Infant formula milk powder rich in basic protein and good in protein digestibility and preparation method of infant formula milk powder

Abstract

The invention discloses infant formula milk powder rich in basic protein and good in protein digestibility and a preparation method of the infant formula milk powder, and belongs to the technical field of dairy product preparation. The preparation method comprises the following steps: degreasing and degerming fresh cow milk, performing basic protein phase transfer, enriching basic protein into a whey phase through filtration of a microfiltration membrane, and further desalting and concentrating the whey phase through ultrafiltration and electrodialysis to form active whey rich in basic protein after sterile concentration; separating the concentrated active whey through a cation exchange column to obtain active whey without basic protein, then eluting the cation exchange column to obtain a basic protein phase, and desalting the basic protein phase; performing milk killing on the trapped fluid filtered by the micro-filtration membrane, and then performing blending, homogenizing, material killing and concentration on the trapped fluid, active whey with basic protein removed and other nutrients to form the infant formula emulsion; and finally, mixing the infant formula emulsion with the basic protein on line, and performing spray drying to obtain the infant formula milk powder which is rich in basic protein and good in protein digestibility.

Description

Technical Field

[0001] The invention relates to an infant formula milk powder which is rich in alkaline protein and has good protein digestibility and a preparation method thereof, and belongs to the technical field of dairy product preparation. Background Art

[0002] Infant formula milk powder is usually made from cow and sheep milk, with appropriate nutrients added to make its total ingredients meet the needs of infant growth and development. In recent years, the research on infant formula milk powder has deepened the exploration of the molecular structure and function of breast milk and the active ingredients of breast milk, such as the strengthening of active lactoferrin. Although the adjustment based on the formula can achieve a composition and function similar to breast milk, the production process of infant formula milk powder requires a series of heat treatment steps, such as sterilization, concentration, spray drying, etc. These heat treatment operations will cause the heat-sensitive alkaline proteins such as lactoferrin and immunoglobulins in milk to denature and lose their biological activity, thereby reducing the quality of infant formula milk powder, and the existing processing technology of infant formula milk powder is almost difficult to retain the activity of functional proteins in fresh milk.

[0003] In addition to active proteins, cow's milk also contains abundant high-abundance proteins, such as α-lactalbumin, β-lactoglobulin, and casein. These proteins are the source of essential amino acids for infants and young children, and can provide the nutritional needs for growth and development of infants and young children; therefore, the good digestion and absorption characteristics of high-abundance proteins are one of the important manifestations of the nutritional value of infant formula. During the heat treatment process, the protein structure will unfold due to heat, the secondary structure will change, the polypeptide chain of the protein will unfold, and the active groups and protein cleavage sites will be exposed, resulting in improved protein digestibility. However, excessive heat treatment may also cause aggregation between proteins, offsetting the improvement in digestion caused by unfolding, and it is necessary to find a balance between the intensity of heat treatment and the improvement in protein digestibility.

[0004] In order to improve the active nutrition of infant formula milk powder protein, currently commercial infant formula milk powder mainly adopts dry or wet process to strengthen basic protein (lactoferrin and immunoglobulin), but most of these ingredients need to rely on import, expensive, and usually only strengthen 1-2 kinds of basic protein at a time, and it is impossible to achieve the retention of most basic protein in whey; even if the retention of most active protein can be achieved by strengthening the method of active whey protein, these ingredients are all spray-dried or freeze-dried products after separation, and the active protein in the whey ingredients will also undergo partial denaturation and reduction of biological activity during the drying process. In addition, the biggest problem in whey protein ingredients is that the content of basic proteins such as lactoferrin is relatively low; because these proteins are all positively charged, they are mainly combined with negatively charged casein in milk, causing most of the basic proteins such as lactoferrin to be trapped in the casein phase when separating whey protein.

[0005] Therefore, how to develop a method for preparing infant formula milk powder that is online enriched with alkaline protein and has good protein digestibility and absorbability is an important problem that needs to be solved urgently in the art. Summary of the invention

[0006] [Technical issues]

[0007] The existing processing technology of infant formula milk powder can hardly retain the activity of alkaline protein in fresh milk;

[0008] The basic proteins in the whey protein ingredients used in infant formula milk powder will partially denature, resulting in reduced biological activity; and the content of basic proteins such as lactoferrin is low, resulting in a huge gap between the prepared infant formula milk powder and breast milk;

[0009] Moderate heat treatment can improve protein digestion and absorption properties, but excessive heat treatment can induce protein aggregation and reduce protein digestibility.

[0010] [Technical solution]

[0011] In order to solve the above problems, the present invention provides an infant formula milk powder rich in alkaline protein and having good protein digestibility and a preparation method thereof; specifically, the present invention firstly performs alkaline protein phase transfer after defatting and sterilizing fresh cow milk, and enriches the alkaline protein to the whey phase through microfiltration membrane filtration, and further desalts and concentrates the whey phase through ultrafiltration and electrodialysis to form aseptically concentrated active whey rich in alkaline protein; the concentrated active whey is separated through a cation exchange column to obtain active whey with alkaline protein removed, and then the cation exchange column is eluted to obtain the alkaline protein phase, which is desalted; the retentate filtered by the microfiltration membrane is milk sterilized (primary sterilization), and then mixed, homogenized, sterilized (secondary sterilization) and concentrated with the active whey with alkaline protein removed and other nutrients to form an infant formula milk; finally, the infant formula milk and the alkaline protein phase are mixed online and spray-dried to obtain an infant formula milk powder rich in alkaline protein and having good protein digestibility.

[0012] The first object of the present invention is to provide a method for preparing an infant formula milk powder containing alkaline protein and good protein digestibility, comprising the following steps:

[0013] (1) adding sodium chloride to the sterilized skim milk to release the alkaline protein, thereby obtaining the skim milk after the alkaline protein phase transfer;

[0014] (2) filtering the skim milk after the alkaline protein phase transfer in step (1) through a membrane to separate the whey protein phase from the casein phase, thereby obtaining a membrane filtration permeate (whey protein rich in alkaline protein) and a membrane filtration retentate (casein); wherein the filtrate used in the membrane filtration is a sodium chloride solution;

[0015] (3) desalting and concentrating the membrane filtration retentate (casein) of step (2) to obtain a concentrated and desalted membrane filtration retentate;

[0016] (4) ultrafiltration and desalination of the membrane filtration permeate (whey protein rich in basic protein) of step (2) to obtain aseptically concentrated active whey rich in basic protein;

[0017] (5) passing the aseptically concentrated active whey rich in basic protein in step (4) through a cation exchange column to transfer the basic protein from the whey to the cation exchange column to obtain a whey phase free of basic protein; then eluting the basic protein on the cation exchange column with a sodium chloride solution to obtain a basic protein phase;

[0018] (6) desalting the alkaline protein phase of step (5) to obtain a desalted alkaline protein phase;

[0019] (7) mixing the concentrated and desalted membrane filtration retentate of step (3) with the whey phase free of alkaline protein of step (5), followed by pasteurization to obtain a protein mixture;

[0020] (8) preparing and fully mixing the protein mixture of step (7) with other nutrients, and then homogenizing, sterilizing and concentrating to obtain an infant formula emulsion;

[0021] (9) The liquid infant formula obtained in step (8) and the desalted alkaline protein obtained in step (6) are mixed, followed by spray drying to obtain an infant formula milk powder rich in alkaline protein and having good protein digestibility.

[0022] In one embodiment of the present invention, the method for preparing the sterilized skim milk in step (1) is as follows:

[0023] After preheating the raw milk to 40-42°C, the milk is centrifuged in a disc centrifuge to obtain cream and skimmed milk; the skimmed milk is then filtered using a 1.4μm microfiltration membrane with the temperature controlled at 40-45°C to remove most of the microorganisms and impurities in the milk to obtain sterilized skimmed milk.

[0024] In one embodiment of the present invention, the concentration of sodium chloride added in step (1) is 200-400 mmol / L, the temperature is 35-39°C, and the pH is 6-7; more preferably, the concentration of sodium chloride added is 300 mmol / L, the temperature is 37°C, and the pH is 6.6.

[0025] In one embodiment of the present invention, in step (1), the alkaline protein is transferred from the casein phase to the whey phase.

[0026] In one embodiment of the present invention, the membrane pore size used in the membrane filtration in step (2) is 90-110nm, the temperature is 35-55°C, the concentration multiple is 2-4, and the number of diafiltrations is 3-5; further preferably, the membrane pore size used in the membrane filtration is 100nm, the temperature is 35-55°C, the concentration multiple is 3, and the number of diafiltrations is 4.

[0027] In one embodiment of the present invention, the operational endpoint of the membrane filtration in step (2) is determined by "the liquid volume in the membrane filtration retentate becomes one third of the initial volume".

[0028] In one embodiment of the present invention, the sodium chloride solution in step (2) is a sodium chloride aqueous solution with a concentration of 200-400 mmol / L.

[0029] In one embodiment of the present invention, after each filtration in the membrane filtration of step (2) reaches the end point of the operation, the filtrate is backfilled to the initial volume and filtered again.

[0030] In one embodiment of the present invention, in step (3), desalination and concentration are performed by filtering with a 0.05-0.15 μm microfiltration membrane, and deionized water is used as a secondary filtration solution; the amount of the secondary filtration solution used is 5-10 times the volume of the retentate, the end point of the desalination operation is determined by "the conductivity value is reduced to the normal retentate conductivity range", and the protein concentration is determined by the Kjeldahl method to determine the total protein concentration in the retentate; further preferably, the pore size of the microfiltration membrane is 0.1 μm.

[0031] In one embodiment of the present invention, the ultrafiltration membrane used in the ultrafiltration in step (4) has a molecular weight cutoff of 9-11 kDa, a concentration multiple of 5-10 times, and an ultrafiltration temperature not exceeding 45°C; further preferably, the ultrafiltration membrane has a molecular weight cutoff of 10 kDa; and the ultrafiltration temperature is 40°C.

[0032] In one embodiment of the present invention, in step (4), desalination is performed by using ultrapure water as the second ultrafiltration solution, and the amount of ultrafiltration solution used is 5-10 times that of the concentrated permeate. After the ultrafiltration is completed, electrodialysis is performed for further desalination; the electrodialysis adopts a working current of 45A, a feed flow rate of 110mL / min, and a feed temperature of 35°C; the endpoint of the desalination operation is determined by "the conductivity value is reduced to the normal permeate conductivity range", and the protein concentration is determined by the Kjeldahl method to determine the total protein concentration in the permeate.

[0033] In one embodiment of the present invention, the filler of the cation exchange column used in step (5) is CM-Sepharose Fast Flow.

[0034] In one embodiment of the present invention, the alkaline protein is eluted with an aqueous solution of sodium chloride in step (5), and the concentration of sodium chloride is 0.4M-1.0M, and more preferably, the concentration of sodium chloride is 0.7M.

[0035] In one embodiment of the present invention, the desalination in step (6) is performed by dialysis using a dialysis bag with a molecular weight cutoff of 7-8 kDa, and the dialysis is performed until the conductivity of the solution reaches a normal range.

[0036] In one embodiment of the present invention, in step (7), the ratio of the concentrated and desalted membrane filtration retentate of step (3) to the whey phase free of alkaline protein of step (5) is 4:6, which is the protein ratio of the two; it is close to the protein ratio in breast milk.

[0037] In one embodiment of the present invention, the sterilization in step (7) is performed at 80-90°C for 10-20s, and more preferably, at 85°C for 15s.

[0038] In one embodiment of the present invention, the protein mixture in step (7) is used as a milk base in the preparation process of infant formula milk powder.

[0039] In one embodiment of the present invention, the nutrients required in the infant formula in step (8) are calculated in accordance with the national food safety standard GB 10765-2021, based on the nutrients required for infants aged 0-6 months, and the content of the nutrients required in the infant formula liquid milk is calculated, with casein liquid, lactose, and vegetable blending oil as the main raw materials, and complex vitamins, minerals, taurine, L-carnitine, etc. are added.

[0040] In one embodiment of the present invention, the nutrients required in the infant formula in step (8) include the following components by dry matter weight: 6.19% whey protein, 4.13% casein, 53.76% lactose, 34.34% vegetable blended oil (by mass percentage, including the following raw material components: 25% coconut oil, 22% OPO, 18% soybean oil, 15% corn oil, 10% low-erucic acid rapeseed oil, 10% high-oleic acid sunflower oil), 1.49% complex minerals (by mass percentage, including the following raw material components: 10.93% calcium citrate, 19.72% potassium chloride, 18.84% sodium citrate, 10.93% calcium citrate, 4.08% magnesium sulfate, 1.25% ferric pyrophosphate, 0.32% zinc sulfate, 0.28% potassium iodate, 0.18% copper sulfate, 0.06% manganese sulfate, 0.05% sodium selenite), 0.31% complex vitamins. % (by mass percentage, including the following raw material components: L-sodium ascorbate 55.67%, dl-α-tocopherol acetate 9.84%, cholecalciferol 3.64%, retinyl acetate 2.58%, niacinamide 2.50%, D-calcium pantothenate 2.03%, cyanocobalamin 1.20%, biotin 1.12%, phytonadione 0.84%, thiamine hydrochloride 0.58%, folic acid 0.47%, riboflavin 0.36%, pyridoxine hydrochloride 0.34%), inositol 0.04%, taurine 0.04%, and L-carnitine 0.01%.

[0041] In one embodiment of the present invention, the required nutrients are added to the infant formula in step (8) as follows:

[0042] Lactose and complex mineral premix are mixed and dissolved in a special container using 40-50°C purified water. Complex vitamin premix and nutritional enhancers (inositol, taurine, L-carnitine) are dissolved separately using 40-45°C purified water. Then, the lactose mineral mixture, complex vitamin mixture and complex vegetable oil are added step by step to the casein solution and mixed thoroughly. The mixing time for adding complex minerals and complex vitamins should be less than 15 minutes. Then, inositol, taurine and L-carnitine are added and mixed thoroughly.

[0043] In one embodiment of the present invention, the homogenization in step (8) is carried out at 25-30 MPa for 1-2 times.

[0044] In one embodiment of the present invention, the sterilization in step (8) is carried out at 90-100°C for 10-20s; more preferably, at 95°C for 15s.

[0045] In one embodiment of the present invention, the concentration in step (8) is evaporation concentration, specifically using a vacuum rotary evaporator with a vacuum degree of 0.07-0.09 MPa, a temperature of 55-57° C., a rotation speed of 50-90 rpm, and rotary evaporation until the solid content reaches 30-40% (mass percentage).

[0046] In one embodiment of the present invention, the spray drying in step (9) is low-temperature and low-pressure spray drying, specifically: the material inlet temperature is not higher than 80°C, and the material outlet temperature is adjusted to 52-55°C by flow regulation.

[0047] In one embodiment of the present invention, the infant formula milk powder containing alkaline protein and good protein digestibility is aseptically filled and stored at room temperature after processing.

[0048] The second object of the present invention is to prepare an infant formula milk powder rich in basic protein and having good protein digestibility by the method of the present invention.

[0049] In one embodiment of the present invention, the basic protein includes lactoferrin, immunoglobulin IgG, lactoperoxidase and the like.

[0050] The third object of the present invention is to use the infant formula milk powder rich in alkaline protein and having good protein digestibility in the field of food.

[0051] The fourth object of the present invention is to provide a method for increasing the content of basic protein in infant formula milk powder and maintaining good protein digestibility, wherein the method uses the infant formula milk powder rich in basic protein and having good protein digestibility of the present invention.

[0052] [Beneficial Effects]

[0053] (1) The preparation method of the infant formula milk powder rich in alkaline protein and good in protein digestibility in the present invention is to add sodium chloride to skim milk to increase the ion concentration, promote the dissociation of casein micelles and alkaline proteins, and more of them are present in the whey phase in a free state; then use a microfiltration membrane to separate the whey protein phase rich in alkaline protein and casein; then use a cation exchange column to adsorb and elute the alkaline protein in the whey protein to obtain an alkaline protein phase. The whey protein phase obtained by this method has a significantly improved alkaline protein enrichment rate compared to the whey protein phase that has not been treated with alkaline protein release, and the alkaline protein phase is separated and enriched by an ion exchange column for subsequent infant formula milk powder processing.

[0054] (2) The present invention uses casein as the milk base in the production process of infant formula milk powder, uses whey from which alkaline protein is separated as the whey protein ingredient, and adds the separated alkaline protein before spray drying to avoid the loss of alkaline protein due to multiple heat treatments. At the same time, the high-abundance milk protein is subjected to a certain intensity of heat treatment to improve digestibility. This process greatly increases the abundance of alkaline protein in the finished infant formula milk powder and maintains good protein digestibility.

[0055] In summary, the infant formula milk powder prepared by the method of the present invention retains more alkaline active proteins and maintains good protein digestibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The present invention is a process flow chart for preparing infant formula milk powder rich in alkaline protein and having good protein digestibility (Example 1 is Route 1, Comparative Example 1 is Route 2).

[0057] Figure 2 This is a process flow chart for preparing infant formula milk powder in Comparative Example 2.

[0058] Figure 3 These are the test results of SDS-PAGE of digestive fluids obtained by re-dissolving and in vitro digestion experiments of infant formula milk powder obtained in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 (Commercially Available 1 and Commercially Available 2).

[0059] Figure 4 These are the test results of polypeptide molecular weight of digestive fluid obtained by re-dissolving and in vitro digestion experiments of infant formula milk powder obtained from Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 (Commercially Available 1 and Commercially Available 2).

[0060] Figure 5 These are the test results of free amino acids in the digestive juice obtained by re-dissolving and in vitro digestion experiments of infant formula milk powder obtained from Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 (Commercially Available 1 and Commercially Available 2).

[0061] Figure 6 The SDS-PAGE test results of different cation exchange column fillers for basic protein separation in Example 2 are shown.

[0062] Figure 7 The SDS-PAGE test results of different cation exchange column eluents for basic protein separation in Example 3 are shown.

[0063] Figure 8The SDS-PAGE results of whey with different pH values ​​after passing through the column (A); the total recovery rate of the received whey and the proportion of eluted protein after whey with different pH values ​​passed through the column (B); the proportion of eluted protein specifically refers to the proportion of protein in the eluate of sodium chloride solution, the black bar graph is the total recovery rate of whey protein, and the gray bar graph is the elution protein ratio; the concentration and recovery rate of lactoferrin in the eluate (C), where the bar graph is the concentration and the line graph is the recovery rate; the macroscopic situation of whey at different pH values ​​(D). DETAILED DESCRIPTION

[0064] 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.

[0065] Test method:

[0066] 1. Active whey extraction:

[0067] Infant formula milk powder and water were dissolved in a mass ratio of 1:8 under magnetic stirring at 45°C for 1 h to obtain a reconstituted milk sample.

[0068] 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE:

[0069] Reduction electrophoresis was performed on 12% separating gel and 4% stacking gel.

[0070] Use 2x sample buffer to mix with reconstituted milk sample solution 1:1 (v / v), add 5% β-mercaptoethanol at the same time, and heat in boiling water bath for 3 minutes after mixing to obtain denatured reconstituted milk protein. Add 10uL of denatured reconstituted milk protein to each loading well.

[0071] The initial voltage was 60 V. When the sample band moved to the junction of the concentrated gel and the separation gel, the voltage was increased to 120 V. SDS-PAGE gel was stained with Coomassie Brilliant Blue (R-250), shaken at room temperature for 1.5 h, and then destained with a destaining solution until the gel background was colorless.

[0072] The images were taken using the Chemi Doc XRS+ chemiluminescent gel imaging system.

[0073] 3. In vitro static digestion:

[0074] Simulated in vitro infant digestion includes gastric and intestinal stages. 63 mL of reconstituted milk samples with different processes and a protein concentration of 11.8 mg / mL were added to a reactor at a constant temperature of 37°C and kept at the constant temperature for 15 minutes.

[0075] For gastric digestion, 1M HCL solution was used to adjust the pH to 5.3, and the ratio of milk sample to gastric digestion fluid was 63:37 (v:v). The gastric digestion fluid contained 94mM NaCl, 13mM KCL, pepsin activity 268U / mL, and lipase activity 19U / mL. The gastric digestion process lasted for 180min, and samples were taken at 0 (including digestive enzymes and enzyme inhibitors), 5, 15, 30, 60, and 120min, respectively. The gastric digestion reaction was terminated by mixing 1M sodium hydroxide solution at a ratio of 1:40.

[0076] For intestinal digestion, the gastric chyme at 120 min of gastric digestion was used;

[0077] Specifically: 1M NaOH solution was used to adjust the pH value to 7 at 60 minutes of gastric digestion to terminate the gastric digestion reaction, and then 1M HCL solution was used to adjust the pH to 6.6. The volume ratio of gastric chyme to intestinal digestive fluid was 63:37, and the intestinal digestive fluid contained 3.1mM ox bile salt, 164mM NaCl, 10mM KCl and 85mM NaHCO3, protease activity of 16U / mL, and lipase activity of 21U / mL. After 0, 1, 5, 15, 30, 60, and 120 minutes of intestinal digestion, enzyme inhibitors containing 0.1M phenylmethylsulfonyl fluoride and 10mM orlistat were mixed with the sample at 1:40 (v:v) to terminate the intestinal digestion reaction.

[0078] Digestion samples were prepared in duplicate. Samples that could not be analyzed immediately were stored at -80°C until further analysis.

[0079] 4. Determination of polypeptide molecular weight:

[0080] A Waters e2695 high performance liquid chromatography system was used to detect the release of peptides during gastrointestinal digestion.

[0081] Take the in vitro digestion samples of each group, centrifuge at 4°C, 13400×g for 30 min, take the supernatant, pass through the membrane, and inject it into a TSK-GELG2000SWxl column (300mm×7.8mm, 5μm); the mobile phase is acetonitrile-water-trifluoroacetic acid (400:600:1, v / v / v), and the flow rate is 0.5mL / min. The column temperature and detection wavelength are set to 30°C and 220nm, respectively.

[0082] 5. Determination of free amino acids:

[0083] The sample pre-treatment before loading is as follows:

[0084] 10% (w / v, mg / mL) TCA solution was added to the gastrointestinal digestion sample at a ratio of 1:1 (v / v), mixed and ultrasonicated for 30 minutes, allowed to stand overnight, and then centrifuged at 4°C and 10,000×g for 30 minutes. The supernatant was filtered through a 0.45 um filter membrane and set aside.

[0085] Agilent 1100 high performance liquid chromatography system was used for detection, and the chromatographic conditions were as follows: Hypersil ODS C18 chromatographic column (4.6 mm×150 mm, 5 μm); column temperature 40°C; detection wavelength 338 nm (262 nm for proline); mobile phase A was 27.6 mmol / L sodium acetate-triethylamine-tetrahydrofuran solution (500:0.11:2.5, v / v / v), mobile phase B was 80.9 mmol / L sodium acetate-methanol-acetonitrile (1:2:2, v / v / v); flow rate 1.0 mL / min; the amino acid content was quantified by the external standard method.

[0086] Materials and instruments:

[0087] BCA kit was from Thermo Fisher Scientific;

[0088] Lactoferrin and immunoglobulin reagent test kits (ElisaKit) were purchased from Bethyl Company, USA;

[0089] The remaining reagents were of analytical grade;

[0090] Cation exchange column filler CM-Sepharose Fast Flow: commercially available;

[0091] The preparation method of sterilized skim milk is as follows:

[0092] After preheating the raw milk to 42°C, it is centrifuged in a disc centrifuge to obtain cream and skimmed milk; the skimmed milk is then filtered using a 1.4μm microfiltration membrane with the temperature controlled at 40°C to remove most of the microorganisms and impurities in the milk to obtain sterilized skimmed milk.

[0093] Commercially available 1, Commercially available 2: Two types of infant formula powder available on the market.

[0094] The solutions involved in the embodiments and comparative examples that do not specify the reaction solvent are water as the solvent, and the reaction temperature that is not specified is room temperature (20-30° C.).

[0095] Example 1 (see Figure 1 Route 1)

[0096] A method for preparing an infant formula milk powder rich in basic protein and having good protein digestibility (such as Figure 1Middle route 1) includes the following steps:

[0097] (1) adding 300 mmol / L sodium chloride to the sterilized skim milk while stirring (500 rpm), controlling the temperature at 37° C. and the pH at 6.6, until the sodium chloride is completely dissolved, thereby obtaining skim milk after alkaline protein phase transfer;

[0098] (2) filtering the skim milk after the alkaline protein phase transfer in step (1) using a 100 nm microfiltration membrane to separate the whey protein phase from the casein phase, thereby obtaining a membrane filtration permeate (whey protein rich in alkaline protein) and a membrane filtration retentate (casein);

[0099] The filtration temperature is controlled at 37±2℃, and the end point of the filtration operation is determined by "the volume of the liquid in the membrane filtration retentate becomes one third of the initial volume", that is, the concentration multiple is 3 times;

[0100] The filtration was performed using a 300 mmol / L sodium chloride aqueous solution as the filtrate. After each filtration reached the end point of the operation, the filtrate was backfilled to the initial volume and re-filtered, and this was repeated 4 times.

[0101] (3) filtering the membrane filtration retentate (casein) of step (2) using a 0.1 μm microfiltration membrane, using deionized water as a secondary diafiltration solution, and the amount of the secondary diafiltration solution used is 8 times the volume of the retentate; the end point of the desalting and concentration operation is determined by "the conductivity value is reduced to the normal retentate conductivity range"; and the total protein concentration in the retentate is determined by the Kjeldahl method;

[0102] (4) The membrane filtration permeate (whey protein rich in basic protein) of step (2) is ultrafiltered and concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, with a concentration multiple of 8 times and an ultrafiltration temperature of 40° C.; ultrapure water is then used as a second ultrafiltration solution, and the amount of ultrafiltration solution used is 8 times that of the concentrated permeate; after the ultrafiltration is completed, electrodialysis (working current of 45 A, feed flow rate of 110 mL / min, feed temperature of 35° C.) is further performed for desalination, and the end point of the desalination operation is determined by "the conductivity value is reduced to the normal permeate conductivity range"; and active whey rich in basic protein after sterile concentration is obtained (this step skips the sterilization unit, and membrane filtration is used to remove microorganisms therein and further concentrate to obtain active whey rich in basic protein);

[0103] (5) adjusting the pH of the aseptically concentrated active whey rich in basic protein in step (4) to 6.9 by 1M HCl solution, then pumping the whey rich in basic protein into a cation exchange column filled with resin filler CM-Sepharose Fast Flow by a peristaltic pump, and collecting the whey flowing out at the end of the column, which is a whey phase without basic protein; then flushing the column with pure water until all the residual whey is discharged, mixing it with the whey received previously, and determining the total protein concentration of the mixed whey by Kjeldahl method; then eluting with a NaCl aqueous solution with a sodium chloride concentration of 0.7M, collecting the liquid flowing out at the end of the column, which is an alkaline protein phase;

[0104] (6) dialyzing the alkaline protein phase for desalination using a dialysis bag with a molecular weight cutoff of 7.5 KDa, wherein the dialysate is ultrapure water, and dialyzing is performed until the conductivity of the alkaline protein phase is in the range of 4-10 mS / m, thereby obtaining a desalted alkaline protein phase;

[0105] (7) mixing the concentrated and desalted membrane filtration retentate (casein) of step (3) with the whey phase without alkaline protein of step (5) in a ratio of casein to whey protein of 4:6, and then pasteurizing the mixture using a tube-sheet combined ultra-high temperature sterilizer at 85° C. for 15 seconds to obtain a protein mixture;

[0106] (8) In accordance with the national food safety standard GB 10765-2021, the nutrients required by infants aged 0-6 months are used as the standard to calculate the content of nutrients required in infant formula liquid milk, with casein liquid, lactose, and vegetable blending oil as the main raw materials, and added with complex vitamins, minerals, taurine, L-9-hydroxy-carnitine, etc.;

[0107] The following components are included by weight of dry matter: whey protein 6.19%, casein 4.13%, lactose 53.76%, vegetable blended oil 34.34% (by mass percentage, including the following raw material components: coconut oil 25%, OPO 22%, soybean oil 18%, corn oil 15%, low-erucic acid rapeseed oil 10%, high-oleic acid sunflower oil 10%), complex minerals 1.49% (by mass percentage, including the following raw material components: calcium citrate 10.93%, potassium chloride 19.72%, sodium citrate 18.84%, calcium citrate 10.93%, magnesium sulfate 4.08%, ferric pyrophosphate 1.25%, zinc sulfate 0.32%, potassium iodate 0.28%, copper sulfate 0.18%, manganese sulfate 0.06%, sodium selenite 0.05%), complex vitamins 0.31 % (by mass percentage, including the following raw material components: L-sodium ascorbate 55.67%, dl-α-tocopherol acetate 9.84%, cholecalciferol 3.64%, retinyl acetate 2.58%, niacinamide 2.50%, D-calcium pantothenate 2.03%, cyanocobalamin 1.20%, biotin 1.12%, phytonadione 0.84%, thiamine hydrochloride 0.58%, folic acid 0.47%, riboflavin 0.36%, pyridoxine hydrochloride 0.34%), inositol 0.04%, taurine 0.04%, L-carnitine 0.01%;

[0108] Lactose and a composite mineral premix are mixed and dissolved in a dedicated container using 45° C. pure water, a composite vitamin premix and a nutritional enhancer (inositol, taurine, and L-carnitine) are dissolved in 45° C. pure water, respectively, and then the lactose mineral mixture, the composite vitamin mixture, and the composite vegetable oil are added step by step to the protein mixture obtained in step (7) and mixed thoroughly. The composite mineral and composite vitamin are added and mixed for 20 minutes, and then inositol, taurine, and L-carnitine are added and mixed thoroughly to obtain an emulsion;

[0109] The whey protein to casein mass ratio is 6:4, close to the protein ratio in breast milk, and the protein content accounts for 15% of the total solids;

[0110] The emulsion was homogenized twice at 25 MPa, sterilized at 95° C. for 15 seconds, and then placed in a vacuum rotary evaporator (vacuum degree of 0.08 MPa, temperature of 56° C., rotation speed of 70 rpm) for rotary evaporation until the solid content reached 35%, thereby obtaining an infant formula emulsion;

[0111] (9) According to the results of Kjeldahl nitrogen determination of protein content, infant formula milk and alkaline protein are mixed; a low-temperature and low-pressure spray drying device is used, the material inlet temperature of alkaline protein is 70°C, and the material outlet temperature is adjusted to 53°C by flow adjustment to obtain infant formula milk powder rich in alkaline protein and good protein digestibility, and the powder is sealed and packaged in a light-proof manner.

[0112] Comparative Example 1 ( Figure 1 Route 2)

[0113] Specific process routes such as Figure 1 Route 2 is different from Example 1 in that:

[0114] The whey phase without alkaline protein in step (5) of Example 1 is transferred to step (8), and the method is changed as follows:

[0115] Step (7): pasteurizing the membrane filtration retentate after concentration and desalination in step (3) at 85° C. for 15 seconds;

[0116] Step (8): Lactose and the composite mineral premix are mixed and dissolved in a dedicated container using 45° C. pure water, and the composite vitamin premix and the nutritional enhancer (inositol, taurine, and L-carnitine) are dissolved in 45° C. pure water, respectively, and then the whey phase not containing alkaline protein, the lactose mineral mixture, the composite vitamin mixture, and the composite vegetable oil are added step by step to the protein mixture obtained in step (7) and mixed thoroughly. The composite mineral and composite vitamin addition and mixing time is 20 minutes, and then inositol, taurine, and L-carnitine are added and mixed thoroughly to obtain an emulsion;

[0117] Among them, the mass ratio of whey protein to casein is 6:4, which is close to the protein ratio in breast milk, and the protein content accounts for 15% of the total solids;

[0118] The emulsion was homogenized twice at 25 MPa, and then sterilized at 95° C. for 15 seconds to obtain an infant formula emulsion.

[0119] Comparative Example 2

[0120] Specific process routes such as Figure 2 As shown, this example is different from Example 1 in that:

[0121] The alkaline protein separation step is not performed, that is, step (4) and step (5) are omitted, and the remaining steps are the same as those in Example 1 to obtain infant formula milk powder.

[0122] Comparative Example 3

[0123] Two types of stage 1 infant formula milk powder were purchased from the market, namely, Commercial 1 (protein 10%, fat 27.2%, carbohydrate 53.7%) and Commercial 2 (Commercial 2 is a lactoferrin-fortified version, with 10% protein, 27.2% fat, and 54.7% carbohydrate).

[0124] The infant formula milk powder obtained in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 was reconstituted to obtain milk samples, and subsequent in vitro digestion experiments were performed. The digestive fluid was subjected to SDS-PAGE, free amino acids, and polypeptide molecular weight index determination. The determination results are as follows:

[0125] Figure 3 The SDS-PAGE results of the digestive fluid obtained by re-dissolving the infant formula milk powder obtained in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 (commercially available 1 and commercially available 2) and simulating the in vitro digestion experiment of infants and young children are shown in Figure 1. G is the gastric digestion part and I is the intestinal digestion part. Figure 3 It can be seen that the reconstituted milk protein in Example 1 is digested slowly under the action of gastric digestion, and some protein bands gradually become lighter. After intestinal digestion, the remaining proteins and polypeptides are rapidly digested, leaving only a few protein bands, and the electrophoresis results are no longer significantly different after 1 minute of intestinal digestion. The difference between the changes in Comparative Example 1 and Example 1 is not large, both are slowly digested in the stomach, and there is no significant difference in the rapid digestion in the intestinal digestion stage. Compared with Example 1 and Comparative Example 1, Comparative Example 2 has a shallower small molecule peptide band at the bottom of the gastric digestion stage, indicating that the protein degradation is less during gastric digestion, and there is no significant difference in the intestinal stage. The gastrointestinal digestion of commercially available 1 and commercially available 2 milk powders in Comparative Example 3 is not significantly different from that of Example 1, Comparative Example 1 and Comparative Example 2. Commercially available 1 does not add lactoferrin, so there is no corresponding band near 80kDa.

[0126] Figure 4 The following are the polypeptide molecular weight test results of the digestive fluid obtained by re-dissolving the infant formula milk powder obtained in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 (commercially available 1 and commercially available 2) and simulating the in vitro digestion experiment of infants. Figure 4It can be seen that: Example 1 is similar to the electrophoresis results, the gastric digestion process is relatively slow, and the results of G5 and G15 are not much different. When the gastric digestion lasts for 120 minutes, about 40% of the proteins or polypeptides with a molecular weight greater than 10,000 kDa are digested; after intestinal digestion, most of the proteins and polypeptides are degraded into polypeptides below 1 kDa, and polypeptides less than 0.5 kDa account for 83.44% of the total polypeptides. In the early stage of gastric digestion, when the gastric digestion lasted for 15 minutes, the proportion of polypeptides greater than 10,000 kDa in Comparative Example 1 decreased by 23.57%, while that in Example 1 was 20.98%. When the gastric digestion lasted for 120 minutes, the values ​​were 33.92% and 38.5% in Example 1 and Comparative Example 1, respectively. That is, Comparative Example 1 was digested faster in the early stage of gastric digestion, but its final digestibility was obviously inferior to that of Example 1. In the intestinal digestion stage, the proportion of polypeptides greater than 10,000 kDa in Example 1 decreased rapidly from 38.01% to 4.28% at the end, while that in Comparative Example 1 decreased from 42.41% to 3.38%. The proportions of small molecular weight polypeptides in the two were similar, that is, compared with Comparative Example 1, the degree of protein degradation in the gastric digestion stage of Example 1 was higher, and the degree of digestion in the intestinal digestion stage was similar. After 120 minutes of gastric digestion in Comparative Example 2, the proportion of polypeptides greater than 10000 kDa decreased from 77.3% to 51.23%, which was relatively high. The gastric digestion was obviously not as good as that in Example 1 and Comparative Example 1. In the intestinal digestion stages I1 and I15, the proportion of low molecular weight peptides in Comparative Example 2 was always lower than that in Example 1 and Comparative Example 1, and the proportion of polypeptides greater than 10000 kDa was always higher than that in Example 1 and Comparative Example 1; at I120, the proportion of polypeptides with a molecular weight of less than 0.5 kDa in Comparative Example 2 was 81.28%, slightly lower than that in Example 1 and Comparative Example 1. It can be seen that the digestibility of Comparative Example 2 is not as good as that of Example 1 and Comparative Example 1. In Comparative Example 3, the molecular weight of polypeptides in Commercially Available Groups 1 and 2 in the gastrointestinal stage did not differ significantly from that in other groups; in summary, except for Comparative Example 2, there was little difference between the other four groups.

[0127] Figure 5 The free amino acid test results of the digestive juice obtained by re-dissolving and in vitro digestion experiments of infant formula milk powder obtained in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 (commercially available 1 and commercially available 2) are shown in FIG. Figure 5It can be seen that the initial free amino acid content in commercially available milk powder is relatively high. In Comparative Example 3, commercially available 1 and commercially available 2 are 19.54 and 23.58 μg / mL, respectively, which are significantly higher than other groups. In the gastric digestion stage, the free amino acid content changes little. After 120 minutes of gastric digestion, the free amino acid content only increases slightly, and the digestion difference between the groups is not significant. In the intestinal digestion stage, only 1 minute later, the free amino acid content increases significantly, and there is no significant difference between the groups. When the intestinal digestion reaches 120 minutes, the free amino acid content of each group is further increased, and the commercially available 1 group has the highest content, which is 370.49 μg / mL, followed by commercially available 2 groups, Example 1 group and Comparative Example 1 group, and they are all significantly higher than Comparative Example 2 group, which is 306.02 μg / mL. The free amino acid results show that in the gastric digestion stage, the degradation of protein is less, while in the intestinal stage, proteins and polypeptides are rapidly degraded, releasing a large amount of free amino acids, and the degree of protein digestion in Comparative Example 2 group is not as good as that in other groups, which is basically consistent with the results of polypeptide molecular weight distribution. The above description shows that the digestibility characteristics of the milk powders of Example 1 and Comparative Example 1 are close to those of commercially available milk powders, and are both better than those of Comparative Example 2.

[0128] Example 2 Exploration of the screening of cation exchange column fillers

[0129] Two gels, CM-Sephadex C-50 and CM-Sepharose Fast Flow, were selected.

[0130] Two groups of 5 mL bovine whey filtered through a 0.45 μm microporous filter membrane were prepared respectively, and the two groups of whey were passed through 1 mL pre-loaded columns of two gels respectively; after the loading was completed, pure water was used to elute the unbound protein, and then 0.3 M NaCl aqueous solution and 0.9 M NaCI aqueous solution were used to elute part of the basic protein and lactoferrin respectively.

[0131] SDS-PAGE was used to compare the effects of alkaline protein separation, and the choice of gel was determined based on the repeatability of the experimental operation.

[0132] The results are as follows Figure 6 As shown in the figure, in the preliminary experiment of separating and purifying basic proteins by two ion exchange resins, after flushing the column with pure water using CM-Sephadex C-50, the washed protein band was darker, indicating that the total amount of protein adsorbed was less, and its repeatability under the elution condition of 0.3M NaCl aqueous solution was poor, which may be related to its unstable adsorption. In addition, the CM-Sephadex C-50 filler has the problem of soft material, and the operation of CM-Sepharose Fast Flow is better in repeatability, relatively simple to operate, and has better separation effect. Therefore, CM-Sepharose Fast Flow was selected as the cation exchange separation resin in subsequent experiments.

[0133] Example 3 Exploring the conditions for cation exchange column to be free of basic protein (eluent concentration)

[0134] A dynamic adsorption experiment was carried out using a CM Sepharose Fast Flow prepacked column (1 mL) at a flow rate of 1 mL / min. The active whey rich in basic protein after aseptic concentration in step (4) of Example 1 was loaded onto the column in an amount of 10 mL, and the column whey was received. After loading, 10 column volumes of ultrapure water were used to balance and receive the column liquid, and then eluted with an aqueous solution with a concentration of 0.1-1.0 M NaCl. Each concentration was eluted to the baseline, and the eluted samples were collected and then subjected to SDS-PAGE experiments.

[0135] The results are as follows:

[0136] Figure 7 The following are the test results of SDS-PAGE for the separation of basic proteins using different cation exchange column eluents. Figure 7 It can be seen that most proteins can be eluted by 0.5M NaCl aqueous solution, and only a very small amount of proteins can be eluted by 0.5M NaCl aqueous solution. Therefore, 0.7M NaCl aqueous solution can be selected as the eluent concentration to elute all basic proteins. A NaCl aqueous solution greater than 0.7M will prolong the time of subsequent dialysis desalination.

[0137] Therefore, 0.7 M NaCl aqueous solution was selected as the optimal condition for the eluent.

[0138] Example 4 Exploring the conditions for cation exchange columns without basic proteins (whey pH)

[0139] A dynamic adsorption experiment was performed using a CM Sepharose Fast Flow pre-packed column (1 mL), with a flow rate controlled at 1 mL / min, and a 6M HCl solution or a 6M NaOH solution was used to adjust the pH of the concentrated whey rich in basic protein obtained in step (4) of Example 1 to 4.5, 4.8, 5.1, 5.4, 5.7, 6.0, 6.3, 6.6, and 6.9. If precipitation occurred, the supernatant was taken after standing for a period of time for column loading, with a sample volume of 10 mL, and the column whey was received. After the loading was completed, 10 column volumes of ultrapure water were used to balance and receive the column liquid, and then eluted by the same volume of 0.7M NaCl aqueous solution, eluted to the baseline at each concentration, and the eluted sample was collected, and then an SDS-PAGE experiment was performed.

[0140] The results are as follows:

[0141] Figure 8The SDS-PAGE results of whey with different pH values ​​after passing through the column (A); the total recovery rate of the whey after passing through the column at different pH values ​​and the proportion of eluted protein (B); the proportion of eluted protein specifically refers to the proportion of protein in the eluate of sodium chloride solution, the black bar graph is the total recovery rate of whey protein, and the gray bar graph is the elution protein ratio; the concentration and recovery rate of lactoferrin in the eluate (C), where the bar graph is the concentration and the line graph is the recovery rate; the macroscopic conditions of whey at different pH values ​​(D). Figure 8 As shown in (A), when the pH is less than 6.0, as the pH decreases, the content of high-abundance whey protein decreases, and the corresponding amount of high-abundance whey protein eluted increases, which indicates that these non-alkaline proteins are more adsorbed on the cation exchange resin. Figure 8 As shown in (B) and (C), as the pH decreases, the recovery rate of lactoferrin in the eluate gradually decreases. When the pH is less than 6.0, the total recovery rate of whey protein decreases, and the proportion of eluted protein increases rapidly, which is consistent with the SDS-PAGE results. As the pH decreases, the recovery rates of whey protein and lactoferrin decrease. This may be due to the aggregation and sedimentation of a small amount of casein and denatured protein in the whey, which entrains part of the whey protein and precipitates together, causing this phenomenon. Figure 8 As shown in (D), when the pH is less than 6.0, precipitation begins to occur.

[0142] Therefore, the whey pH of 6.9 was selected as the optimal condition.

[0143] Comparative Example 4

[0144] A conventional method for preparing infant formula milk powder comprises the following steps:

[0145] (1) Raw milk indicators:

[0146] Take fresh milk and measure the protein, lactose, milk fat and minerals in the milk;

[0147] (2) Calculate the nutrients required in infant formula according to the national food safety standard GB 10765-2021;

[0148] (3) Skim milk treatment:

[0149] After preheating the raw milk to 42°C, the milk was centrifuged in a disc centrifuge to obtain cream and skim milk;

[0150] (4) One-time sterilization:

[0151] The skim milk was pasteurized at 85°C for 15 seconds;

[0152] (5) Formula milk preparation:

[0153] According to the national food safety standard GB 10765-2021, the nutrients for infants aged 0 to 6 months are used as the standard to calculate the content of nutrients required in infant formula liquid milk. Skim milk, lactose, and vegetable blending oil are used as the main raw materials, and complex vitamins, minerals, taurine, L-carnitine, etc. are added. The following components are included by dry matter weight: casein 4.98% (based on 80% of total milk protein), concentrated whey protein powder 7.95%, lactose 56.38%, vegetable blending oil 27.16% (by mass percentage, including the following raw material components: coconut oil 25%, OPO 22%, soybean oil 18%, corn oil 15%, low-erucic acid rapeseed oil 10%, high-oleic acid sunflower oil 10%), compound minerals 1.80% (by mass percentage, including the following raw material components: calcium citrate 10.93%, potassium chloride 19.72%, sodium citrate 18.84%, calcium citrate 10.93%, magnesium sulfate 4.08%, ferric pyrophosphate 1.25%, zinc sulfate 0.32%, potassium iodate 0.28%, copper sulfate 0.18%, manganese sulfate 0.06%, sodium selenite 0.05%), compound vitamins 0.38 % (by mass percentage, including the following raw material components: 55.67% L-sodium ascorbate, 9.84% dl-α-tocopheryl acetate, 3.64% cholecalciferol, 2.58% retinyl acetate, 2.50% niacinamide, 2.03% D-calcium pantothenate, 1.20% cyanocobalamin, 1.12% biotin, 0.84% ​​phytonadione, 0.58% thiamine hydrochloride, 0.47% folic acid, 0.36% riboflavin, 0.34% pyridoxine hydrochloride), 0.05% inositol, 0.05% taurine, 0.01% L-carnitine;

[0154] Mix and dissolve lactose and compound mineral premix in a special container with 45°C pure water, dissolve compound vitamin premix and nutritional enhancer (inositol, taurine, L-carnitine) in 45°C pure water, then add the lactose mineral mixture, compound vitamin mixture and compound vegetable oil to the casein solution step by step and mix thoroughly. The compound mineral and compound vitamin addition and mixing time is 20 minutes, then add inositol, taurine and L-carnitine and mix thoroughly. (Note: The ratio of whey protein to casein is 3:2, which is close to the protein ratio in breast milk, and the protein content accounts for 15% of the total solids.)

[0155] (6) Homogenization:

[0156] The infant formula milk was homogenized at a homogenization pressure of 25 MPa for 2 times;

[0157] (7) Secondary sterilization:

[0158] Secondary sterilization of infant formula at 95°C for 15 seconds;

[0159] (8) Evaporation concentration:

[0160] The infant formula milk is evaporated and concentrated by a vacuum rotary evaporator with a vacuum degree of 0.08 MPa, a temperature of 56° C., and a rotation speed of 70 rpm, and the rotary evaporation is performed until the solid content reaches 35%;

[0161] (9) Spray drying:

[0162] A low-temperature and low-pressure spray drying device is used, the material inlet temperature is 75°C, and the material outlet temperature is adjusted to 54°C by flow regulation to obtain a milk powder product, which is then sealed and packaged in a light-proof manner.

[0163] The products obtained in Example 1 and Comparative Example 4 were subjected to performance tests, and the test results are as follows:

[0164] The content of undenatured whey in the whey extracted in Example 1 is significantly higher than that in Comparative Example 4; moreover, the retention rate of whey active protein in the infant formula milk powder obtained by the method of Example 1 is much higher than that in Comparative Example 1.

[0165] Comparative Example 5 Effect of Selection of Different Microfiltration Filtrates on the Enrichment Rate of Active Whey Lactoferrin

[0166] The washing filtrate sodium chloride solution in step (2) of Example 1 was adjusted to deionized water, and the rest was kept the same as Example 1 to obtain a membrane filtration permeate.

[0167] The obtained product was subjected to performance test, and the test results are as follows:

[0168] The lactoferrin content and corresponding enrichment rate of the diafiltration permeate obtained by comparative example 5 using deionized water diafiltration are very low. This may be because when using deionized water to diafiltration the retentate, the ionic strength (sodium chloride concentration) in the retentate decreases, causing lactoferrin to be unable to continue phase separation, and the lactoferrin content in the permeate decreases rapidly. Therefore, sodium chloride solution needs to be used for diafiltration.

[0169] Comparative Example 6 Effect of lactoferrin phase separation step on lactoferrin enrichment rate in active whey

[0170] The alkaline protein phase separation step in step (1) of Example 1, i.e., the operation of adding sodium chloride, was omitted, and the rest was kept the same as in Example 1 to obtain a membrane filtration permeate.

[0171] The obtained product was subjected to performance test, and the test results are as follows:

[0172] In Comparative Example 6, the step of adding sodium chloride for phase separation was omitted, and the lactoferrin enrichment rate was greatly reduced.

[0173] Comparative Example 7 Effect of microfiltration temperature on lactoferrin enrichment rate in active whey

[0174] The filtration temperature in step (2) of Example 1 was adjusted to 30° C., and the other conditions were kept the same as in Example 1 to obtain a membrane permeate.

[0175] Comparative Example 8 Effect of microfiltration temperature on the enrichment rate of lactoferrin in active whey

[0176] The filtration temperature in step (2) of Example 1 was adjusted to 45° C., and the other conditions were kept the same as in Example 1 to obtain a membrane permeate.

[0177] The membrane filtration permeate obtained in Comparative Examples 7 and 8 was subjected to performance tests, and the test results are as follows:

[0178] The lactoferrin enrichment rate in the whey of Comparative Examples 7 and 8 is lower than that in Example 1. The microfiltration temperature affects the content and enrichment rate of lactoferrin in the microfiltration permeate, i.e., the active whey. This may be because the temperature condition affects the charge attraction strength between lactoferrin and casein micelles in the milk system. Among the three temperature conditions of 30, 37, and 45°C, the lactoferrin enrichment rate at 37°C is the highest. Considering the conditions in actual operation, 37°C is selected for better effect.

[0179] Comparative Example 9: Timing of adding sodium chloride

[0180] The timing of adding sodium chloride is changed to adding it to casein, as follows:

[0181] (1) The sterilized skim milk is filtered through a 0.1 μm microfiltration membrane to separate the whey protein phase from the casein phase, thereby obtaining a membrane filtration permeate and a membrane filtration retentate (casein); the filtration temperature is controlled at 37±2° C., the pH is 6.6, and the end point of the filtration operation is determined by “the liquid volume in the membrane filtration retentate becomes one third of the initial volume”; the filtration is performed using a deionized water solution as a filtration solution, and after each filtration reaches the end point of the operation, the filtration solution is backfilled to the initial volume and re-filtered, and the process is repeated 4 times; and the permeate obtained from each filtration is collected;

[0182] (2) The membrane filtration retentate (casein) is further filtered using a 0.1 μm microfiltration membrane, and a sodium chloride solution containing 450 mmol is backfilled to the volume before step (1) to make a final sodium chloride concentration of 300 mmol, and the filtration is continued under this condition; subsequently, a 300 mmol sodium chloride solution is used as a second filtration solution; the end point of the filtration operation is determined by "the liquid volume in the membrane filtration retentate becomes one-third of the initial volume". After each filtration reaches the end point of the operation, the filtration solution is backfilled to the initial volume and filtered again, and the above filtration is repeated 4 times; and the permeate obtained from each filtration is collected.

[0183] The whey protein content in the sterilized skim milk in step (1) and the microfiltration permeate obtained in steps (2) and (3) was determined using an ELISA kit, and the whey protein enrichment rate was calculated.

[0184] turn out:

[0185] Under the condition of adding sodium chloride at a concentration of 300 mmol, the lactoferrin enrichment rate in the membrane permeate obtained after phase separation of lactoferrin in casein in Comparative Example 9 is lower than that in Example 1, and the process is more complicated, so the timing of adding sodium chloride is to add sodium chloride in skim milk.

Claims

1. A method for preparing an infant formula milk powder containing alkaline protein and good protein digestibility, characterized in that: The steps include: (1) adding sodium chloride to the sterilized skim milk to release the alkaline protein, thereby obtaining the skim milk after the alkaline protein phase transfer; (2) filtering the skim milk after the alkaline protein phase transfer in step (1) through a membrane to separate the whey protein phase from the casein phase, thereby obtaining a membrane filtration permeate (whey protein rich in alkaline protein) and a membrane filtration retentate (casein); wherein the filtrate used in the membrane filtration is a sodium chloride solution; (3) desalting and concentrating the membrane filtration retentate (casein) of step (2) to obtain a concentrated and desalted membrane filtration retentate; (4) ultrafiltration and desalination of the membrane filtration permeate (whey protein rich in basic protein) of step (2) to obtain aseptically concentrated active whey rich in basic protein; (5) passing the aseptically concentrated active whey rich in basic protein in step (4) through a cation exchange column to transfer the basic protein from the whey to the cation exchange column to obtain a whey phase free of basic protein; then eluting the basic protein on the cation exchange column with a sodium chloride solution to obtain a basic protein phase; (6) desalting the alkaline protein phase of step (5) to obtain a desalted alkaline protein phase; (7) mixing the concentrated and desalted membrane filtration retentate of step (3) with the whey phase free of alkaline protein of step (5), followed by pasteurization to obtain a protein mixture; (8) preparing and fully mixing the protein mixture of step (7) with other nutrients, and then homogenizing, sterilizing and concentrating to obtain an infant formula emulsion; (9) The liquid infant formula obtained in step (8) and the desalted alkaline protein obtained in step (6) are mixed, followed by spray drying to obtain an infant formula milk powder rich in alkaline protein and having good protein digestibility.

2. The method according to claim 1, characterized in that In step (1), the concentration of sodium chloride added is 200-400 mmol / L, the temperature is 35-39° C., and the pH is 6-7.

3. The method according to claim 1, characterized in that In step (2), the membrane filtration uses a membrane pore size of 90-110 nm, a temperature of 35-55° C., a concentration multiple of 2-4, and a diafiltration number of 3-5.

4. The method according to claim 1, characterized in that The sodium chloride solution in step (2) is a sodium chloride aqueous solution with a concentration of 200-400 mmol / L.

5. The method according to claim 1, characterized in that The filler of the cation exchange column used in step (5) is CM-Sepharose Fast Flow.

6. The method according to claim 1, characterized in that In step (5), the alkaline protein is eluted with an aqueous solution of sodium chloride, and the concentration of sodium chloride is 0.4M-1.0M.

7. The method according to claim 1, characterized in that The spray drying in step (9) is low-temperature and low-pressure spray drying, specifically: the material inlet temperature is not higher than 75°C, and the material outlet temperature is adjusted to 52-55°C by flow regulation.

8. Infant formula milk powder rich in basic protein and having good protein digestibility prepared by the method according to any one of claims 1 to 7.

9. Use of the infant formula milk powder rich in alkaline protein and having good protein digestibility as claimed in claim 8 in the food field.

10. A method for increasing the content of alkaline protein in infant formula milk powder and maintaining good protein digestibility, characterized in that: The infant formula milk powder rich in alkaline protein and having good protein digestibility as claimed in claim 8 is used.

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