Lactoferrin-rich active whey powder, high-activity infant formula, and preparation and use thereof

Abstract

Lactoferrin-rich active whey powder, a high-activity infant formula, and a preparation and use thereof. Active whey containing high content of lactoferrin is prepared by means of a sodium chloride ion dissociation technique, and then is added to concentrated infant formula to improve activity; and active whey powder containing high content of lactoferrin can also be prepared by means of spray drying. In addition, lactoferrin and other basic proteins and whey proteins containing no lactoferrin and other basic proteins can also be separated by means of an ion exchange technique, wherein the basic proteins are mixed with the infant formula, and the whey proteins containing no lactoferrin and other basic proteins undergo secondary sterilization to improve digestibility, so as to obtain high-activity, easily digestible infant formula.

Description

An active whey powder rich in lactoferrin, a highly active infant formula milk powder, and its preparation and application. Technical Field

[0001] This invention relates to an active whey powder rich in lactoferrin, a highly active infant formula milk powder, and their preparation and application, belonging to the field of dairy product preparation technology. Background Technology

[0002] Commercially available whey protein products, such as WPC and WPI, are primarily derived from whey, a byproduct of cheese processing. During the heat treatment, fermentation, and subsequent sterilization processes in cheese production, the natural structure and active ingredients of whey protein are easily damaged, resulting in low lactoferrin content in whey protein formulations. This leads to a situation where, although the final product has a high protein content, it generally suffers from low lactoferrin content and difficulty in maintaining its activity. Furthermore, the difference in particle size between casein (50-600 nm micelles) and whey protein (2-5 nm) in cow's milk makes membrane filtration a potential alternative for directly separating whey protein from skim milk; however, this process still faces the problem of low lactoferrin content. Therefore, developing an active whey powder rich in lactoferrin while maintaining its biological activity to meet the market demand for high-quality nutritional foods is of great significance.

[0003] Currently, commercially available infant formula primarily utilizes dry / wet processes to fortify basic protein active ingredients such as lactoferrin and immunoglobulins. However, this approach has several limitations: the relevant ingredients are reliant on imports and are costly; each fortification only allows for the retention of a single active protein, failing to preserve multiple active proteins in whey; even with fortification using active whey proteins to retain multiple components, the subsequent sterilization, concentration, and spray drying processes can still lead to denaturation and decreased bioactivity of some proteins. Furthermore, existing whey protein ingredients generally suffer from low levels of basic proteins such as lactoferrin. Therefore, developing an online enrichment technology for high-lactoferrin active whey combined with aseptic post-addition is crucial for better preserving the activity of functional proteins in infant formula. α-lactalbumin and β-lactoglobulin in whey are high-grade globular proteins with low digestibility; appropriate heat treatment can effectively improve their digestibility. Therefore, developing an online enrichment method for basic proteins such as lactoferrin with good protein digestibility and absorption is a critical problem that urgently needs to be solved in this field. Summary of the Invention [Technical Issues]

[0004] Existing whey powder processing techniques can hardly preserve the activity of functional proteins in whey, resulting in a significant difference between the prepared whey powder and actual whey. Conventional whey powder products are derived from casein processing byproducts, and lactoferrin binds to casein via electrostatic interactions, leading to low lactoferrin content in whey powder. Existing infant formula processing techniques can hardly preserve the activity of functional proteins in fresh milk. Active proteins in whey protein ingredients used in infant formula undergo partial denaturation and reduced biological activity. Furthermore, the low content of basic proteins such as lactoferrin results in a significant difference between the prepared infant formula and breast milk. Moderate heat treatment can improve protein digestibility and absorption, but excessive heat treatment can cause protein aggregation, leading to reduced protein digestibility.

[0005] [Technical Solution]

[0006] To address the aforementioned problems, this invention provides an active whey powder rich in lactoferrin, a highly active infant formula milk powder, and their preparation and application. Specifically, this invention involves adding sodium chloride to sterilized skim milk to dissociate lactoferrin, followed by membrane filtration to obtain a membrane filtration permeate (whey protein rich in lactoferrin), and finally ultrafiltration, desalting, and spray drying to obtain whey powder rich in lactoferrin.

[0007] This invention involves first defatting and sterilizing fresh cow's milk, followed by lactoferrin phase transfer. Lactoferrin is then enriched into the whey phase through microfiltration. The whey phase is further desalted and concentrated via ultrafiltration and electrodialysis to form sterile, concentrated, lactoferrin-rich active whey. The microfiltration retentate is then desalted, milk-killed (first sterilization), blended with other nutrients, homogenized, milk-killed (second sterilization), and concentrated. The final liquid is then mixed online with the sterile, concentrated, lactoferrin-rich active whey and spray-dried to obtain infant formula milk powder with high lactoferrin content and active whey protein.

[0008] This invention involves first defatting and sterilizing fresh milk, followed by alkaline protein phase transfer. The alkaline proteins are then enriched in the whey phase through microfiltration. The whey phase is further desalted and concentrated via ultrafiltration and electrodialysis to form a sterile, concentrated, alkaline protein-rich active whey. This active whey is then separated using a cation exchange column to remove alkaline proteins. The alkaline protein phase is then eluted from the cation exchange column and desalted. The microfiltration retentate is desalted and mixed with the alkaline protein-removed active whey. This mixture is then subjected to primary sterilization (milking), followed by blending with other nutrients, homogenization, secondary sterilization (milking), and concentration to form an infant formula milk powder. Finally, the infant formula milk powder is mixed online with the sterile alkaline protein phase and spray-dried to obtain an infant formula milk powder rich in alkaline proteins with good protein digestibility.

[0009] The first objective of this invention is to provide a method for preparing active whey powder rich in lactoferrin, comprising the following steps:

[0010] (1) Dissociation of lactoferrin: Add sodium chloride to the sterilized skim milk while stirring, control the temperature at 35-39℃ and the pH at 6-7, until the sodium chloride is completely dissolved to obtain skim milk after lactoferrin phase transfer; wherein, the concentration of sodium chloride added is 200-400 mmol / L.

[0011] (2) Enrichment of lactoferrin: Skim milk after lactoferrin phase transfer is subjected to membrane filtration to obtain membrane filtration permeate (whey protein phase rich in lactoferrin); wherein, the washing filtrate used for membrane filtration is sodium chloride solution.

[0012] (3) Aseptic concentration: The membrane filtration permeate is subjected to ultrafiltration and desalting to obtain aseptically concentrated active whey rich in lactoferrin.

[0013] (4) Spray drying: Active whey rich in lactoferrin is spray dried to obtain whey powder rich in lactoferrin.

[0014] Further, in step (1), the concentration of sodium chloride added is 300 mmol / L, the temperature is 37℃, and the pH is 6.6; the stirring speed is 300-800 rpm; lactoferrin is transferred from the casein phase to the whey phase. In step (2), the membrane filtration uses a membrane pore size of 90-110 nm, a temperature of 30-55℃, a concentration factor of 2-4, and 3-5 washing cycles; the endpoint of membrane filtration is determined by "the liquid volume in the membrane filtration retentate becomes one-third of the initial volume"; the sodium chloride solution is an aqueous sodium chloride solution with a concentration of 200-400 mmol / L; after each filtration reaches the endpoint, the washing filtrate is backfilled to the initial volume and filtration is repeated. In step (3), the ultrafiltration membrane used has a molecular weight cutoff of 9-11 kDa, a concentration factor of 5-10 times, and an ultrafiltration temperature not exceeding 45℃. For desalination, ultrapure water is used as the secondary ultrafiltration solution, with the amount of ultrafiltration solution used being 5-10 times that of the concentrated permeate. After ultrafiltration, electrodialysis is performed for further desalination. The electrodialysis uses a working current of 45A, a feed flow rate of 110 mL / min, and a feed temperature of 35℃. The desalination endpoint is determined by the decrease in conductivity to the normal permeate conductivity range. The protein concentration is determined using the Kjeldahl method to measure the total protein concentration in the permeate. In step (4), the spray drying parameters are: material inlet temperature of 60-80℃ and material outlet temperature of 52-55℃. The active whey powder rich in lactoferrin is stored in a sealed container at room temperature after processing.

[0015] The second objective of this invention is to prepare active whey powder rich in lactoferrin using the method described in this invention.

[0016] The third objective of this invention is to provide a method for increasing the lactoferrin content in whey powder, with the same steps as in "A method for preparing active whey powder rich in lactoferrin".

[0017] A fourth objective of this invention is to provide a method for simultaneously preparing alkaline powders such as lactoferrin and whey powders that do not contain alkaline proteins such as lactoferrin, comprising the following steps:

[0018] (1) Dissociation of lactoferrin: Add sodium chloride to the sterilized skim milk while stirring, control the temperature at 35-39℃ and the pH at 6-7, until the sodium chloride is completely dissolved to obtain skim milk after lactoferrin phase transfer; wherein, the concentration of sodium chloride added is 200-400 mmol / L.

[0019] (2) Enrichment of lactoferrin: Skim milk after lactoferrin phase transfer is subjected to membrane filtration to obtain membrane filtration permeate (whey protein phase rich in lactoferrin); wherein, the washing filtrate used for membrane filtration is sodium chloride aqueous solution.

[0020] (3) Aseptic concentration: The membrane filtration permeate is subjected to ultrafiltration and desalting to obtain aseptically concentrated active whey rich in lactoferrin.

[0021] (4) Further enriching basic proteins such as lactoferrin and collecting whey without lactoferrin and other basic proteins: The sterile concentrated active whey rich in lactoferrin is passed through a cation exchange column to transfer basic proteins such as lactoferrin from the whey to the cation exchange column, thus obtaining whey without lactoferrin and other basic proteins; then, the basic proteins such as lactoferrin on the cation exchange column are eluted with an aqueous solution of sodium chloride to remove salt and obtain basic proteins such as lactoferrin;

[0022] (5) Drying: Lactoferrin and other alkaline proteins are freeze-dried to obtain lactoferrin and other alkaline protein powder; whey without lactoferrin and other alkaline proteins is spray-dried to obtain whey powder without lactoferrin and other alkaline proteins.

[0023] Furthermore, the parameters for steps (1)-(3) are the same as those in "A method for preparing active whey powder rich in alkaline proteins such as lactoferrin". In step (4), the packing material for the cation exchange column is CM-Sepharose Fast Flow; the concentration of the sodium chloride aqueous solution is 0.9-1.1M; desalination is performed by dialysis, and the dialysis bag specification is 10Kda. In step (5), the parameters for spray drying are: material inlet temperature at 60-80℃, material outlet temperature at 52-55℃; the parameters for freeze drying are: drying at -80℃ for 24h, and vacuum degree at 30Pa.

[0024] The fifth objective of this invention is to obtain lactoferrin and other alkaline protein powders and whey powders without lactoferrin and other alkaline proteins prepared by the method described in this invention.

[0025] The sixth objective of this invention is to provide a method for preparing infant formula with highly active immune proteins, comprising the following steps:

[0026] (1) Add sodium chloride to the sterilized skim milk to dissociate lactoferrin and obtain skim milk after lactoferrin phase transfer;

[0027] (2) The skim milk after the lactoferrin phase transfer obtained in step (1) is filtered through a membrane to separate the whey protein phase from the casein phase, resulting in a membrane filtration permeate (whey protein rich in lactoferrin) and a membrane filtration retentate (casein); wherein, the washing solution used for membrane filtration is a sodium chloride solution.

[0028] (3) Desalinate and sterilize the membrane filtration retentate obtained in step (2) to obtain desalinated and sterilized membrane filtration retentate;

[0029] (4) The membrane permeate obtained in step (2) is subjected to ultrafiltration and desalting to obtain sterile concentrated active whey rich in lactoferrin.

[0030] (5) The desalted and sterilized membrane filtration retentate obtained in step (3) is used as casein solution. The nutrients required for infant formula are added to the casein solution and mixed thoroughly. The mixture is then homogenized, sterilized, and evaporated to concentrate the solution to obtain a casein solution containing the ingredients.

[0031] (6) The casein solution containing ingredients obtained in step (5) is concentrated to obtain a concentrate; then the concentrate is mixed with the sterile concentrated active whey rich in lactoferrin obtained in step (4) and spray-dried to obtain infant formula milk powder with high active immune protein.

[0032] Further, the preparation method of the sterilized skim milk in step (1) is as follows: After preheating the raw milk to 40-42℃, centrifuge it using a disc centrifuge to obtain cream and skim milk; then filter the skim milk using a 1.4μm microfiltration membrane at a temperature controlled at 40-45℃ to remove most of the microorganisms and impurities from the milk, thus obtaining sterilized skim milk. The concentration of sodium chloride added is 200-400mmol / L, the temperature is 35-39℃, and the pH is 6-7. In step (2), the membrane filtration uses a membrane pore size of 90-110nm, a temperature of 35-55℃, a concentration factor of 2-4, and 3-5 washing cycles; the endpoint of membrane filtration is determined by "the liquid volume in the membrane filtration retentate becomes one-third of the initial volume"; the sodium chloride solution is an aqueous sodium chloride solution with a concentration of 200-400mmol / L; after each filtration reaches the endpoint, the washing filtrate is backfilled to the initial volume and filtered again. In step (3), desalination is performed using a 0.05-0.15μm microfiltration membrane, with deionized water as the wash solution. The amount of wash solution used is 5-10 times the volume of the retentate. The endpoint of the desalination operation is determined by the "conductivity value decreasing to the normal retentate conductivity range". The protein concentration is determined by the Kjeldahl method to measure the total protein concentration in the retentate. More preferably, the pore size of the microfiltration membrane is 0.1μm. Sterilization is performed at 80-90℃ for 10-20s. In step (4), the ultrafiltration membrane used has a molecular weight cutoff of 9-11 kDa, a concentration factor of 5-10 times, and an ultrafiltration temperature not exceeding 45°C. For desalination, ultrapure water is used as the ultrafiltration solution, and the amount of ultrafiltration solution used is 5-10 times that of the concentrated permeate. After ultrafiltration, electrodialysis is performed for further desalination. The electrodialysis uses a working current of 45A, a feed flow rate of 110 mL / min, and a feed temperature of 35°C. The desalination operation endpoint is determined by the "conductivity value decreasing to the normal permeate conductivity range". The protein concentration is determined by the Kjeldahl method to measure the total protein concentration in the permeate.

[0033] Furthermore, in step (5), the casein solution is used as the milk base in the preparation of infant formula; the nutrients required in infant formula are calculated according to the national food safety standard GB 10765-2021, taking the nutrients required by infants aged 0-6 months as the standard, and the content of nutrients required in liquid infant formula is calculated. Casein solution, lactose, and vegetable blended oil are the main raw materials, and compound vitamins, minerals, taurine, L-carnitine, etc. are added; the nutrients required in infant formula include the following components by dry matter weight: 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), 1.49% compound minerals (by weight 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%), 0.31% compound vitamins. % (by weight, including the following raw material components: L-ascorbic acid sodium 55.67%, dl-α-tocopherol acetate 9.84%, cholecalciferol 3.64%, retinyl acetate 2.58%, nicotinamide 2.50%, D-calcium pantothenate 2.03%, cyanocobalamin 1.20%, biotin 1.12%, phytonabinone 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%.

[0034] Further, in step (5), the required nutrients in the infant formula are added as follows: lactose and compound mineral premix are dissolved in purified water at 40-50℃ in a special container; compound vitamin premix and nutritional fortifiers (inositol, taurine, and L-carnitine) are dissolved in purified water at 40-45℃ respectively; then, the above-mentioned lactose-mineral mixture, compound vitamin mixture, and compound vegetable oil are added to the casein solution in steps and mixed thoroughly. The mixing time for adding compound minerals and compound vitamins should be less than 15 minutes. Then, inositol, taurine, and L-carnitine are added and mixed thoroughly. In step (5), homogenization is performed 1-2 times at 25-30 MPa; sterilization is performed at 90-100℃ for 10-20 seconds; more preferably, treatment is performed at 95℃ for 15 seconds.

[0035] Further, in step (6), the concentration is evaporative concentration, specifically using a vacuum rotary evaporator with a vacuum degree of 0.07-0.09 MPa, a temperature of 55-57°C, and a rotation speed of 50-90 rpm, evaporating until the solid content reaches 30-40% (mass percentage). After mixing the concentrate with aseptically concentrated active whey rich in lactoferrin, the mass ratio of whey protein to casein reaches ≥6:4, close to the protein ratio in breast milk, and the protein content accounts for 10-15% (mass percentage) of the total solids. Spray drying is low-temperature, 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 adjusting the flow rate.

[0036] Furthermore, infant formula milk powder with highly active immune proteins requires aseptic filling after processing and cold chain transportation or low-temperature refrigeration.

[0037] The seventh objective of this invention is to produce infant formula milk powder with highly active immune proteins prepared by the method described in this invention.

[0038] The eighth objective of this invention is to provide a method for increasing the content of active proteins in infant formula, wherein the method uses infant formula with highly active immune proteins as described in this invention.

[0039] The ninth objective of this invention is to provide a method for preparing an infant formula milk powder with basic protein and good protein digestibility, comprising the following steps:

[0040] (1) Add sodium chloride to the sterilized skim milk to dissociate the basic proteins and obtain skim milk after basic protein phase transfer;

[0041] (2) After the basic protein phase transfer in step (1), the skim milk is filtered through a membrane to separate the whey protein phase from the casein phase, resulting in a membrane filtration permeate (whey protein rich in basic protein) and a membrane filtration retentate (casein); wherein, the washing solution used for membrane filtration is a sodium chloride solution.

[0042] (3) Desalt and concentrate the membrane filtration retentate (casein) from step (2) to obtain concentrated and desalted membrane filtration retentate;

[0043] (4) The membrane filtration permeate (whey protein rich in basic protein) from step (2) is subjected to ultrafiltration and desalting to obtain sterile concentrated active whey rich in basic protein.

[0044] (5) Pass the sterile concentrated active whey rich in basic protein from step (4) through a cation exchange column to transfer the basic protein from the whey to the cation exchange column, and obtain a whey phase without basic protein; then use sodium chloride solution to elute the basic protein on the cation exchange column to obtain a basic protein phase;

[0045] (6) Desalt the alkaline protein phase from step (5) to obtain the desalted alkaline protein phase;

[0046] (7) The concentrated and desalted membrane filtration retentate from step (3) is mixed with the whey phase without alkaline protein from step (5), and then pasteurized to obtain a protein mixture.

[0047] (8) The protein mixture from step (7) is mixed with other nutrients and thoroughly mixed, then homogenized, sterilized and concentrated to obtain infant formula milk.

[0048] (9) The liquid infant formula milk from step (8) and the desalted alkaline protein from step (6) are mixed and then spray-dried to obtain infant formula milk powder rich in alkaline protein and with good protein digestibility.

[0049] Further, in step (1), the concentration of added sodium chloride is 200-400 mmol / L, the temperature is 35-39℃, and the pH is 6-7. In step (2), the membrane filtration uses a membrane pore size of 90-110 nm, a temperature of 35-55℃, a concentration factor of 2-4, and 3-5 washes. The endpoint of membrane filtration is determined by the point at which the liquid volume in the filtrate becomes one-third of the initial volume. The sodium chloride solution is an aqueous sodium chloride solution with a concentration of 200-400 mmol / L. After each filtration reaches the endpoint, the wash filtrate is backfilled to the initial volume for filtration again. In step (3), desalting and concentration are performed using a 0.05-0.15 μm microfiltration membrane, with deionized water as the wash solution. The amount of wash solution used is 5-10 times the volume of the retentate. The endpoint of the desalting operation is determined by the "conductivity value decreasing to the normal retentate conductivity range". The protein concentration is determined by the Kjeldahl method to measure the total protein concentration in the retentate. More preferably, the pore size of the microfiltration membrane is 0.1 μm. In step (4), the ultrafiltration membrane used has a molecular weight cutoff of 9-11 kDa, a concentration factor of 5-10 times, and an ultrafiltration temperature not exceeding 45°C. For desalination, ultrapure water is used as the ultrafiltration solution, and the amount of ultrafiltration solution used is 5-10 times that of the concentrated permeate. After ultrafiltration, electrodialysis is performed for further desalination. The electrodialysis uses a working current of 45A, a feed flow rate of 110 mL / min, and a feed temperature of 35°C. The desalination operation endpoint is determined by the "conductivity value decreasing to the normal permeate conductivity range". The protein concentration is determined by the Kjeldahl method to measure the total protein concentration in the permeate.

[0050] Further, in step (5), the packing material of the cation exchange column is CM-Sepharose Fast Flow; the alkaline protein is eluted with an aqueous solution of sodium chloride, with a sodium chloride concentration of 0.4M-1.0M. In step (6), desalting is performed by dialysis using a dialysis bag with a molecular weight cutoff of 7-8 kDa, dialysis is performed until the conductivity of the solution reaches the normal range. In step (7), the ratio of the concentrated and desalted membrane filtration retentate from step (3) to the whey phase without alkaline protein from step (5) is 4:6, which is the protein ratio of the two; close to the protein ratio in breast milk. Sterilization is performed at 80-90℃ for 10-20s, and more preferably at 85℃ for 15s. The protein mixture in step (7) is used as the milk base in the preparation of infant formula. In step (8), the nutrients required in the infant formula are calculated according to the national food safety standard GB 10765-2021, using the nutrients required by infants aged 0-6 months as the standard. The main raw materials are casein liquid, lactose, and vegetable blended oil, with added compound vitamins, minerals, taurine, L-carnitine, etc. In step (8), homogenization is performed 1-2 times at 25-30 MPa. Sterilization is performed at 90-100℃ for 10-20 seconds; for further optimization, it is performed at 95℃ for 15 seconds. Concentration is performed by evaporation concentration, specifically using a vacuum rotary evaporator with a vacuum degree of 0.07-0.09 MPa, a temperature of 55-57℃, and a rotation speed of 50-90 rpm, until the solid content reaches 30-40% (mass percentage).

[0051] Furthermore, in step (9), the spray drying is low-temperature, low-pressure spray drying, specifically: the material inlet temperature is not higher than 80℃, and the material outlet temperature is adjusted to 52-55℃ through flow rate regulation. The alkaline protein infant formula with good protein digestibility is aseptically filled and stored at room temperature after processing.

[0052] The tenth objective of this invention is to prepare an infant formula milk powder rich in alkaline protein and with good protein digestibility using the method described in this invention.

[0053] The eleventh objective of this invention is to provide a method for increasing the content of alkaline protein in infant formula and maintaining good protein digestibility. The method uses the infant formula described in this invention, which is rich in alkaline protein and has good protein digestibility. [Beneficial Effects]

[0054] (1) In this invention, the whey powder rich in lactoferrin is prepared by adding sodium chloride to skim milk to increase the ion concentration, promoting the dissociation of casein micelles from the lactoferrin complex, and causing the lactoferrin adsorbed on the casein micelles to dissociate from the lactoferrin complex, existing more in a free state in the whey phase; then, whey protein rich in lactoferrin is obtained by separation using a microfiltration membrane. The whey powder prepared by the method of this invention has a significantly higher lactoferrin enrichment rate compared to whey protein that has not undergone lactoferrin dissociation treatment.

[0055] (2) Compared with whey obtained from cheese processing by-products, the whey powder prepared by the present invention avoids the heating and fermentation process in cheese production and retains active proteins such as lactoferrin in whey very well.

[0056] (3) The lactoferrin powder prepared by the present invention has a purity of more than 98% and a recovery rate of more than 70%. Because the membrane filtration sterilization is used in the preparation process, the activity of lactoferrin is well preserved. Based on the functions of lactoferrin such as enhancing immunity, promoting iron absorption and regulating intestinal health, the lactoferrin powder prepared by the present invention has important application value in nutritional supplementation, disease prevention and treatment.

[0057] (4) The whey powder prepared by the present invention, which does not contain lactoferrin, retains most of the whey protein and the protein purity is as high as 90% or more, and can be used as a nutritional ingredient and for scientific research. In addition, the whey powder is almost unaffected by heat treatment, and the protein activity is well preserved, which enhances its nutritional and functional activity in food.

[0058] (5) The method for preparing infant formula with highly active immune proteins in this invention involves adding sodium chloride to skim milk to increase the ion concentration, promoting the dissociation of casein micelles and lactoferrin complexes, and causing lactoferrin adsorbed on casein micelles to dissociate and exist in the whey phase in a more free state; then, a microfiltration membrane is used to separate the whey protein phase rich in lactoferrin from the casein. The whey protein phase obtained by this method has a significantly higher lactoferrin enrichment rate compared to the whey protein phase without lactoferrin dissociation treatment.

[0059] (6) In this invention, casein is used as the milk base in the formulation and production process of infant formula milk powder. Whey protein is added and mixed before spray drying to avoid the loss of active proteins in whey protein due to repeated heat treatment, thus greatly improving the abundance of active proteins in the finished infant formula milk powder.

[0060] (7) The method for preparing infant formula milk powder rich in basic proteins and with good protein digestibility in this invention involves adding sodium chloride to skim milk to increase the ion concentration, promoting the dissociation of casein micelles and basic proteins, and allowing them to exist in the whey phase in a more free state. Then, a microfiltration membrane is used to separate the whey protein phase rich in basic proteins from the casein. Next, a cation exchange column is used to adsorb, elute, and enrich the basic proteins in the whey protein to obtain the basic protein phase. The whey protein phase obtained by this method has a significantly higher basic protein enrichment rate compared to the whey protein phase without basic protein dissociation treatment, and the basic protein phase is separated and enriched by the ion exchange column for subsequent infant formula milk powder processing.

[0061] (8) In this invention, casein is used as the milk base in the formulation and production process of infant formula milk powder, and whey from which basic proteins are separated is used as whey protein ingredient. The separated basic proteins are added before spray drying to avoid the loss of basic proteins due to multiple heat treatments. At the same time, the high-abundance milk protein is subjected to heat treatment of a certain intensity, which improves digestibility. This process greatly improves the abundance of basic proteins in the finished infant formula milk powder and maintains good protein digestibility.

[0062] In summary, the whey powder prepared by the method of the present invention retains more lactoferrin, and the prepared infant formula milk powder retains more alkaline active proteins and maintains good protein digestibility. Attached Figure Description

[0063] Figure 1 is a process flow diagram of the method for preparing active whey powder rich in lactoferrin according to the present invention.

[0064] Figure 2 shows the retention rate of undenatured whey from raw milk in Example 1 and Comparative Example 1.

[0065] Figure 3 shows the test results of lactoferrin (A) and immunoglobulin IgG (B) in the whey powder of Example 1 and Comparative Example 1.

[0066] Figure 4 shows the test results of lactoferrin content in whey powder of Example 1 and Comparative Examples 2-6.

[0067] Figure 5 is an HPLC chromatogram of the lactoferrin sample obtained after reconstitution of the lactoferrin powder prepared in Example 2.

[0068] Figure 6 is an SDS-PAGE image of the whey sample without lactoferrin obtained after reconstitution of the whey powder without lactoferrin prepared in Example 2.

[0069] Figure 7 shows the SDS-PAGE images of whey proteins prepared in Example 2 and Comparative Example 7.

[0070] Figure 8 shows the recovery rate of lactoferrin in whey prepared in Example 2 and Comparative Example 7.

[0071] Figure 9 shows the test results of separating and purifying lactoferrin using two ion exchange resins in Example 3.

[0072] Figure 10 is a process flow diagram of the method for preparing highly active immune proteins in infant formula milk powder in Example 3.

[0073] Figure 11 is a process flow diagram of conventional infant formula milk powder in Comparative Example 8.

[0074] Figure 12 shows the content and retention rate of undenatured whey protein in raw milk in Example 4 and Comparative Example 8; where (a) is the content of undenatured whey protein and (b) is the retention rate of undenatured whey protein.

[0075] Figure 13 shows a comparison of the process routes for infant formula milk powder in Example 4 and Comparative Example 9.

[0076] Figure 14 shows the content of lactoferrin (a) and immunoglobulin IgG (b) in milk powder in Example 4 and Comparative Examples 8 and 9.

[0077] Figure 15 is a process flow diagram for preparing infant formula milk powder rich in alkaline protein and with good protein digestibility (Example 5 is route 1, and Comparative Example 10 is route 2).

[0078] Figure 16 is a process flow diagram of the preparation of infant formula milk powder in Comparative Example 11.

[0079] Figure 17 shows the SDS-PAGE test results of the digestive fluid obtained from the reconstitution and in vitro digestion experiments of the infant formula milk powders obtained in Example 5, Comparative Example 10, Comparative Example 11, and Comparative Example 12 (commercially available 1 and commercially available 2).

[0080] Figure 18 shows the test results of the polypeptide molecular weight of the digestive fluid obtained from the reconstitution and in vitro digestion experiments of the infant formula milk powders obtained in Example 5, Comparative Example 10, Comparative Example 11, and Comparative Example 12 (commercially available 1 and commercially available 2).

[0081] Figure 19 shows the test results of free amino acids in the digestive fluid obtained from the reconstitution and in vitro digestion experiments of the infant formula milk powders obtained in Example 5, Comparative Example 10, Comparative Example 11, and Comparative Example 12 (commercially available 1 and commercially available 2).

[0082] Figure 20 shows the SDS-PAGE test results of different cation exchange column packings for basic protein separation in Example 6.

[0083] Figure 21 shows the test results of SDS-PAGE for basic protein separation using different cation exchange column eluents in Example 7.

[0084] Figure 22 shows the SDS-PAGE results of whey passing through the column at different pH (A); the total recovery rate of the received whey and the proportion of eluted protein after passing whey through the column at different pH (B); where the proportion of eluted protein specifically refers to the protein content in the sodium chloride solution eluent, the black bar graph represents the total recovery rate of whey protein, and the gray bar graph represents the eluted protein ratio; the concentration and recovery rate of lactoferrin in the eluent (C), where the bar graph represents the concentration and the line graph represents the recovery rate; the macroscopic condition of whey at different pH (D). Detailed Implementation

[0085] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0086] Test method:

[0087] 1. Active whey extract:

[0088] Whey powder and water were dissolved at a mass ratio of 1:8 and magnetically stirred at 45℃ for 1 hour to obtain a whey sample. The whey sample was then poured into a centrifuge tube and centrifuged at 8000g at 4℃ for 15 minutes. The upper layer of milk fat was scraped off with a small spatula. The pH of the obtained whey was adjusted to 4.6 and then placed at 4℃ for 1 hour to allow casein and denatured whey protein to precipitate completely. During this period, the pH was monitored and adjusted as needed. The supernatant sample was transferred to an ultracentrifuge tube and centrifuged at 31200 rpm at room temperature for 1.5 hours. After centrifugation, the sample separated into three layers (the uppermost layer was residual milk fat, the middle layer was whey, and the lower layer was aggregated casein micelles). The middle layer of whey sample was carefully pipetted into a clean centrifuge tube for later use. The diluted whey was used for subsequent index determination.

[0089] 2. Determination of undenatured whey: The protein content of undenatured whey was determined according to the Kjeldahl method.

[0090] 3. Determination of lactoferrin content:

[0091] Bovine lactoferrin standards were serially diluted to concentrations between 500-7.8 ng / mL. Samples were then diluted 500-2000 times according to the standard curve concentration range (depending on the standard curve range). 100 μL of the graded concentration standards and previously extracted active whey (diluted 10000 times) were added to each well, with three replicates. After reacting at room temperature for 1 hour, the wells were washed and dried. 100 μL of horseradish peroxidase-conjugated goat anti-bovine lactoferrin detection antibody was added, and the plate was incubated at room temperature for 1 hour, followed by five washes. 100 μL of LTMB (enzyme substrate) was added, and the 96-well plate was incubated in the dark at room temperature for 15 minutes. 100 μL of stop solution (0.18 mol H2SO4) was added, and the plate was shaken for 5 seconds before the absorbance was read at 450 nm. Finally, a 4-parameter curve was fitted using Soft-Max Pro to calculate the lactoferrin concentration in the samples, with the sample dilutions serving as a blank control.

[0092] 4. Immunoglobulin G content determination: Same as the lactoferrin content determination method, but the active whey sample is diluted 10,000 times.

[0093] 5. Determination of lactoferrin purity:

[0094] The purity of LFs was determined by RP-HPLC. Sample preparation involved mixing equal volumes of LFs solution with buffer (0.1M Bis-Tris propane, 8M urea, 20mM DTT, 1.3% sodium citrate, adjusted to pH 7) and filtering through a 0.22μm organic membrane. RP-HPLC analysis was performed on a C430 nm 3.5μm particle size column, equilibrated in 95% mobile phase A (0.1% trifluoroacetic acid solution) and 5% mobile phase B (0.1% trifluoroacetic acid acetonitrile solution). Absorbance was monitored at 280 nm. The column temperature was set at 60℃, the injection volume was 30μL, and the bound protein was eluted with a linear gradient of 38-60% mobile phase B at a flow rate of 1mL / min for 20min.

[0095] 6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE):

[0096] Reduction electrophoresis was performed on a 12% separating gel and a 4% stacking gel. The reconstituted milk sample solution was mixed with 2x sample buffer at a 1:1 (v / v) ratio, and 5% β-mercaptoethanol was added to the final volume. The mixture was then heated in a boiling water bath for 3 min to obtain denatured reconstituted milk protein. 10 μL of the denatured reconstituted milk protein was added to each well. The initial voltage was 60 V, and the voltage was increased to 120 V when the sample bands moved to the interface between the stacking and separating gels. The SDS-PAGE gel was stained with Coomassie Brilliant Blue (R-250), shaken at room temperature for 1.5 h, and then destained with destaining solution until the gel background was colorless. Images were taken using a Chemi Doc XRS+ chemiluminescent gel imaging system.

[0097] 7. In vitro static digestion:

[0098] Simulated in vitro infant digestion included gastric and intestinal stages. 63 mL of reconstituted milk samples (10.0 mg / mL protein) prepared using different processes were added to a reactor at a constant temperature of 37°C and incubated for 15 min. For gastric digestion, the pH was adjusted to 5.3 using 1M HCl solution, with a milk sample to gastric digestion fluid ratio of 63:37 (v:v). The gastric digestion fluid contained 94 mM NaCl, 13 mM KCl, pepsin activity of 268 U / mL, and lipase activity of 19 U / mL. The gastric digestion process lasted 180 min, with samples taken at 0 (containing digestive enzymes and enzyme inhibitors), 5, 15, 30, 60, and 120 min. The gastric digestion reaction was terminated using a 1:40 mixture containing 1M sodium hydroxide solution. For intestinal digestion, gastric chyme was used at 120 min of gastric digestion; specifically, the gastric digestion reaction was terminated at 60 min by adjusting the pH to 7 using 1M NaOH solution, and then the pH was adjusted to 6.6 using 1M HCl solution. The volume ratio of gastric chyme to intestinal digestive fluid was 63:37. The intestinal digestive fluid contained 3.1 mM ox bile salts, 164 mM NaCl, 10 mM KCl, and 85 mM NaHCO3, with a protease activity of 16 U / mL and a lipase activity of 21 U / mL. After intestinal digestion for 0, 1, 5, 15, 30, 60, and 120 min, the digestion reaction was terminated by mixing the sample with an enzyme inhibitor containing 0.1 M benzosulfonyl fluoride and 10 mM orlistat at a ratio of 1:40 (v:v). Two copies of the digested sample were prepared. Samples that could not be analyzed immediately were stored at -80°C until further analysis.

[0099] 8. Determination of polypeptide molecular weight:

[0100] The dissociation of peptides during gastrointestinal digestion was detected using a Waters e2695 high-performance liquid chromatography (HPLC) system. Samples from each group were digested in vitro, centrifuged at 13400×g for 30 min at 4℃, and the supernatant was collected, filtered through a membrane, and injected into a TSK-GELG2000SWxl column (300 mm × 7.8 mm, 5 μm). The mobile phase was acetonitrile-water-trifluoroacetic acid (400:600:1, v / v / v), and the flow rate was 0.5 mL / min. The column temperature and detection wavelength were set at 30℃ and 220 nm, respectively.

[0101] 9. Determination of free amino acids:

[0102] The sample pretreatment before processing is as follows: 10% (w / v, mg / mL) TCA solution is added to the gastrointestinal digested sample at a ratio of 1:1 (v / v), mixed well, sonicated for 30 min, allowed to stand overnight, and then centrifuged at 4℃ and 10000×g for 30 min. The supernatant is filtered through a 0.45 μm filter membrane for later use. The amino acid content was detected using an Agilent 1100 high-performance liquid chromatography system under the following chromatographic conditions: Hypersil ODS C18 column (4.6 mm × 150 mm, 5 μm); column temperature 40 ℃; 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; and the external standard method was used to quantify the amino acid content.

[0103] Raw materials used in the examples and comparative examples:

[0104] The preparation method of sterilized skim milk is as follows:

[0105] Raw milk is preheated to 42°C and then centrifuged in a disc centrifuge to obtain cream and skim milk. The skim milk is then filtered through a 1.4μm microfiltration membrane at a temperature controlled at 40°C to remove most of the microorganisms and impurities from the milk, resulting in sterilized skim milk.

[0106] Lactoferrin and Immunoglobulin ELISA Kit: Bethyl Laboratories, USA; normal permeate conductivity range: 4-7 mS / m; cation exchange column packing material: CM-Sepharose Fast Flow: commercially available; Commercially available 1 and 2: two commercially available infant formulas. All other reagents were of analytical grade. In the examples and comparative examples, solutions without a specified reaction solvent used water, and reactions without a specified temperature refer to room temperature (20-30°C).

[0107] Example 1

[0108] A method for preparing active whey powder rich in lactoferrin (as shown in Figure 1) includes the following steps:

[0109] (1) Dissociation of lactoferrin:

[0110] In the sterilized skim milk, while stirring (500 rpm), 300 mmol / L sodium chloride is added, the temperature is controlled at 37℃ and the pH is 6.6, until the sodium chloride is completely dissolved, and skim milk after lactoferrin phase transfer is obtained.

[0111] (2) Enrichment of lactoferrin:

[0112] Skim milk after lactoferrin phase transfer was filtered through a 100 nm microfiltration membrane to separate the whey protein phase from the casein phase, yielding a membrane filtration permeate (whey protein rich in lactoferrin). The filtration temperature was controlled at 37±2℃, and the filtration endpoint was determined by the point at which the liquid volume in the membrane filtration retentate decreased to one-third of the initial volume, i.e., a concentration factor of 3 times. A 300 mmol / L sodium chloride aqueous solution was used as the washing solution for filtration. After each filtration reached the endpoint, the washing solution was refilled to the initial volume and filtration was repeated 4 times.

[0113] (3) Aseptic concentration:

[0114] The permeate (whey protein rich in lactoferrin) was concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, at a concentration factor of 8 times and an ultrafiltration temperature of 40°C. Ultrapure water was then used as the secondary ultrafiltration solution, with a volume eight times that of the concentrated permeate. After ultrafiltration, electrodialysis (operating current 45 A, feed flow rate 110 mL / min, feed temperature 35°C) was performed for further desalination. The desalination endpoint was determined by the reduction of conductivity to the normal range of permeate conductivity. This yielded sterile, concentrated, lactoferrin-rich active whey (this step skipped the sterilization unit; microorganisms were removed through membrane concentration to obtain lactoferrin-rich active whey). The total protein concentration in the permeate was determined using the Kjeldahl method.

[0115] (4) Spray drying:

[0116] The aseptically concentrated active whey rich in lactoferrin was spray-dried (material inlet temperature 75℃, material outlet temperature 53℃) to obtain active whey powder rich in lactoferrin; then sealed and packaged in a light-proof container.

[0117] Comparative Example 1 (Control Group)

[0118] Several WPC concentrated whey protein products were purchased from the market and labeled as WPC1, WPC2, WPC3 and WPC4, respectively. Their main components are protein (≥80%), fat (≤8%), ash (≤6%) and moisture (≤5%). The WPC concentrated whey protein products were mixed with warm water at a mass ratio of 1:8 at a water temperature of 45°C. While maintaining the temperature, the mixture was stirred continuously at a speed of 500 rpm. After reconstitution, the indicators were compared with the whey powder in Example 1.

[0119] The obtained whey powder was reconstituted, and the protein and active lactoferrin content of the reconstituted whey were determined. The test results are as follows:

[0120] Figure 2 shows the retention rates of undenatured whey in Example 1 and Comparative Example 1. As can be seen from Figure 2, the retention rates of undenatured whey protein in Example 1, WPC1, WPC2, WPC3 and WPC4 are 94.06%, 72.09%, 94.64%, 99.84% and 96.44%, respectively. Except for WPC1, the vast majority of whey powder did not undergo denaturation.

[0121] Figure 3 shows the content test results of lactoferrin (A) and immunoglobulin IgG (B) in the whey powder of Example 1 and Comparative Example 1. As can be seen from Figure 3, the active lactoferrin content in the concentrated whey powder of Example 1 is significantly higher than that in the samples of Comparative Example 1, reaching 1997.23 mg / 100g protein. In Comparative Example 1, the highest lactoferrin content was found in WPC1 at 844.99 mg / 100g protein, followed by WPC2, WPC3, and WPC4 at 221.05, 94.32, and 193.40 mg / 100g protein, respectively. The concentrated whey powder of Example 1 and Comparative Example 1 showed the highest active IgG content in WPC1, at 3669.80 and 3253.35 mg / 100g protein, respectively, significantly exceeding the levels of WPC2, WPC3, and WPC4 (573.27, 2315.19, and 2678.69 mg / 100g protein, respectively). The results above show that the active lactoferrin and immunoglobulin content of the concentrated whey powder in Example 1 is higher than that of the WPC products in each group in Comparative Example 1, indicating that the process used in Example 1 can effectively enrich and increase the content of lactoferrin and immunoglobulin in whey.

[0122] Comparative Example 2: Effect of different washing solutions on the enrichment rate of lactoferrin in whey during microfiltration

[0123] The sodium chloride solution used as the washing filtrate in step (2) of Example 1 was changed to deionized water, while other steps remained the same as in Example 1, to obtain whey powder.

[0124] Comparative Example 3: Effect of lactoferrin phase separation step on lactoferrin enrichment rate in whey

[0125] Omit the lactoferrin phase separation step (i.e., the addition of sodium chloride) in Comparative Example 2 step (1), and keep everything else the same as in Comparative Example 2 to obtain whey powder.

[0126] Comparative Example 4: Effect of microfiltration temperature (30℃) on the enrichment rate of lactoferrin in whey

[0127] Adjust the filtration temperature in step (2) of Comparative Example 3 to 30°C, while keeping everything else the same as Comparative Example 3, to obtain whey powder.

[0128] Comparative Example 5: Effect of microfiltration temperature (45℃) on the enrichment rate of lactoferrin in whey

[0129] Adjust the filtration temperature in step (2) of Comparative Example 3 to 45℃, and keep everything else the same as Comparative Example 3 to obtain whey powder.

[0130] Comparative Example 6: Effect of Sodium Chloride Addition Timing on Lactoferrin Accumulation in Whey

[0131] The timing of sodium chloride addition has been changed to adding it to casein, as detailed below:

[0132] (1) The sterilized skim milk was filtered using a 0.1 μm microfiltration membrane to separate the whey protein phase from the casein phase, resulting in membrane filtration permeate and membrane filtration retentate (casein). The filtration temperature was controlled at 37±2℃ and the pH was 6.6. The endpoint of the filtration operation was determined by the point that "the volume of liquid in the membrane filtration retentate becomes one-third of the initial volume". Deionized water was used as the washing solution for filtration. After each filtration reached the endpoint, the washing solution was backfilled to the initial volume and the filtration was repeated 4 times. The permeate obtained from each filtration was collected.

[0133] (2) The membrane filtration retentate (casein) is further filtered using a 0.1 μm microfiltration membrane. A sodium chloride solution containing 450 mmol is used to backfill the volume before step (1) to make the final sodium chloride concentration 300 mmol. Under this condition, the filtration is continued. A 300 mmol sodium chloride solution is used as the washing solution for subsequent washing. The end point of the filtration operation is determined by the "volume of liquid in the membrane filtration retentate becomes one-third of the initial volume". After each filtration reaches the end point, the washing solution is backfilled to the initial volume and the filtration is repeated. The above filtration is repeated 4 times. The permeate obtained from each filtration is collected.

[0134] The whey protein content in the skim milk in step (1) and the microfiltration permeate obtained in step (2) was determined using an ELISA kit, and the whey protein enrichment rate was calculated.

[0135] Figure 4 shows the lactoferrin content results in whey powder of Example 1 and Comparative Examples 2-6. As can be seen from Figure 4, the specific lactoferrin contents in whey powder of Example 1 and Comparative Examples 2-6 are 1997.23, 1111.84, 493.93, 187.91, 286.75, and 1214.03 mg / 100g protein, respectively. From the results of Example 1, Comparative Example 1, and Comparative Example 2 in Figure 4, it can be seen that replacing the washing filtrate with deionized water significantly reduced the lactoferrin content in the whey powder. If the step of adding sodium chloride to the skim milk is further omitted, the lactoferrin content decreases even further. In Comparative Examples 3-5, after omitting the lactoferrin phase separation step, the whey powder obtained by washing with deionized water at 30°C, 37°C, and 45°C yielded the highest lactoferrin content (493.93 mg / 100g protein) in the final whey powder product obtained at 37°C, indicating that 37°C is the optimal condition. In Comparative Example 6, changing the timing of sodium chloride addition resulted in less effective filtration of lactoferrin from the casein retentate compared to Example 1, with the final whey powder containing 1214.03 mg / 100g protein. In conclusion, Example 1 yields a better whey powder rich in lactoferrin.

[0136] Example 2

[0137] A method for preparing active whey powder rich in lactoferrin includes the following steps:

[0138] (1) Same as steps (1)-(3) in Example 1, to obtain the sterilized, concentrated and desalted active whey phase;

[0139] (4) Further enrich lactoferrin and collect whey without lactoferrin:

[0140] Aseptically concentrated lactoferrin-rich active whey was pumped through a peristaltic pump into a cation exchange column filled with resin packing material (CM-Sepharose Fast Flow). The whey flowing out at the end of the column was collected; this whey was active whey without lactoferrin. The column was then flushed with pure water until all residual whey was discharged. The cation exchange column was then eluted with a 0.3M sodium chloride aqueous solution; the eluted portion consisted of non-lactoferrin impurities. The liquid flowing out at the end of the column was collected and refilled into the column-passed whey. The cation exchange column was then eluted with a 1.0M sodium chloride aqueous solution, and the liquid flowing out at the end of the column was collected. Finally, the liquid phase flowing out at the end of the column was dialyzed using a dialysis bag with a molecular weight cutoff of 10 kDa. The dialysate was ultrapure water, and dialysis was performed until the conductivity of the lactoferrin phase reached the normal range, thus obtaining lactoferrin.

[0141] (5) Drying:

[0142] Lactoferrin samples were freeze-dried (temperature -80℃, time 36h, and vacuum degree 30pa), and whey without lactoferrin was spray-dried (material inlet temperature 75℃, material outlet temperature 53℃) to obtain whey powder without lactoferrin; then sealed and packaged in a light-proof container.

[0143] The lactoferrin powder and whey powder obtained above were reconstituted. The purity of the lactoferrin powder was tested, and the whey powder without lactoferrin was tested by SDS-PAGE. The test results are as follows:

[0144] Figure 5 shows the HPLC chromatogram of the lactoferrin sample obtained after reconstitution of the lactoferrin powder prepared in Example 2. As can be seen from Figure 5, the lactoferrin sample reconstituted in Example 2 has high purity; calculations show that the lactoferrin peak area accounts for more than 98% of the total area.

[0145] Figure 6 shows the SDS-PAGE image of the whey sample without lactoferrin obtained after reconstitution of the whey powder without lactoferrin prepared in Example 2. As can be seen from Figure 6, the lactoferrin band should originally be at around 80 kDa. After column separation, the whey powder no longer contains lactoferrin, but the main α-lactalbumin and β-lactoglobulin, as well as a small amount of low-abundance whey protein, are still retained.

[0146] The above results demonstrate that the lactoferrin powder and lactoferrin-free whey powder described in Example 2 have excellent performance.

[0147] Comparative Example 7: Preparation of whey powder by acid precipitation

[0148] A method for preparing whey powder includes the following steps:

[0149] (1) Fresh milk is preheated and then centrifuged to remove fat, resulting in skimmed milk;

[0150] (2) Skim milk was filtered through a 1.4 μm pore size membrane to obtain sterilized milk, and samples were taken;

[0151] (3) Take a portion of the sterilized milk obtained in step (2), adjust the pH to 4.6 with 6M HCl solution, centrifuge at 4℃ and 10000g for 15 minutes, and take the supernatant;

[0152] (4) Adjust the pH of the supernatant obtained in step (2) to 6.8 using 6M NaOH solution, and then centrifuge at 4℃ and 10000g for 15 minutes to collect the supernatant (whey);

[0153] (5) Slowly add sodium chloride to the remaining sterilized milk in step (2) to make the final concentration 0.3M, perform 100nm membrane separation, and sample the whey obtained from the first filtration. The remaining operations are the same as step (2) of Example 2.

[0154] The whey obtained from the first filtration in steps (4) and (5) was subjected to SDS-PAGE analysis. The recovery rates of all lactoferrin obtained in steps (4) and (5) were determined using an ELISA kit. The results are as follows:

[0155] Figure 7 shows the SDS-PAGE images of whey proteins prepared in Example 2 and Comparative Example 7. As can be seen from Figure 7, compared to the whey obtained by acid precipitation in Comparative Example 7, the whey obtained by membrane filtration in Example 2 has a darker band of lactoferrin, indicating a higher lactoferrin content.

[0156] Figure 8 shows the recovery rates of lactoferrin in the whey prepared in Example 2 and Comparative Example 7. As can be seen from Figure 8, the whey obtained by membrane filtration in Example 2 has a higher lactoferrin recovery rate than the acid-precipitated whey in Comparative Example 7, at 70.64% and 45.80%, respectively. During acid precipitation, casein micelles precipitate and carry a large amount of lactoferrin, leading to a decrease in the overall lactoferrin content and thus reducing the recovery rate. Therefore, membrane filtration in Example 2 is a better choice.

[0157] Example 3: Screening of cation exchange column packing material

[0158] Two types of gels, CM-Sephadex C-50 and CM-Sepharose FastFlow, were selected. Two sets of 5 mL bovine whey were prepared, and each set of whey was passed through a 1 mL pre-packed column of one of the two gels. After loading, unbound proteins were eluted with pure water, followed by elution with 0.3 M NaCl aqueous solution and 0.9 M NaCl aqueous solution to remove some impurities and lactoferrin. The separation effect was compared by SDS-PAGE, and the choice of gel was determined based on the reproducibility of the experimental procedure.

[0159] Figure 9 shows the test results of separating and purifying lactoferrin using two ion exchange resins in Example 3. As can be seen from Figure 9, after rinsing the column with pure water using CM-Sephadex C-50, the protein bands eluted were darker, indicating a lower total amount of adsorbed protein, and its reproducibility under 0.3M NaCl aqueous solution elution conditions was poor. The protein eluted by CM-Sephadex C-50 in 0.9M NaCl aqueous solution contained impurities, indicating lower lactoferrin purity compared to CM-Sepharose Fast Flow. Furthermore, CM-Sepharose Fast Flow showed better operational reproducibility, was relatively simple to operate, and achieved better separation results. Therefore, CM-Sepharose Fast Flow was selected as the ion exchange resin for separation.

[0160] Example 4 (as shown in Figure 10)

[0161] A method for preparing infant formula with highly active immune proteins includes the following steps:

[0162] (1) Add 300 mmol / L sodium chloride to the sterilized skim milk while stirring, keep the temperature at 37°C and the pH at 6.6 until the sodium chloride is completely dissolved to obtain skim milk after lactoferrin phase transfer.

[0163] (2) The skim milk obtained after the lactoferrin phase transfer in step (1) was filtered using a 0.1 μm microfiltration membrane to separate the membrane filtration permeate (whey protein rich in lactoferrin) and the membrane filtration retentate (casein); the filtration temperature was controlled at 37±2℃, and the filtration endpoint was determined by the "liquid volume in the membrane filtration retentate becoming one-third of the initial volume", that is, the concentration factor was 3 times; filtration was performed using a 300 mmol / L sodium chloride aqueous solution as the washing filtrate. After each filtration reached the endpoint, the washing filtrate was backfilled to the initial volume and filtration was repeated 4 times;

[0164] (3) The membrane filtration retentate (casein) obtained in step (2) was washed and desalted with deionized water using a 0.1 μm microfiltration membrane; the end point of the desalting operation was determined by the "conductivity value decreasing to the normal conductivity range of the retentate"; the total protein concentration in the retentate was determined by the Kjeldahl method; and then sterilized at 85°C for 15 s to obtain the desalted and sterilized membrane filtration retentate.

[0165] (4) The membrane filtration permeate (whey protein) obtained in step (2) was concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, with a concentration factor of 8 times and an ultrafiltration temperature of 40°C. Then, ultrapure water was used as the ultrafiltration solution, and the amount of ultrafiltration solution used was 8 times that of the concentrated permeate. After ultrafiltration, electrodialysis (working current of 45A, feed flow rate of 110 mL / min, feed temperature of 35°C) was performed to further desalinate the protein. The endpoint of the desalination operation was determined by the "conductivity value decreasing to the normal conductivity range of the permeate". Sterile concentrated active whey rich in lactoferrin was obtained. The total protein concentration in the permeate was determined by the Kjeldahl method.

[0166] (5) In accordance with the national food safety standard GB 10765-2021, and based on the nutritional requirements of infants aged 0-6 months, the content of required nutrients in infant formula liquid milk was calculated. Casein, lactose, and blended vegetable oil were the main raw materials, with added compound vitamins, minerals, taurine, L-carnitine, etc. The dry matter weight included the following components: whey protein 6.19%, casein 4.13%, lactose 53.76%, and blended vegetable 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), 1.49% compound minerals (by weight 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%), 0.31% compound vitamins. % (by weight, including the following raw material components: L-ascorbic acid sodium 55.67%, dl-α-tocopherol acetate 9.84%, cholecalciferol 3.64%, retinyl acetate 2.58%, nicotinamide 2.50%, D-calcium pantothenate 2.03%, cyanocobalamin 1.20%, biotin 1.12%, phytonabinone 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%;

[0167] Lactose and compound mineral premix were dissolved in purified water at 45°C in a dedicated container. Compound vitamin premix and nutritional fortifiers (inositol, taurine, and L-carnitine) were dissolved separately in purified water at 45°C. Then, the above lactose-mineral mixture, compound vitamin mixture, and compound vegetable oil were added to the casein solution step by step and mixed thoroughly. The addition and mixing time for compound minerals and compound vitamins was 20 minutes. Then, inositol, taurine, and L-carnitine were added and mixed thoroughly to obtain an emulsion. The emulsion was homogenized twice at 25 MPa and then sterilized at 95°C for 15 seconds to obtain a casein solution containing the ingredients.

[0168] (6) The casein solution containing the ingredients was placed in a vacuum rotary evaporator (vacuum degree of 0.08 MPa, temperature of 56°C, rotation speed of 70 rpm) and rotary evaporated until the solid content reached 35%; according to the Kjeldahl protein content determination results, the casein solution containing the ingredients was mixed with sterile concentrated active whey rich in lactoferrin, so that the whey protein to casein protein mass ratio reached 6:4 and the protein content accounted for 10% of the total solids; finally, the powder was sprayed using a low temperature and low pressure spray drying device with an inlet temperature of 75°C and an outlet temperature of 53°C to obtain infant formula milk powder with high active immune protein, which was sealed and packaged in a light-proof container.

[0169] Comparative Example 8 (as shown in Figure 11)

[0170] The conventional method for preparing infant formula includes the following steps:

[0171] (1) Raw milk indicators: Take fresh milk and measure the protein, lactose, milk fat and minerals in the milk;

[0172] (2) Calculate the required nutrients in infant formula milk according to the national food safety standard GB 10765-2021;

[0173] (3) Skimmed milk purification process: After preheating the raw milk to 42°C, the milk is centrifuged in a disc centrifuge to obtain cream and skimmed milk;

[0174] (4) First sterilization: pasteurize the skim milk at 85°C for 15 seconds;

[0175] (5) Formula milk preparation: Refer to the same formula as step (5) in Example 4;

[0176] Lactose and the compound mineral premix were dissolved in purified water at 45°C in a dedicated container. The compound vitamin premix and nutritional fortifiers (inositol, taurine, and L-carnitine) were dissolved separately in purified water at 45°C. Then, the lactose-mineral mixture, compound vitamin mixture, and compound vegetable oil were added to the casein solution in steps and mixed thoroughly. The compound mineral and compound vitamin mixtures were added and mixed for 20 minutes. Then, inositol, taurine, and L-carnitine were added and mixed thoroughly. (Note: The whey protein to casein ratio is 3:2, close to the protein ratio in breast milk, and the protein content accounts for 10% of the total solids.)

[0177] (6) Homogenization: Homogenize the above-mentioned infant formula milk at a pressure of 25 MPa, and homogenize twice.

[0178] (7) Secondary sterilization: The infant formula milk is subjected to secondary sterilization at 95℃ for 15 seconds.

[0179] (8) Evaporation and concentration: The above-mentioned infant formula milk is evaporated and concentrated using a vacuum rotary evaporator with a vacuum degree of 0.08 MPa, a temperature of 55-57°C, and a rotation speed of 70 rpm. The solid content is evaporated until it reaches 35%.

[0180] (9) Spray drying: A low-temperature and low-pressure spray drying device is used. The material inlet temperature is 75℃, and the material outlet temperature is adjusted to 53℃ to obtain milk powder products, which are then sealed and packaged in a light-proof manner.

[0181] The products obtained in Example 4 and Comparative Example 8 were subjected to performance tests, and the test results are as follows:

[0182] Figure 12 shows the content and retention rate of undenatured whey in Example 4 and Comparative Example 8, where (a) represents the content of undenatured whey protein and (b) represents the retention rate of undenatured whey protein. As can be seen from Figure 12, the content of undenatured whey in the whey extracted in Example 4 was 12.57 mg / mL, significantly higher than the 7.13 mg / mL in Comparative Example 8; the retention rate of active whey protein in the infant formula milk powder of Example 4 reached 89.95%, higher than the 58.85% in Comparative Example 8. This indicates that active proteins in milk undergo significant loss after conventional infant formula processing.

[0183] Comparative Example 9: Timing of Addition of Aseptically Concentrated Active Whey Rich in Lactoferrin

[0184] In Example 4, the timing of adding active whey in step (6) was adjusted to the homogenization step in step (5), while other steps remained the same as in Example 4, resulting in milk powder; the specific process route is shown in Figure 13.

[0185] The products obtained in Example 4, Comparative Examples 8 and 9 were subjected to performance tests, and the test results are as follows:

[0186] Figure 14 shows the lactoferrin (a) and immunoglobulin IgG (b) content in the milk powder of Example 4 and Comparative Examples 8 and 9. As can be seen from Figure 14, regarding lactoferrin content, Comparative Example 8 had extremely low levels, Comparative Example 9 had less than 20 mg / 100g protein, and Example 4 had approximately 150 mg / 100g protein. Regarding IgG content, it was not detected in Comparative Example 8, less than 20 mg / 100g protein in Comparative Example 9, and approximately 250 mg / 100g protein in Example 4. The results show that conventional infant formula processing methods can hardly retain lactoferrin, while the milk powder of Example 4 had high levels of both lactoferrin and immunoglobulins, significantly higher than commonly fortified infant formulas on the market.

[0187] The lactoferrin and IgG content of the milk powder in Comparative Example 9 was higher than that in Example 4; the method in Example 4 can effectively increase the content of active proteins in infant formula, thereby improving the content of active proteins in infant formula.

[0188] Example 5 (see Route 1 in Figure 15)

[0189] A method for preparing infant formula milk powder rich in basic protein and with good protein digestibility includes the following steps:

[0190] (1) Same as steps (1)-(4) in Example 4

[0191] (5) The aseptically concentrated active whey from step (4) was adjusted to pH 6.9 with 1M HCl solution. Then, the active whey was pumped into a cation exchange column filled with CM-Sepharose Fast Flow resin packing using a peristaltic pump, and the whey flowing out at the end of the column was collected. This whey was a whey phase without basic proteins. The column was then flushed with pure water until all the residual whey was discharged. The whey was then mixed with the previously received whey, and the total protein concentration of the mixed whey was determined by the Kjeldahl method. Then, the whey was eluted with a 0.7M NaCl aqueous solution, and the liquid flowing out at the end of the column was collected. This liquid was a basic protein phase.

[0192] (6) The basic protein phase was dialyzed and desalted using a dialysis bag with a molecular weight cutoff of 7.5 kDa. The dialysate was ultrapure water. The dialysate was dialyzed until the conductivity of the basic protein phase was in the range of 4-10 mS / m, and the desalted basic protein phase was obtained.

[0193] (7) The concentrated and desalted membrane filtration retentate (casein) from step (3) and the whey phase without alkaline protein from step (5) are mixed at a protein content ratio of 4:6 for casein and whey protein. Then, pasteurize the mixture using a tube-plate combined ultra-high temperature sterilizer at 85°C for 15 seconds to obtain a protein mixture.

[0194] (8) Refer to the same formula and steps as in Example 4, step (5);

[0195] (9) Based on the Kjeldahl nitrogen content determination results, infant formula milk powder and basic protein are mixed; a low temperature and low pressure spray drying device is used, the material inlet temperature is 70℃ for basic protein, and the material outlet temperature is adjusted to 53℃ by flow rate adjustment to obtain infant formula milk powder rich in basic protein and with good protein digestibility, which is then sealed and packaged in a light-proof container.

[0196] Comparative Example 10 (Route 2, Figure 15)

[0197] The difference from Example 5 is that:

[0198] In Example 5, the whey phase without basic proteins in step (5) is transferred to step (8), with the following method modification:

[0199] Step (8): Dissolve lactose and compound mineral premix in purified water at 45°C in a special container. Dissolve compound vitamin premix and nutritional fortifiers (inositol, taurine, L-carnitine) in purified water at 45°C. Then add the whey phase without alkaline protein, lactose mineral mixture, compound vitamin mixture, and compound vegetable oil to the protein mixture obtained in step (7) and mix thoroughly. The mixing time for adding compound minerals and compound vitamins is 20 minutes. Then add inositol, taurine, and L-carnitine and mix thoroughly to obtain an emulsion.

[0200] The protein content 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.

[0201] The emulsion was homogenized twice at 25 MPa, and then sterilized at 95°C for 15 seconds to obtain infant formula emulsion.

[0202] Comparative Example 11

[0203] The specific process route is shown in Figure 16. The difference between this example and Example 5 is that:

[0204] The alkaline protein separation step was omitted, i.e. steps (4) and (5) were omitted. The remaining steps were the same as in Example 5, and infant formula milk powder was obtained.

[0205] Comparative Example 12

[0206] I purchased two stage 1 infant formula milk powders from the market: one is commercially available 1 (10% protein, 27.2% fat, and 53.7% carbohydrates) and the other is commercially available 2 (commercially available 2 is a lactoferrin-fortified version with 10% protein, 27.2% fat, and 54.7% carbohydrates).

[0207] The infant formula milk powders obtained in Examples 5, 10, 11, and 12 were reconstituted to obtain milk samples, which were then subjected to subsequent in vitro digestion experiments. The digestion solutions were analyzed by SDS-PAGE, and the free amino acid and polypeptide molecular weights were determined. The results are as follows:

[0208] Figure 17 shows the SDS-PAGE results of the digestive fluids obtained from the reconstituted infant formula milk powders of Example 5, Comparative Example 10, Comparative Example 11, and Comparative Example 12 (commercially available 1 and 2) after in vitro digestion experiments simulating infant digestion. G represents the gastric digestion portion, and I represents the intestinal digestion portion. As can be seen from Figure 17, the reconstituted milk protein of Example 5 digested slowly in the stomach, with some protein bands gradually fading. After intestinal digestion, the remaining proteins and peptides were rapidly digested, leaving only a few protein bands, and the electrophoresis results showed no significant difference after 1 minute of intestinal digestion. Comparative Example 10 showed little difference from Example 5, both exhibiting slow gastric digestion, while the rapid intestinal digestion stage showed no significant difference. Compared to Example 5 and Comparative Example 10, Comparative Example 16 showed lighter small peptide bands at the bottom during the gastric digestion stage, indicating less protein degradation during gastric digestion, with no significant difference in the intestinal stage. The gastrointestinal digestion of commercially available 1 and 2 milk powders in Comparative Example 12 showed no significant difference from Example 5, Comparative Example 10, and Comparative Example 11. Commercially available product 1 does not contain lactoferrin, therefore there is no corresponding band around 80kDa.

[0209] Figure 18 shows the polypeptide molecular weight test results of the digestive fluid obtained from the reconstituted infant formula milk powders of Examples 5, 10, 11, and 12 (commercially available 1 and 2) in simulated in vitro digestion experiments. As can be seen from Figure 18, the gastric digestion process in Example 5 was relatively slow. After 120 minutes of gastric digestion, approximately 40% of proteins or polypeptides with a molecular weight greater than 10,000 kDa were digested. After intestinal digestion, most proteins and polypeptides were degraded into polypeptides with a molecular weight less than 1 kDa, with polypeptides less than 0.5 kDa accounting for 83.44% of the total polypeptides. In the initial stage of gastric digestion in Comparative Example 10, after 15 minutes of gastric digestion, the proportion of peptides greater than 10,000 kDa decreased by 23.57%, compared to 20.98% in Example 5. However, after 120 minutes of gastric digestion, this value was 33.92% in Example 5 and 38.5% in Comparative Example 10. This indicates that Comparative Example 10 digested faster in the initial stage of gastric digestion, but its final digestibility was significantly lower than that of Example 5. In the intestinal digestion stage, the proportion of peptides greater than 10,000 kDa in Example 5 rapidly decreased from 38.01% to 4.28% at the end, while in Comparative Example 10 it decreased from 42.41% to 3.38%. The proportions of small molecular weight peptides in both examples were similar. This indicates that compared to Comparative Example 10, Example 5 showed a higher degree of protein degradation in the gastric digestion stage, while the degree of digestion in the intestinal digestion stage was similar. After 120 minutes of gastric digestion, the proportion of peptides greater than 10,000 kDa in Comparative Example 11 decreased from 77.3% to 51.23%, a relatively high proportion, indicating that gastric digestion was significantly less efficient than in Examples 5 and Comparative Example 10. During the intestinal digestion stages I1 and I15, the proportion of low-molecular-weight peptides in Comparative Example 11 was consistently lower than in Examples 5 and Comparative Example 10, while the proportion of peptides greater than 10,000 kDa was consistently higher. At I120, the proportion of peptides with a molecular weight less than 0.5 kDa in Comparative Example 11 was 81.28%, slightly lower than in Examples 5 and Comparative Example 10. This suggests that the digestibility of Comparative Example 11 is less than that of Examples 5 and Comparative Example 10. In Comparative Example 12, the changes in peptide molecular weight in the gastrointestinal stages of commercially available groups 1 and 2 were not significantly different from other groups. In summary, except for Comparative Example 11, the differences between the other four groups were not significant.

[0210] Figure 19 shows the test results of free amino acids in the digestive fluids obtained from the reconstitution and in vitro digestion experiments of infant formula milk powders obtained in Example 5, Comparative Example 10, Comparative Example 11, and Comparative Example 12 (commercially available 1 and 2). As can be seen from Figure 19, the initial free amino acid content in the commercially available milk powders was relatively high. In Comparative Example 12, the levels of commercially available 1 and 2 were 19.54 and 23.58 μg / mL, respectively, significantly higher than the other groups. During gastric digestion, the free amino acid content changed little; after 120 minutes of gastric digestion, the free amino acid content increased only slightly, and the differences in digestion among the groups were not significant. During intestinal digestion, the free amino acid content increased significantly after only 1 minute, with no significant differences among the groups. When intestinal digestion reached 120 minutes, the free amino acid content in each group further increased, with the highest content in commercially available 1 group at 370.49 μg / mL, followed by commercially available 2 group, Example 5 group, and Comparative Example 10, all significantly higher than Comparative Example 11 at 306.02 μg / mL. The results for free amino acids indicate that protein degradation is minimal during gastric digestion, while proteins and peptides rapidly degrade in the intestines, releasing a large amount of free amino acids. Comparative Example 11 showed lower protein digestibility compared to the other groups, which is consistent with the peptide molecular weight distribution results. These findings demonstrate that the digestibility characteristics of the milk powders in Example 5 and Comparative Example 10 are similar to those of commercially available milk powders, and both are superior to Comparative Example 11.

[0211] Example 6 explores the screening of cation exchange column packing materials.

[0212] Two gels were selected: CM-Sephadex C-50 and CM-Sepharose Fast Flow.

[0213] Two sets of 5 mL bovine whey were prepared, each filtered through a 0.45 μm microporous membrane. The whey was then loaded onto 1 mL pre-packed columns containing two different gels. After loading, unbound proteins were eluted with pure water, followed by partial elution of basic proteins and lactoferrin with 0.3 M NaCl and 0.9 M NaCl aqueous solutions, respectively. The separation efficiency of basic proteins was compared using SDS-PAGE, and the choice of gel was determined based on the reproducibility of the experimental procedure.

[0214] The results are shown in Figure 20. In the preliminary experiment of separating and purifying basic proteins using two ion exchange resins, the protein bands washed off with CM-Sephadex C-50 after rinsing the column with pure water were darker, indicating a lower total protein adsorption. Furthermore, its reproducibility under 0.3M NaCl aqueous solution elution conditions was poor, which may be related to its adsorption instability. In addition, the CM-Sephadex C-50 packing material is relatively soft, while CM-Sepharose Fast Flow offers better reproducibility, is simpler to operate, and provides better separation. Therefore, CM-Sepharose Fast Flow was selected as the cation exchange resin for subsequent experiments.

[0215] Example 7 explores the conditions for cation exchange columns without basic proteins (elution buffer concentration).

[0216] Dynamic adsorption experiments were performed using a CM Sepharose Fast Flow pre-packed column (1 mL) with a flow rate of 1 mL / min. The active whey rich in basic proteins, which was aseptically concentrated in step (4) of Example 5, was loaded onto the column in a volume of 10 mL. The whey was collected after loading. After loading, the liquid was equilibrated with 10 column volumes of ultrapure water and collected. Then, the liquid was eluted with an aqueous solution of 0.1-1.0 M NaCl. Each concentration was eluted to the baseline and the eluted sample was collected. SDS-PAGE experiments were then performed.

[0217] The results are as follows:

[0218] Figure 21 shows the SDS-PAGE test results for basic protein separation using different cation exchange column eluents. As can be seen from Figure 21, the vast majority of proteins can be eluted with 0.5M NaCl aqueous solution, with only a very small amount eluted. Therefore, a 0.7M NaCl aqueous solution is sufficient to elute all basic proteins. A NaCl aqueous solution concentration greater than 0.7M will prolong the subsequent dialysis desalting time. Therefore, a 0.7M NaCl aqueous solution is selected as the optimal eluent.

[0219] Example 8 explores the conditions (whey pH) for cation exchange columns without basic proteins.

[0220] Dynamic adsorption experiments were performed using a CM Sepharose FastFlow pre-packed column (1 mL) at a flow rate of 1 mL / min. The pH of the concentrated whey rich in basic proteins obtained in step (4) of Example 5 was adjusted to 4.5, 4.8, 5.1, 5.4, 5.7, 6.0, 6.3, 6.6, and 6.9 using 6M HCl or 6M NaOH solution. If precipitation occurred, the supernatant was collected after standing for a period of time and then loaded onto the column at a volume of 10 mL. The whey was collected after column loading. After loading, the liquid was equilibrated with 10 column volumes of ultrapure water and collected. The liquid was then eluted with the same volume of 0.7M NaCl aqueous solution, eluting to baseline at each concentration. The eluted samples were collected and then subjected to SDS-PAGE experiments.

[0221] The results are as follows:

[0222] Figure 22 shows the SDS-PAGE results of whey passing through the column at different pH values ​​(A); the total recovery rate of the received whey and the proportion of eluted protein after passing whey through the column at different pH values ​​(B); where the proportion of eluted protein specifically refers to the protein content in the sodium chloride solution eluent, the black bars represent the total whey protein recovery rate, and the gray bars represent the eluted protein ratio; the concentration and recovery rate of lactoferrin in the eluent (C), where the bars represent the concentration and the line graph represents the recovery rate; and the macroscopic characteristics of whey at different pH values ​​(D). As shown in Figure 22, when the pH is less than 6.0, the content of high-abundance whey protein decreases with decreasing pH, corresponding to an increase in the amount of high-abundance whey protein eluted, indicating that these non-basic proteins are more adsorbed onto the cation exchange resin. With decreasing pH, the recovery rate of lactoferrin in the eluent gradually decreases. When the pH is less than 6.0, the total whey protein recovery rate decreases, and the proportion of eluted protein increases rapidly, consistent with the SDS-PAGE results. As pH decreases, the recovery rates of whey protein and lactoferrin decrease, possibly due to the aggregation and precipitation of small amounts of casein and denatured proteins in the whey, which carry some whey protein with them. Precipitation begins to occur at pH below 6.0. Therefore, a whey pH of 6.9 is selected as the optimal condition.

Claims

1. A method for preparing active whey powder rich in lactoferrin, characterized in that, Includes the following steps: (1) Dissociation of lactoferrin: Add sodium chloride to the sterilized skim milk while stirring, maintaining a temperature of 35-39°C and a pH of 6-7, until the sodium chloride is completely dissolved, to obtain skim milk after lactoferrin phase transfer; The concentration of sodium chloride added is 200-400 mmol / L; (2) Enrichment of lactoferrin: Skim milk after lactoferrin phase transfer was filtered through a membrane to obtain membrane permeate (whey protein phase rich in lactoferrin); The washing solution used in membrane filtration is sodium chloride solution; (3) Aseptic concentration: The permeate from the membrane filter was subjected to ultrafiltration and desalting to obtain sterile concentrated active whey rich in lactoferrin. (4) Spray drying: Active whey rich in lactoferrin is spray-dried to obtain whey powder rich in lactoferrin.

2. The active whey powder rich in lactoferrin prepared by the method of claim 1.

3. A method for increasing the lactoferrin content in whey powder, characterized in that, Includes the following steps: (1) Dissociation of lactoferrin: Add sodium chloride to the sterilized skim milk while stirring, maintaining a temperature of 35-39°C and a pH of 6-7, until the sodium chloride is completely dissolved, to obtain skim milk after lactoferrin phase transfer; The concentration of sodium chloride added is 200-400 mmol / L; (2) Enrichment of lactoferrin: Skim milk after lactoferrin phase transfer was filtered through a membrane to obtain membrane permeate (whey protein rich in lactoferrin); The washing solution used in membrane filtration is sodium chloride solution; (3) Aseptic concentration: The permeate from the membrane filter was subjected to ultrafiltration and desalting to obtain sterile concentrated active whey rich in lactoferrin. (4) Spray drying: Active whey rich in lactoferrin is spray-dried to obtain whey powder rich in lactoferrin.

4. A method for simultaneously preparing lactoferrin powder and lactoferrin-free whey powder, characterized in that, Includes the following steps: (1) Dissociation of lactoferrin: Add sodium chloride to the sterilized skim milk while stirring, maintaining a temperature of 35-39°C and a pH of 6-7, until the sodium chloride is completely dissolved, to obtain skim milk after lactoferrin phase transfer; The concentration of sodium chloride added is 200-400 mmol / L; (2) Enrichment of lactoferrin: Skim milk after lactoferrin phase transfer was filtered through a membrane to obtain membrane permeate (whey protein phase rich in lactoferrin); The washing solution used in membrane filtration is sodium chloride solution; (3) Aseptic concentration: The permeate from the membrane filter was subjected to ultrafiltration and desalting to obtain sterile concentrated active whey rich in lactoferrin. (4) Further enrich lactoferrin and collect whey without lactoferrin: The sterile, concentrated whey rich in lactoferrin was passed through a cation exchange column to transfer lactoferrin from the whey to the column, resulting in whey without lactoferrin. Subsequently, the lactoferrin on the cation exchange column was eluted with an aqueous sodium chloride solution to remove salt and obtain lactoferrin. (5) Drying: Lactoferrin was freeze-dried to obtain lactoferrin powder; whey without lactoferrin was spray-dried to obtain whey powder without lactoferrin.

5. Lactoferrin powder and whey powder without lactoferrin prepared by the method of claim 4.

6. A method for preparing infant formula with highly active immune proteins, characterized in that, Includes the following steps: (1) Add sodium chloride to the sterilized skim milk to dissociate lactoferrin and obtain skim milk after lactoferrin phase transfer; (2) The skim milk after the lactoferrin phase transfer obtained in step (1) is filtered through a membrane to separate the whey protein phase from the casein phase, resulting in a membrane filtration permeate (whey protein rich in lactoferrin) and a membrane filtration retentate (casein); wherein, the washing solution used for membrane filtration is a sodium chloride solution. (3) The membrane filtration retentate obtained in step (2) is desalted, concentrated and sterilized to obtain a membrane filtration retentate after desalting, concentration and sterilization. (4) The membrane permeate obtained in step (2) is subjected to ultrafiltration and desalting to obtain sterile concentrated active whey rich in lactoferrin. (5) The desalted and sterilized membrane filtration retentate obtained in step (3) is used as casein solution. The nutrients required for infant formula are added to the casein solution and mixed thoroughly. The mixture is homogenized and sterilized to obtain a casein solution containing the ingredients. (6) The casein solution containing ingredients obtained in step (5) is concentrated to obtain a concentrate; then the concentrate is mixed with the sterile concentrated active whey rich in lactoferrin obtained in step (4), concentrated and spray-dried to obtain infant formula milk powder with high active immune protein.

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

8. The method according to claim 6, characterized in that, In step (6), the spray drying is low-temperature, low-pressure spray drying.

9. Infant formula milk powder with highly active immune proteins prepared by the method according to any one of claims 6-8.

10. A method for increasing the content of active proteins in infant formula, characterized in that, Infant formula milk powder using the highly active immune protein described in claim 9.

11. A method for preparing infant formula milk powder with basic protein and good protein digestibility, characterized in that, Includes the following steps: (1) Add sodium chloride to the sterilized skim milk to dissociate the basic proteins and obtain skim milk after basic protein phase transfer; (2) After the basic protein phase transfer in step (1), the skim milk is filtered through a membrane to separate the whey protein phase from the casein phase, resulting in a membrane filtration permeate (whey protein rich in basic protein) and a membrane filtration retentate (casein); wherein, the washing solution used for membrane filtration is a sodium chloride solution. (3) Desalt and concentrate the membrane filtration retentate (casein) from step (2) to obtain concentrated and desalted membrane filtration retentate; (4) The membrane filtration permeate (whey protein rich in basic protein) from step (2) is subjected to ultrafiltration and desalting to obtain sterile concentrated active whey rich in basic protein. (5) Pass the sterile concentrated active whey rich in basic protein from step (4) through a cation exchange column to transfer the basic protein from the whey to the cation exchange column, and obtain a whey phase without basic protein; then use sodium chloride solution to elute the basic protein on the cation exchange column to obtain a basic protein phase; (6) Desalt the alkaline protein phase from step (5) to obtain the desalted alkaline protein phase; (7) The concentrated and desalted membrane filtration retentate from step (3) is mixed with the whey phase without alkaline protein from step (5), and then pasteurized to obtain a protein mixture. (8) The protein mixture from step (7) is mixed with other nutrients and thoroughly mixed, then homogenized, sterilized and concentrated to obtain infant formula milk. (9) The liquid infant formula milk from step (8) and the desalted alkaline protein from step (6) are mixed and then spray-dried to obtain infant formula milk powder rich in alkaline protein and with good protein digestibility.

12. The method according to claim 11, characterized in that, In step (1), the concentration of sodium chloride added is 200-400 mmol / L, the temperature is 35-39℃, and the pH is 6-7.

13. The method according to claim 11, characterized in that, The packing material used in step (5) for the cation exchange column is CM-Sepharose Fast Flow.

14. The method according to claim 11, characterized in that, In step (5), the alkaline protein is washed away with an aqueous solution of sodium chloride, with a sodium chloride concentration of 0.4M-1.0M.

15. The method according to claim 11, characterized in that, In step (9), the spray drying is low-temperature, low-pressure spray drying.

16. The infant formula milk powder rich in basic protein and with good protein digestibility prepared by the method of any one of claims 11-15.

17. A method for increasing the basic protein content in infant formula while maintaining good protein digestibility, characterized in that, The infant formula milk powder rich in alkaline protein and with good protein digestibility as described in claim 16 was used.

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