Preparation method of low-mineralization whole-casein recombinant micelle and stable emulsion of low-mineralization whole-casein recombinant micelle

Abstract

The invention discloses a preparation method of a low-mineralization whole casein recombinant micelle and a stable emulsion thereof, and belongs to the technical field of dairy product processing. The preparation method comprises the following steps: aiming at animal latex bundle-state casein ingredients, carrying out decalcification treatment by adopting ion exchange resin to obtain decalcified micelle-state casein; and regulating and controlling recombination environments such as casein concentration, ion species and addition concentration by regulating and controlling recombination modes such as ion addition sequence, addition batch, addition time interval, recombination pH and temperature, and further screening the decalcification rate of the micelle-state casein, thereby preparing the low-mineralization full-casein recombinant micelle. And the mineralization degree and the micelle structure are close to those of human casein micelles. Afterwards, anhydrous milk fat is added into the whole casein recombinant micelle solution, and emulsion with stable low-mineralization whole casein recombinant micelles is prepared; the improvement of protein and lipid digestibility and calcium bioavailability is realized, the fatty acid content of calcium soap is reduced, and the full utilization of animal milk casein is realized.

Description

Technical Field

[0001] The invention relates to a method for preparing a low-mineralized whole casein recombinant micelle and a stable emulsion thereof, and belongs to the technical field of dairy product processing. Background Art

[0002] Infant formula is the best breast milk substitute when breastfeeding is not possible or insufficient, and can provide infants with a variety of nutrients such as protein, lipids, and minerals. In the preparation process of infant formula, an emulsion is prepared by a homogenization process, and the surface of the fat droplets is coated with protein, which can reduce the mutual aggregation of fat droplets or the separation of water and oil phases. The presence of casein in infant formula will affect the stability of the emulsion, and at the same time affect the flocculation behavior and digestion and absorption performance of infant formula in gastrointestinal fluid, thereby affecting the growth and development of infants.

[0003] Casein in milk mainly exists in the form of micelle structure, and casein subcomponents and colloidal calcium phosphate form the skeleton structure inside the micelle through calcium ion bridging. Casein used in infant formula milk powder mainly comes from cow's milk or goat's milk, which is quite different from human milk casein in terms of mineralization degree and structural characteristics, and thus there are also great differences in emulsification and digestibility. The mineralization degree of cow's milk casein micelles is high, and the molar ratio of micellar calcium to micellar casein can reach 18-19, while the ratio in human milk is only 7-9, which also makes the particle size of human milk micelles smaller and the internal structure looser, which is easier to digest and absorb. Patent CN 115316488B shows that decalcified cow's milk casein micelles are prepared by adding calcium ion chelating agents or ion exchange, which are more likely to form small and loose clot structures during gastric digestion, thereby greatly improving the digestibility of protein. Casein micelles are in dynamic equilibrium with the solution environment, and their mineralization degree and structure are affected by the solution ion environment. When decalcified casein micelles are used in the production of infant formula, in order to meet the national standards (GB 10765-2021 "National Food Safety Standard Infant Formula", GB 10766-2021 "National Food Safety Standard Older Infant Formula", GB 10767-2021 "National Food Safety Standard Toddler Formula") for the mineral level of infant formula, it is also necessary to add minerals such as calcium salts and phosphates. The addition conditions of these minerals (such as level and method) will further affect the mineralization degree and structure of casein micelles, and then affect the digestibility of infant formula. In order to achieve the improvement of the digestibility of infant formula, how to regulate the conditions for mineral addition will be particularly important, and no relevant reports have been seen.

[0004] Milk contains α s -, β-, and κ-casein, the ratio of the three is about 50:35:15; human milk contains β-, α s-, κ-casein, the ratio of the three is about 68:12:20. In patent CN 114601013B, the selective separation technology is used to separate and purify the bovine milk casein subcomponents, and then compounded according to the ratio of each subcomponent in human milk casein micelles, and citrate ions, calcium ions and phosphate ions are added according to the level of each mineral salt ion in human milk to prepare recombinant casein micelles, which are similar to natural human milk micelles in casein composition, mineralization degree and structure, and digestibility is also relatively close. The preparation process is complicated, industrial production is difficult and costly, and it will also lead to α s -The surplus of casein cannot realize the full utilization of casein. Bovine milk casein micelle is a natural spherical polymer, in which α s -, β-, and κ-caseins contain 8-13, 5, and 1 phosphate groups, respectively. The different casein compositions, phosphorylation degrees (i.e., charge numbers), and aggregation states result in different interactions with ions, which results in different schemes and conditions for reconstructed micelles, different micelle structural characteristics, and different conditions for emulsion preparation.

[0005] During the processing and storage of infant formula, the stability of the emulsion will affect the stability of lipids as well as the uniformity and solubility of the product. The stability of the emulsion is related to the properties of the lipid droplet size and potential, the viscoelasticity of the interfacial membrane, etc., which are in turn related to the composition of the stable particles, the number of phosphate groups and the particle size. Increasing the homogenization pressure can improve the stability of the emulsion by reducing the lipid droplet size. However, the average particle size of bovine milk casein micelles is large (150-200nm), and it is not easy to quickly adsorb to the interface and stabilize the interface. In addition, the dense clots formed by bovine milk casein micelles in gastric juice will wrap the lipid droplets, thereby affecting the digestion and absorption of lipids. The fatty acids released by lipid digestion, especially long-chain saturated fatty acids, are easily combined with calcium ions in the solution to form calcium soaps, thereby reducing the bioavailability of lipids and calcium, and also affecting the gastrointestinal health of infants and young children. Bovine milk α s -, β-, and κ-casein have different numbers of phosphate groups, and the phosphopeptides produced by their digestion have different abilities to compete for calcium binding, and thus different abilities to inhibit the formation of calcium soaps. In addition, the citrate ions and phosphate ions in the system will also compete for calcium binding, thereby affecting the availability of lipids and calcium.

[0006] In summary, using animal milk whole casein as raw material, regulating the ion environment will affect the mineralization degree, particle size and other structural characteristics of the recombinant casein micelles, thereby affecting the structure and stability of the stabilized emulsion, and at the same time affecting the digestion and absorption of protein, lipid and calcium. This is of great significance for achieving full utilization of casein resources, and no relevant reports have been seen. Summary of the invention

[0007] [Technical issues]

[0008] When decalcified animal milk casein micelles are used in the production of infant formula, minerals need to be added to meet the national standards for the mineral level of infant formula.

[0009] In order to achieve structural simulation of human milk micelles by casein micelles and thus improve the protein digestibility of infant formula milk powder, it is particularly important to regulate the level and method of mineral addition, but no relevant reports have been found.

[0010] Furthermore, in order to improve the stability of the emulsion during the processing and storage of infant formula milk powder, and at the same time improve the bioavailability of lipids and calcium, how to add minerals and how to prepare the emulsion will be particularly important, and there have been no relevant reports.

[0011] [Technical solution]

[0012] In order to solve the above problems, the present invention uses ion exchange resin to decalcify the animal latex micellar casein ingredients to obtain decalcified micellar casein; and referring to the mineral ion composition of human milk, by adjusting the ion addition sequence, addition batch, addition time interval, reconstitution pH and temperature and other reconstitution methods, the casein concentration, ion type and addition concentration and other reconstitution environments are adjusted, and then the decalcification rate of micellar casein is screened to prepare low-mineralized whole casein reconstituted micelles, which are close to human milk casein micelles in mineralization degree and micellar structure; then, the present invention adds anhydrous milk fat to the low-mineralized whole casein reconstituted micelle solution, stirs for balance, shears and homogenizes, and performs high-pressure homogenization, by adjusting the milk fat addition sequence and emulsification temperature, and further screening the decalcification rate and the concentrations of calcium ions, citrate ions and phosphate ions, to prepare a stable emulsion of low-mineralized whole casein reconstituted micelles. The emulsion prepared by the invention is stable, has a small lipid droplet size and a low negative potential, improves protein and lipid digestibility and calcium bioavailability, reduces the content of calcium soap fatty acids, and realizes full utilization of animal milk casein.

[0013] The first object of the present invention is to provide a method for preparing low-mineralized whole casein recombinant micelles, comprising the following steps:

[0014] The temperature of the decalcified micellar casein solution was adjusted to 37-57°C and the pH to 6.6-7.2; calcium ions, citrate ions and phosphate ions were then added, and the temperature was adjusted to 25°C and the pH to 7.0, and the volume was fixed to obtain a low-mineralized whole casein reconstituted micellar solution, wherein α s1 -、α s2 The mass proportions of -, β-, and κ-casein are 5-45%, 5-25%, 25-75%, and 5-25%, respectively.

[0015] In one embodiment of the present invention, the calcium desorption rate of the decalcified micellar casein solution is 42%-81%; preferably, the calcium desorption rate is 42%-61%.

[0016] In one embodiment of the present invention, the decalcified micellar casein solution is a decalcified micellar casein aqueous solution with a mass concentration of 0.8%-2.8%; so that the casein mass concentration in the prepared low-mineralized whole casein recombinant micellar solution is 0.4%-1.4%.

[0017] In one embodiment of the present invention, the method for preparing the decalcified micellar casein solution is:

[0018] The sterilized skim milk is taken and subjected to microfiltration and diafiltration treatment to obtain a micellar casein retentate; a cation exchange resin is then added to perform a decalcification treatment at room temperature, the resin is removed by gauze filtration, and the solution is diluted to obtain a decalcified micellar casein solution;

[0019] Among them, the addition amount of cation exchange resin is 48.0-146.7g / 100g casein, and the decalcification rate of micellar casein is controlled to be 42%-81%; preferably, the addition amount of cation exchange resin is 48.0-86.7g / 100g casein, and the decalcification rate of micellar casein is controlled to be 42%-61%.

[0020] In one embodiment of the present invention, calcium ions are added in the form of a calcium ion solution; the calcium ions are introduced by one or both of calcium chloride and calcium nitrate, the concentration of the calcium ion solution is 31.4-51.4 mM, and the calcium ion concentration in the low-mineralized whole casein recombinant micelle solution is 6-9 mM.

[0021] In one embodiment of the present invention, citrate ions are added in the form of a citrate ion solution; the citrate ions are introduced by one or both of sodium citrate and potassium citrate, and the concentration of the citrate ion solution is 199-499 mM; the molar ratio of calcium ions to citrate ions in the solution of low-mineralized whole casein recombinant micelles is 1:0.27-1:0.67.

[0022] In one embodiment of the present invention, phosphate ions are added in the form of a phosphate ion solution; the phosphate ions are introduced by one or both of disodium hydrogen phosphate and dipotassium hydrogen phosphate, the concentration of the phosphate ion solution is 0-36.2 mM, and the molar ratio of calcium ions to phosphate ions in the solution of low-mineralized whole casein recombinant micelles is 1:0.17-1:0.90.

[0023] In one embodiment of the present invention, the addition of calcium ions, citrate ions and phosphate ions involves three orders of adding the three ions, including sequentially adding a citrate ion solution, a calcium ion solution and a phosphate ion solution, or sequentially adding a citrate ion solution, a phosphate ion solution and a calcium ion solution, or sequentially adding a phosphate ion solution, a citrate ion solution and a calcium ion solution.

[0024] In one embodiment of the present invention, the addition of calcium ions, citrate ions and phosphate ions involves 3-5 addition batches of the three ions, that is, the citrate ion solution, the calcium ion solution and the phosphate ion solution are equally divided into 3-5 portions (either by volume or by mass), and each portion of the ion solution is added in sequence according to the order of ion addition, and the above addition order is repeated 2-4 times until all the ion solutions are added.

[0025] In one embodiment of the present invention, the addition of calcium ions, citrate ions and phosphate ions involves a time interval of 15-20 minutes after the addition of the three ions, that is, after adding one portion of ion solution, it takes 15-20 minutes to balance before adding the next portion of ion solution.

[0026] In one embodiment of the present invention, the pH is adjusted to 6.6-7.2 by using 0.05-0.1 M NaOH solution, KOH solution or HCl solution.

[0027] In one embodiment of the present invention, the pH value is adjusted to 7.0 by using 0.05-0.1 M NaOH solution, KOH solution or HCl solution.

[0028] In one embodiment of the present invention, the volume is adjusted by adding water.

[0029] The second object of the present invention is the low-mineralized whole casein reconstituted micellar solution prepared by the method of the present invention.

[0030] The third object of the present invention is to provide a casein ingredient, which is obtained by drying the low-mineralized whole casein recombinant micelle solution of the present invention.

[0031] In one embodiment of the present invention, the preparation method of casein ingredient is as follows:

[0032] The low-mineralized whole casein recombinant micellar solution is concentrated by nanofiltration or reverse osmosis at 35-55° C. and 3-5 MPa, and then spray-dried or freeze-dried to obtain a casein ingredient.

[0033] The fourth object of the present invention is to provide a method for preparing a low-mineralized whole casein recombinant micelle-stabilized emulsion, comprising the following steps:

[0034] Anhydrous milk fat is added to the low-mineralized whole casein recombinant micelle solution of the present invention, the temperature is maintained at 40-60°C, the mixture is stirred and balanced at 300-500rpm for 10-20min, shear homogenized at a speed of 20000-30000rpm for 5-10min and high-pressure homogenized at 30-50MPa for 3-5 times, and the temperature is then adjusted to 25°C to obtain a stable emulsion of low-mineralized whole casein recombinant micelles.

[0035] In one embodiment of the present invention, the dosage ratio of the low-mineralized whole casein reconstituted micelle solution and anhydrous milk fat is 20 mL:0.7 g.

[0036] In one embodiment of the present invention, the stirring balance, shear homogenization and high pressure homogenization are all maintained at 40-60°C.

[0037] In one embodiment of the present invention, when the molar ratio of calcium ions to citrate ions in the low-mineralized whole casein recombinant micellar solution is 1:0.57-1:0.67 and the molar ratio of calcium ions to phosphate ions is 1:0.17-1:0.33, the formation of calcium soap is further reduced and the bioaccessibility of calcium is improved, thereby achieving the effect of synergistically promoting calcium and fatty acid absorption.

[0038] The fifth object of the present invention is the low-mineralized whole casein recombinant micelle-stabilized emulsion prepared by the method of the present invention.

[0039] In one embodiment of the present invention, the fat concentration in the emulsion stabilized by low-mineralized whole casein recombinant micelles is 3-4%, where % is mass percentage.

[0040] The sixth object of the present invention is to provide an infant formula powder, which uses the solution of low-mineralized whole casein recombinant micelles, casein ingredients or low-mineralized whole casein recombinant micelles stabilized emulsion described in the present invention.

[0041] The seventh objective of the present invention is to use the solution of the low-mineralized whole casein recombinant micelles, the casein ingredients or the emulsion stabilized by the low-mineralized whole casein recombinant micelles described in the present invention in the field of dairy product processing.

[0042] The eighth object of the present invention is to provide a method for improving the calcium bioaccessibility of a whole casein recombinant micelle-stabilized emulsion, which uses the solution of the low-mineralized whole casein recombinant micelles, the casein ingredients or the emulsion stabilized by the low-mineralized whole casein recombinant micelles described in the present invention.

[0043] In one embodiment of the present invention, when the molar ratio of calcium ions to citrate ions is 1:0.57-1:0.67, and the molar ratio of calcium ions to phosphate ions is 1:0.17-1:0.33, a synergistic effect of promoting calcium absorption is exhibited.

[0044] The ninth object of the present invention is to provide a method for reducing the amount of calcium soap fatty acid generated in an emulsion stabilized by whole casein recombinant micelles after gastrointestinal digestion, which uses the solution of low-mineralized whole casein recombinant micelles, casein ingredients or emulsion stabilized by low-mineralized whole casein recombinant micelles described in the present invention.

[0045] In one embodiment of the present invention, when the molar ratio of calcium ions to citrate ions is 1:0.57-1:0.67, and the molar ratio of calcium ions to phosphate ions is 1:0.17-1:0.33, the effect of synergistically reducing the formation of calcium soap is exhibited.

[0046] [Beneficial Effects]

[0047] (1) The present invention simulates the ionic environment of human milk by replenishing mineral salt ions into decalcified micellar casein, and further regulates the micelle decalcification rate, the method of adding mineral salt ions, the pH and temperature during reconstitution, and the concentrations of calcium ions, citrate ions, phosphate ions and casein, to prepare low-mineralized whole casein reconstituted micelles with low mineralization degree, small average particle size and high hydration rate, which are structurally similar to human milk casein micelles.

[0048] (2) The present invention adds anhydrous milk fat to the low-mineralized whole casein recombinant micellar solution, and prepares a stable emulsion by regulating the milk fat addition order and the emulsification temperature, and further regulating the decalcification rate and the concentrations of calcium ions, citrate ions and phosphate ions. The lipid droplet size is small and the negative potential is low, which can inhibit the water-oil phase stratification during the processing of infant formula milk powder and is conducive to the re-dissolution of infant formula milk powder.

[0049] (3) The low-mineralized whole casein recombinant micelle-stabilized emulsion prepared by the present invention can reduce the flocculation of the emulsion in the stomach stage and the encapsulation effect of the flocculation structure on the lipid droplets, while improving the digestibility of casein and lipids, thereby achieving full utilization of animal milk casein resources.

[0050] (4) The low-mineralized whole casein recombinant micellar emulsion prepared by the present invention can reduce the content of calcium soap fatty acids by reducing the combination of calcium ions and released free fatty acids during digestion, which is beneficial for alleviating intestinal health problems such as constipation and gastrointestinal discomfort in infants and young children; it can also improve the bioaccessibility of calcium, which plays an important role in promoting the bone development of infants and young children.

[0051] (5) The present invention further achieves a synergistic absorption-promoting effect by combining increasing the concentration of citrate ions with decreasing the concentration of phosphate ions, thereby further reducing the formation of calcium soaps in feces and increasing the bioavailability of fatty acids and calcium. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1These are photos of the appearance of digested products formed by the casein micelle-stabilized emulsions in Example 12 and Comparative Examples 4-6 after being digested in a simulated infant stomach for 0 and 60 minutes. DETAILED DESCRIPTION

[0053] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0054] The raw materials used in the embodiments and comparative examples are:

[0055] 1. Preparation of micellar casein

[0056] Fresh milk was skimmed by disc centrifugation and sterilized by 1.4μm ceramic membrane to obtain sterilized skim milk. Then, micellar casein was separated by microfiltration using 100nm ceramic membrane, where the concentration was 3 times, and 2 volumes of deionized water were added and then diafiltration was performed 3 times. The microfiltration and diafiltration temperature was set at 45℃, and the retentate obtained was micellar casein (MCC) retentate.

[0057] 2. Preparation of anhydrous milk fat

[0058] Fresh milk was skimmed by disc centrifugal degreasing to obtain cream, which was matured at 4°C for 12 hours and then broken by a fat breaker until obvious clots were formed. After the buttermilk was removed, it was washed twice with precooled deionized water, and the butter clots were collected by gauze filtration, heated to dissolve at 60°C, and then centrifuged at 8000×g for 30 minutes to collect the upper layer of anhydrous milk fat.

[0059] 3. Separation of micellar phase and whey phase

[0060] The casein micelle solution was taken and ultracentrifuged at 150,000×g for 60 min at room temperature. The resulting precipitate and supernatant were the micelle phase and whey phase, respectively.

[0061] 4. Separation of mixed micelle phase and precipitate phase

[0062] The product was removed and centrifuged at 10,000 × g for 60 min at 4°C. The intermediate phase obtained was the mixed micelle phase, and the precipitate contained calcium soap.

[0063] In the examples and comparative examples, % without specific meaning refers to mass percentage, and solutions without specific solvents refer to water as solvent; w / v is 10 mg / mL; and the room temperature is 20-30°C.

[0064] The test methods used in the embodiments and comparative examples are:

[0065] 1. Determination of decalcification rate

[0066] The determination of calcium content refers to the first method of national standard 5009.268-2016 "National Food Safety Standard Determination of Multiple Elements in Food", that is, inductively coupled plasma mass spectrometry (ICP-MS) is used for determination.

[0067] Take the MCC retentate, add water to dilute it to a MCC solution with a casein concentration of 0.8% (w / v, 8 mg / mL), ultracentrifuge it at 150,000×g for 60 min at room temperature, and then centrifuge the supernatant at 2000×g for 60 min using a 10 kDa ultrafiltration centrifuge tube to obtain an ultrafiltration permeate.

[0068] The total calcium content in the MCC solution and the soluble calcium content in the ultrafiltration permeate were determined separately, and the difference between the two was the micellar calcium content.

[0069] The calculation formula of casein micelle decalcification rate is as follows:

[0070]

[0071] 2. Turbidity determination

[0072] The turbidity of the casein micelle solution was measured by a spectrophotometer, wherein the detection wavelength was 400 nm and the sample cell thickness was 10 mm.

[0073] 3. Determination of casein composition

[0074] The casein composition was determined using an e2695 high performance liquid chromatograph (Waters Corp., Milford, MA, USA) with an XBridge BEH C18 column (250 mm × 4.6 mm) and a detection wavelength of 220 nm.

[0075] 4. Determination of free casein

[0076] The casein micelle solution and the supernatant were taken and the casein content was determined by Kjeldahl method with a conversion factor of 6.38.

[0077] The formula for calculating the percentage of free casein is:

[0078]

[0079] 5. Determination of mineralization degree

[0080] The micellar calcium content in the casein micellar solution was determined by ICP-MS, and the casein content was determined by the Kjeldahl method. The calculation formula for the micellar mineralization degree is as follows:

[0081]

[0082] 6. Determination of micelle size

[0083] The particle size distribution of casein micelles was determined using a Malvern nanometer particle size potentiometer (Malvern Instruments Ltd., Malvern, Worcestershire, UK), and the refractive index of the protein particles was set to 1.57.

[0084] 7. Determination of micelle hydration rate

[0085] The micellar phase after ultracentrifugation was taken, dried at 103°C for 7 h and the dry basis moisture content of the precipitate was determined, which was the micellar hydration rate.

[0086] 8. Determination of lipid droplet size and potential

[0087] The lipid droplet size distribution was determined using a Microtrac S3500 laser particle size analyzer (Microtrac Inc., Largo, FL, USA). The refractive index of the dispersed phase was set to 1.33 and the refractive index of the oil phase was set to 1.46. The lipid droplet potential was determined using a Malvern nanoparticle size potentiometer.

[0088] 9. Determination of emulsion stability

[0089] The emulsion stability was determined using a LUMisizer stability analyzer (LUM. GmbH, Berlin, Germany), with a centrifugal speed of 2000 rpm, a test temperature of 25°C, a scanning time interval of 10 s, and a scanning number of 200 times.

[0090] 10. Determination of in vitro gastrointestinal digestion in infants

[0091] The casein micelle solution was taken and mixed with simulated gastric digestive fluid (724.3U / mL pepsin, 51.4U / mL gastric lipase, 94mM sodium chloride, 13mM potassium chloride, pH 5.3) at a volume ratio of 63:37, and then the pH of the mixture was adjusted to 5.3 with 1M HCl solution, and digested at 37°C for 60 minutes. After the gastric digestion, the pH of the gastric digest was adjusted to 6.6 with 1M NaOH solution, and mixed with simulated intestinal digestive fluid (42.1U / mL trypsin, 236.8U / mL pancreatic lipase, 249mM sodium chloride, 10mM potassium chloride, 8.2mM bile salt, pH 6.6) at a volume ratio of 62:38, and digested at 37°C for 60 minutes, and the pH of the intestinal digest was maintained at 6.6 by adding 1M NaOH solution. The digesta at 0 min and 60 min of the gastric digestion stage were collected, placed in a small transparent dish and photographed using a camera.

[0092] 11. Determination of absorption in young mice

[0093] One-week-old Sprague-Dawley rats were purchased and fed for one week in a constant temperature and humidity environment. After that, the rats were fasted for 6 hours, weighed, and gavaged with micellar emulsion samples at a volume / body weight ratio of 1 mL:36 g, and the feces of the rats were collected.

[0094] 12. Determination of free amino content

[0095] The digested sample was precipitated with 6.2% (w / v) trichloroacetic acid solution and centrifuged at 10000×g for 30 min. Then 150 μL of the supernatant was mixed with 3 mL of o-phthalaldehyde solution and reacted for 15 min in the dark. The absorbance was measured at 340 nm. A standard curve was drawn with L-leucine as the standard, and the results were expressed in μmol / mg protein.

[0096] 13. Determination of lipolysis degree

[0097] The fat components of the digest were extracted using chloroform-methanol solution. After the chloroform solvent was removed by rotary evaporation, the fat was dissolved using n-hexane-isopropanol-formic acid mobile phase.

[0098] The analysis of fat digestion products was performed using a Waters e2695 high performance liquid chromatograph and a Waters 2414 differential refractometer detector, and the chromatographic column used was a Sepax HP silica gel column (250 mm×4.6 mm).

[0099] The calculation formula of lipolysis degree is as follows:

[0100]

[0101] Where [TAG], [DAG], [MAG] and [FFA] are the molar concentrations of triglycerides, diglycerides, monoglycerides and free fatty acids, respectively.

[0102] 14. Determination of fatty acid content of calcium soap

[0103] Take the precipitate phase obtained after centrifugation of the digest in the raw material preparation method 4, or the feces of young rats in animal experiments, and add n-hexane, n-hexane-isopropanol (3:2, v / v) and water in sequence to remove the fat components that are not involved in the formation of calcium soap. Then, n-hexane is used to extract the calcium soap fatty acid, and then 14% (v / v) boron trifluoride-methanol solution is added at 70°C for methyl esterification, and then n-hexane is added to extract the fatty acid methyl ester component, and the analysis is performed using a gas chromatography-mass spectrometer.

[0104] 15. Determination of Calcium Bioaccessibility

[0105] ICP-MS was used to determine the calcium content in the digestate and mixed micelle phase after intestinal digestion. The calculation formula for calcium bioaccessibility is as follows:

[0106]

[0107] 16. Determination of relative fecal calcium content

[0108] ICP-MS was used to determine the total amount of calcium in the feces and intragastric emulsion of young mice. The calculation formula for the relative content of fecal calcium is as follows:

[0109]

[0110] Example 1 Low-mineralized whole casein reconstituted micelles

[0111] A method for preparing low-mineralized whole casein recombinant micelles comprises the following steps:

[0112] AmberliteSR1LNa strong acidic cation exchange resin was added to MCC retentate (casein concentration 7.5%, w / v) at 6.5 g / 100 g retentate, and decalcified for 4 h at room temperature. The resin was then removed by filtering with 4 layers of gauze to obtain a decalcified MCC retentate, which was then diluted with water to obtain an MCC solution with a casein concentration of 0.8% (w / v) and a decalcification rate of 61%;

[0113] Take 10mL of decalcified MCC solution, adjust the pH to 7.0 with 0.05M NaOH solution, and maintain the temperature at 37°C; divide into three batches and add one portion of potassium citrate solution, one portion of calcium chloride solution, and one portion of dipotassium hydrogen phosphate solution in each batch in turn. After adding each portion of salt solution, balance for 15 minutes before adding the next portion of salt solution. Add 67μL of 277mM potassium citrate solution, 1mL41.4 mM calcium chloride solution, and 1mL 7.9mM dipotassium hydrogen phosphate solution in each batch in turn. Finally, adjust the temperature to 25°C, adjust the pH to 7.0 with 0.05M HCl solution, add water to make the solution dilute to 20mL, and obtain a low-mineralized whole casein reconstituted micelle solution;

[0114] The casein concentration in the solution of low-mineralized whole casein reconstituted micelles is 0.4%, the citrate ion (Cit) concentration is 2.8 mM, the calcium ion (Ca) concentration is 7.5 mM, and the phosphate ion (Pi) concentration is 2.5 mM.

[0115] Comparative Example 1 β+κ-casein reinforced recombinant micelles

[0116] A method for preparing β+κ-casein fortified recombinant micelles comprises the following steps:

[0117] Take sterilized skim milk, add 6M HCl solution to adjust its pH to 4.6, centrifuge at 10000×g for 15 minutes, and obtain bovine casein precipitate. Redissolve the precipitate in deionized water, and add 2M NaOH solution to adjust its pH to 11 to obtain a casein solution with a casein concentration of 2.5% (w / v). Then add 4M calcium chloride solution to adjust the final calcium ion concentration of the casein solution to 65mM, and centrifuge at 10000×g for 15 minutes to obtain a supernatant enriched with κ-casein and a supernatant enriched with α s - and β-casein precipitation. Then add 6M HCl solution, adjust the pH of the supernatant to 3.8 and centrifuge at 10000×g for 15 minutes to obtain a precipitate enriched with κ-casein. s - and β-casein precipitates were redissolved in water, and 2M NaOH solution was added to adjust the pH to 7.0 so that the concentration of casein was 2.5% (w / v). The temperature of the reconstituted solution was lowered to 4°C, and 6M HCl solution was added to adjust its pH to 4.6, and it was kept at 4°C for 6h, followed by centrifugation at 10000×g for 15min. The obtained supernatant was warmed to 35°C and centrifuged again for 15min to obtain a precipitate enriched with β-casein. The β-casein and κ-casein precipitates were taken and mixed according to a casein mass ratio of 3:7, so that the β-casein and κ-casein components in the casein accounted for 68% and 20%, respectively, and then dialyzed at 4°C for 72h using a dialysis membrane with a molecular weight cutoff of 7kDa, and Amberlite SR1LNa ion exchange resin was added to the dialysis external solution at 0.5g resin / g casein, and freeze-dried after completion to obtain a lyophilized powder.

[0118] The lyophilized powder was reconstituted with water to obtain a composite casein solution with a casein concentration of 0.8% (w / v), and potassium citrate solution, calcium chloride solution and dipotassium hydrogen phosphate solution were sequentially added to the composite casein solution in the manner of Example 1, wherein the concentration of potassium citrate solution added in each batch was 280 mM, the concentration of calcium chloride solution was 50.0 mM, and the concentration of dipotassium hydrogen phosphate solution was 16.6 mM, so that the final concentrations of casein and each ion were consistent with those in Example 1, and a β+κ-casein fortified recombinant micelle solution was obtained.

[0119] Comparative Example 2: Recombinant micelles of bovine milk sodium caseinate

[0120] A method for preparing bovine milk sodium caseinate recombinant micelles comprises the following steps:

[0121] Sodium caseinate was taken and redissolved in water to form a sodium caseinate solution with a casein concentration of 0.8% (w / v). A saline solution was added in the manner of Example 1, wherein the concentration of potassium citrate solution added in each batch was 280 mM, the concentration of calcium chloride solution was 47.7 mM, and the concentration of dipotassium hydrogen phosphate solution was 16.3 mM, so that the final concentrations of casein and each ion were consistent with those in Example 1, and a bovine milk sodium caseinate reconstituted micelle solution was obtained.

[0122] Comparative Example 3: Bovine casein micelles

[0123] A method for preparing bovine casein micelles comprises the following steps:

[0124] Take the MCC retentate and dilute it with water to form an MCC solution with a casein concentration of 0.8% (w / v). Prepare an ion mixture containing potassium citrate, calcium chloride and dipotassium hydrogen phosphate, wherein the concentration of potassium citrate is 8.9mM, the concentration of calcium chloride is 13.5mM, and the concentration of dipotassium hydrogen phosphate is 3.8mM. Take 10mL of MCC solution, adjust the pH to 7.0 with 0.05M NaOH solution, maintain the temperature at 37°C, slowly add 6.2mL of the ion mixture, then adjust the temperature to 25°C, adjust the pH to 7.0 with 0.05M HCl solution and make up to 20mL to obtain a bovine casein micellar solution, wherein the final concentration of casein and each ion is consistent with Example 1.

[0125] The micelles obtained in Example 1 and Comparative Examples 1-3 were subjected to performance tests, and the test results are as follows:

[0126] Human milk is the gold standard for the development of infant formula. The casein content, calcium ion, citrate ion, and phosphate ion content in human milk are approximately 0.4% and 7.5, 2.8, and 2.5 mM, respectively. Therefore, casein recombinant micelles were prepared based on the casein and ion content in human milk.

[0127] As shown in Table 1, the α s1 -、α s2 The mass proportions of α-, β-, and κ-casein are 40.0%, 6.8%, 38.1%, and 15.1%, respectively. Its casein composition is similar to that of the reconstituted micelle solution of bovine milk sodium caseinate and the micelle solution of bovine milk casein. In comparison, the β+κ-casein fortified reconstituted micelle solution contains a lower proportion of α s-casein and a higher proportion of β- and κ-casein. The mineralization degree and average particle size of different casein micelles are consistent with β+κ-casein fortified recombinant micelles < low mineralized whole casein recombinant micelles < bovine milk sodium caseinate recombinant micelles < bovine milk casein micelles, indicating that increasing the proportion of β+κ-casein or using MCC with a certain decalcification rate to prepare recombinant micelles can effectively reduce the level of micellar calcium in casein micelles and promote the formation of small-size micelles.

[0128] Table 1 Physicochemical properties of casein micelles in Example 1, Comparative Examples 1, 2 and 3

[0129] index Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 <![CDATA[α S1 -Casein (%)]]> <![CDATA[40.0±0.4 a ]]> <![CDATA[12.2±0.6 b ]]> <![CDATA[40.4±1.0 a ]]> <![CDATA[40.1±0.3 a ]]> <![CDATA[α S2 -Casein (%)]]> <![CDATA[6.8±0.6 b ]]> <![CDATA[0 c ]]> <![CDATA[7.9±0.8 a ]]> <![CDATA[6.4±0.5 b ]]> β-Casein (%) <![CDATA[38.1±0.2 b ]]> <![CDATA[68.1±1.2 a ]]> <![CDATA[37.3±0.7 b ]]> <![CDATA[38.3±0.4 b ]]> κ-Casein (%) <![CDATA[15.1±0.2 b ]]> <![CDATA[19.7±0.7 a ]]> <![CDATA[14.4±0.6 b ]]> <![CDATA[15.2±0.6 b ]]> Mineralization degree (mM / mM) <![CDATA[15.7±0.4 c ]]> <![CDATA[14.2±0.4 d ]]> <![CDATA[16.8±0.6 b ]]> <![CDATA[18.5±0.5 a ]]> Average particle size (nm) <![CDATA[65.7±1.7 c ]]> <![CDATA[62.3±1.2 d ]]> <![CDATA[73.4±0.7 b ]]> <![CDATA[145.4±2.1 a ]]> Hydration rate (g water / g dry micelle) <![CDATA[2.94±0.13 a ]]> <![CDATA[3.06±0.15 a ]]> <![CDATA[2.82±0.06 a ]]> <![CDATA[1.92±0.17 b ]]>

[0130] Example 2 Low-mineralized whole casein recombinant micelles: Effect of decalcification rate

[0131] The method for preparing whole casein recombinant micelles comprises the following steps:

[0132] Adding cation exchange resin to the MCC retentate for decalcification, wherein the amount of the resin added is 0, 1.3, 3.6, 6.5 or 11.0 g / 100 g of the retentate, filtering to remove the resin, and obtaining MCC retentate with decalcification rates of 0, 20, 42, 61, and 81%, respectively;

[0133] Deionized water was added to the MCC retentate with different decalcification rates to adjust the casein concentration to 0.8% (w / v). Referring to the method in Example 1, the concentrations of calcium chloride solution added to the MCC solution with different decalcification rates were 27.9, 32.4, 37.1, 41.4 and 45.7 mM, respectively, and the final concentrations of casein and each ion were kept consistent with those in Example 1, to obtain a series of full casein reconstituted micelle solutions.

[0134] The obtained series of whole casein reconstituted micelle solutions were subjected to performance tests, and the test results are as follows:

[0135] As shown in Table 2, with the increase of decalcification rate, especially when the decalcification rate reaches 61%-81%, the mineralization degree and average particle size of the prepared recombinant micelles are significantly reduced, while the free casein percentage and hydration rate are significantly increased (P<0.05), indicating that the structure of the micelles is gradually loose. The decalcification treatment causes the dissociation of the casein micelle structure, and the re-addition of calcium ions, phosphate ions and citrate ions can promote the re-formation of the new structure of the micelles. The initial dissociation degree of the micelles has a greater influence on the structure of the recombinant micelles. The mineralization degree of human milk casein micelles is only 7-9mM / mM, and the micelle particle size is about 60-80nm. Therefore, in order to better simulate the structure of human milk casein micelles, MCC solution with a decalcification rate of 42%-81% can be preferably used for the preparation of whole casein recombinant micelles.

[0136] Table 2 Physicochemical properties of whole casein reconstituted micelles in Example 2

[0137]

[0138] Example 3 Low-mineralized whole casein reconstituted micelles: Effect of ion addition order

[0139] The three salt solutions in Example 1 were adjusted to be added in one batch, and the order of adding the salt solutions was calcium chloride-potassium citrate-dipotassium hydrogen phosphate (Ca-Cit-Pi), calcium chloride-dipotassium hydrogen phosphate-potassium citrate (Ca-Pi-Cit), potassium citrate-calcium chloride-dipotassium hydrogen phosphate (Cit-Ca-Pi), potassium citrate-dipotassium hydrogen phosphate-calcium chloride (Cit-Pi-Ca), dipotassium hydrogen phosphate-potassium citrate-calcium chloride (Pi-Cit-Ca) or dipotassium hydrogen phosphate-calcium chloride-potassium citrate (Pi-Ca-Cit), and the remaining steps were consistent with Example 1 to obtain a series of whole casein reconstituted micelle solutions.

[0140] The preparation of casein recombinant micelles involves complex interactions between casein components and different ions, so the way ions are added has a great influence on the structure of the formed micelles.

[0141] As shown in Table 3, the recombinant micelles obtained by adding Cit-Ca-Pi, Cit-Pi-Ca and Pi-Cit-Ca have the lowest turbidity and mineralization degree and the smallest particle size, followed by Pi-Ca-Cit and Ca-Cit-Pi, while the recombinant micelles obtained by adding Ca-Pi-Cit have the highest turbidity and mineralization degree and the largest particle size, indicating that the citrate ions added first can chelate part of the calcium ions, thereby reducing the binding of calcium ions to casein, while the calcium ions added first preferentially bind to casein, resulting in a significant increase in the degree of mineralization of the micelles. Therefore, in order to better simulate the structure of human milk casein micelles, the addition order of Cit-Ca-Pi, Cit-Pi-Ca and Pi-Cit-Ca can be preferred for the preparation of whole casein recombinant micelles and subsequent experimental optimization.

[0142] Table 3 Physicochemical properties of whole casein recombinant micelles in Example 3

[0143] index Cit-Ca-Pi Cit-Pi-Ca Pi-Cit-Ca Pi-Ca-Cit Ca-Cit-Pi Ca-Pi-Cit Turbidity <![CDATA[0.61±0.02 d ]]> <![CDATA[0.66±0.02 c ]]> <![CDATA[0.69±0.03 c ]]> <![CDATA[0.85±0.01 b ]]> <![CDATA[0.87±0.02 b ]]> <![CDATA[1.01±0.02 a ]]> Degree of mineralization <![CDATA[17.2±0.4 b ]]> <![CDATA[17.2±0.2 b ]]> <![CDATA[17.6±0.4 ab ]]> <![CDATA[17.6±0.6 ab ]]> <![CDATA[17.7±0.3 ab ]]> <![CDATA[18.0±0.3 a ]]> Average particle size (nm) <![CDATA[86.2±1.2 f ]]> <![CDATA[90.1±0.5 e ]]> <![CDATA[92.5±0.8 d ]]> <![CDATA[98.4±1.4 c ]]> <![CDATA[102.5±1.1 b ]]> <![CDATA[107.3±1.6 a ]]>

[0144] Example 4 Low-mineralized whole casein reconstituted micelles: Effect of ion addition batch

[0145] The three salt solutions in Example 1 were adjusted to be added in one batch, two batches, three batches, four batches or five batches respectively, and the remaining steps were kept consistent with Example 1 to obtain a series of whole casein reconstituted micelle solutions.

[0146] As shown in Table 4, with the increase in the number of batches of salt solution added, the turbidity and mineralization of the formed whole casein recombinant micelles were significantly reduced and the average particle size gradually decreased (P<0.05), and reached the lowest at 3-5 batches. The addition of small amounts and multiple times helps the calcium ions to fully interact with citrate ions and phosphate ions, thereby reducing the binding of calcium ions to casein. Therefore, in order to better simulate the structure of human milk casein micelles, it is preferred that the salt solution be added in 3-5 batches for the preparation of whole casein recombinant micelles and subsequent experimental optimization.

[0147] Table 4 Physicochemical properties of whole casein reconstituted micelles in Example 4

[0148] index One batch Two batches Three batches (Example 1) Four batches Five batches Turbidity <![CDATA[0.61±0.02 a ]]> <![CDATA[0.42±0.03 b ]]> <![CDATA[0.35±0.02 c ]]> <![CDATA[0.34±0.01 c ]]> <![CDATA[0.34±0.01 c ]]> Degree of mineralization <![CDATA[17.2±0.4 a ]]> <![CDATA[16.9±0.3 a ]]> <![CDATA[15.7±0.4 b ]]> <![CDATA[15.2±0.3 b ]]> <![CDATA[15.1±0.2 b ]]> Average particle size (nm) <![CDATA[86.2±1.2 a ]]> <![CDATA[77.6±0.8 b ]]> <![CDATA[65.7±1.7 c ]]> <![CDATA[65.0±1.1 c ]]> <![CDATA[64.7±1.4 c ]]>

[0149] Example 5 Low-mineralized whole casein reconstituted micelles: Effect of ion addition time interval

[0150] The time intervals between the two additions of the salt solution in Example 1 were adjusted to 0, 5, 10, 15 or 20 min respectively, and the remaining steps were kept consistent with Example 1 to obtain a series of whole casein recombinant micelle solutions.

[0151] As shown in Table 5, with the increase of the time interval for adding the salt solution, the turbidity and mineralization degree of the formed whole casein reconstituted micelles were significantly reduced, and the average particle size was significantly reduced (P<0.05), and reached the minimum when the time interval was 15-20min. The slow addition of the salt solution is also conducive to the full interaction between different ions, so that more calcium ions are distributed in the whey phase, thereby reducing the binding of calcium to casein and reducing the mineralization degree of the micelles. Therefore, in order to better simulate the structure of human milk casein micelles, it is preferred that the time interval for adding the salt solution is 15-20min for the preparation of whole casein reconstituted micelles and subsequent experimental optimization.

[0152] Table 5 Physicochemical properties of whole casein recombinant micelles in Example 5

[0153] index 0min 5min 10min 15min (Example 1) 20min Turbidity <![CDATA[0.41±0.01 a ]]> <![CDATA[0.39±0.02 ab ]]> <![CDATA[0.38±0.01 b ]]> <![CDATA[0.35±0.02 c ]]> <![CDATA[0.35±0.01 c ]]> Degree of mineralization <![CDATA[17.1±0.5 a ]]> <![CDATA[16.8±0.3 a ]]> <![CDATA[16.4±0.3 ab ]]> <![CDATA[15.7±0.4 bc ]]> <![CDATA[15.2±0.5 c ]]> Average particle size (nm) <![CDATA[76.5±1.1 a ]]> <![CDATA[73.3±0.7 b ]]> <![CDATA[71.5±1.2 b ]]> <![CDATA[65.7±1.7 c ]]> <![CDATA[65.3±0.6 c ]]>

[0154] Example 6 Low-mineralized whole casein reconstituted micelles: Effect of pH during reconstitution

[0155] During the preparation of whole casein recombinant micelles, the pH during reconstitution (i.e., the pH of the decalcified MCC solution) was adjusted to 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4 or 7.6, respectively. After adding the salt solution, the pH was adjusted to 7.0. The remaining steps were consistent with Example 1 to obtain a series of whole casein recombinant micelle solutions.

[0156] As shown in Table 6, when the pH value is 6.2 and 6.4, the turbidity and average particle size of the recombinant micelles are large and the mineralization degree is low. With the increase of pH value, the turbidity and average particle size gradually decrease, while the mineralization degree gradually increases, and the mineralization degree reaches the maximum at pH 7.6. This may be because the electrostatic repulsion between casein molecules gradually increases with the increase of pH, while the solubility of calcium phosphate gradually decreases, resulting in an increase in the mineralization degree of the recombinant micelles.

[0157] Taking into account the mineralization degree and average particle size of micelles, the optimal reconstitution pH value is 6.6-7.2 for the preparation of whole casein reconstituted micelles and subsequent experimental optimization.

[0158] Table 6 Physicochemical properties of whole casein recombinant micelles in Example 6

[0159] pH Turbidity Degree of mineralization Average particle size (nm) 6.2 <![CDATA[0.48±0.02 a ]]> <![CDATA[14.4±0.3 e ]]> <![CDATA[79.6±0.6 a ]]> 6.4 <![CDATA[0.44±0.03 b ]]> <![CDATA[14.9±0.5 de ]]> <![CDATA[77.8±1.0 ab <!-- 11 -->]]> 6.6 <![CDATA[0.40±0.02 c ]]> <![CDATA[15.2±0.6 cd ]]> <![CDATA[75.8±1.4 b ]]> 6.8 <![CDATA[0.37±0.03 cd ]]> <![CDATA[15.4±0.4 cd ]]> <![CDATA[71.4±0.8 c ]]> 7.0 (Example 1) <![CDATA[0.35±0.02 de ]]> <![CDATA[15.7±0.4 bc ]]> <![CDATA[65.7±1.7 d ]]> 7.2 <![CDATA[0.32±0.02 ef ]]> <![CDATA[15.9±0.3 abc ]]> <![CDATA[65.2±1.5 d ]]> 7.4 <![CDATA[0.32±0.01 ef ]]> <![CDATA[16.3±0.4 ab ]]> <![CDATA[65.0±1.3 d ]]> 7.6 <![CDATA[0.30±0.02 f ]]> <![CDATA[16.6±0.3 a ]]> <![CDATA[64.5±0.7 d ]]>

[0160] Example 7 Low-mineralized whole casein reconstituted micelles: Effect of temperature during reconstitution

[0161] During the preparation of whole casein recombinant micelles, the temperature during reconstitution (i.e., the temperature of the decalcified MCC solution) was maintained at 17, 27, 37, 47, 57, 67, 77 or 87° C., and the temperature was adjusted to 25° C. after the salt solution was added. The remaining steps were consistent with Example 1 to obtain a series of whole casein recombinant micelle solutions.

[0162] As shown in Table 7, when the reconstitution temperature was 17°C, the turbidity and mineralization degree of the formed whole casein reconstituted micelles were the lowest and the average particle size was the smallest. As the temperature gradually increased, the turbidity and mineralization degree gradually increased and the particle size gradually increased, and reached the maximum at 77°C and 87°C (P<0.05). The reconstitution temperature mainly affects the structure of the reconstituted micelles by affecting the solubility of micellar calcium phosphate and the hydrophobic interaction between casein molecules. As the temperature increases, the hydrophobic interaction between casein molecules is enhanced, thereby promoting the aggregation of casein and the increase of particle size. Higher reconstitution temperatures will also lead to a decrease in the solubility of calcium phosphate, thereby causing more calcium ions to transfer from the whey phase to the micelle phase and causing an increase in the degree of mineralization of the micelles. Therefore, in order to better simulate the structure of human milk casein micelles, the reconstitution temperature of 37-57°C can be preferably used for the preparation of whole casein reconstituted micelles and subsequent experimental optimization.

[0163] Table 7 Physicochemical properties of whole casein recombinant micelles in Example 7

[0164] Temperature(℃) Turbidity Degree of mineralization Average particle size (nm) 17 <![CDATA[0.28±0.02 e ]]> <![CDATA[14.3±0.5 f ]]> <![CDATA[63.4±0.7 f ]]> 27 <![CDATA[0.33±0.04 de ]]> <![CDATA[15.4±0.6 e ]]> <![CDATA[65.1±1.3 ef ]]> 37 (Example 1) <![CDATA[0.35±0.02 d ]]> <![CDATA[15.7±0.4 de ]]> <![CDATA[65.7±1.7 e ]]> 47 <![CDATA[0.38±0.02 cd ]]> <![CDATA[16.2±0.3 cd ]]> <![CDATA[71.1±1.4 d ]]> 57 <![CDATA[0.42±0.04 bc ]]> <![CDATA[16.5±0.4 bc ]]> <![CDATA[75.5±0.7 c ]]> 67 <![CDATA[0.47±0.03 b ]]> <![CDATA[16.8±0.3 abc ]]> <![CDATA[77.5±1.5 c ]]> 77 <![CDATA[0.57±0.04 a ]]> <![CDATA[17.1±0.5 ab ]]> <![CDATA[84.0±0.8 b ]]> 87 <![CDATA[0.61±0.03 a ]]> <![CDATA[17.3±0.3 a ]]> <![CDATA[86.6±0.9 a ]]>

[0165] Example 8 Low-mineralized whole casein recombinant micelles: Effect of calcium ion concentration

[0166] The concentration of the calcium chloride solution in Example 1 was adjusted to 21.4, 31.4, 41.4, 51.4 or 61.4 mM, so that the calcium ion concentration in the whole casein reconstituted micelle solution was 4.5, 6, 7.5, 9 or 10.5 mM, while the final concentrations of casein, citrate ions and phosphate ions remained the same as in Example 1.

[0167] The addition of calcium ions can promote the formation of micellar calcium phosphate and the self-assembly of casein molecules. As shown in Table 8, when the calcium ion concentration in the whole casein recombinant micelle solution is 4.5mM, the degree of mineralization of the micelle is low and the average particle size is small, and the hydration rate is high. Along with the increase of calcium ion concentration, the degree of mineralization and the average particle size of the prepared whole casein recombinant micelles significantly increase, and reach the maximum when the concentration is 10.5mM, and the hydration rate of the micelles significantly decreases (P < 0.05), indicating that a large particle size and densely structured recombinant micelle is gradually formed. Therefore, in order to better simulate the structure of human milk casein micelles, the concentration of calcium chloride solution can be preferably 31.4-51.4mM, so that the calcium ion concentration in the whole casein recombinant micelle solution is 6-9mM.

[0168] Table 8 Physicochemical properties of whole casein recombinant micelles in Example 8

[0169] index 4.5mM 6.0mM 7.5mM (Example 1) 9.0mM 10.5mM Mineralization degree (mM / mM) <![CDATA[12.7±0.5 d ]]> <![CDATA[14.2±0.6 c ]]> <![CDATA[15.7±0.4 b ]]> <![CDATA[16.8±0.5 a ]]> <![CDATA[17.6±0.6 a ]]> Average particle size (nm) <![CDATA[54.3±1.2 e ]]> <![CDATA[62.2±0.7 d ]]> <![CDATA[65.7±1.7 c ]]> <![CDATA[77.6±1.3 b ]]> <![CDATA[106.4±1.8 a ]]> Hydration rate (g water / g dry micelle) <![CDATA[5.22±0.15 a ]]> <![CDATA[3.46±0.07 b ]]> <![CDATA[2.94±0.13 c ]]> <![CDATA[2.45±0.10 d ]]> <![CDATA[2.16±0.15 e ]]>

[0170] Example 9 Low-mineralized whole casein reconstituted micelles: Effect of citrate ion concentration

[0171] The concentration of the potassium citrate solution in Example 1 was adjusted to 49, 124, 199, 277, 349, 424 or 499 mM, respectively, so that the molar ratio of calcium ions to citrate ions in the whole casein reconstituted micelle solution was 1:0.07, 1:0.17, 1:0.27, 1:0.37, 1:0.47, 1:0.57 or 1:0.67, respectively, while the final concentrations of casein, calcium ions and phosphate ions remained the same as in Example 1.

[0172] The addition of citrate ions can affect the structure of reconstituted micelles by affecting the distribution of calcium ions in the whey phase and the micelle phase. As shown in Table 9, with the decrease in the molar ratio of calcium ions to citrate ions, that is, the increase in the concentration of citrate ions, the mineralization degree and average particle size of the whole casein reconstituted micelles are significantly reduced, while the hydration rate is significantly increased (P<0.05), and the hydration rate reaches the maximum when the ratio is 1:0.57 and 1:0.67. The addition of citrate ions can chelate calcium ions to make them preferentially distributed in the whey phase, thereby reducing the formation of micellar calcium phosphate and making the reconstituted micelle structure looser. Therefore, in order to better simulate the structure of human milk casein micelles, the concentration of potassium citrate solution can be preferably 199-499mM, so that the molar ratio of calcium ions to citrate ions in the whole casein reconstituted micelle solution is 1:0.27-1:0.67.

[0173] Table 9 Physicochemical properties of whole casein recombinant micelles in Example 9

[0174] Ca:Cit Mineralization degree (mM / mM) Average particle size (nm) Hydration rate (g water / g dry micelle) 1:0.07 <![CDATA[22.0±1.3 a ]]> <![CDATA[84.1±3.2 a ]]> <![CDATA[2.19±0.11 d ]]> 1:0.17 <![CDATA[17.7±1.1 b ]]> <![CDATA[76.5±1.2 b ]]> <![CDATA[2.40±0.18 d ]]> 1:0.27 <![CDATA[17.6±0.5 b ]]> <![CDATA[69.5±1.6 c ]]> <![CDATA[2.77±0.13 c ]]> 1:0.37 (Example 1) <![CDATA[15.7±0.4 c ]]> <![CDATA[65.7±1.7 d ]]> <![CDATA[2.94±0.13 bc ]]> 1:0.47 <![CDATA[12.1±0.6 d ]]> <![CDATA[64.0±1.0 d ]]> <![CDATA[3.19±0.06 b ]]> 1:0.57 <![CDATA[10.1±0.7 e ]]> <![CDATA[60.0±1.6 e ]]> <![CDATA[3.62±0.02 a ]]> 1:0.67 <![CDATA[10.0±0.8 e ]]> <![CDATA[54.7±4.4 f ]]> <![CDATA[3.72±0.10 a ]]>

[0175] Example 10 Low-mineralized whole casein reconstituted micelles: Effect of phosphate ion concentration

[0176] The concentration of the dipotassium hydrogen phosphate solution in Example 1 was adjusted to 0, 7.9, 17.2, 26.5, 36.2 or 47.2 mM, respectively, so that the molar ratio of calcium ions to phosphate ions in the whole casein reconstituted micelle solution was 1:0.17, 1:0.33, 1:0.52, 1:0.71, 1:0.90 or 1:1.12, respectively, while the final concentrations of casein, citrate ions and calcium ions remained the same as in Example 1.

[0177] On the one hand, the addition of phosphate ions can form micellar calcium phosphate with calcium ions, promoting the reorganization of the casein micelle structure. On the other hand, the phosphate ions distributed in the whey phase will also compete with the casein micelles for calcium binding. As shown in Table 10, as the molar ratio of calcium ions to phosphate ions decreases, that is, the amount of phosphate ions added increases, the average particle size of the prepared whole casein recombinant micelles decreases slightly, and reaches the minimum when the molar ratio is 1:0.90 and 1:1.12.

[0178] Table 10 Physicochemical properties of whole casein recombinant micelles in Example 10

[0179] index 1:0.17 1:0.33 (Example 1) 1:0.52 1:0.71 1:0.90 1:1.12 Average particle size (nm) <![CDATA[69.1±1.6 a ]]> <![CDATA[65.7±1.7 b ]]> <![CDATA[65.5±1.2 b ]]> <![CDATA[64.4±1.1 b ]]> <![CDATA[62.0±1.7 c ]]> <![CDATA[60.3±0.9 c ]]>

[0180] Example 11 Low-mineralized whole casein reconstituted micelles: Effect of casein concentration

[0181] The casein concentration of the decalcified MCC solution in Example 1 was adjusted to 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8 or 3.2%, and the concentrations of the potassium citrate solution, calcium chloride solution and dipotassium hydrogen phosphate solution were adjusted to 138, 20.7 and 3.9 mM, 277, 41.4 and 7.9 mM, 415, 62.1 and 11.8 mM, and 553, 82.8 and 15.7 mM, respectively. 692, 103.5, 19.7 mM; 830, 124.2, 23.6 mM; 968, 144.9, 27.5 mM; 1107, 165.6, 31.5 mM, so that the mass concentration of casein in the whole casein recombinant micelle solution is 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 or 1.6%, respectively, and the ratio of casein to the final concentration of each ion is kept consistent with that in Example 1.

[0182] As shown in Table 11, when the casein concentration of the recombinant micellar solution is 0.2% and 0.4%, its turbidity and mineralization are low and the particle size is small. As the casein concentration increases, the turbidity, mineralization and average particle size of the micellar solution gradually increase, and reach a maximum when the casein concentration is 1.6%. The increase in casein concentration increases the binding of protein to calcium, and more calcium ions are distributed in the micellar phase, resulting in an increase in the formation of micellar calcium phosphate and an increase in the degree of micellar mineralization. On the whole, in order to better simulate the structure of human milk casein micelles, the casein concentration of the decalcified MCC solution can be preferably 0.8%-2.8%, so that the casein mass concentration of the whole casein recombinant micellar solution is 0.4%-1.4%.

[0183] Table 11 Physicochemical properties of whole casein recombinant micelles in Example 11

[0184] Casein concentration (%) Turbidity Degree of mineralization Average particle size (nm) 0.2 <![CDATA[0.33±0.04 d ]]> <![CDATA[15.0±0.6 e ]]> <![CDATA[63.7±2.1 e ]]> 0.4 (Example 1) <![CDATA[0.35±0.02 d ]]> <![CDATA[15.7±0.4 de ]]> <![CDATA[65.7±1.7 e ]]> 0.6 <![CDATA[0.38±0.02 d ]]> <![CDATA[16.1±0.5 cd ]]> <![CDATA[68.4±1.5 d ]]> 0.8 <![CDATA[0.44±0.03 c ]]> <![CDATA[16.9±0.6 bc ]]> <![CDATA[76.2±1.3 c ]]> 1.0 <![CDATA[0.62±0.05 b ]]> <![CDATA[17.3±0.5 ab ]]> <![CDATA[85.8±0.6 b ]]> 1.2 <![CDATA[0.64±0.04 ab ]]> <![CDATA[17.5±0.4 ab ]]> <![CDATA[86.2±1.4 b ]]> 1.4 <![CDATA[0.66±0.03 ab ]]> <![CDATA[17.7±0.4 ab ]]> <![CDATA[87.1±1.0 b ]]> 1.6 <![CDATA[0.69±0.03 a ]]> <![CDATA[18.1±0.3 a ]]> <![CDATA[90.4±0.6 a ]]>

[0185] Example 12 Low-mineralized whole casein reconstituted micelle-stabilized emulsion

[0186] A method for preparing a low-mineralized whole casein recombinant micelle-stabilized emulsion comprises the following steps:

[0187] 0.7 g of anhydrous milk fat was added to 20 mL of the solution of low-mineralized whole casein recombinant micelles prepared in Example 1, and the temperature was maintained at 50°C, i.e., stirring equilibrium was performed at 50°C for 15 min, shear homogenization was performed at 50°C and 21500 rpm for 5 min, followed by high-pressure homogenization at 50°C and 30 MPa for 3 times, and finally the temperature was adjusted to 25°C to obtain a stable emulsion of low-mineralized whole casein recombinant micelles with a fat concentration of 3.5% (w / v).

[0188] Comparative Example 4 β+κ-casein reinforced recombinant micelle-stabilized emulsion

[0189] The "low-mineralized whole casein recombinant micelle solution prepared in Example 1" in Example 12 was adjusted to "β+κ-casein fortified recombinant micelle solution prepared in Comparative Example 1", and the rest was kept consistent with Example 12 to obtain an emulsion.

[0190] Comparative Example 5 Emulsion stabilized by reconstituted micelles of bovine milk sodium caseinate

[0191] The "solution of low-mineralized whole casein recombinant micelles prepared in Example 1" in Example 12 was adjusted to "the solution of bovine sodium caseinate recombinant micelles prepared in Comparative Example 2", and the rest was kept consistent with Example 12 to obtain an emulsion.

[0192] Comparative Example 6 Emulsion stabilized by bovine casein micelles

[0193] The "solution of low-mineralized whole casein recombinant micelles prepared in Example 1" in Example 12 was adjusted to "the bovine milk casein micelle solution prepared in Comparative Example 3", and the rest was kept consistent with Example 12 to obtain an emulsion.

[0194] The obtained emulsion was subjected to performance test, and the test results are as follows:

[0195] As shown in Table 12, compared with bovine milk casein micelles, the lipid droplet size of the emulsions stabilized by the other three recombinant micelles is smaller (P<0.05). The zeta-potential value of the emulsion is consistent with the low-mineralized whole casein recombinant micelle emulsion < bovine milk sodium caseinate recombinant micelle emulsion < bovine milk casein micelle emulsion < β+κ-casein fortified recombinant micelle emulsion. In the instability index of the emulsion, the low-mineralized whole casein recombinant micelle emulsion is significantly lower than the β+κ-casein fortified recombinant micelle emulsion and the bovine milk casein micelle emulsion (P<0.05). The stability of the emulsion is related to the lipid droplet size and zeta-potential. The low-mineralized whole casein recombinant micelle emulsion has a small particle size and a high absolute value of zeta-potential, so it shows better emulsion stability. The α s -The casein level decreases and the isoelectric point increases, resulting in a decrease in the absolute value of the zeta-potential of the emulsion and a decrease in the stability of the emulsion. The particle size of the reconstituted micellar emulsion of bovine milk sodium caseinate is slightly increased compared to the reconstituted micellar emulsion of low-mineralized whole casein, while the emulsion stability is reduced. The particle size of bovine milk casein micelles is the largest, which is not conducive to the emulsification of lipid droplets. Therefore, the emulsion formed has the largest lipid droplet size and the worst emulsion stability.

[0196] like Figure 1As shown in Table 12, the low-mineralized whole casein recombinant micelle emulsion and the β+κ-casein fortified recombinant micelle emulsion formed small and loose gastric clots at 0 min of digestion, and further degraded after 60 min of digestion. The sodium caseinate recombinant micelle emulsion formed slightly larger gastric clots during gastric digestion, while the bovine milk casein micelle emulsion formed large and dense gastric clots, and small clots still remained after 60 min of digestion. Compared with the bovine milk casein micelle emulsion, the low-mineralized whole casein recombinant micelle emulsion, the casein fortified recombinant micelle emulsion, and the sodium caseinate recombinant micelle emulsion all showed higher free amino content and lipolysis degree (P<0.05). In comparison, the calcium soap fatty acid content of the low-mineralized whole casein recombinant micellar emulsion was lower and the calcium bioaccessibility was significantly higher (P<0.05), indicating that the preparation of low-mineralized whole casein recombinant micelles using micellar casein with a certain decalcification rate and using it to stabilize the emulsion effectively improved the stability of the emulsion, the digestibility of protein and fat, and the bioavailability of calcium.

[0197] Table 12 Physicochemical properties and digestibility of the emulsions in Example 12, Comparative Examples 4, 5 and 6

[0198] index Example 12 Comparative Example 4 Comparative Example 5 Comparative Example 6 Lipid droplet size (μm) <![CDATA[0.66±0.14 b ]]> <![CDATA[0.62±0.11 b ]]> <![CDATA[0.77±0.09 b ]]> <![CDATA[1.32±0.25 a ]]> Zeta-potential <![CDATA[-22.7±1.6 d ]]> <![CDATA[-14.3±0.8 a ]]> <![CDATA[-20.2±1.1 c ]]> <![CDATA[-17.8±1.3 b ]]> Instability Index <![CDATA[0.08±0.01 c ]]> <![CDATA[0.12±0.01 b ]]> <![CDATA[0.10±0.01 bc ]]> <![CDATA[0.18±0.02 a ]]> Free amino content (μmol / mg) <![CDATA[0.65±0.01 a ]]> <![CDATA[0.68±0.03 a ]]> <![CDATA[0.63±0.02 a ]]> <![CDATA[0.51±0.04 b ]]> Degree of lipolysis (%) <![CDATA[48.4±1.1 ab ]]> <![CDATA[49.2±0.8 a ]]> <![CDATA[46.5±1.3 b ]]> <![CDATA[41.6±1.2 c ]]> Calcium soap fatty acid content (mg / g fat) <![CDATA[49.3±1.2 b ]]> <![CDATA[60.4±1.8 a ]]> <![CDATA[51.8±1.4 b ]]> <![CDATA[59.1±0.9 a ]]> Calcium bioaccessibility (%) <![CDATA[27.6±1.5 a ]]> <![CDATA[14.5±1.2 c ]]> <![CDATA[23.8±1.2 b ]]> <![CDATA[15.3±2.6 c ]]>

[0199] Example 13 Emulsion stabilized by reconstituted micelles of low-mineralized whole casein: Effect of the order of milk fat addition

[0200] The whole casein micelle-stabilized emulsion was prepared by referring to the methods in Examples 1 and 12, but the order of the homogenization treatment and the ion addition steps was adjusted;

[0201] That is, a decalcified MCC solution having a casein concentration of 0.8% (w / v) is first mixed with anhydrous milk fat and homogenized before adding a salt solution to obtain a decalcified MCC emulsion, and then a potassium citrate solution, a calcium chloride solution and a dipotassium hydrogen phosphate solution are gradually added to the decalcified MCC emulsion in three batches to obtain an emulsion stabilized by full casein micelles;

[0202] Alternatively, after adding the first batch of salt solution to the decalcified MCC solution, the solution is mixed with anhydrous milk fat and homogenized, and then the remaining two batches of salt solution are added to obtain an emulsion stabilized by whole casein micelles;

[0203] Alternatively, after adding the second batch of salt solution to the decalcified MCC solution, the solution is mixed with anhydrous milk fat and homogenized, and then the remaining batch of salt solution is added to obtain an emulsion stabilized by whole casein micelles;

[0204] Alternatively, after the third batch of salt solution is added to the decalcified MCC solution, the solution is mixed with anhydrous milk fat and homogenized to obtain an emulsion stabilized by whole casein micelles.

[0205] As shown in Table 13, compared with the low-mineralized whole casein recombinant micelle emulsion prepared by adding ions first and then homogenizing, the whole casein micelle emulsion prepared by homogenizing first and then adding ions (before ion addition, after the first batch of ion addition, after the second batch of ion addition) has a large lipid droplet size and poor stability, and the free amino content and lipolysis degree after digestion are significantly lower (P<0.05), and the formation of calcium soap fatty acids is increased and the calcium bioaccessibility is reduced. This may be because the method of adding mineral ions after homogenization is not conducive to the formation of recombinant micelles, and a large amount of calcium ions free in the whey phase are more likely to induce casein flocculation during gastric digestion, resulting in a decrease in protein and lipid digestibility.

[0206] This indicates that when preparing emulsions stabilized by whole casein reconstituted micelles, it is more desirable to add mineral ions first and then milk fat.

[0207] Table 13 Physicochemical properties and digestibility of the emulsion in Example 13

[0208]

[0209] Example 14 Decalcified casein micelle-stabilized emulsion: Effect of decalcification rate

[0210] A cation exchange resin was added to the MCC retentate for decalcification, wherein the amount of the resin added was 0, 1.3, 3.6, 6.5 or 11.0 g / 100 g of the retentate, and the decalcification was carried out at room temperature for 4 hours, and then the resin was removed by filtering with 4 layers of gauze to obtain MCC retentate with a decalcification rate of 0, 20, 42, 61, and 81%. Then, the decalcified MCC solution with a casein concentration of 0.4% (w / v) was diluted with water, and the decalcified MCC solution was mixed with anhydrous milk fat in the manner of Example 12 to prepare a decalcified MCC stable emulsion.

[0211] As shown in Table 14, different amounts of resin added correspond to MCC with a calcium depletion rate of 0%-81%. With the increase of calcium depletion rate, the lipid droplet size of the decalcified MCC stable emulsion decreased significantly and the emulsion stability was significantly improved. However, when the calcium depletion rate reached 81%, the lipid droplet size increased compared with the 61% decalcified micellar emulsion, and the emulsion stability decreased significantly, indicating that micelle size that was too large or too small was not conducive to the emulsification of lipid droplets and the stability of the emulsion. After digestion, the free amino content and lipolysis degree of the emulsion increased significantly with the increase of calcium depletion rate, while the calcium soap fatty acid content decreased significantly (P<0.05). The above results indicate that decalcification may reduce the aggregation of micellar casein in the gastrointestinal stage by reducing the calcium ion bridging effect. The loosely structured clots also reduce the encapsulation of fat, accelerate the action of digestive enzymes on the substrate, and thus improve the digestibility of protein and lipid. At the same time, the lowered calcium level also reduces the combination and precipitation of calcium with free fatty acids, especially saturated long-chain fatty acids. On the one hand, this can improve the bioavailability of fatty acids. On the other hand, the reduced amount of calcium soap formation can also reduce constipation, gastrointestinal discomfort and other intestinal health problems caused by this in infants.

[0212] Table 14 Physicochemical properties and digestibility of the emulsion in Example 14

[0213] Resin addition amount (g / 100g retentate) 0 1.3 3.6 6.5 11 Resin addition amount (g / 100g casein) 0 17.3 48.0 86.7 146.7 Decalcification rate (%) 0 20 42 61 81 Lipid droplet size (μm) <![CDATA[1.25±0.18 a ]]> <![CDATA[0.93±0.14 b ]]> <![CDATA[0.81±0.15 b ]]> <![CDATA[0.78±0.06 b ]]> <![CDATA[0.88±0.04 b ]]> Instability Index <![CDATA[0.16±0.02 a ]]> <![CDATA[0.13±0.01 b ]]> <![CDATA[0.11±0.01 bc ]]> <![CDATA[0.10±0.01 c ]]> <![CDATA[0.13±0.02 b ]]> Free amino content (μmol / mg) <![CDATA[0.43±0.01 d ]]> <![CDATA[0.49±0.02 c ]]> <![CDATA[0.56±0.01 b ]]> <![CDATA[0.59±0.01 a ]]> <![CDATA[0.61±0.01 a ]]> Degree of lipolysis (%) <![CDATA[42.3±1.1 d ]]> <![CDATA[45.1±0.4 c ]]> <![CDATA[47.8±0.6 b ]]> <![CDATA[48.9±0.6 ab ]]> <![CDATA[49.8±1.1 a <!-- 16 -->]]> Calcium soap fatty acid content (mg / g fat) <![CDATA[34.8±2.3 a ]]> <![CDATA[26.1±1.4 b ]]> <![CDATA[19.5±2.4 c ]]> <![CDATA[14.3±0.5 d ]]> <![CDATA[6.3±1.3 e ]]>

[0214] Example 15 Low-mineralized whole casein reconstituted micelle-stabilized emulsion: Effect of decalcification rate

[0215] The series of whole casein recombinant micelle solutions in Example 2 were prepared according to the method in Example 12 to obtain a whole casein recombinant micelle-stabilized emulsion.

[0216] The emulsion test results are shown in Table 15.

[0217] Table 15 Physicochemical properties and digestibility of the emulsion in Example 15

[0218]

[0219] As shown in Table 15, the lipid droplet size of the reconstituted micellar emulsion decreased significantly with the increase of decalcification rate, and the emulsion stability was significantly improved (P<0.05). When the decalcification rate reached 42%, the free amino content, lipolysis degree and calcium bioaccessibility of the prepared reconstituted micellar emulsion increased significantly, while the content of calcium soap fatty acids formed decreased significantly (P<0.05). The mineralization degree of the reconstituted micelles prepared by decalcified MCC decreased and the free casein level increased, thereby reducing the aggregation of casein during digestion, promoting the formation of gastrointestinal clots with smaller particles and looser structure, and thus accelerating the digestibility of protein and lipid. In addition, the increase in protein hydrolysis may reduce calcium precipitation by promoting the formation of peptides and their binding with free calcium. On the other hand, the increase in fat digestion products may also increase the bioaccessibility of free fatty acids by promoting the formation of mixed micelles, thereby reducing the formation of calcium soaps and improving the bioavailability of calcium. In summary, low-mineralized whole casein reconstituted micelles may further affect their digestion characteristics by affecting the structure and stability of the emulsion. In order to improve the digestibility and bioavailability of micelles, when using decalcified MCC solution to prepare an emulsion stabilized by whole casein recombinant micelles, the decalcification rate can be preferably 42%-81%, and the most preferred decalcification rate is 42%-61%.

[0220] Example 16 Low-mineralized whole casein reconstituted micelle-stabilized emulsion: Effect of calcium ion concentration

[0221] The series of whole casein recombinant micelle solutions in Example 8 were prepared according to the method in Example 12 to obtain a whole casein recombinant micelle-stabilized emulsion.

[0222] As shown in Table 16, the increase in calcium ion concentration significantly reduced the amount of free amino group formation and lipolysis degree after emulsion digestion. This may be because the calcium ion bridging effect caused the reconstituted micelle emulsion to form a denser clot during the gastrointestinal digestion stage, thereby limiting the effect of digestive enzymes on proteins and lipids. The content of calcium soap fatty acids increased significantly with the increase of calcium ion concentration (P<0.05), while the calcium bioaccessibility decreased significantly with the increase of calcium ion concentration (P<0.05). Therefore, considering the micelle digestibility and bioaccessibility, the concentration of the calcium ion solution added can be preferably 31.4-51.4mM, and the calcium ion concentration of the whole casein reconstituted micelle solution is controlled to be 6-9mM for the preparation of reconstituted micelle stable emulsion.

[0223] Table 16 Digestibility of the emulsion in Example 16

[0224]

[0225] Example 17 Low-mineralized whole casein reconstituted micelle-stabilized emulsion: Effect of citrate ion concentration

[0226] The series of whole casein recombinant micelle solutions in Example 9 were prepared according to the method in Example 12 to obtain a whole casein recombinant micelle-stabilized emulsion.

[0227] As shown in Table 17, as the molar ratio of calcium ions to citrate ions decreases, the free amino content and lipolysis degree of the prepared recombinant micelle emulsion after digestion gradually increase, and reach the maximum at a molar ratio of 1:0.57 and 1:0.67, indicating that the addition of citrate ions promotes the digestion of proteins and lipids. The experiment found that when the molar ratio of calcium ions to citrate ions is lower than 1:0.27, that is, the concentration of citrate ions is increased, the amount of calcium soap fatty acid formed is significantly reduced and the calcium bioaccessibility is significantly increased (P<0.05), indicating that the chelation of calcium ions and citrate ions reduces the formation and precipitation of calcium soaps in the intestinal digestion stage. In summary, the addition of citrate ions helps to improve the digestibility and bioavailability of the recombinant micelle emulsion, so the molar ratio of calcium ions to citrate ions can be preferably 1:0.27-1:0.67.

[0228] Table 17 Digestibility of the emulsion in Example 17

[0229]

[0230] Example 18 Low-mineralized whole casein reconstituted micelle-stabilized emulsion: Effect of phosphate ion concentration

[0231] The whole casein recombinant micelle solutions in Example 10 were prepared according to the method in Example 12 to obtain a whole casein recombinant micelle-stabilized emulsion;

[0232] Alternatively, the concentration of the potassium citrate solution in Example 1 is adjusted to 499 mM and the concentration of the dipotassium hydrogen phosphate solution is adjusted to 0 mM, so that the molar ratio of calcium ions to citrate ions in the whole casein reconstituted micelle solution is 1:0.67 and the molar ratio of calcium ions to phosphate ions is 1:0.17, and then the emulsion is prepared according to the method in Example 12.

[0233] As shown in Table 18, the free amino content and lipolysis degree of the emulsion increased with the decrease of the molar ratio of calcium ions to phosphate ions during digestion, while the calcium bioaccessibility decreased significantly with the decrease of the molar ratio (P<0.05). This may be due to the high pH in the intestinal digestion stage, and the solubility of calcium ions gradually decreased after combining with phosphate ions, and may further combine with bile salts to form precipitation. Increasing the molar ratio of calcium ions to phosphate ions and reducing the molar ratio of calcium ions to citrate ions, that is, reducing the concentration of phosphate ions and increasing the concentration of citrate ions, the obtained emulsion not only has high protein and lipid digestibility, but also synergistically improves calcium bioaccessibility, that is, its calcium bioaccessibility is not only significantly higher than that of the 1:0.33 group, but also its increment (8.2%) is higher than the sum of the increment (6%) of the 1:0.67 group in Example 17 and the increment (0.6%) of the 1:0.17 group in Example 18. Taking into account the micellar digestibility and the bioavailability of mineral salts, the preferred molar ratio of calcium ions to phosphate ions is 1:0.17-1:0.90.

[0234] Table 18 Digestibility of the emulsion in Example 18

[0235] Ca:Pi Ca:Cit Free amino content (μmol / mg) Degree of lipolysis (%) Calcium bioaccessibility (%) 1:0.17 1:0.67 <![CDATA[0.68±0.02 ab ]]> <![CDATA[49.8±0.8 ab ]]> <![CDATA[35.8±1.2 a ]]> 1:0.17 1:0.37 <![CDATA[0.62±0.01 c ]]> <![CDATA[46.6±1.0 c ]]> <![CDATA[28.2±1.2 b ]]> 1:0.33 (Example 12) 1:0.37 <![CDATA[0.65±0.01 b ]]> <![CDATA[48.4±1.1 bc ]]> <![CDATA[27.6±1.5 b ]]> 1:0.52 1:0.37 <![CDATA[0.66±0.02 ab ]]> <![CDATA[49.7±1.1 ab ]]> <![CDATA[21.9±1.0 c ]]> 1:0.71 1:0.37 <![CDATA[0.67±0.02 ab ]]> <![CDATA[50.1±1.0 ab ]]> <![CDATA[20.0±1.3 cd ]]> 1:0.90 1:0.37 <![CDATA[0.69±0.01 a ]]> <![CDATA[50.6±1.0 a ]]> <![CDATA[16.8±3.0 d ]]> 1:1.12 1:0.37 <![CDATA[0.69±0.02 a ]]> <![CDATA[51.2±0.7 a ]]> <![CDATA[12.4±3.7 e ]]>

[0236] Example 19 Emulsion stabilized by low-mineralized whole casein reconstituted micelles: Effect of emulsification temperature

[0237] The “maintaining temperature at 50° C.” in Example 12 is adjusted to “maintaining temperature at 20, 30, 40, 50, 60, 70 or 80° C.” and the rest are kept consistent with Example 12 to obtain an emulsion.

[0238] As shown in Table 19, as the temperature increases during the preparation of the emulsion, the lipid droplet size and instability index of the emulsion show a trend of first decreasing and then increasing. Too low a temperature causes the lipid to be in a solid state, while too high a temperature causes the lipid viscosity to decrease and the fluidity to be too high, which is not conducive to the formation of a stable emulsion. In summary, an emulsification temperature of 40-60°C can be selected for the preparation of recombinant micelle-stabilized emulsions.

[0239] Table 19 Physicochemical properties of the emulsion in Example 19

[0240] Temperature(℃) Lipid droplet size (μm) Instability Index 20 <![CDATA[0.89±0.08 a ]]> <![CDATA[0.12±0.01 a ]]> 30 <![CDATA[0.75±0.06 ab ]]> <![CDATA[0.10±0.01 ab ]]> 40 <![CDATA[0.68±0.12 b ]]> <![CDATA[0.08±0.01 b ]]> 50 (Example 12) <![CDATA[0.66±0.14 b ]]> <![CDATA[0.08±0.01 b ]]> 60 <![CDATA[0.72±0.07 ab ]]> <![CDATA[0.09±0.01 b ]]> 70 <![CDATA[0.77±0.05 ab ]]> <![CDATA[0.10±0.02 ab ]]> 80 <![CDATA[0.86±0.10 a ]]> <![CDATA[0.12±0.02 a ]]>

[0241] Example 20 Comparison of bioavailability of emulsion fatty acids and calcium

[0242] The emulsions in Example 12, Example 15 (0% decalcified reconstitution), Example 17 (Ca:Cit=1:0.67), Example 18 (Ca:Pi=1:0.17), Example 18 (Ca:Pi=1:0.17, Ca:Cit=1:0.67), Comparative Example 4 and Comparative Example 6 were respectively gavaged into 2-week-old SD rats, and the feces of the rats were collected for determination.

[0243] As shown in Table 20, after the low-mineralized whole casein recombinant micelle stabilized emulsion in Example 12 was digested and absorbed in young mice, the calcium soap fatty acid content and relative fecal calcium content in the feces formed were both low. In comparison, the calcium soap fatty acid content and relative fecal calcium content of the recombinant micelle emulsion prepared using 0% decalcified MCC (Example 15) were significantly increased (P<0.05). In addition, increasing the citrate ion concentration (Example 17) or reducing the phosphate ion concentration (Example 18, Ca:Pi=1:0.17) further reduced the formation of calcium soap fatty acids and fecal calcium (P<0.05). The β+κ-casein reinforced recombinant micelle emulsion (Comparative Example 4) and the bovine milk casein micelle emulsion (Comparative Example 6) showed the highest calcium soap fatty acid formation and relative fecal calcium content. At the same time, increasing the citrate ion concentration and reducing the phosphate ion concentration (Example 18, Ca:Pi=1:0.17, Ca:Cit=1:0.67) also exhibited a synergistic effect. It not only significantly reduced the formation of calcium soap and fecal calcium compared to the whole casein reconstituted micellar emulsion in Example 12, but also achieved a reduction in the relative content of calcium soap fatty acids and fecal calcium (21.2 mg / g feces dry basis and 8.9%, respectively), which was higher than the sum of the reductions achieved by increasing the citrate ion concentration (Example 17) alone (12.7 mg / g feces dry basis and 4.8%, respectively) and reducing the phosphate ion concentration (Example 18, Ca:Pi=1:0.17) alone (4.4 mg / g feces dry basis and 2.1%, respectively). In summary, when preparing the whole casein recombinant micelle-stabilized emulsion, increasing the concentration of citrate ions or decreasing the concentration of phosphate ions can both improve the calcium absorption rate of the emulsion and reduce the formation of calcium soap, and the combination of the two also shows a synergistic effect of promoting absorption.

[0244] Table 20 Bioavailability of fatty acids and calcium in emulsions

[0245]

[0246] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 low-mineralized whole casein recombinant micelles, characterized in that: The steps include: The temperature of the decalcified micellar casein solution is adjusted to 37-57°C and the pH is 6.6-7.2; calcium ions, citrate ions and phosphate ions are then added, and the temperature is adjusted to 25°C and the pH is 7.0, and the volume is constant to obtain a low-mineralized whole casein reconstituted micellar solution, wherein α s1 -、α s2 The mass proportions of -, β-, and κ-casein are 5-45%, 5-25%, 25-65%, and 5-25%, respectively.

2. The method according to claim 1, characterized in that The decalcification rate of the decalcified micellar casein solution is 42%-81%; the decalcified micellar casein solution is a decalcified micellar casein aqueous solution with a mass concentration of 0.8%-2.8%; the mass concentration of casein in the prepared low-mineralized whole casein recombinant micellar solution is 0.4%-1.4%; the molar ratio of calcium ions to citrate ions is 1:0.27-1:0.67, and the molar ratio of calcium ions to phosphate ions is 1:0.17-1:0.

90.

3. The low-mineralized whole casein reconstituted micellar solution prepared by the method according to claim 1 or 2.

4. A casein ingredient, characterized in that: The low-mineralized whole casein recombinant micelle solution according to claim 3 is dried to obtain the product.

5. A method for preparing an emulsion stabilized by low-mineralized whole casein recombinant micelles, characterized in that: The steps include: Anhydrous milk fat is added to the low-mineralized whole casein recombinant micelle solution of claim 3, the temperature is maintained at 40-60°C, the mixture is stirred and balanced at 300-500rpm for 10-20min, shear homogenized at 20000-30000rpm for 5-10min and high-pressure homogenized at 30-50MPa for 3-5 times, and the temperature is then adjusted to 25°C to obtain a stable emulsion of low-mineralized whole casein recombinant micelles.

6. The low-mineralized whole casein reconstituted micelle-stabilized emulsion prepared by the method according to claim 5.

7. An infant formula powder, characterized in that: The low-mineralized whole casein recombinant micelle solution according to claim 3, the casein ingredient according to claim 4 or the low-mineralized whole casein recombinant micelle stabilized emulsion according to claim 6 is used.

8. Use of the low-mineralized whole casein recombinant micelle solution according to claim 3, the casein ingredient according to claim 4, or the low-mineralized whole casein recombinant micelle stabilized emulsion according to claim 6 in the field of dairy product processing.

9. A method for improving the calcium bioaccessibility of an emulsion stabilized by whole casein recombinant micelles, characterized in that: The low-mineralized whole casein recombinant micelle solution according to claim 3, the casein ingredient according to claim 4 or the low-mineralized whole casein recombinant micelle stabilized emulsion according to claim 6 is used, and when the molar ratio of calcium ions to citrate ions is 1:0.57-1:0.67 and the molar ratio of calcium ions to phosphate ions is 1:0.17-1:0.33, a synergistic effect of promoting calcium absorption is exhibited.

10. A method for reducing the amount of calcium soap fatty acid generated in an emulsion stabilized by whole casein recombinant micelles after gastrointestinal digestion, characterized in that: The low-mineralized whole casein recombinant micelle solution according to claim 3, the casein ingredient according to claim 4 or the low-mineralized whole casein recombinant micelle stabilized emulsion according to claim 6 is used, and when the molar ratio of calcium ions to citrate ions is 1:0.57-1:0.67 and the molar ratio of calcium ions to phosphate ions is 1:0.17-1:0.33, the effect of synergistically reducing the formation of calcium soap is shown.

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