Casein micelles with phosphorylation characteristics simulating human milk and preparation method thereof

Abstract

The present invention discloses a casein micelle with phosphorylation characteristics simulating human milk and a preparation method thereof, belonging to the technical field of dairy product processing. The present invention first uses animal milk casein as raw material, adopts selective precipitation technology to separate casein components, mixes each casein component according to the ratio of human milk casein, and realizes the preparation of composite casein; then dephosphorylates the composite casein to different degrees, and according to the phosphorylation degree and distribution of human milk casein, mixes composite caseins with different phosphorylation degrees to realize the preparation of human milk casein with phosphorylation characteristics; finally, according to the assembly process of natural micelles in mammary cells, simulates human milk by assembling the initial ion environment or simulating human milk by the assembly process (continuous) to reconstruct the simulated human milk casein micelles, so that its casein composition, casein phosphorylation characteristics, mineralization degree, structure and digestibility are similar to those of human milk casein micelles.

Description

Technical Field

[0001] The invention relates to a casein micelle with phosphorylation characteristics simulating human milk and a preparation method thereof, belonging to the technical field of dairy product processing. Background Art

[0002] Breast milk is the best natural food for infants; however, with socioeconomic development, insufficient breastfeeding is becoming increasingly common, making infant formula the best alternative to breast milk. The proteins in infant formula primarily come from bovine and goat milk, which differ significantly from human milk in casein composition and phosphorylation. Bovine and goat milk caseins are primarily α- and β-casein, while human milk caseins are primarily β-casein. β-casein in bovine and goat milk is highly phosphorylated, containing five phosphate groups (5P); β-casein in human milk is less phosphorylated and exhibits a polymorphic phosphorylation pattern, containing zero to five phosphate groups (0-5P). These differences in casein from bovine, goat, and human milk lead to differences in the mineralization and structure of casein micelles, which in turn leads to significant differences in the digestibility of the micelles and the types and functions of the peptides produced by digestion. Therefore, to better meet the digestive, assimilation, and growth and development needs of infants and young children, the development of formula milk needs to consider the composition and phosphorylation of casein, as well as the mineralization and structure of casein micelles.

[0003] Human milk casein micelles are assembled within mammary cells: first, casein is synthesized by ribosomes on the endoplasmic reticulum and transported to the Golgi apparatus, where it undergoes partial phosphorylation under the action of calcium-ATP-dependent kinases. Subsequently, mineral ions such as calcium, phosphate, and citrate are continuously transported into the Golgi apparatus and bind to casein to form casein micelles. The formation of human milk micelles is a complex, continuous process. The Golgi apparatus provides the initial ionic environment for micelle formation, and the subsequent transport of mineral ions further induces casein assembly. Therefore, to prepare highly simulated human milk casein micelles, multiple aspects must be considered, including the initial ionic environment of assembly, casein composition and phosphorylation, and the composition, concentration, and order of addition of mineral ions, in order to closely mimic the in vivo formation process of human milk micelles.

[0004] Enzymatic dephosphorylation can regulate the phosphorylation of bovine and sheep milk casein, but its selectivity for sites is low, and the phosphorylation of human milk casein shows a multi-distribution pattern. Therefore, it is difficult to achieve simultaneous simulation of the phosphorylation degree and distribution of human milk casein by optimizing the dephosphorylation conditions alone. In patent CN105695542A, partially dephosphorylated bovine milk casein was prepared by optimizing the dephosphorylation time of bovine milk whole casein, but its β-casein phosphorylation distribution showed four variants of 1-4P, which still had a large difference in phosphorylation distribution from human milk β-casein and could not simulate human milk casein in terms of phosphorylation distribution.

[0005] Casein phosphorylation (degree and distribution), as well as the complex assembly formation mechanism in vivo (initial ionic environment and continuous process, etc.), all greatly affect the interaction between caseins and between caseins and mineral salt ions, ultimately affecting the structure of casein micelles. In patent CN114601013A, simulated human milk micelles were prepared by simulating the composition of human milk casein. However, the degree and distribution of phosphorylation of human milk casein, as well as the initial ionic environment and continuous process of assembly in vivo, were not taken into account. As a result, the prepared micelles still had significant differences from human milk micelles in terms of casein phosphorylation characteristics, mineralization degree, structure, digestibility, etc. Summary of the Invention

[0006] [Technical Issues]

[0007] Currently, using animal milk, such as cow's milk or goat's milk, as casein raw materials, it is difficult to simultaneously simulate the phosphorylation level and distribution of human milk casein by optimizing enzymatic dephosphorylation conditions alone. Furthermore, no research has comprehensively considered the casein composition, phosphorylation level and distribution, and the initial ionic environment and continuous assembly process of human milk casein micelles to achieve a highly simulated human milk casein micelle preparation. Only when casein micelles mimic human milk in multiple aspects, such as casein composition and phosphorylation characteristics, micelle mineralization level, and micelle structure, can they be more beneficial for digestion, absorption, and growth and development in infants and young children.

[0008] [Technical solution]

[0009] In order to solve the above problems, the present invention first uses animal milk casein as raw material, adopts selective precipitation technology to separate casein components, and mixes each casein component according to the ratio of human milk casein to achieve the preparation of compound casein; then the compound casein is dephosphorylated to different degrees, and according to the phosphorylation degree and distribution of human milk casein, the compound caseins with different phosphorylation degrees are mixed to achieve the preparation of human milk casein with phosphorylation characteristics simulating human milk; finally, according to the assembly process of natural micelles in mammary cells, the initial ion environment of the assembly is simulated by simulating human milk or the assembly process (continuous) simulates human milk, and the simulated human milk casein micelles are reconstructed so that the casein composition, casein phosphorylation characteristics, mineralization degree, structure and digestibility are similar to those of human milk casein micelles.

[0010] The first object of the present invention is to provide a method for preparing casein micelles having phosphorylation characteristics simulating human milk, comprising the following steps:

[0011] (1) Compound caseins with phosphorylation degrees of 2.74, 1.85 and 0.73 were mixed in a mass ratio of 1:2:0.4 to obtain simulated human milk casein; wherein, κ-casein, α-casein and α-casein in the simulated human milk casein were s1-Casein and β-casein account for 17-23%, 9-15%, and 65-71% respectively, and do not contain α s2 -Casein; β-casein phosphorylation distribution shows 6 variants from 0 to 5P;

[0012] (2) Preparation method 1: prepare an initial salt solution containing calcium chloride, potassium citrate, magnesium chloride, and dipotassium hydrogen phosphate, wherein the concentration of citrate ions is 1.35-2.7 mM, the concentration of calcium ions is 4.8-5.2 mM, the concentration of magnesium ions is 1.0-1.2 mM, and the concentration of phosphate ions is 1.0-2.2 mM, and the pH is adjusted to 6.7-7.2; add simulated human milk casein to the initial salt solution to a concentration of 0.1-0.2 mM, mix well, and adjust the pH to 6.7-7.2; continue to add the mixed solution in two batches in succession Calcium chloride, magnesium chloride, and dipotassium hydrogen phosphate are added, with the calcium ion concentration added in each batch being 0.4-1.1 mM, the magnesium ion concentration being 0.05-0.2 mM, and the phosphate ion concentration being 0.2-0.65 mM, and the pH being adjusted to 6.7-7.2 to obtain a solution simulating human milk casein micelles; wherein the casein concentration is 0.1-0.2 mM, the citrate ion concentration is 1.35-2.7 mM, the calcium ion concentration is 5.6-7.2 mM, the magnesium ion concentration is 1.1-1.6 mM, and the phosphate ion concentration is 1.4-3.5 mM;

[0013] Preparation method 2: Prepare an initial salt solution containing calcium chloride, potassium citrate, magnesium chloride, and dipotassium hydrogen phosphate, wherein the concentration of citrate ions is 1.35-2.7 mM, the concentration of calcium ions is 5.6-7.2 mM, the concentration of magnesium ions is 1.1-1.6 mM, and the concentration of phosphate ions is 1.4-3.5 mM, and the pH is adjusted to 5.8-5.9; add simulated human milk casein to the initial salt solution to a concentration of 0.1-0.2 mM, mix well, and adjust the pH to 5.8 -5.9; adding urea to the mixed solution to a concentration of 2.5-3.0 mM, adding urease to an enzyme activity of 1.0 U / mL, and slowly raising the pH of the solution to 6.7-7.2 after equilibrium to obtain a solution simulating human milk casein micelles; wherein the casein concentration is 0.1-0.2 mM, the citrate ion concentration is 1.35-2.7 mM, the calcium ion concentration is 5.6-7.2 mM, the magnesium ion concentration is 1.1-1.6 mM, and the phosphate ion concentration is 1.4-3.5 mM.

[0014] In one embodiment of the present invention, the preparation method of compound casein in step (1) is:

[0015] ① Centrifuge animal milk at 20-30°C and 2000-4000g for 20-40 minutes to obtain skim milk; add hydrochloric acid solution (4-8M) to a pH of 4.3-4.9, and centrifuge at 5000-15000g for 10-20 minutes; take the casein precipitate and redissolve it in water, add NaOH solution (1-3M) to a pH of 10-12, and obtain a casein reconstituted solution; wherein the mass concentration of casein is 2-3%. Animal milk includes cow's milk, goat's milk, camel's milk, horse's milk, donkey's milk, etc.

[0016] ② Add calcium chloride to the casein solution to a concentration of 50-70 mM, centrifuge at 5000-15000 g for 10-20 min, and obtain the α-enriched s - and β-casein precipitate, and the supernatant enriched with κ-casein; hydrochloric acid solution was added to the supernatant enriched with κ-casein to pH 3-4, and centrifuged to obtain a precipitate enriched with κ-casein; and α-casein was taken. s - and β-casein precipitate are redissolved in water, NaOH solution is added to pH 6.5-7.5, so that the mass concentration of casein is 2-3%; cooled to 0-8°C, hydrochloric acid solution is added to pH 4.3-4.9, maintained at 0-8°C for 4-8 hours, centrifuged, and the supernatant is collected, heated to 30-40°C, and centrifuged to obtain a precipitate enriched with β-casein;

[0017] ③ The precipitate enriched with κ-casein and the precipitate enriched with β-casein were mixed at a casein mass ratio of 10:7, and dialyzed at 0-8°C for 48-96 hours using a dialysis membrane with a molecular weight cutoff of 5000-10000Da. Ion exchange resin Amberlite SR1LNa (0.4-0.6g resin / g casein) was added to the dialyzed external fluid. After the dialysis, the mixture was freeze-dried to obtain a composite casein.

[0018] In one embodiment of the present invention, the preparation method of compound casein with phosphorylation degrees of 2.74, 1.85 and 0.73 in step (1) is as follows:

[0019] Compound casein was prepared into a compound casein solution; bovine intestinal alkaline phosphatase was prepared into an enzyme solution; the compound casein solution and the enzyme solution were then mixed in a volume ratio of 1:1 and incubated in a water bath at 37°C for 1, 15, and 180 minutes, respectively; the enzyme was then inactivated, dialyzed, and freeze-dried to obtain compound caseins with phosphorylation degrees of 2.74, 1.85, and 0.73;

[0020] The concentration of the compound casein solution is 4-6 mg / mL, the concentration of the enzyme solution is 0.3-0.5 U / mL; the solvent of the enzyme solution is Tris-HCl buffer with a pH of 8.0; the solvent of the casein solution is water; the enzyme is inactivated by maintaining the high temperature at 80°C for 10 minutes; and the dialysis is performed by dialyzing the inactivated enzyme hydrolyzate using a dialysis membrane with a molecular weight cutoff of 5000-10000 Da at 4°C for 72 hours.

[0021] The second object of the present invention is to provide casein micelles with phosphorylation characteristics that mimic those of human milk, prepared by the method of the present invention.

[0022] The third object of the present invention is the application of the casein micelles with phosphorylation characteristics simulating human milk in the field of dairy product processing.

[0023] In one embodiment of the present invention, the application includes providing casein ingredients for the research and development and production of infant formula powder.

[0024] In one embodiment of the present invention, the application is to ultrafilter a solution of phosphorylated characteristic mimicking human milk casein micelles through a 5-20 kDa organic membrane, concentrate it to 20-50 times, take the concentrate, and obtain casein ingredients by spray drying or freeze drying for the research and development and production of infant formula powder.

[0025] The fourth object of the present invention is to provide a method for improving the similarity of simulated human milk casein micelles with respect to casein phosphorylation degree, distribution and mineralization degree with human milk casein, which uses the casein micelles with phosphorylation characteristics simulating human milk described in the present invention.

[0026] [Beneficial Effects]

[0027] (1) The method for preparing simulated human milk casein micelles provided by the present invention simulates the natural micelle assembly mechanism in mammary cells, that is, by simulating human milk by assembling the initial ionic environment or simulating human milk by the assembly process (continuous), thereby achieving a high degree of in vitro simulation of the micelle assembly process, thereby achieving a highly simulated preparation of human milk casein micelles.

[0028] (2) The simulated human milk casein micelles provided by the present invention are similar to human milk casein in terms of casein composition, phosphorylation degree and distribution, and β-casein presents a multi-phosphorylated distribution form of 0-5P.

[0029] (3) The simulated human milk casein micelles provided by the present invention are similar to human milk casein micelles in terms of structural characteristics such as the proportion of free calcium, the proportion of free casein, the degree of mineralization, the average particle size, and the hydration rate.

[0030] (4) The simulated human milk casein micelles provided by the present invention form fine flocculent particles in gastric juice, which are conducive to the hydrolysis of pepsin. The degree of casein hydrolysis is similar to that of human milk casein micelles. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the urea electrophoresis diagram of the dephosphorylated composite casein in Example 1.

[0032] Figure 2 The urea electrophoresis diagrams of the compound casein and the casein in Example 2 and Comparative Examples 1, 2, and 3 are shown.

[0033] Figure 3 This is the effect of calcium ion concentration on the absorbance of the recombinant casein micelle solution in Example 5.

[0034] Figure 4 This is the effect of phosphate ion concentration on the absorbance of the recombinant casein micelle solution in Example 6. DETAILED DESCRIPTION

[0035] 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.

[0036] Test method:

[0037] 1. Casein separation

[0038] Take the casein micelle solution, add hydrochloric acid solution (2M) to pH 4.3 (human milk source) or pH 4.6 (cow milk source), centrifuge at 10000g for 15 minutes, and the resulting precipitate is casein.

[0039] 2. Separation of micellar phase and whey phase

[0040] The casein micelle solution was centrifuged at 25°C and 150,000 g for 1 h. The resulting precipitate and supernatant were the micelle phase and whey phase, respectively.

[0041] 3. Determination of casein content

[0042] Casein content was determined using the Kjeldahl method with a conversion factor of 6.38.

[0043] 4. Determination of casein composition

[0044] The casein composition was determined using an e2695 high performance liquid chromatograph with an XBridge BEH C18 column (250 mm × 4.6 mm) and a detection wavelength of 220 nm.

[0045] 5. Determination of calcium, magnesium, potassium and sodium ion content

[0046] Referring to the national standard GB5009.268-2016 "National Food Safety Standard Determination of Multiple Elements in Food", inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the content of calcium, magnesium, potassium and sodium ions.

[0047] The molar ratio of calcium to casein in the micellar phase is the degree of mineralization of the casein micelles.

[0048] 6. Determination of casein phosphorylation degree

[0049] The casein micelle solution was treated with a MARS microwave digestion system to obtain a digestion solution.

[0050] The protein in the casein micelle solution was precipitated with trichloroacetic acid to obtain a supernatant.

[0051] Referring to the national standard GB 5413.22-2010 "Determination of phosphorus in infant foods and dairy products", the phosphate ion content in the digestion solution and supernatant was determined, followed by the total phosphorus and inorganic phosphorus content, respectively. The difference between the two is the organic phosphorus content, and the molar ratio of organic phosphorus to total casein is the degree of casein phosphorylation.

[0052] 7. Determination of Casein Phosphorylation Distribution

[0053] Urea-PAGE was used to analyze the phosphorylation distribution of casein;

[0054] Specifically, the casein micelle solution was mixed with sample buffer (62.5 mM Tris-HCl, 8 M urea, 5% β-mercaptoethanol, pH 7.6) in an equal volume ratio, the sample volume was 30 μL, and the mass concentrations of the stacking gel and the separation gel were 4% and 8%, respectively.

[0055] 8. Determination of absorbance

[0056] The absorbance of the casein micelle solution was measured by a spectrophotometer with a detection wavelength of 400 nm and a sample cell thickness of 10 mm.

[0057] 9. Determination of micelle particle size

[0058] The particle size distribution of casein micelles was determined using a Malvern nanometer potentiometer, and the refractive index of the protein particles was set to 1.57.

[0059] 10. In vitro gastric digestion in infants and young children

[0060] The casein micelle solution was mixed with simulated gastric digestive fluid (94 mM sodium chloride, 13 mM potassium chloride, pH 5.3) in a volume ratio of 63:37. The pH was then slowly adjusted to 5.3 with hydrochloric acid solution (1 M). Pepsin was added with stirring to a concentration of 268 U / mL. The digestion temperature was 37°C, the time was 0-60 min, and the digestion reaction was terminated using the pepsin inhibitor Pepstatin A.

[0061] 11. Determination of protein hydrolysis degree

[0062] 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.

[0063] 0-10 mM L-leucine solution was used as a standard;

[0064] 100 μL of standard or gastric digest (60 min) was mixed with 2 mL of OPA reagent, reacted for 2 min in the dark, and the absorbance was measured at 340 nm;

[0065] The free amino group content of gastric digesta was calculated according to the standard curve:

[0066]

[0067] h=(Serine-NH2-β) / α

[0068] Among them, h tot =8.2, α=1.039, β=0.383.

[0069] The raw materials used in the embodiment are:

[0070] The preparation method of compound casein comprises the following steps:

[0071] Fresh milk was centrifuged at 25°C and 3000 g for 30 min to obtain skimmed milk;

[0072] Hydrochloric acid solution (6M) was added to skim milk to pH 4.6, and the mixture was centrifuged at 10,000 g for 15 minutes to obtain a bovine milk casein precipitate; the bovine milk casein precipitate was redissolved in deionized water, and NaOH solution (2M) was added to pH 11 to obtain a casein reconstituted solution; wherein the casein concentration was 2.5%;

[0073] Calcium chloride was added to the casein solution to a concentration of 65 mM and centrifuged at 10,000 g for 15 min to obtain the α-enriched s- and β-casein precipitate, and the supernatant enriched with κ-casein; hydrochloric acid solution (6M) was added to the supernatant enriched with κ-casein to pH 3.8, and centrifuged at 10000g for 15min to obtain a precipitate enriched with κ-casein; the supernatant enriched with α s - and β-casein precipitates were redissolved in deionized water, and NaOH solution (2 M) was added to pH 7.0 so that the mass concentration of casein was 2.5%; the temperature was lowered to 4°C, HCl solution (6 M) was added to pH 4.6, and the mixture was kept at 4°C for 6 hours, centrifuged at 10,000 g for 15 minutes, and the supernatant enriched with β-casein was taken, the temperature was raised to 35°C, and centrifuged at 10,000 g for 15 minutes to obtain a precipitate enriched with β-casein;

[0074] κ-casein and β-casein precipitates were mixed at a casein mass ratio of 10:7, and dialyzed at 4°C for 72 hours using a dialysis membrane with a molecular weight cutoff of 7000 Da to remove salt. Ion exchange resin Amberlite SR1LNa (0.5 g resin / g casein) was added to the dialyzed fluid. After dialysis, the mixture was freeze-dried to obtain the reconstituted casein.

[0075] Comparative Example 1 Human milk casein

[0076] Fresh human milk (mature milk 3-8 months after birth, n=10) was centrifuged at 25°C and 3000g for 30 minutes to obtain skimmed human milk; hydrochloric acid solution (2M) was added to pH 4.3, and the resulting precipitate was centrifuged at 10000g for 15 minutes to obtain human milk casein.

[0077] The protein compositions of bovine milk casein precipitate, precipitate enriched with κ-casein, precipitate enriched with β-casein, composite casein, and human milk casein are shown in Table 1.

[0078] As shown in Table 1, the κ-casein, α-casein and s1 -Casein, α s2 The relative contents of -casein and β-casein are close to the relative contents of each casein subtype in human milk casein, achieving a compound that simulates the composition of human milk casein.

[0079] Table 1 Protein composition of compound casein

[0080] sample κ-casein <![CDATA[α s1 -Casein]]> <![CDATA[α s2 -Casein]]> β-casein Bovine milk casein precipitation (%) 14.5±0.4 40.0±0.2 10.0±0.1 35.5±0.5 κ-casein precipitation (%) 35.7±0.5 20.2±0.7 - 44.1±0.6 β-casein precipitation (%) - - - 100 Compound casein (%) 20.4±0.8 12.5±0.4 - 67.1±1.0 Human milk casein (%) 20.7±0.2 11.7±0.5 - 67.6±0.6

[0081] Example 1 Dephosphorylation of compound casein: Regulation of different phosphorylation degrees of casein

[0082] The dephosphorylation method of compound casein comprises the following steps:

[0083] The compound casein was dissolved in water to obtain a casein solution with a concentration of 5 mg / mL; the calf intestinal alkaline phosphatase was dissolved in Tris-HCl buffer with a pH of 8.0 to obtain an enzyme solution with a concentration of 0.4 U / mL;

[0084] Take the compound casein solution and the calf intestinal alkaline phosphatase solution, mix them in a volume ratio of 1:1, and keep them in a 37°C water bath for 0, 1, 2, 4, 6, 8, 10, 15, 30, 60, 90, 120, and 180 minutes respectively to perform different degrees of dephosphorylation treatment; then keep them at 80°C for 10 minutes to inactivate the enzyme; then use a dialysis membrane with a molecular weight cutoff of 7000Da to dialyze at 4°C for 72 hours to remove salt, and freeze-dry after the dialysis to obtain the dephosphorylated compound casein.

[0085] The phosphorylation degree and phosphorylation distribution of the above dephosphorylated composite casein are shown in Tables 2 and Figure 1 .

[0086] It can be seen from Table 2 that as the dephosphorylation time increases, the phosphorylation degree of the compound casein decreases from 4.56 to 0.73 mM / mM.

[0087] from Figure 1 It can be seen that as the dephosphorylation time increases, the overall mobility of the casein electrophoretic band gradually decreases due to the removal of the phosphate groups, and the bands show a multi-distribution pattern, corresponding to the composite caseins containing different numbers of phosphate groups.

[0088] When the dephosphorylation time was 15 minutes, the phosphorylation degree of the reconstituted casein reached 1.85, close to that of human milk casein. The phosphorylation distribution of the β-casein showed six variants, ranging from 0 to 5P, but the 0P and 5P variants were present in very low amounts, and the ratio of the six variants differed significantly from that of human milk. Therefore, simply controlling the dephosphorylation time cannot achieve a phosphorylation distribution pattern consistent with human milk.

[0089] Table 2 Phosphorylation degree of dephosphorylated compound casein

[0090]

[0091] Example 2 Preparation of simulated human milk casein: Casein composition, phosphorylation degree and phosphorylation distribution all simulate human milk

[0092] The preparation method of simulated human milk casein comprises the following steps:

[0093] The composite casein dephosphorylated for 1, 15, and 180 min in Example 1, whose phosphorylation degrees were 2.74, 1.85, and 0.73, respectively, were mixed at a mass ratio of 1:2:0.4 to obtain simulated human milk casein with a phosphorylation degree of 1.98.

[0094] Comparative Example 2 Recombinant Casein 1: Casein phosphorylation distribution does not simulate human milk

[0095] The recombinant caseins dephosphorylated for 1, 30, and 120 min in Example 1, whose phosphorylation degrees were 2.74, 1.47, and 0.77, respectively, were mixed at a mass ratio of 1:1:0.4 to obtain recombinant casein 1 with a phosphorylation degree of 1.88.

[0096] Comparative Example 3 Recombinant Casein 2: The degree of casein phosphorylation did not simulate human milk

[0097] The recombinant caseins dephosphorylated for 1, 8, and 180 min in Example 1, whose phosphorylation degrees were 2.74, 2.48, and 0.73, respectively, were mixed at a mass ratio of 1.5:1:1 to obtain recombinant casein 2 with a phosphorylation degree of 2.10.

[0098] The phosphorylation degree and phosphorylation distribution of casein in Example 2 and Comparative Examples 1, 2, and 3 were characterized. The test results are shown in Tables 3 and Figure 2 :

[0099] From Table 3 and Figure 2 It can be seen that: human milk casein phosphorylation degree is 1.92, and β-casein phosphorylation distribution presents 6 kinds of variants of 0-5P. In embodiment 2, simulated human milk casein phosphorylation degree is 1.98, and β-casein phosphorylation distribution presents 6 kinds of variants of 0-5P, and the proportioning of 6 kinds of variants is close to human milk, that is, the casein composition, phosphorylation degree and phosphorylation distribution of simulated human milk casein are similar to human milk casein. In comparative example 2, the phosphorylation degree of recombinant casein 1 is 1.88, which is closer to the human milk casein phosphorylation degree, but its β-casein phosphorylation distribution presents 5 kinds of variants of 0-4P, which is inconsistent with the human milk β-casein phosphorylation distribution. In comparative example 3, although the β-casein phosphorylation distribution of recombinant casein 2 presents 6 kinds of variants of 0-5P, its phosphorylation degree is 2.10, which is much higher than the human milk casein phosphorylation degree; and in recombinant casein 2, 0P and 5P contents are higher, and the proportioning of 6 kinds of variants is larger than human milk difference. Therefore, the phosphorylation distribution and phosphorylation degree of Comparative Examples 2 and 3 failed to achieve simultaneous simulation of the phosphorylation degree and distribution of human milk casein.

[0100] When enzymatically regulating the phosphorylation level of casein, initially, the highly phosphorylated variant of β-casein predominates. With continued dephosphorylation, the moderately phosphorylated variant (2-4P) predominates. With longer dephosphorylation times, the highly dephosphorylated, hypophosphorylated variant predominates. Because enzymatic dephosphorylation is site-selective and human milk β-casein exhibits a polymorphic phosphorylation pattern, simply controlling the time required to achieve the simultaneous presence of both hypophosphorylated and hyperphosphorylated variants in a compound casein is not feasible.

[0101] Table 3 Phosphorylation levels of human milk casein, simulated human milk casein, and recombinant caseins 1 and 2

[0102]

[0103] Example 3 Preparation of Simulated Human Milk Casein Micelles 1: Assembling the Initial Ionic Environment Simulating Human Milk

[0104] The simulated human milk casein in Example 2 was used to prepare simulated human milk casein micelles, wherein the casein phosphorylation degree was 1.98, the β-casein phosphorylation distribution pattern was a polyphosphorylation distribution with 0-5 phosphate groups, and the citrate, calcium, magnesium, and phosphate ions and their concentrations in the human milk whey phase were used as the initial ion environment, and the citrate, calcium, magnesium, and phosphate ions and their total concentrations in human milk were used as the final ion environment.

[0105] Simulated human milk casein micelles 1 were prepared as follows:

[0106] An initial salt solution containing potassium citrate, calcium chloride, magnesium chloride, and dipotassium hydrogen phosphate was prepared, wherein the concentration of citrate ion was 2.3 mM, the concentration of calcium ion was 5.0 mM, the concentration of magnesium ion was 1.2 mM, and the concentration of phosphate ion was 2.1 mM; the pH was adjusted to 7.0 using 0.1 mM sodium hydroxide;

[0107] The simulated human milk casein of Example 2 was added to the initial saline solution to a concentration of 0.17 mM and stirred for 30 minutes; the pH was readjusted to 7.0;

[0108] Calcium chloride, magnesium chloride, and potassium hydrogen phosphate were added to the mixed solution in two batches, with the calcium ion concentration in each batch being 1.05 mM, the magnesium ion concentration being 0.15 mM, and the phosphate ion concentration being 0.35 mM;

[0109] Finally, the pH is adjusted to 7.0 to obtain the casein micelle solution simulating human milk;

[0110] The casein micelle solution simulating human milk contained a casein concentration of 0.17 mM, a citrate ion concentration of 2.3 mM, a calcium ion concentration of 7.1 mM, a magnesium ion concentration of 1.5 mM, and a phosphate ion concentration of 2.8 mM.

[0111] Example 4 Preparation of Simulated Human Milk Casein Micelles 2: Assembly Process (Continuous) Simulated Human Milk

[0112] The simulated human milk casein in Example 2 was used to prepare simulated human milk casein micelles, wherein the casein phosphorylation degree was 1.98, and the β-casein phosphorylation distribution pattern was a polyphosphorylated distribution with 0-5 phosphate groups; the citrate, calcium, magnesium, and phosphate ions in human milk and their total concentrations were used as the ionic environment; the initial pH was 5.8, and the pH was slowly increased by urea hydrolysis to achieve continuous assembly of simulated human milk casein micelles 2, the steps being as follows:

[0113] An initial salt solution containing potassium citrate, calcium chloride, magnesium chloride, and dipotassium hydrogen phosphate was prepared, wherein the concentration of citrate ion was 2.3 mM, the concentration of calcium ion was 7.1 mM, the concentration of magnesium ion was 1.5 mM, and the concentration of phosphate ion was 2.8 mM; the pH was adjusted to 5.8 using 0.1 mM HCl;

[0114] The simulated human milk casein of Example 2 was added to the initial salt solution to a concentration of 0.17 mM and stirred for 30 min; after the pH was readjusted to 5.8, urea was added to the mixed solution to a concentration of 2.5 mM, urease (1.0 U / mL) was added, and the pH of the solution was slowly raised to 7.0 after 60 min of equilibrium to obtain the simulated human milk casein micellar solution;

[0115] The concentration of casein in the solution simulating human milk casein micelles was 0.17 mM, the concentration of citrate ions was 2.3 mM, the concentration of calcium ions was 7.1 mM, the concentration of magnesium ions was 1.5 mM, and the concentration of phosphate ions was 2.8 mM.

[0116] Comparative Example 4 Human Casein Micelles

[0117] Fresh human milk (mature milk 3-8 months after birth, n=10) was centrifuged at 25°C and 3000g for 30 minutes to obtain skimmed human milk, which is a solution of natural human milk casein micelles.

[0118] Comparative Example 5 Recombinant Casein Micelles 1: Assembly Initial Ionic Environment and Process (Discontinuous) Not Simulating Human Milk

[0119] Recombinant casein micelles were prepared using the simulated human milk casein in Example 2, wherein the casein phosphorylation degree was 1.98, and the β-casein phosphorylation distribution pattern was a multi-phosphorylation distribution with 0-5 phosphate groups;

[0120] Recombinant casein micelles 1 were prepared as follows:

[0121] The simulated human milk casein in Example 2 was dissolved in water to a concentration of 0.17 mM and stirred for 30 min; the pH was adjusted to 7.0;

[0122] Potassium citrate, calcium chloride, magnesium chloride, and dipotassium hydrogen phosphate were added to a simulated human milk casein solution in three batches, with the concentration of citrate ion added in each batch being 0.77 mM, calcium ion concentration being 2.37 mM, magnesium ion concentration being 0.50 mM, and phosphate ion concentration being 0.93 mM;

[0123] Adjusting the pH to 7.0 to obtain the recombinant casein micelle 1;

[0124] The recombinant casein micelle 1 solution has a casein concentration of 0.17 mM, a citrate ion concentration of 2.3 mM, a calcium ion concentration of 7.1 mM, a magnesium ion concentration of 1.5 mM, and a phosphate ion concentration of 2.8 mM.

[0125] Table 4 shows the physicochemical properties and digestibility test results of the micelles obtained in Examples 3 and 4 and Comparative Examples 4 and 5. It can be seen from Table 4 that compared with the recombinant casein micelle 1, the average particle size, free calcium percentage, free casein percentage, degree of mineralization and hydration rate of the simulated human milk casein micelles 1 and 2 are closer to those of the human milk casein micelles.

[0126] A human milk casein micelle solution was maintained at 50°C for 30 minutes, followed by immediate centrifugation at 150,000g for 1 hour. The precipitate was then reconstituted in the ultrafiltration permeate of skimmed human milk to obtain a reconstituted human milk casein micelle solution with a casein concentration of 0.17 mM. This solution was then used for comparative gastric digestibility studies. Compared to recombinant casein micelle 1, the hydrolysis degrees of simulated human milk casein micelles 1 and 2 were closer to those of human milk casein micelles. During digestion, it was also observed that, like human milk casein micelles, both simulated human milk casein micelles 1 and 2 exhibited a more delicate flocculent structure, which facilitates hydrolysis by pepsin.

[0127] Table 4 Physicochemical properties of casein micelle solutions in Examples 3, 4 and Comparative Examples 4, 5

[0128]

[0129] Comparative Example 6 Recombinant Casein Micelles 2: Casein Phosphorylation Distribution Does Not Simulate Human Milk Micelles

[0130] The recombinant casein dephosphorylated for 15 minutes in Example 1 had a phosphorylation degree of 1.85, and recombinant casein micelles 2 were prepared according to the method of Example 3; wherein the casein composition and phosphorylation degree of the dephosphorylated recombinant casein simulated human milk, but the phosphorylation distribution did not simulate human milk.

[0131] The performance of the micelles obtained in Example 3 and Comparative Examples 4 and 6 was tested. The test results are shown in Table 5 below:

[0132] Table 5 Physicochemical properties of casein micelle solutions in Example 3, Comparative Examples 4 and 6

[0133]

[0134] It can be seen from Table 5 that compared with recombinant casein micelles 2, the average particle size, free calcium percentage, free casein percentage, mineralization degree, hydration rate and hydrolysis degree of simulated human milk casein micelles 1 are closer to those of human milk casein micelles. During the digestion process, it was also observed that the flocculent particles formed by recombinant casein micelles 2 are larger, which is not conducive to the hydrolysis of pepsin.

[0135] Comparative Example 7 Recombinant Casein Micelles 3: Casein phosphorylation degree does not simulate human milk - high phosphorylation

[0136] The non-dephosphorylated casein of Example 1 was used to prepare recombinant casein micelles 3 according to the method of Example 3.

[0137] Comparative Example 8 Recombinant Casein Micelles 4: Casein phosphorylation degree does not simulate human milk - low phosphorylation

[0138] The recombinant casein micelle 4 was prepared according to the method of Example 3 using the recombinant casein micelle that was dephosphorylated for 180 min and had a phosphorylation degree of 0.73 in Example 1.

[0139] The performance of the micelles obtained in Comparative Examples 4, 7, 8 and Example 3 was tested. The test results are shown in Table 6 below:

[0140] Table 6 shows that compared to recombinant casein micelles 3 and 4, the average particle size, free calcium percentage, free casein percentage, mineralization level, hydration rate, and degree of hydrolysis of simulated human milk casein micelle 1 are closer to those of human milk casein micelles. During digestion, it was also observed that the flocculent particles formed by recombinant casein micelles 3 and 4 were larger, which is not conducive to hydrolysis by pepsin. With the removal of phosphate groups, the calcium ion bridges formed by the binding of casein and calcium decrease, resulting in more calcium and casein distributed in the serum and a decrease in the degree of micelle mineralization. When phosphate groups are largely removed, the net negative charge of casein is greatly reduced, the electrostatic repulsion between caseins decreases, and the hydrophobic binding interaction increases, thus promoting the substantial aggregation of casein.

[0141] Table 6 Physicochemical properties of casein micelle solutions in Example 3, Comparative Examples 4, 7, and 8

[0142]

[0143] Comparative Example 9 Recombinant Casein Micelles 5: No Magnesium Ions Added

[0144] Take the simulated human milk casein in Example 2, omit the magnesium chloride in Example 3, and keep the other steps consistent with Example 3 to obtain recombinant casein micelles 5.

[0145] The obtained protein micelles were subjected to performance tests, and the test results are shown in Table 7 below:

[0146] It can be seen from Table 7 that compared with recombinant casein micelles 5, the average particle size, solution absorbance and hydration rate of simulated human milk casein micelles 1 are closer to those of human milk casein micelles, indicating that although the content of divalent magnesium ions in human milk is lower than that of divalent calcium ions, they are also important for the assembly of the simulated human milk micelle structure.

[0147] Table 7 Physicochemical properties of casein micelle solutions in Example 3, Comparative Examples 4 and 9

[0148]

[0149] Example 5 Key ion regulation in the preparation of simulated human milk casein micelles: calcium and magnesium ion concentrations

[0150] The simulated human milk casein in Example 2 was used, the calcium ion concentration in Example 3 was adjusted to 1.1-11.1 mM, and the molar ratio of calcium and magnesium ions was fixed to 7.1:1.5; the other steps were consistent with Example 3 to obtain a series of recombinant casein micelle solutions.

[0151] The absorbance of the obtained series of recombinant casein micelle solutions was tested, and the results are shown in Figure 3 :

[0152] from Figure 3 It can be seen that when the calcium ion concentration is 5.6-7.2 mM, the absorbance of the solution is within the absorbance range of human milk casein micelle solution. Therefore, it is preferred to prepare simulated human milk casein micelles with a calcium ion concentration of 5.6-7.2 mM and a magnesium ion concentration of 1.1-1.6 mM.

[0153] Example 6 Key ion regulation in the preparation of simulated human milk casein micelles: phosphate ion concentration

[0154] The simulated human milk casein in Example 2 was taken, and the final concentration of phosphate ions in Example 3 was adjusted to 0-14 mM. The other steps were kept consistent with Example 3 to obtain a series of recombinant casein micelle solutions.

[0155] The absorbance of the obtained series of recombinant casein micelle solutions was tested, and the results are shown in Figure 4 :

[0156] from Figure 4 It can be seen that when the phosphate ion concentration is between 1.4-3.5 mM, the absorbance of the solution is within the absorbance range of human milk casein micelle solution. Therefore, a phosphate ion concentration of 1.4-3.5 mM is preferably used to prepare simulated human milk casein micelles.

[0157] Comparative Example 10 Preparation of recombinant casein micelles 6

[0158] (1) Dephosphorylation of compound casein:

[0159] Dissolve the compound casein in water to obtain a casein solution with a concentration of 5 mg / mL; dissolve potato acid phosphatase in Tris-HCl buffer with a pH of 6.8 to obtain an enzyme solution with a concentration of 0.48 U / mL;

[0160] The compound casein solution and potato acid phosphatase solution were mixed in a volume ratio of 1:1 and kept in a 37°C water bath for 2, 30, and 180 minutes, respectively, followed by keeping at 80°C for 10 minutes to inactivate the enzyme. The mixture was then dialyzed for 72 hours at 4°C using a dialysis membrane with a molecular weight cutoff of 7000 Da to remove salts. After dialysis, the mixture was freeze-dried to obtain compound caseins with phosphorylation degrees of 2.72, 1.83, and 0.72.

[0161] (2) Preparation of recombinant casein 3:

[0162] Compound caseins with phosphorylation degrees of 2.72, 1.83, and 0.72 were mixed at a mass ratio of 1:2:0.4 to obtain recombinant casein 3 with a phosphorylation degree of 1.96;

[0163] (3) Preparation of recombinant casein micelles 6:

[0164] An initial salt solution containing potassium citrate, calcium chloride, magnesium chloride, and dipotassium hydrogen phosphate was prepared, wherein the concentration of citrate ion was 2.3 mM, the concentration of calcium ion was 5.0 mM, the concentration of magnesium ion was 1.2 mM, and the concentration of phosphate ion was 2.1 mM; the pH was adjusted to 7.0 using 0.1 mM sodium hydroxide;

[0165] Add recombinant casein 3 to the initial salt solution to a concentration of 0.17 mM and stir for 30 minutes; adjust the pH to 7.0;

[0166] Calcium chloride, magnesium chloride, and potassium hydrogen phosphate were added to the mixed solution in two batches, with the calcium ion concentration in each batch being 1.05 mM, the magnesium ion concentration being 0.15 mM, and the phosphate ion concentration being 0.35 mM;

[0167] Finally, the pH is readjusted to 7.0 to obtain the solution of recombinant casein micelles 6;

[0168] The casein concentration in the solution of recombinant casein micelle 6 was 0.17 mM, the citrate ion concentration was 2.3 mM, the calcium ion concentration was 7.1 mM, the magnesium ion concentration was 1.5 mM, and the phosphate ion concentration was 2.8 mM.

[0169] Table 8 shows the performance test results of the micelles obtained in Example 3 and Comparative Examples 4 and 10. It can be seen from Table 8 that compared with recombinant casein micelles 6, the average particle size, free calcium ratio, free casein ratio, mineralization degree and hydration rate of the simulated human milk casein micelles 1 are closer to those of human milk casein micelles. Among them, the urea electrophoresis band of the dephosphorylated composite casein prepared by potato acid phosphatase shows a diffuse background. The higher the degree of dephosphorylation, the more diffuse it is. This may be related to the proteolytic side reaction of potato acid phosphatase. During dephosphorylation, the primary structure of the protein molecule changes, thereby affecting the ability of recombinant casein 3 to assemble and form micelles.

[0170] Table 8 Physicochemical properties of casein micelle solutions in Example 3 and Comparative Examples 4 and 10

[0171]

[0172] 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 preparing casein micelles with phosphorylation characteristics simulating human milk, characterized in that: The steps include: (1) Compound caseins with phosphorylation degrees of 2.74, 1.85 and 0.73 were mixed in a mass ratio of 1:2:0.4 to obtain simulated human milk casein; wherein, κ-casein, α s1 -Casein and β-casein account for 17-23%, 9-15% and 65-71% respectively, and do not contain α s2 -Casein; β-casein phosphorylation distribution shows 6 variants from 0 to 5P; (2) The method for preparing the human milk-simulating casein micelles comprises: An initial salt solution containing calcium chloride, potassium citrate, magnesium chloride, and dipotassium hydrogen phosphate is prepared, wherein the concentration of citrate ions is 1.35-2.7 mM, the concentration of calcium ions is 4.8-5.2 mM, the concentration of magnesium ions is 1.0-1.2 mM, and the concentration of phosphate ions is 1.0-2.2 mM, and the pH is adjusted to 6.7-7.2; simulated human milk casein is added to the initial salt solution to a concentration of 0.1-0.2 mM, mixed evenly, and the pH is readjusted to 6.7-7.2; calcium chloride, magnesium chloride, and dipotassium hydrogen phosphate are continuously added to the mixed solution in two batches, with the calcium ion concentration of each batch being 0.4-1.1 mM, the magnesium ion concentration being 0.05-0.2 mM, and the phosphate ion concentration being 0.2-0.65 mM, and the pH is readjusted to 6.7-7.2, to obtain a solution of simulated human milk casein micelles; wherein the casein concentration is 0.1-0.2 mM, citrate ion concentration is 1.35-2.7 mM, calcium ion concentration is 5.6-7.2 mM, magnesium ion concentration is 1.1-1.6 mM, and phosphate ion concentration is 1.4-3.5 mM; Alternatively, the method for preparing the human milk-simulating casein micelles comprises: An initial salt solution containing calcium chloride, potassium citrate, magnesium chloride, and dipotassium hydrogen phosphate is prepared, wherein the concentration of citrate ions is 1.35-2.7 mM, the concentration of calcium ions is 5.6-7.2 mM, the concentration of magnesium ions is 1.1-1.6 mM, and the concentration of phosphate ions is 1.4-3.5 mM, and the pH is adjusted to 5.8-5.9; simulated human milk casein is added to the initial salt solution to a concentration of 0.1-0.2 mM, mixed evenly, and the pH is readjusted to 5.8-5.9; urea is added to the mixed solution to a concentration of 2.5-3.0 mM, urease is added to an enzyme activity of 1.0 U / mL, and the pH of the solution is slowly raised to 6.7-7.2 after equilibrium to obtain a solution of simulated human milk casein micelles; wherein the casein concentration is 0.1-0.2 mM, citrate ion concentration is 1.35-2.7 mM, calcium ion concentration is 5.6-7.2 mM, magnesium ion concentration is 1.1-1.6 mM, and phosphate ion concentration is 1.4-3.5 mM.

2. The method according to claim 1, characterized in that The preparation method of compound casein with phosphorylation degrees of 2.74, 1.85 and 0.73 in step (1) is as follows: Compound casein was prepared into a compound casein solution; bovine intestinal alkaline phosphatase was prepared into an enzyme solution; the compound casein solution and the enzyme solution were then mixed in a volume ratio of 1:1 and incubated in a water bath at 37°C for 1, 15, and 180 min, respectively; the enzyme was then inactivated, dialyzed, and freeze-dried to obtain compound caseins with phosphorylation degrees of 2.74, 1.85, and 0.

73.

3. The method according to claim 2, wherein The concentration of the reconstituted casein solution is 4-6 mg / mL.

4. The method according to claim 2, wherein The concentration of the enzyme solution is 0.3-0.5 U / mL.

5. The method according to claim 2, characterized in that The solvent of the enzyme solution is Tris-HCl buffer with a pH of 8.0; the solvent of the composite casein solution is water.

6. Casein micelles with phosphorylation characteristics mimicking human milk, prepared by the method according to any one of claims 1 to 5.

7. Use of the casein micelles with phosphorylation characteristics simulating human milk according to claim 6 in the field of preparing dairy products.

8. The use according to claim 7, characterized in that The application includes providing casein ingredients for the research and development and production of infant formula powder.

9. The use according to claim 8, characterized in that The application is to ultrafilter a solution of casein micelles with phosphorylated characteristics simulating human milk through a 5-20 kDa organic membrane, concentrate it to 20-50 times, and then take the concentrated liquid, spray drying or freeze drying it to obtain casein ingredients for the research and development and production of infant formula powder.

10. A method for improving the similarity of simulated human milk casein micelles to human milk casein in terms of casein phosphorylation degree and distribution, and mineralization degree, characterized in that: The casein micelles with phosphorylation characteristics simulating human milk as claimed in claim 6 are used.

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