3) Optimization of low-temperature plasma treatment time: Ovotransferrin with a volume of 1 mL and a concentration of 6 mg / mL, NaHCO3 with a volume of 0.5 mL and a concentration of 150 mM, FeSO4 solution with a volume of 0.5 mL and a concentration of 30 mM, and the effect of citric acid-sodium citrate buffer with a PH value of 7.0 on the chelation reaction. After mixing evenly, it was transferred to a reaction dish. The low-temperature plasma power was selected as 495 W. The effects of different low-temperature plasma treatment times of 10 s, 20 s, 30 s, and 40 s on the iron chelation amount were investigated respectively. After dialysis, it was made up to 8 mL. It was measured by atomic absorption spectrophotometry. The results are as follows Figure 8 As shown, the treatment time of 20 s is the best.

[0052] (4) Response surface optimization: Based on the quadratic polynomial regression analysis, the prediction model of the iron chelation amount (Y) of ovotransferrin Fe 2+ is as follows (the results are shown in Table 2):

[0053] Y = 9.09 - 0.175A - 0.748B - 0.2435C + 0.1651AB + 0.0031BC - 0.5203A 2 - 0.2511B 2 - 0.

[0054] 5889C 2

[0055] The P-value of the model is less than 0.01, and the lack-of-fit term P > 0.05, indicating that the equation has a good fitting degree. There is a high correlation (R 2 = 0.9690) between the actual value and the predicted value, indicating that the model can better describe the relationship between the Fe chelation amount of ovotransferrin and the pH value, low-temperature plasma power, and treatment time, and can be used to predict the optimal process conditions. From the P-values, the power (A), pH (C), interaction term (AB), and all quadratic terms (A 2+ 、B 2 、B 2 、C 2 ) all significantly affect the Fe chelation amount of ovotransferrin (P < 0.05). The F-value analysis results show that the order of the influence degree on the Fe chelation amount of ovotransferrin is: pH (C) > power (A) > time (B). 2+ As shown in the attachment, there is a highly significant interaction between the power (A) and the treatment time (B) (p < 0.01), while the interaction of other factors is not significant. From the above results, the optimal conditions of the response surface are: the optimal low-temperature plasma treatment power is 449.644 W, the optimal treatment time is 25.392 s, the optimal pH is 7.145, and the predicted Fe chelation amount is Fe 2+ / OVT = 9.138 (mg / g). Due to equipment limitations, the selected optimal conditions are: the low-temperature plasma treatment power is 460 W, the optimal treatment time is 25 s, the optimal pH is 7.0, and the Fe chelation amount is 9.082 mg / g.

[0056] As attached Figure 9 shown, there is a highly significant interaction between the power (A) and the treatment time (B) (p < 0.01), while the interaction of other factors is not significant. From the above results, the optimal conditions of the response surface are: the optimal low-temperature plasma treatment power is 449.644 W, the optimal treatment time is 25.392 s, the optimal pH is 7.145, and the predicted Fe chelation amount is Fe 2+ / OVT = 9.138 (mg / g). Due to equipment limitations, the selected optimal conditions are: the low-temperature plasma treatment power is 460 W, the optimal treatment time is 25 s, the optimal pH is 7.0, and the Fe chelation amount is 9.082 mg / g.

[0057] Table 2 Analysis of variance of the model regression equation

[0058]

[0059] The present invention has been described above by way of examples, but the present invention is not limited to the above specific examples. Any modification or variation based on the present invention falls within the scope of protection required by the present invention.

Claims

1. A method for increasing the ferrous chelation capacity of ovotransferrin by low-temperature plasma, characterized in that: The method comprises the following steps: S1: pre-treating fresh egg white to prepare a protein solution; purifying by cation exchange chromatography to prepare a pure solution of ovotransferrin; desalting the protein by dialysis, and freeze-drying to obtain a freeze-dried protein powder; S2: Prepare a protein solution of a certain concentration, add NaHCO3 and a certain concentration of FeSO4 solution to the solution in sequence, adjust the pH value of the mixed solution, and maintain a certain temperature condition; S3: transferring the above mixed solution to a reaction vessel, treating it with a low-temperature plasma of a certain power, and preparing a high-chelating amount of ovotransferrin ferrous chelate within a certain reaction time range; S4: dialyzing the chelate to remove unreacted small molecules, and determining the iron content of the ovotransferrin ferrous chelate by flame atomic absorption spectrometry.

2. The method for increasing the ferrous iron chelation capacity of ovotransferrin by low-temperature plasma according to claim 1, characterized in that: In step S2, the concentration of ovotransferrin is 6.00-30.00 mg / mL, the concentration of FeSO4 solution is 6.00-30.00 mM, and the pH value of the mixed solution is 6.0-9.

0.

3. The method for increasing the ferrous iron chelation capacity of ovotransferrin by low-temperature plasma according to claim 1, characterized in that: The power of the low-temperature plasma in step S3 is 385W-550W, and the reaction time is 10s-40s.

4. The high-chelation ovotransferrin ferrous chelate prepared by the method for increasing the ferrous chelation amount of ovotransferrin by low-temperature plasma according to any one of claims 1 to 3, characterized in that: Application in the preparation of iron supplement products.

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