A method for improving the digestibility of alpha-lactalbumin

Abstract

The invention discloses a method for improving the digestibility of alpha-lactalbumin, and belongs to the technical field of dairy product processing. The method for complete decalcification of alpha-lactalbumin of the present invention comprises the following steps: (1) the pH of alpha-lactalbumin powder solution is adjusted to 2.0-3.0, balanced at 20-30 DEG C for 1h and above, and a mixed solution is obtained; or, the pH of alpha-lactalbumin powder solution is adjusted to 7.5-9.0 to obtain alpha-lactalbumin solution; the pH of EDTA solution is adjusted to 7.5-9.0 to obtain EDTA solution; the alpha-lactalbumin solution and EDTA solution of the same pH are mixed according to a volume ratio of 1:1, the pH is adjusted to the same as before mixing, balanced at 20-30 DEG C for 1h and above, and a mixed solution is obtained; (2) the mixed solution is subjected to ultrafiltration and concentration, acid precipitation, and completely decalcified alpha-lactalbumin is obtained. The fully decalcified α-lactalbumin prepared by the present invention significantly improves digestibility in infants and young children. The fully decalcified α-lactalbumin contains no residual EDTA, making it suitable for use in infant foods. Furthermore, the present invention combines fully decalcified α-lactalbumin with moderate heat treatment to achieve a synergistically improved digestibility.

Description

Technical Field

[0001] The invention relates to a method for improving the digestibility of alpha-lactalbumin, and belongs to the technical field of dairy product processing. Background Art

[0002] Breast milk is the ideal food for infants and young children, providing them with essential energy and nutrients. Alpha-lactalbumin is the primary protein in breast milk, accounting for approximately 50% of the total protein content. Alpha-lactalbumin is rich in essential amino acids such as tryptophan, lysine, and phenylalanine. It has been shown to regulate sleep rhythms, alleviate depression, and induce tumor cell apoptosis, making it crucial for infant growth and development. Insufficient breastfeeding is common, and infant formula is often used as a breast milk substitute. The development of infant formula uses breast milk as the gold standard, aiming to fully mimic its composition. The protein in infant formula primarily comes from bovine and goat milk, which differ significantly from breast milk in protein composition, with a lower proportion of alpha-lactalbumin in bovine and goat milk. Therefore, to make infant formula more similar in protein composition to breast milk, it is necessary to supplement with alpha-lactalbumin.

[0003] Compared to α-lactalbumin in breast milk, α-lactalbumin from bovine and goat milk is difficult to digest, and infants and young children who consume bovine and goat milk-based infant formula are prone to gastrointestinal health issues such as bloating and decreased appetite. α-lactalbumin from breast milk and bovine and goat milk differ in amino acid sequence, leading to differences in molecular structure. α-lactalbumin is a calcium-binding protein that exists in a calcium-bound state in natural milk. Its compact globular molecular structure hinders the contact of digestive enzymes with hydrolysis sites, resulting in poor digestibility. Therefore, α-lactalbumin from bovine and goat milk is not suitable for direct use in foods for infants and young children, who may have limited gastrointestinal function.

[0004] Conventional methods for improving the digestibility of α-lactalbumin from bovine and goat milk include high-pressure treatment, heat treatment, and enzymatic hydrolysis. For example, patent CN115594748A discloses that high-pressure treatment can alter the molecular structure of α-lactalbumin, thereby improving its digestibility. However, high-pressure treatment is costly and energy-intensive, and its discontinuous nature makes it unsuitable for industrial application. These methods also present challenges such as high cost and energy consumption, nutritional losses, and limited improvement.

[0005] Removing calcium ions from α-lactalbumin can alter its molecular structure, potentially improving its digestibility. α-lactalbumin has a strong calcium ion binding capacity, and its structure is sensitive to changes in environmental conditions. Current decalcification methods, which are suitable for other proteins like casein, are not suitable for α-lactalbumin decalcification. These methods can lead to irreversible changes in amino acid residues or structure, insufficient decalcification, and significant amounts of residual decalcification reagents. Each α-lactalbumin molecule has a strong calcium ion binding site that can bind one calcium ion. This residual calcium means that some α-lactalbumin remains bound to calcium ions, making it difficult to digest and absorb. Furthermore, residual decalcification reagents can pose problems such as food safety and food sensory issues. These issues limit the application of the resulting α-lactalbumin in foods with high raw material requirements, such as infant formula. Summary of the Invention

[0006] [Technical Issues]

[0007] The α-lactalbumin in cow and sheep milk-based infant formula is a calcium ion binding protein with a tight globular molecular structure. It is difficult for infants and young children to digest and absorb, and can easily cause gastrointestinal health problems.

[0008] Currently, there are no methods for preparing completely decalcified α-lactalbumin, and research on improving α-lactalbumin digestibility is limited. Infants and young children with gastrointestinal impairment have difficulty digesting calcium-bound α-lactalbumin. Residual calcium means some α-lactalbumin is bound to calcium ions, making it difficult to digest and absorb. Therefore, completely decalcifying α-lactalbumin can maximize its digestibility in the infant gastrointestinal tract and better promote its growth and development.

[0009] [Technical solution]

[0010] In order to solve the above problems, the present invention decalcifies α-lactalbumin by ultrafiltration concentration and acid precipitation under specific conditions at low pH alone or after adding disodium ethylenediaminetetraacetic acid (EDTA) to obtain completely decalcified α-lactalbumin. The completely decalcified α-lactalbumin is then subjected to a moderate heat treatment to prepare α-lactalbumin with high digestibility, which can be used in food for people with insufficient gastrointestinal function, such as infants and young children.

[0011] The first object of the present invention is to provide a method for completely decalcifying α-lactalbumin, comprising the following steps:

[0012] (1) adjusting the pH of the α-lactalbumin powder solution to 2.0-3.0, and equilibrating at 20-30° C. for 1 hour or more to obtain a mixed solution;

[0013] or

[0014] The pH of the α-lactalbumin powder solution is adjusted to 7.5-9.0 to obtain an α-lactalbumin solution; the pH of the EDTA solution is adjusted to 7.5-9.0 to obtain an EDTA solution; the α-lactalbumin solution and the EDTA solution of the same pH are mixed in a volume ratio of 1:1, the pH is adjusted to the same as before mixing, and the mixture is equilibrated at 20-30° C. for 1 hour or more to obtain a mixed solution;

[0015] (2) The mixed solution is ultrafiltered and concentrated, and acid precipitated to obtain completely decalcified α-lactalbumin.

[0016] In one embodiment of the present invention, the α-lactalbumin powder solution in step (1) is an α-lactalbumin powder aqueous solution with a concentration of 0.7-2 mM.

[0017] In one embodiment of the present invention, in step (1), the pH is adjusted to 2.0-3.0 using aqueous hydrochloric acid solution with a concentration of 0.5-2.5M.

[0018] In one embodiment of the present invention, in step (1), the pH is adjusted to 7.5-9.0 using a sodium hydroxide aqueous solution with a concentration of 0.5-2.5M.

[0019] In one embodiment of the present invention, the EDTA solution in step (1) is an EDTA aqueous solution with a concentration of 1.4-22.4 mM.

[0020] In one embodiment of the present invention, the molar ratio of α-lactalbumin to EDTA in step (1) is 1:1-16.

[0021] In one embodiment of the present invention, the ultrafiltration concentration in step (2) is performed by cyclic ultrafiltration concentration at 20-30° C. using an ultrafiltration membrane with a molecular weight cutoff of 8-10 kDa, the ultrafiltration times are 6-8 times, and the volume concentration multiple is 3-5 times.

[0022] In one embodiment of the present invention, the acid precipitation in step (2) is performed by adjusting the pH of the retentate after ultrafiltration and concentration to 4.7-5.1, equilibrating at 20-30° C. for 1 hour or more, and performing acid precipitation; after acid precipitation, centrifugation is performed and the precipitate is collected to obtain the completely decalcified α-lactalbumin.

[0023] The second object of the present invention is the completely decalcified α-lactalbumin prepared by the method of the present invention.

[0024] A third object of the present invention is to provide a method for improving the digestibility of α-lactalbumin, comprising the following steps:

[0025] The completely decalcified α-lactalbumin is redissolved, the pH is adjusted to 7.0, and the solution is equilibrated at 20-30° C. for 1 hour or longer. The solution is then heat treated at 60-80° C. for 5-20 minutes and rapidly cooled to room temperature to obtain α-lactalbumin with high digestibility.

[0026] The fourth object of the present invention is to provide α-lactalbumin with high digestibility prepared by the method of the present invention.

[0027] A fifth object of the present invention is to provide an infant formula milk powder, which uses the α-lactalbumin with high digestibility described in the present invention.

[0028] The sixth object of the present invention is the use of the completely decalcified α-lactalbumin and the α-lactalbumin with high digestibility in the food field.

[0029] [Beneficial Effects]

[0030] (1) The method for completely decalcifying α-lactalbumin described in the present invention is to remove more than 92% of the bound calcium in α-lactalbumin by EDTA, then remove the free calcium and EDTA in the system by ultrafiltration, and finally purify it by acid precipitation to obtain 100% decalcified α-lactalbumin; the subsequent use in infant food can maximize its digestibility in the gastrointestinal tract of infants and better promote the growth and development of infants.

[0031] (2) The completely decalcified α-lactalbumin prepared by the present invention has a significant improvement effect on infant gastric digestion, and there is no residual EDTA in the completely decalcified α-lactalbumin, which is suitable for use in infant food.

[0032] (3) The present invention combines completely decalcified α-lactalbumin with moderate heat treatment to achieve a synergistic effect of improving digestibility. DETAILED DESCRIPTION

[0033] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0034] Test method:

[0035] 1. Determination of α-lactalbumin content

[0036] The α-lactalbumin solution was analyzed by Waters e2695 high performance liquid chromatography using an XBridgeBEH C8 (250 mm×4.6 mm) column and a detection wavelength of 220 nm.

[0037] 2. Separation of α-lactalbumin-bound calcium and free calcium

[0038] Take the α-lactalbumin solution, place it in an ultrafiltration centrifuge tube with a molecular weight cutoff of 10kDa, centrifuge it at 3000g for 40 minutes, and collect the permeate. The calcium in the α-lactalbumin solution is the total calcium, the calcium in the permeate is the free calcium, and the bound calcium is calculated by subtracting the free calcium from the total calcium.

[0039] 3. Determination of calcium content

[0040] The determination of calcium content refers to the first method of the national standard 5009.268-2016 "National Food Safety Standard Determination of Multiple Elements in Food" and is determined by inductively coupled plasma mass spectrometry.

[0041] 4. Determination of denaturation temperature of α-lactalbumin

[0042] The denaturation temperature of α-lactalbumin was determined using a differential scanning microcalorimeter with a heating rate of 1°C / min and a heating range of 5-95°C.

[0043] 5. Determination of EDTA content

[0044] The determination of EDTA content refers to the national standard 5009.278-2016 "National Food Safety Standard Determination of Ethylenediaminetetraacetate in Food", the chromatographic column used is Plus-C18 (250mm×4.6mm), and the detection wavelength is 254nm.

[0045] 6. Simulate infant gastric digestion

[0046] An α-lactalbumin solution was mixed with simulated gastric digestive fluid (94 mM sodium chloride, 13 mM potassium chloride, pH 5.3) at a volume ratio of 63:37. The pH was then adjusted to 5.3 with a hydrochloric acid solution (1 M). Pepsin was added with stirring to a concentration of 268 U / mL. The digestion temperature was 37°C for 60 min, and the reaction was terminated with the inhibitor Pepstatin A to obtain a gastric digestate.

[0047] 7. Determination of free amino content

[0048] Weigh 3.810 g of sodium tetraborate, 0.088 g of dithiothreitol, 0.100 g of SDS, and 0.080 g of OPA, and dissolve them to a volume of 100 mL to obtain OPA reagent;

[0049] Mix 100 μL of gastric digest with 2 mL of OPA reagent. Incubate in the dark for 2 minutes and measure absorbance at 340 nm. Draw a standard curve using L-leucine as the standard. Express the results in μmol / mg protein.

[0050] The raw materials used in the embodiments and comparative examples are:

[0051] Cow milk α-lactalbumin powder: α-lactalbumin content is 95.3% (w / w), calcium content is 1 (M / M protein);

[0052] Water: deionized water;

[0053] M / M: moles of calcium per mole of protein;

[0054] The solutions in the Examples and Comparative Examples, where no solvent is specified, all use water as the solvent.

[0055] Example 1 Removal of α-lactalbumin-bound calcium: Effect of different pH values ​​at a 1:1 molar ratio of EDTA to α-lactalbumin

[0056] Dissolve bovine milk α-lactalbumin powder in deionized water to a concentration of 1.4 mM, and adjust the pH to 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 using a 2M HCl solution or a 2M NaOH solution to obtain an α-lactalbumin solution;

[0057] Dissolve EDTA (disodium ethylenediaminetetraacetate) powder in deionized water to a concentration of 1.4 mM, and adjust the pH to 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 using 2 M HCl solution or 2 M NaOH solution to obtain an EDTA solution.

[0058] The α-lactalbumin solution and EDTA solution of the same pH as above were mixed in a volume ratio of 1:1, the pH was adjusted to the same as before mixing, and the mixture was equilibrated at 25° C. for 1 h to obtain a mixed solution;

[0059] The concentration of α-lactalbumin in the mixed solution is 0.7 mM, and the molar ratio of EDTA to α-lactalbumin is 1:1.

[0060] The calcium-bound content of α-lactalbumin in the mixed solution prepared above is shown in Table 1.

[0061] Table 1 shows that the bound calcium content of α-lactalbumin gradually decreases with increasing pH. When the pH increases to 7.5 and above, the bound calcium content of α-lactalbumin is less than 0.5 (M / M protein), indicating that more than half of the bound calcium has been removed. The higher the pH, the stronger the EDTA's decalcification ability, and the lower the amount of DETA required for decalcification, which facilitates subsequent EDTA removal from the system.

[0062] Separately, bovine milk α-lactalbumin powder was dissolved in deionized water to a concentration of 0.7 mM, and the pH was adjusted to 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 using 2 M HCl solution or 2 M NaOH solution. The solution was equilibrated at 25°C for 1 h, and then the pH was adjusted to 7.0 using 2 M NaOH solution or 2 M HCl solution. DSC testing was then performed, and the denaturation temperatures are shown in Table 1.

[0063] As can be seen from Table 1, with the increase of the first adjusted pH, the denaturation temperature remained unchanged between 5.0 and 9.0, and then decreased, indicating that excessively high pH would cause irreversible changes in the amino acid residues or structure of α-lactalbumin.

[0064] In summary, α-lactalbumin can be decalcified using EDTA at pH 7.5-9.0, with pH 9.0 being preferred.

[0065] Table 1 Calcium content and denaturation temperature of α-lactalbumin in Example 1

[0066]

[0067] Example 2 Removal of α-lactalbumin-bound calcium: Effect of different EDTA to α-lactalbumin molar ratios at pH 9.0

[0068] Cow milk α-lactalbumin powder was dissolved in deionized water to a concentration of 1.4 mM, and the pH was adjusted to 9.0 using 2 M NaOH solution to obtain an α-lactalbumin solution;

[0069] EDTA powder was dissolved in deionized water to a concentration of 0, 0.7, 1.05, 1.4, 2.8, 5.6, 11.2, or 22.4 mM, and the pH was adjusted to 9.0 with 2 M NaOH solution to obtain EDTA solution.

[0070] The α-lactalbumin solution and EDTA solutions of different concentrations were mixed at a volume ratio of 1:1, the pH was adjusted to the same as before mixing, and the mixture was equilibrated at 25° C. for 1 h to obtain a mixed solution;

[0071] The concentration of α-lactalbumin in the mixed solution is 0.7 mM, and the molar ratio of EDTA to α-lactalbumin is 0:1, 0.5:1, 0.75:1, 1:1, 2:1, 4:1, 8:1 or 16:1.

[0072] The calcium-bound content of α-lactalbumin in the mixed solution prepared above is shown in Table 2.

[0073] As can be seen from Table 2, with the increase of EDTA addition, the bound calcium content of α-lactalbumin gradually decreased; when the EDTA addition amount was increased to 1 (M / M protein), the bound calcium content of α-lactalbumin was less than 0.2 (M / M protein), that is, more than 80% of the bound calcium of α-lactalbumin was removed; when the EDTA addition amount was increased to 4 (M / M protein), the bound calcium content of α-lactalbumin was less than 0.05, that is, more than 95% of the bound calcium of α-lactalbumin was removed; when the EDTA addition amount was increased to 8 (M / M protein) or above, the removal of bound calcium of α-lactalbumin no longer increased significantly.

[0074] In summary, at pH 9.0, the EDTA addition amount can be selected from 1 to 16 (M / M protein) to decalcify α-lactalbumin, with the preferred addition amount being 4 (M / M protein).

[0075] Table 2 Calcium content of α-lactalbumin in Example 2

[0076]

[0077] Comparative Example 1 Removal of α-lactalbumin-bound calcium: Effects of different equilibrium temperatures and times

[0078] Cow milk α-lactalbumin powder was dissolved in deionized water to a concentration of 1.4 mM, and the pH was adjusted to 9.0 using 2 M NaOH solution to obtain an α-lactalbumin solution;

[0079] Dissolve EDTA powder in deionized water to a concentration of 5.6 mM and adjust the pH to 9.0 with 2 M NaOH solution to obtain an EDTA solution.

[0080] The α-lactalbumin solution and the EDTA solution were mixed at a volume ratio of 1:1, the pH was adjusted to the same as before mixing, and the mixture was equilibrated at 4° C. and 25° C. for 12 h, respectively, to obtain a mixed solution;

[0081] The concentration of α-lactalbumin in the mixed solution is 0.7 mM, and the molar ratio of EDTA to α-lactalbumin is 4:1.

[0082] The calcium-bound contents of α-lactalbumin in the mixed solutions prepared in Example 2 and Comparative Example 1 are shown in Table 3.

[0083] It can be seen from Table 3 that when the equilibrium time is 12h, the bound calcium content of α-lactalbumin at 25°C is lower than that at 4°C; when the equilibrium temperature is 25°C, there is no significant difference in the bound calcium content of α-lactalbumin when the equilibrium time is 1h and 12h.

[0084] In summary, when EDTA is used to decalcify α-lactalbumin, the optimal equilibrium temperature is 25°C and the equilibrium time is 1 h.

[0085] Table 3 Calcium content of α-lactalbumin in Example 2 and Comparative Example 1

[0086]

[0087] Example 3 Removal of α-lactalbumin-bound calcium: Effect of different pH values ​​alone

[0088] Bovine milk α-lactalbumin powder was dissolved in deionized water to a concentration of 0.7 mM, and the pH was adjusted to 7.0, 6.0, 5.0, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5 or 1.0 using 2 M HCl solution, and equilibrated at 25° C. for 1 h to obtain a mixed solution.

[0089] The calcium-bound content of α-lactalbumin in the mixed solution prepared above is shown in Table 4.

[0090] As can be seen from Table 4, the bound calcium content of α-lactalbumin gradually decreases with decreasing pH. When the pH is less than 3 or below, the bound calcium content of α-lactalbumin is less than 0.2 (M / M protein), indicating that more than 80% of the bound calcium of α-lactalbumin is removed.

[0091] Separately, bovine milk α-lactalbumin powder was dissolved in deionized water to a concentration of 0.7 mM. The pH was adjusted to 7.0, 6.0, 5.0, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, or 1.0 using 2 M HCl solution. The mixture was equilibrated at 25°C for 1 h, and then the pH was adjusted to 7.0 using 2 M NaOH solution. DSC testing was then performed, and the denaturation temperatures are shown in Table 4.

[0092] As can be seen from Table 4, as the pH value adjusted for the first time decreases, the denaturation temperature remains unchanged between 2.0 and 7.0, and then decreases, indicating that excessively low pH may cause irreversible changes in the amino acid residues or structure of α-lactalbumin.

[0093] In summary, the pH can be adjusted to 2.0-3.0 to decalcify α-lactalbumin, with the preferred pH being 2.0.

[0094] Table 4 Calcium content and denaturation temperature of α-lactalbumin in Example 3

[0095]

[0096] Comparative Example 2 Removal of α-lactalbumin-bound calcium: Effect of different molar ratios of sodium citrate to α-lactalbumin

[0097] Cow milk α-lactalbumin powder was dissolved in deionized water to a concentration of 1.4 mM, and the pH was adjusted to 7.0 using 2 M HCl solution to obtain an α-lactalbumin solution;

[0098] Sodium citrate powder was dissolved in deionized water to a concentration of 0, 2.8, 5.6, 11.2, 22.4, 44.8, or 89.6 mM, and the pH was adjusted to 7.0 with 2 M HCl solution to obtain a sodium citrate solution;

[0099] The α-lactalbumin solution and sodium citrate solutions of different concentrations were mixed at a volume ratio of 1:1, the pH was adjusted to the same as before mixing, and the mixture was equilibrated at 25° C. for 1 h to obtain a mixed solution;

[0100] The concentration of α-lactalbumin in the mixed solution is 0.7 mM, and the molar ratio of sodium citrate to α-lactalbumin is 0:1, 2:1, 4:1, 8:1, 16:1, 32:1 or 64:1.

[0101] The calcium-bound content of α-lactalbumin in the mixed solution prepared above is shown in Table 5.

[0102] It can be seen from Table 5 that as the amount of sodium citrate added increases, the content of α-lactalbumin-bound calcium decreases slightly.

[0103] In summary, sodium citrate has a weak ability to remove calcium bound to α-lactalbumin and is not selected for decalcification of α-lactalbumin.

[0104] Table 5 Bound calcium content of α-lactalbumin in Comparative Example 2

[0105]

[0106] Example 4: Effect of different ultrafiltration times at pH 9.0 and a molar ratio of EDTA to α-lactalbumin of 4:1 on crude decalcified α-lactalbumin

[0107] Cow milk α-lactalbumin powder was dissolved in deionized water to a concentration of 1.4 mM, and the pH was adjusted to 9.0 using 2 M NaOH solution to obtain an α-lactalbumin solution;

[0108] Dissolve EDTA powder in deionized water to a concentration of 5.6 mM and adjust the pH to 9.0 with 2 M NaOH solution to obtain an EDTA solution.

[0109] The α-lactalbumin solution and the EDTA solution were mixed in a volume ratio of 1:1, the pH was adjusted to the same as before mixing, and the mixture was equilibrated at 25° C. for 1 hour to obtain a mixed solution; wherein the concentration of α-lactalbumin in the mixed solution was 0.7 mM, and the molar ratio of EDTA to α-lactalbumin was 4:1;

[0110] The mixed solution was subjected to cyclic ultrafiltration at 25°C using an ultrafiltration membrane (tangential flow) with a molecular weight cutoff of 10 kDa until the volume concentration factor reached 4. Deionized water 3 times the volume of the retentate was added, and the pH was adjusted to 9.0 using 2M NaOH solution. The above ultrafiltration step was repeated 7 times, and the retentate was collected.

[0111] The residual calcium and EDTA contents in the retentate prepared above are shown in Table 6.

[0112] Table 6 shows that the residual EDTA content gradually decreases with increasing ultrafiltration cycles. No EDTA is detected in the retentate at 6 or higher ultrafiltration cycles. The residual calcium content first decreases and then increases with increasing ultrafiltration cycles, reaching its lowest level at 6 ultrafiltration cycles. α-lactalbumin has a strong calcium-binding capacity. When no EDTA is detected in the system, it readily binds to trace amounts of calcium in the added deionized water, leading to an increase in residual calcium.

[0113] In summary, at pH 9.0, after decalcification of α-lactalbumin with an EDTA addition amount of 4 (M / M protein), the decalcified α-lactalbumin can be cruded by ultrafiltration for 6-8 times, with 6 being the preferred ultrafiltration times.

[0114] Table 6 Residual calcium and EDTA in the retentate of Example 4

[0115]

[0116] Note: “-” means not detected.

[0117] Example 5: Crude decalcified α-lactalbumin: Effect of different ultrafiltration times at pH 2.0

[0118] Bovine milk α-lactalbumin powder was dissolved in deionized water to a concentration of 0.7 mM, the pH was adjusted to 2.0 using 2 M HCl solution, and the mixture was equilibrated at 25° C. for 1 h to obtain a mixed solution;

[0119] The mixed solution was subjected to cyclic ultrafiltration at 25°C using an ultrafiltration membrane (tangential flow) with a molecular weight cutoff of 10 kDa until the volume concentration factor reached 4. Deionized water (3 times the volume of the retentate) was added and the pH was adjusted to 2.0 using a 2M HCl solution. The ultrafiltration step was repeated 7 times, and the retentate was collected.

[0120] The residual calcium content in the retentate prepared above is shown in Table 7.

[0121] Table 7 shows that as the number of ultrafiltration cycles increases to 6, the residual calcium gradually decreases; as the number of ultrafiltration cycles continues to increase, the residual calcium remains unchanged. The proportions of bound and free calcium in the α-lactalbumin solution are in equilibrium. Influenced by pH, free calcium is gradually removed with increasing ultrafiltration cycles, and the bound calcium gradually reaches equilibrium with the trace amount of free calcium in the added deionized water.

[0122] In summary, after decalcifying α-lactalbumin by adjusting the pH to 2.0 alone, the ultrafiltration times can be selected to be 6-8 to crude the decalcified α-lactalbumin, wherein the preferred ultrafiltration times are 6.

[0123] Table 7 Calcium Residue in Retentate of Example 5

[0124]

[0125] Example 6 Purification of decalcified α-lactalbumin: Effect of acid precipitation pH

[0126] The retentate after six ultrafiltration cycles in Example 4 was taken and the pH was adjusted to 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 or 5.2 using 2M HCl solution. The mixture was equilibrated at 25°C for 1 hour and then centrifuged at 10,000 g for 30 minutes to collect the precipitate.

[0127] Comparative Example 3 Direct acid precipitation of α-lactalbumin

[0128] Bovine milk α-lactalbumin powder was dissolved in deionized water to a concentration of 2.8 mM, and the pH was adjusted to 5.0 using 2 M HCl solution. The mixture was equilibrated at 25° C. for 1 h, and then centrifuged at 10,000 g for 30 min to collect the precipitate.

[0129] The α-lactalbumin recovery rate and the residual calcium content in the precipitates prepared in Example 6 and Comparative Example 3 are shown in Table 8.

[0130] Table 8 α-lactalbumin recovery and calcium residue in the precipitate in Example 6 and Comparative Example 3

[0131]

[0132] Note: “-” means not detected.

[0133] As shown in Table 8, in Example 6, the recovery of α-lactalbumin initially increased and then decreased with increasing pH. At pH 4.7-5.1, the recovery rate exceeded 90%, and reached its highest at pH 5.0. Between pH 4.6 and 5.2, no calcium was detected in the precipitate. In Comparative Example 3, the recovery rate of α-lactalbumin in the precipitate was low, and the residual calcium content was high. After decalcification, the α-lactalbumin molecule becomes less stable, becoming more sensitive to low pH and more susceptible to precipitate formation, thus requiring purification.

[0134] In summary, at pH 9.0, α-lactalbumin is decalcified using EDTA addition of 4 (M / M protein), and then the decalcified α-lactalbumin is cruded using ultrafiltration times 6. The decalcified α-lactalbumin can then be refined using acid precipitation at a pH of 4.7-5.1, with a preferred pH of 5.0.

[0135] Comparative Example 4: Purification of decalcified α-lactalbumin: Effect of different ultrafiltration modes

[0136] Bovine milk α-lactalbumin powder was dissolved in a solution of 1 mM EDTA, 20 mM Tris-HCl, pH 8.5, and equilibrated at 4°C overnight to obtain a mixed solution; wherein the concentration of α-lactalbumin in the mixed solution was 0.7 mM;

[0137] The mixed solution was centrifuged and ultrafiltered at 4°C using a 10 kDa molecular weight cutoff ultrafiltration tube (flow-through) until the volume concentration reached 4 times. Deionized water (3 times the volume of the retentate) was added and the pH was adjusted to 8.5 using 2 M NaOH solution. The ultrafiltration step was repeated 5 times, and the retentate was collected.

[0138] The retentate was taken, the pH was adjusted to 5.0 with 2M HCl solution, equilibrated at 25°C for 1 hour, and then centrifuged at 10,000 g for 30 minutes to collect the precipitate.

[0139] The residual calcium and EDTA contents in the retentate and precipitate prepared above are shown in Table 9.

[0140] Table 9 shows that the residual calcium and EDTA levels in both the retentate and precipitate are high. Direct-flow ultrafiltration is prone to concentration polarization on the membrane surface, especially at low temperatures and high viscosity, which hinders the transmembrane transport of free calcium and EDTA.

[0141] In summary, direct current ultrafiltration has a weak decalcification capacity, and even combined with acid precipitation, it cannot achieve calcium-free detection, so it is not selected for the purification of decalcified α-lactalbumin.

[0142] Table 9 Calcium Residue and EDTA Residue in the Retentate and Precipitate in Comparative Example 4

[0143]

[0144] Example 7 Simulated gastric digestion of decalcified α-lactalbumin: Effect of different heat treatment temperatures

[0145] The decalcified α-lactalbumin purified in Example 6 was dissolved in deionized water to a concentration of 12 mg / mL, the pH was adjusted to 7.0 with a 2M NaOH solution, and the mixture was equilibrated at 25° C. for 1 h to obtain a mixed solution;

[0146] The mixed solution was taken and heated at 30, 40, 50, 60, 70, 80, 90 or 100° C. for 10 min, and then quickly cooled to 25° C. to obtain a heat-treated mixed solution.

[0147] Comparative Example 5 Simulated gastric digestion of α-lactalbumin: Effects of different heat treatment temperatures

[0148] Bovine milk α-lactalbumin powder was dissolved in deionized water to a concentration of 12 mg / mL, the pH was adjusted to 7.0 with 2 M HCl solution, and the mixture was equilibrated at 25°C for 1 h to obtain a mixed solution;

[0149] The mixed solution was taken and heated at 30, 40, 50, 60, 70, 80, 90 or 100° C. for 10 min, and then quickly cooled to 25° C. to obtain a heat-treated mixed solution.

[0150] The free amino group contents of the mixed solutions prepared in Example 7 and Comparative Example 5 and the heat-treated mixed solutions after simulated infant gastric digestion are shown in Table 10. The peptide bonds of protein molecules are hydrolyzed by digestive enzymes to release free amino groups, and the free amino group content can indicate the digestibility of the protein.

[0151] As can be seen from Table 10, as the heat treatment temperature increases, the free amino group content (μmol / mg protein) first increases and then decreases. The content of free amino groups released by decalcified α-lactalbumin reaches its maximum at 60-80°C, and the content of free amino groups released by α-lactalbumin reaches its maximum at 80-90°C. The content of free amino groups released by decalcified α-lactalbumin is higher than that of α-lactalbumin.

[0152] Compared with unheat-treated α-lactalbumin, 70°C heat treatment alone increased the free amino group content by 0.035 μmol / mg protein, decalcification alone increased the free amino group content by 0.123 μmol / mg protein, and the combination of 70°C heat treatment and decalcification increased the free amino group content by 0.293 μmol / mg protein, which was greater than the sum of the effects of 70°C heat treatment and decalcification alone, i.e., 0.158 μmol / mg protein. This indicates that decalcification and 70°C heat treatment have a synergistic effect in improving the digestibility of α-lactalbumin.

[0153] Similarly, it can be analyzed that decalcification and heat treatment at 60 and 80 °C also have a synergistic effect in improving the digestibility of α-lactalbumin;

[0154] Decalcification treatment partially unfolds the molecular structure of α-lactalbumin, and moderate heat treatment further unfolds the molecular structure of α-lactalbumin, which is conducive to the hydrolysis of digestive enzymes; excessive heat treatment may induce protein aggregation induced by disulfide bonds or hydrophobic effects, which is not conducive to the hydrolysis of digestive enzymes.

[0155] In summary, it is preferred to combine decalcification with heat treatment at 60-80°C to synergistically improve the digestibility of α-lactalbumin, making it easier for infants and young children and other people with insufficient gastrointestinal function to digest and absorb.

[0156] Table 10 Free amino group content of the mixed solution after gastric digestion in Example 7 and Comparative Example 5 (μmol / mg protein)

[0157]

[0158] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for improving the digestibility of α-lactalbumin, characterized in that: The steps include: (1) Adjusting the pH of the α-lactalbumin powder solution to 2.0-3.0 and equilibrating at 20-30°C for 1 h or longer to obtain a mixed solution; or adjusting the pH of the α-lactalbumin powder solution to 7.5-9.0 to obtain an α-lactalbumin solution; Adjusting the pH of the EDTA solution to 7.5-9.0 to obtain an EDTA solution; Mixing an α-lactalbumin solution and an EDTA solution of the same pH in a volume ratio of 1:1, adjusting the pH to the same as before mixing, and equilibrating at 20-30° C. for 1 h or more to obtain a mixed solution; wherein the molar ratio of α-lactalbumin to EDTA is 1:2-16; EDTA solution is an aqueous solution of EDTA with a concentration of 1.4-22.4 mM; (2) ultrafiltration and concentration of the mixed solution obtained in step (1) and acid precipitation to obtain completely decalcified α-lactalbumin; Among them, ultrafiltration concentration is to use an ultrafiltration membrane with a molecular weight cutoff of 8-10 kDa to perform cyclic ultrafiltration concentration at 20-30°C, the ultrafiltration times are 6-8 times, and the volume concentration multiple is 3-5 times; Acid precipitation pH is 4.7-5.1; (3) The completely decalcified α-lactalbumin obtained in step (2) is redissolved, the pH is adjusted to 7.0, and the solution is equilibrated at 20-30°C for 1 hour or longer, followed by heat treatment at 60-80°C for 5-20 minutes, and then rapidly cooled to room temperature to obtain α-lactalbumin with high digestibility.

2. The method according to claim 1, characterized in that In step (1), the pH of the α-lactalbumin powder solution is adjusted to 2.0 and equilibrated at 20-30° C. for 1 hour or more.

3. The method according to claim 1, characterized in that The equilibrium in step (1) was carried out at 25°C for 1 h.

4. The method according to claim 1, wherein The α-lactalbumin powder solution in step (1) is an α-lactalbumin powder aqueous solution with a concentration of 0.7-2 mM.

5. The method according to claim 1, wherein The concentration of the EDTA aqueous solution in step (1) is 4 mM.

6. The method according to claim 1, characterized in that The number of ultrafiltrations in step (2) is 6.

7. The method according to claim 1, wherein The pH of the acid precipitation in step (2) is 5.

0.

8. α-lactalbumin with high digestibility prepared by the method according to any one of claims 1 to 7.

9. An infant formula milk powder, characterized in that: The highly digestible α-lactalbumin according to claim 8 is used.

10. Use of the highly digestible α-lactalbumin according to claim 8 in the field of food preparation.

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