Infant formula milk powder containing high-activity immune protein and preparation method of infant formula milk powder

Abstract

The invention discloses infant formula milk powder with high-activity immune protein and a preparation method of the infant formula milk powder, and belongs to the technical field of preparation of dairy products. The preparation method comprises the following steps: degreasing and degerming fresh milk, transferring lactoferrin phase combination, enriching lactoferrin to a whey phase through filtration of a microfiltration membrane, further desalting and concentrating the whey phase through ultrafiltration and electrodialysis, and forming active whey rich in lactoferrin after sterile concentration; the trapped fluid filtered by the microfiltration membrane is subjected to milk killing (primary sterilization), then is blended with other nutrients, is homogenized, is subjected to material killing (secondary sterilization) and is concentrated, and finally, the treated trapped fluid and the active whey which is subjected to sterile concentration and is rich in lactoferrin are mixed on line and then are subjected to evaporation concentration and spray drying; the infant formula milk powder rich in active whey protein with high lactoferrin content is obtained.

Description

Technical Field

[0001] The invention relates to an infant formula milk powder containing high-activity immune protein and a preparation method thereof, belonging to the technical field of dairy product preparation. Background Art

[0002] Breast milk is the most natural, safest and most comprehensive food, and is considered the gold standard for infant food. It not only provides infants with all the nutrients they need for growth and development, but also contains a wealth of natural immune active substances, such as lactoferrin, immunoglobulins (including IgA, IgG and IgM, etc.), lactoperoxidase and other low-abundance proteins. These low-abundance active proteins play an important role in enhancing infant immunity, improving cognitive ability, strengthening physical fitness, and reducing the incidence of diseases such as asthma. For example, lactoferrin participates in regulating iron absorption, promoting intestinal cell proliferation and differentiation and other physiological processes, and can effectively reduce the risk of respiratory and gastrointestinal infections in infants. IgA, IgG and IgM are important immune antibodies in the human mucosa and play an important role in protecting newborns from intestinal and respiratory microbial infections. The World Health Organization recommends that breastfeeding should be for more than 6 months, but due to work, health and other reasons, many mothers are unable to breastfeed. The exclusive breastfeeding rate of more than 6 months in my country is only 29%; this means that most infants need to rely on infant formula to meet their own growth and development needs.

[0003] Infant formula milk powder is usually made from cow and sheep milk, with appropriate nutrients added to make its total ingredients meet the needs of infant growth and development. At present, infant formula milk powder has completed the breast milk-like basic nutritional components, including protein, lipids, carbohydrates, vitamins and minerals. Some infant formula milk powders also add 1,3-dioleyl-2-palmitoyl triglyceride (OPO), casein phosphopeptide (CPP), probiotics, prebiotics (HMOs) and other ingredients to improve the quality of milk powder, but there are still large differences between infant formula milk powder feeding and breastfeeding. Previous studies have shown that the probability of infant formula-fed babies suffering from diarrhea, respiratory diseases, otitis media, asthma, allergies and other diseases is much higher than that of breastfeeding, and the incidence of metabolic diseases in adulthood is also significantly higher than that of breastfeeding. The main difference between infant formula milk powder feeding and breastfeeding is closely related to the difference in the activity of its functional components. Among these functional components, protein is the cornerstone of life activities.

[0004] The optimization of protein in infant formula milk powder initially focused on exploring the types of breast milk nutrients. Specifically, the protein content was adjusted first; then the ratio and function of breast milk nutrients were explored, and then the ratio of whey protein and casein was adjusted and the ratio of α-lactalbumin was increased. In recent years, the research on infant formula milk powder has deepened the exploration of the molecular domain functions of breast milk and the active components of breast milk, such as the strengthening of active lactoferrin. Although the adjustment based on the formula can achieve a composition and function similar to that of breast milk, the production process of infant formula milk powder requires a series of heat treatment steps, such as sterilization, concentration, spray drying, etc. These heat treatment operations will cause the heat-sensitive active proteins such as lactoferrin and immunoglobulins in milk to denature and lose their biological activity. The loss of these active proteins greatly reduces the quality of infant formula milk powder, and the existing processing technology of infant formula milk powder is almost difficult to retain the activity of functional proteins in fresh milk.

[0005] In order to improve the active nutrition of infant formula milk powder, the infant formula milk powder currently on the market mainly adopts dry or wet processes to strengthen immune active proteins (such as lactoferrin and immunoglobulin), but these ingredients all need to rely on imports, are expensive, and can only strengthen one immune active protein at a time, and cannot achieve the retention of most active proteins in whey; even if the method of strengthening active whey protein can retain most active proteins, these ingredients are all separated and then spray-dried or freeze-dried products, and the active proteins in the whey ingredients will also undergo partial denaturation and reduction of biological activity during the drying process.

[0006] In addition, the biggest problem in whey protein ingredients is the low content of alkaline proteins such as lactoferrin. Because these proteins are positively charged and mainly bind to negatively charged casein in milk, most of the alkaline proteins such as lactoferrin are retained in the casein phase when separating whey protein.

[0007] Therefore, how to develop a method for preparing infant formula milk powder by online enrichment of active whey protein with high content of alkaline proteins such as lactoferrin is an important problem to be solved in the art. Summary of the invention

[0008] [Technical issues]

[0009] The existing processing technology of infant formula milk powder can hardly retain the activity of functional proteins in fresh milk;

[0010] The active proteins in the whey protein ingredients used in infant formula will undergo partial denaturation and reduced biological activity; and the content of basic proteins such as lactoferrin is low, resulting in a huge gap between the prepared infant formula and breast milk.

[0011] [Technical solution]

[0012] In order to solve the above problems, the present invention provides an infant formula milk powder with high active immune protein and a preparation method thereof; specifically, the present invention firstly defats and sterilizes fresh cow milk, then transfers it in combination with lactoferrin, and enriches lactoferrin to a whey phase through microfiltration membrane filtration, and further desalts and concentrates the whey phase through ultrafiltration and electrodialysis to form aseptically concentrated active whey rich in lactoferrin; the retentate filtered by the microfiltration membrane is milk-sterilized (primary sterilization), and then mixed, homogenized, sterilized (secondary sterilization) and concentrated with other nutrients, and finally the treated retentate is mixed online with the aseptically concentrated active whey rich in lactoferrin, and then spray-dried to obtain an infant formula milk powder with active whey protein rich in high content of lactoferrin.

[0013] The first object of the present invention is to provide a method for preparing an infant formula milk powder containing highly active immune proteins, comprising the following steps:

[0014] (1) adding sodium chloride to the sterilized skim milk to release lactoferrin, thereby obtaining skim milk after lactoferrin phase transfer;

[0015] (2) filtering the skim milk obtained in step (1) after the lactoferrin phase transfer to separate the whey protein phase from the casein phase through a membrane filter to obtain a membrane filtration permeate (lactoferrin-rich whey protein) and a membrane filtration retentate (casein); wherein the wash filtrate used in the membrane filtration is a sodium chloride solution;

[0016] (3) desalting and sterilizing the membrane filtration retentate obtained in step (2) to obtain a desalted and sterilized membrane filtration retentate;

[0017] (4) ultrafiltration and desalination of the membrane filtration permeate obtained in step (2) to obtain aseptically concentrated active whey rich in lactoferrin;

[0018] (5) using the desalted and sterilized membrane filtration retentate obtained in step (3) as a casein solution, adding the nutrients required in the infant formula to the casein solution, mixing thoroughly, homogenizing, sterilizing and evaporating to obtain a casein solution containing ingredients;

[0019] (6) Concentrating the casein solution containing ingredients obtained in step (5) to obtain a concentrate; then mixing the concentrate with the aseptically concentrated active whey rich in lactoferrin obtained in step (4), and spray drying the mixture to obtain an infant formula milk powder containing highly active immune proteins.

[0020] In one embodiment of the present invention, the method for preparing the sterilized skim milk in step (1) is as follows:

[0021] After preheating the raw milk to 40-42°C, the milk is centrifuged in a disc centrifuge to obtain cream and skimmed milk; the skimmed milk is then filtered using a 1.4μm microfiltration membrane with the temperature controlled at 40-45°C to remove most of the microorganisms and impurities in the milk to obtain sterilized skimmed milk.

[0022] In one embodiment of the present invention, the concentration of sodium chloride added in step (1) is 200-400 mmol / L, the temperature is 35-39°C, and the pH is 6-7; more preferably, the concentration of sodium chloride added is 300 mmol / L, the temperature is 37°C, and the pH is 6.6.

[0023] In one embodiment of the present invention, in step (1), lactoferrin is transferred from the casein phase to the whey phase.

[0024] In one embodiment of the present invention, the membrane pore size used in the membrane filtration in step (2) is 90-110nm, the temperature is 35-55°C, the concentration multiple is 2-4, and the number of diafiltrations is 3-5; further preferably, the membrane pore size used in the membrane filtration is 100nm, the temperature is 35-55°C, the concentration multiple is 3, and the number of diafiltrations is 4.

[0025] In one embodiment of the present invention, the operational endpoint of the membrane filtration in step (2) is determined by "the liquid volume in the membrane filtration retentate becomes one third of the initial volume".

[0026] In one embodiment of the present invention, the sodium chloride solution in step (2) is a sodium chloride aqueous solution with a concentration of 200-400 mmol / L.

[0027] In one embodiment of the present invention, after each filtration in the membrane filtration of step (2) reaches the end point of the operation, the filtrate is backfilled to the initial volume and filtered again.

[0028] In one embodiment of the present invention, the desalination in step (3) is performed by filtering with a 0.05-0.15 μm microfiltration membrane, and deionized water is used as a secondary filtration solution; the amount of the secondary filtration solution used is 5-10 times the volume of the retentate, the end point of the desalination operation is determined by "the conductivity value is reduced to the normal retentate conductivity range", and the protein concentration is determined by the Kjeldahl method to determine the total protein concentration in the retentate; further preferably, the pore size of the microfiltration membrane is 0.1 μm.

[0029] In one embodiment of the present invention, the sterilization in step (3) is performed at 80-90°C for 10-20s, and more preferably, at 85°C for 15s.

[0030] In one embodiment of the present invention, the ultrafiltration membrane used in the ultrafiltration in step (4) has a molecular weight cutoff of 9-11 kDa, a concentration multiple of 5-10 times, and an ultrafiltration temperature not exceeding 45°C; further preferably, the ultrafiltration membrane has a molecular weight cutoff of 10 kDa; and the ultrafiltration temperature is 40°C.

[0031] In one embodiment of the present invention, in step (4), desalination is performed using ultrapure water as a second ultrafiltration solution, and the amount of ultrafiltration solution used is 5-10 times that of the concentrated permeate. After the ultrafiltration is completed, electrodialysis is performed for further desalination; the electrodialysis uses a working current of 45A, a feed flow rate of 110mL / min, and a feed temperature of 35°C; the end point of the desalination operation is determined by "the conductivity value is reduced to the normal permeate conductivity range", and the protein concentration is determined by the Kjeldahl method to determine the total protein concentration in the permeate.

[0032] In one embodiment of the present invention, the casein solution in step (5) is used as a milk base in the preparation process of infant formula milk powder.

[0033] In one embodiment of the present invention, the nutrients required in the infant formula in step (5) are calculated according to the national food safety standard GB 10765-2021, based on the nutrients required for infants aged 0-6 months, and the content of the nutrients required in the infant formula liquid milk is calculated, with casein liquid, lactose, and vegetable blending oil as the main raw materials, and complex vitamins, minerals, taurine, L-carnitine, etc. are added.

[0034] In one embodiment of the present invention, the nutrients required in the infant formula in step (5) include the following components by dry matter weight: whey protein 6.19%, casein 4.13%, lactose 53.76%, vegetable blending oil 34.34% (by mass percentage, including the following raw material components: coconut oil 25%, OPO 22%, soybean oil 18%, corn oil 15%, low-erucic acid rapeseed oil 10%, high-oleic acid sunflower oil 10%), complex minerals 1.49% (by mass percentage, including the following raw material components: calcium citrate 10.93%, potassium chloride 19.72%, sodium citrate 18.84%, calcium citrate 10.93%, magnesium sulfate 4.08%, ferric pyrophosphate 1.25%, zinc sulfate 0.32%, potassium iodate 0.28%, copper sulfate 0.18%, manganese sulfate 0.06%, sodium selenite 0.05%), complex vitamins 0.31 % (by mass percentage, including the following raw material components: L-sodium ascorbate 55.67%, dl-α-tocopherol acetate 9.84%, cholecalciferol 3.64%, retinyl acetate 2.58%, niacinamide 2.50%, D-calcium pantothenate 2.03%, cyanocobalamin 1.20%, biotin 1.12%, phytonadione 0.84%, thiamine hydrochloride 0.58%, folic acid 0.47%, riboflavin 0.36%, pyridoxine hydrochloride 0.34%), inositol 0.04%, taurine 0.04%, and L-carnitine 0.01%.

[0035] In one embodiment of the present invention, the required nutrients are added to the infant formula in step (5) as follows:

[0036] Lactose and complex mineral premix are mixed and dissolved in a special container using 40-50°C purified water. Complex vitamin premix and nutritional enhancers (inositol, taurine, L-carnitine) are dissolved separately using 40-45°C purified water. Then, the lactose mineral mixture, complex vitamin mixture and complex vegetable oil are added step by step to the casein solution and mixed thoroughly. The mixing time for adding complex minerals and complex vitamins should be less than 15 minutes. Then, inositol, taurine and L-carnitine are added and mixed thoroughly.

[0037] In one embodiment of the present invention, the homogenization in step (5) is carried out at 25-30 MPa for 1-2 times.

[0038] In one embodiment of the present invention, the sterilization in step (5) is carried out at 90-100°C for 10-20s; more preferably, at 95°C for 15s.

[0039] In one embodiment of the present invention, the concentration in step (6) is evaporation concentration, specifically using a vacuum rotary evaporator with a vacuum degree of 0.07-0.09 MPa, a temperature of 55-57° C., a rotation speed of 50-90 rpm, and rotary evaporation until the solid content reaches 30-40% (mass percentage).

[0040] In one embodiment of the present invention, after the concentrate in step (6) is mixed with the aseptically concentrated lactoferrin-rich active whey, the mass ratio of whey protein to casein reaches ≥6:4, which is close to the protein ratio in breast milk, and the protein content accounts for 10-15% (mass percentage) of the total solids.

[0041] In one embodiment of the present invention, the spray drying in step (6) is low-temperature and low-pressure spray drying, specifically: the material inlet temperature is not higher than 80°C, and the material outlet temperature is adjusted to 52-55°C by flow regulation.

[0042] In one embodiment of the present invention, the infant formula milk powder with high-activity immune protein needs cold chain transportation or low-temperature refrigeration after aseptic filling after processing.

[0043] The second object of the present invention is the infant formula milk powder with high-activity immune protein prepared by the method of the present invention.

[0044] In one embodiment of the present invention, the highly active immune protein includes lactoferrin, immunoglobulin IgG and the like.

[0045] The third object of the present invention is the application of the infant formula milk powder containing the highly active immune protein of the present invention in the food field.

[0046] The fourth object of the present invention is to provide a method for increasing the active protein content in infant formula milk powder, wherein the method uses the infant formula milk powder with high active immune protein of the present invention.

[0047] [Beneficial Effects]

[0048] (1) The method for preparing infant formula milk powder with high-activity immune protein in the present invention is to add sodium chloride to skim milk to increase the ion concentration, promote the dissociation of casein micelles and lactoferrin complexes, release lactoferrin wrapped in casein micelles from the lactoferrin complexes, and more lactoferrin is present in the whey phase in a free state; then, a whey protein phase rich in lactoferrin and casein are separated by a microfiltration membrane. The whey protein phase obtained by this method has a significantly improved lactoferrin enrichment rate compared to a whey protein phase that has not been treated for lactoferrin release.

[0049] (2) The present invention uses casein as the milk base in the production process of infant formula milk powder, and adds whey protein to mix before spray drying, thereby avoiding the loss of active protein in whey protein due to multiple heat treatments, and greatly improving the abundance of active protein in the finished infant formula milk powder.

[0050] In summary, more active proteins are retained in the infant formula milk powder prepared by the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The present invention is a process flow chart of the method for preparing infant formula milk powder with high-activity immune protein.

[0052] Figure 2 It is the process flow chart of conventional infant formula milk powder in reference example 1.

[0053] Figure 3 The graphs are the undenatured whey content and retention rate of raw milk in Example 1 and Comparative Example 1; wherein (a) is the undenatured whey protein content; and (b) is the undenatured whey protein retention rate.

[0054] Figure 4 The process routes of the infant formula milk powder of Example 1 and Reference Example 2 are compared.

[0055] Figure 5 Graph showing the lactoferrin (a) and immunoglobulin IgG (b) contents in milk powder in Example 1, Comparative Example 1 and Comparative Example 2.

[0056] Figure 6 The lactoferrin content and enrichment rate in the microfiltration permeate (active whey) in Example 1, Reference Example 3 and Reference Example 4 are shown in the figure. The bar graph is the lactoferrin content, and the line graph is the total enrichment rate of lactoferrin, wherein (a) is Example 1, (b) is Reference Example 3, and (c) is Reference Example 4.

[0057] Figure 7 The lactoferrin content and enrichment rate in the microfiltration permeate of Control Example 5 and Control Example 6, the bar graph is the lactoferrin content, and the line graph is the cumulative enrichment rate of lactoferrin, where (a) is Control Example 5 and (b) is Control Example 6.

[0058] Figure 8 The figure shows the ferritin content and enrichment rate in the microfiltration permeate in Control Example 7. The bar graph shows the lactoferrin content, and the line graph shows the cumulative enrichment rate of lactoferrin. DETAILED DESCRIPTION

[0059] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0060] Materials and instruments:

[0061] BCA kit was purchased from Thermo Fisher Scientific;

[0062] Lactoferrin and immunoglobulin reagent test kits (ElisaKit) were purchased from Bethyl Company, USA;

[0063] The remaining reagents were of analytical grade;

[0064] The ultracentrifuge was a Beckman ultracentrifuge.

[0065] Test method:

[0066] 1. Active whey extraction:

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

[0068] Then pour the milk sample into a centrifuge tube, centrifuge at 8000g for 15 minutes at 4°C, scrape off the upper layer of milk fat with a small spoon, adjust the pH of the skim milk to 4.6, and then let it stand at 4°C for 1 hour to allow the casein and denatured whey protein to completely precipitate. During this period, pay attention to monitoring and adjusting the pH;

[0069] The supernatant sample was transferred to an ultra-high speed centrifuge tube and centrifuged at 31200 rpm and room temperature for 1.5 h. After centrifugation, the sample was divided into three layers (the top layer was residual milk fat, the middle layer was whey, and the bottom layer was aggregated casein micelles);

[0070] Use a pipette to carefully draw the whey sample from the middle layer into a clean centrifuge tube for later use;

[0071] The extracted whey was diluted for subsequent index determination.

[0072] 2. Determination of undenatured whey:

[0073] According to the method provided by the BCA kit, the 2mg / mL bovine serum albumin standard was graded diluted with PBS (0.025mg / mL-2.0mg / mL). The provided A and B working solutions were mixed at a mass ratio of 1:50 and set aside. The whey was diluted 10 times with PBS, 25μL of whey and a series of standards were placed in a 96-well plate, and 200μL of mixed working solution was added to each well using a dispenser. After mixing, it was sealed with plastic wrap to prevent liquid evaporation. After incubation in the dark at 37°C for 30 minutes, it was taken out and the absorbance at a wavelength of 562nm was read using an ELISA reader. Three replicate wells were set for each sample, and distilled water was used as a blank control.

[0074] 3. Determination of lactoferrin content:

[0075] Take the bovine lactoferrin standard and dilute it in a gradient to make its concentration between 500-7.8ng / mL, then dilute the sample 500-2000 times according to the standard curve concentration range (determined by different standard curve ranges). Add 100μL of gradient concentration standard and previously extracted active whey (active whey diluted 1000 times), set up 3 replicate wells, react at room temperature for 1h, repeat the well washing operation and pat dry; add 100μL horseradish peroxidase-conjugated goat anti-bovine lactoferrin detection antibody (200000 times dilution) and incubate at room temperature for 1h, repeat the plate washing 5 times; add 100μLTMB (enzyme substrate) and place the 96-well plate in the dark and incubate at room temperature for 15min, add 100μL stop solution (0.18mol H2 S After 400 μL of PBS, the absorbance at 450 nm was read after oscillating for 5 seconds in a microplate reader. Finally, Soft-Max Pro was used to perform 4-parameter curve fitting of the standard curve and calculate the concentration of active protein in the sample, with the sample dilution as the blank control.

[0076] 4. Determination of immunoglobulin G content:

[0077] Same as the method for determining lactoferrin content, the active whey sample was diluted 10,000 times.

[0078] Example 1

[0079] A method for preparing infant formula milk powder with high-activity immune protein comprises the following steps:

[0080] (1) preheating raw milk to 42° C. and centrifuging the milk with a disc centrifuge to obtain cream and skim milk; filtering the skim milk with a 1.4 μm microfiltration membrane at a temperature controlled at 40° C. to remove most of the microorganisms and impurities in the milk, thereby obtaining sterilized skim milk; adding 300 mmol / L of sodium chloride to the sterilized skim milk while stirring, and controlling the temperature at 37° C. and the pH at 6.6 until the sodium chloride is completely dissolved, thereby obtaining skim milk after lactoferrin phase transfer;

[0081] (2) filtering the skim milk after the lactoferrin phase transfer obtained in step (1) using a 100 nm microfiltration membrane to separate the whey protein phase from the casein phase to obtain a membrane filtration permeate (lactoferrin-rich whey protein) and a membrane filtration retentate (casein); wherein the filtration temperature is controlled at 37±2° C., and the operation endpoint of the filtration is determined by “the liquid volume in the membrane filtration retentate becomes one-third of the initial volume”, that is, the concentration multiple is 3 times; the filtration is performed using a 300 mmol / L sodium chloride aqueous solution as a filtrate wash, and after each filtration reaches the operation endpoint, the filtrate wash is backfilled to the initial volume and filtered again, and the process is repeated 4 times;

[0082] (3) filtering the membrane filtration retentate (casein) obtained in step (2) using a 0.1 μm microfiltration membrane, using deionized water as a secondary filtration solution, and the amount of the secondary filtration solution used is 8 times the volume of the retentate; the end point of the desalination operation is determined by "the conductivity value is reduced to the normal retentate conductivity range"; the total protein concentration in the retentate is determined by the Kjeldahl method; and then sterilizing at 85° C. for 15 seconds to obtain a desalted and sterilized membrane filtration retentate;

[0083] (4) the membrane filtration permeate (lactoferrin-rich whey protein) obtained in step (2) is ultrafiltered and concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, the concentration multiple is 8 times, and the ultrafiltration temperature is 40° C.; then ultrapure water is used as a second ultrafiltration solution, and the amount of ultrafiltration solution used is 8 times that of the concentrated permeate; after the ultrafiltration is completed, electrodialysis (working current is 45A, feed flow rate is 110 mL / min, feed temperature is 35° C.) is further desalted, and the end point of the desalination operation is determined by "the conductivity value is reduced to the normal permeate conductivity range"; the active whey rich in lactoferrin after sterile concentration is obtained; and the total protein concentration in the permeate is determined by the Kjeldahl method;

[0084] (5) In accordance with the national food safety standard GB 10765-2021, the nutrients required by infants aged 0-6 months are used as the standard to calculate the content of nutrients required in infant formula liquid milk, with casein liquid, lactose, and vegetable blending oil as the main raw materials, and added with complex vitamins, minerals, taurine, L-carnitine, etc.;

[0085] The following components are included by weight of dry matter: whey protein 6.19%, casein 4.13%, lactose 53.76%, vegetable blended oil 34.34% (by mass percentage, including the following raw material components: coconut oil 25%, OPO 22%, soybean oil 18%, corn oil 15%, low-erucic acid rapeseed oil 10%, high-oleic acid sunflower oil 10%), complex minerals 1.49% (by mass percentage, including the following raw material components: calcium citrate 10.93%, potassium chloride 19.72%, sodium citrate 18.84%, calcium citrate 10.93%, magnesium sulfate 4.08%, ferric pyrophosphate 1.25%, zinc sulfate 0.32%, potassium iodate 0.28%, copper sulfate 0.18%, manganese sulfate 0.06%, sodium selenite 0.05%), complex vitamins 0.31 % (by mass percentage, including the following raw material components: L-sodium ascorbate 55.67%, dl-α-tocopherol acetate 9.84%, cholecalciferol 3.64%, retinyl acetate 2.58%, niacinamide 2.50%, D-calcium pantothenate 2.03%, cyanocobalamin 1.20%, biotin 1.12%, phytonadione 0.84%, thiamine hydrochloride 0.58%, folic acid 0.47%, riboflavin 0.36%, pyridoxine hydrochloride 0.34%), inositol 0.04%, taurine 0.04%, L-carnitine 0.01%;

[0086] Lactose and a composite mineral premix are mixed and dissolved in a special container using 45° C. pure water, a composite vitamin premix and a nutrient enhancer (inositol, taurine, and L-carnitine) are respectively dissolved in 45° C. pure water, and then the lactose mineral mixture, the composite vitamin mixture, and the composite vegetable oil are added step by step to the casein solution and fully mixed. The composite mineral and composite vitamin are added and mixed for 20 minutes, and then inositol, taurine, and L-carnitine are added and fully mixed to obtain an emulsion; the emulsion is homogenized twice at 25 MPa, and then sterilized at 95° C. for 15 seconds to obtain a casein solution containing ingredients;

[0087] (6) placing the casein solution containing the ingredients in a vacuum rotary evaporator (vacuum degree of 0.08 MPa, temperature of 56°C, rotation speed of 70 rpm) and rotary evaporating until the solid content reaches 35%; according to the result of Kjeldahl nitrogen determination of protein content, mixing the casein solution containing the ingredients with aseptically concentrated active whey rich in lactoferrin, so that the whey protein and casein mass ratio reaches 6:4, which is close to the protein ratio in breast milk, and the protein content accounts for 15% of the total solids; finally, using a low-temperature and low-pressure spray drying device, the material inlet temperature is 75°C, and the material outlet temperature is adjusted to 53°C by flow regulation to obtain an infant formula milk powder with high active immune protein, and sealing and light-proof packaging.

[0088] Comparative Example 1

[0089] Conventional preparation method of infant formula milk powder (such as Figure 2 ), comprising the following steps:

[0090] (1) Raw milk indicators: Take fresh milk and measure the protein, lactose, milk fat and minerals in the milk;

[0091] (2) Calculate the nutrients required in infant formula according to the national food safety standard GB 10765-2021;

[0092] (3) Skimmed milk treatment: After preheating the raw milk to 42°C, the milk is centrifuged in a disc centrifuge to obtain cream and skimmed milk;

[0093] (4) Primary sterilization: pasteurize skim milk at 85°C for 15 seconds;

[0094] (5) Formula milk preparation: In accordance with the national food safety standard GB 10765-2021, the nutrients for infants aged 0 to 6 months are used as the standard to calculate the content of nutrients required in infant formula liquid milk. Skim milk, lactose, and vegetable blending oil are used as the main raw materials, and complex vitamins, minerals, taurine, L-carnitine, etc. are added. The following components are included by dry matter weight: casein 4.98% (based on 80% of total milk protein), concentrated whey protein powder 7.95%, lactose 56.38%, vegetable blending oil 27.16% (based on mass percentage, including the following raw material components: coconut oil 25%, OPO 22%, soybean oil 18%, corn oil 15%, low-erucic acid rapeseed oil 10%, high-oleic acid sunflower oil 10%), compound minerals 1.80% (by mass percentage, including the following raw material components: calcium citrate 10.93%, potassium chloride 19.72%, sodium citrate 18.84%, calcium citrate 10.93%, magnesium sulfate 4.08%, ferric pyrophosphate 1.25%, zinc sulfate 0.32%, potassium iodate 0.28%, copper sulfate 0.18%, manganese sulfate 0.06%, sodium selenite 0.05%), compound vitamins 0.38 % (by mass percentage, including the following raw material components: 55.67% L-sodium ascorbate, 9.84% dl-α-tocopheryl acetate, 3.64% cholecalciferol, 2.58% retinyl acetate, 2.50% niacinamide, 2.03% D-calcium pantothenate, 1.20% cyanocobalamin, 1.12% biotin, 0.84% ​​phytonadione, 0.58% thiamine hydrochloride, 0.47% folic acid, 0.36% riboflavin, 0.34% pyridoxine hydrochloride), 0.05% inositol, 0.05% taurine, 0.01% L-carnitine;

[0095] Mix and dissolve lactose and compound mineral premix in a special container with 45°C pure water, dissolve compound vitamin premix and nutritional enhancer (inositol, taurine, L-carnitine) in 45°C pure water, then add the lactose mineral mixture, compound vitamin mixture and compound vegetable oil to the casein solution step by step and mix thoroughly. The compound mineral and compound vitamin addition and mixing time is 20 minutes, then add inositol, taurine and L-carnitine and mix thoroughly. (Note: The ratio of whey protein to casein is 3:2, which is close to the protein ratio in breast milk, and the protein content accounts for 15% of the total solids.)

[0096] (6) Homogenization: homogenize the above infant formula milk at a homogenization pressure of 25 MPa for 2 times;

[0097] (7) Secondary sterilization: Secondary sterilization of infant formula milk at 95°C for 15 seconds;

[0098] (8) Evaporation and concentration: The 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, and rotary evaporation is performed until the solid content reaches 35%;

[0099] (9) Spray drying: A low-temperature and low-pressure spray drying device was used, with the material inlet temperature at 75°C and the material outlet temperature at 54°C adjusted by flow rate to obtain a milk powder product, which was then sealed and packaged in a light-proof manner.

[0100] The products obtained in Example 1 and Reference Example 1 were subjected to performance tests, and the test results are as follows:

[0101] Figure 3 The following is a graph showing the undenatured whey content and retention rate of raw milk in Example 1 and Comparative Example 1, wherein (a) is the undenatured whey protein content; (b) is the undenatured whey protein retention rate. Figure 3 It can be seen that the content of undenatured whey in the whey extracted in Example 1 is 12.57 mg / mL, which is significantly higher than 7.13 mg / mL in Control Example 1; in addition, the retention rate of whey active protein in infant formula milk powder obtained by the method of Example 1 is 89.95%, which is higher than 58.85% in Control Example 1. It can be seen that the active protein in milk will be significantly lost after being processed by conventional infant formula milk powder process.

[0102] Comparative Example 2 Timing of adding active whey rich in lactoferrin after aseptic concentration

[0103] The timing of adding active whey in step (6) of Example 1 is adjusted to the homogenization step in step (5), and the rest is the same as in Example 1 to obtain milk powder;

[0104] For specific process routes, see Figure 4 ;

[0105] The products obtained from Example 1, Comparative Example 1 and Comparative Example 2 were subjected to performance tests, and the test results are as follows:

[0106] Figure 5 Graph showing the contents of lactoferrin (a) and immunoglobulin IgG (b) in milk powder in Example 1, Comparative Example 1 and Comparative Example 2. Figure 5 It can be seen from (a) that the lactoferrin content in the milk powder of Control Example 1 is very low, the lactoferrin content in the milk powder of Control Example 2 is relatively low, less than 20 mg / 100 g, and the lactoferrin content in the milk powder of Example 1 is about 150 mg / 100 g. Figure 5 It can be seen from (b) that the immunoglobulin content in the milk powder of Control Example 1 was not detected, the immunoglobulin content in the milk powder of Control Example 2 was relatively small, less than 20 mg / 100 g, and the immunoglobulin content in the milk powder of Example 1 was about 250 mg / 100 g.

[0107] The results showed that the conventional processing method of infant formula milk powder could hardly retain lactoferrin, and immunoglobulins were completely lost, while the lactoferrin and immunoglobulin contents in the milk powder of Example 1 were relatively high, even significantly higher than those of the commonly fortified infant formula milk powder on the market.

[0108] The lactoferrin content and immunoglobulin IgG content in the infant formula milk powder obtained in Control Example 2 are significantly less than those in Example 1. This is because the active protein in the active whey is subjected to heat loss after the material killing step, and the activity is significantly reduced. The active whey must be added after the material killing step. It can be seen that the method of Example 1 can effectively increase the active protein content in the infant formula milk powder and achieve the improvement of the active protein content in the infant formula milk powder.

[0109] Control Example 3 Effect of different filtration solutions on the enrichment rate of active whey lactoferrin

[0110] The washing filtrate sodium chloride solution in step (2) of Example 1 was adjusted to deionized water, and the rest was kept the same as Example 1 to obtain a membrane filtration permeate.

[0111] Control Example 4 Effect of Lactoferrin Phase Separation Step on Lactoferrin Enrichment Rate in Active Whey

[0112] The lactoferrin phase separation step in step (1) of control example 3, i.e., the operation of adding sodium chloride, was omitted, and the rest was kept the same as control example 3 to obtain a membrane filtration permeate.

[0113] The membrane filtration permeate obtained in Example 1 and Comparative Examples 3 and 4 was subjected to performance tests, and the test results are as follows:

[0114] Figure 6The results of lactoferrin content and enrichment rate in the microfiltration permeate of Example 1 (a), Comparative Example 3 (b) and Comparative Example 4 (c) are shown. The bar graph shows the lactoferrin content and the line graph shows the cumulative enrichment rate of lactoferrin. Filter 1 represents the first microfiltration of skim milk, and Wash 1-4 represents the number of times the filtration was performed using the filtrate. The permeate was collected for each filtration to determine the ferritin content and calculate the enrichment rate. Figure 6 It can be seen that the lactoferrin content and corresponding enrichment rate of the filtration liquid obtained by the deionized water filtration in reference example 3 are very low. This may be because when the retentate is filtrationed with deionized water, the ionic strength (sodium chloride concentration) in the retentate decreases, causing lactoferrin to be unable to continue phase separation, and the lactoferrin content in the filtration liquid decreases rapidly. Therefore, sodium chloride solution needs to be used for filtration. The addition of sodium chloride for phase separation step is omitted in reference example 4, and its lactoferrin enrichment rate is only 17%; while the addition of sodium chloride for phase separation step in embodiment 1 can effectively improve the lactoferrin content and enrichment rate in the filtration liquid (whey) obtained by microfiltration, and the lactoferrin enrichment rate is increased from about 17% to about 40%. The lactoferrin phase separation step, i.e., adding sodium chloride to improve the ionic strength of the milk system, can effectively improve the lactoferrin content and enrichment rate in the microfiltration filtration liquid.

[0115] Control Example 5 Effect of microfiltration temperature on the enrichment rate of lactoferrin in active whey

[0116] The filtration temperature in step (2) of control example 4 was adjusted to 30°C, and the other conditions were kept the same as those of control example 4 to obtain a membrane permeate.

[0117] Comparative Example 6

[0118] The filtration temperature in step (2) of control example 4 was adjusted to 45°C, and the other conditions were kept the same as control example 4 to obtain a membrane permeate.

[0119] The membrane filtration permeate obtained in Reference Example 5 and Reference Example 6 was subjected to performance testing, and the test results are as follows:

[0120] Figure 7 The lactoferrin content and enrichment rate of the membrane permeate of Control Example 5 (a) and Control Example 6 (b) are as follows; Figure 7 The results of control example 4 Figure 6 As shown in (c), the lactoferrin enrichment rate in whey of control examples 5 and 6 is lower than that of control example 4. The microfiltration temperature will affect the content and enrichment rate of lactoferrin in the microfiltration permeate, i.e., the active whey. This may be because the temperature condition affects the charge attraction strength between lactoferrin and casein micelles in the milk system. Among the three temperature conditions of 30, 37 and 45°C, the lactoferrin enrichment rate at 37°C is the highest. Considering the conditions in actual operation, 37°C is selected for better results.

[0121] Comparative Example 7

[0122] The timing of adding sodium chloride is changed to adding it to casein, as follows:

[0123] (1) Preheating raw milk to 42° C., centrifuging the milk with a disc centrifuge to obtain cream and skim milk; then filtering the skim milk with a 1.4 μm microfiltration membrane at a temperature controlled at 40° C. to remove most of the microorganisms and impurities in the milk, thereby obtaining sterilized skim milk;

[0124] (2) filtering skim milk with a 0.1 μm microfiltration membrane to separate the whey protein phase from the casein phase, and obtaining a membrane filtration permeate and a membrane filtration retentate (casein); the filtration temperature is controlled at 37±2° C., the pH is 6.6, and the end point of the filtration operation is determined by “the liquid volume in the membrane filtration retentate becomes one-third of the initial volume”; the filtration is performed using a deionized water solution as a filtration solution, and after each filtration reaches the end point of the operation, the filtration solution is backfilled to the initial volume and re-filtered, and the process is repeated 4 times; and the permeate obtained from each filtration is collected;

[0125] (3) The membrane filtration retentate (casein) is further filtered using a 0.1 μm microfiltration membrane, and a sodium chloride solution containing 450 mmol is backfilled to the volume before step (2) to make a final sodium chloride concentration of 300 mmol, and the filtration is continued under this condition; subsequently, a 300 mmol sodium chloride solution is used as a second filtration solution; the end point of the filtration operation is determined by "the liquid volume in the membrane filtration retentate becomes one-third of the initial volume". After each filtration reaches the end point of the operation, the filtration solution is backfilled to the initial volume and filtered again, and the above filtration is repeated 4 times; and the permeate obtained from each filtration is collected.

[0126] The whey protein content in the skim milk in step (1) and the microfiltration permeate obtained in steps (2) and (3) is determined using an ELISA kit, and the whey protein enrichment rate is calculated.

[0127] Figure 8 The lactoferrin content and enrichment rate results of the membrane permeate of Control Example 7 are shown in the figure. The bar graph shows the lactoferrin content and the line graph shows the cumulative enrichment rate of lactoferrin. Filter 1 represents the first microfiltration of skim milk, washes 1-4 represent the number of times of filtration with deionized water, and salt washes 1-4 represent the number of times of filtration with sodium chloride solution. The permeate is collected for each filtration to determine the ferritin content and calculate the enrichment rate.

[0128] contrast Figure 8 and Figure 6 The results in (a) show that when the concentration of sodium chloride added is 300 mmol, the lactoferrin enrichment rate in the membrane permeate obtained after lactoferrin phase separation in casein in Control Example 7 is lower than that in Example 1, and the process is more complicated. Therefore, the timing of adding sodium chloride is to add sodium chloride to skim milk.

[0129] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing infant formula milk powder with highly active immune protein, characterized in that: The steps include: (1) adding sodium chloride to the sterilized skim milk to release lactoferrin, thereby obtaining skim milk after lactoferrin phase transfer; (2) filtering the skim milk obtained in step (1) after the lactoferrin phase transfer to separate the whey protein phase from the casein phase through a membrane filter to obtain a membrane filtration permeate (lactoferrin-rich whey protein) and a membrane filtration retentate (casein); wherein the wash filtrate used in the membrane filtration is a sodium chloride solution; (3) desalting and sterilizing the membrane filtration retentate obtained in step (2) to obtain a desalted and sterilized membrane filtration retentate; (4) ultrafiltration and desalination of the membrane filtration permeate obtained in step (2) to obtain aseptically concentrated active whey rich in lactoferrin; (5) using the desalted and sterilized membrane filtration retentate obtained in step (3) as a casein solution, adding the nutrients required in the infant formula to the casein solution, fully mixing, homogenizing, and sterilizing to obtain a casein solution containing ingredients; (6) Concentrating the casein solution containing ingredients obtained in step (5) to obtain a concentrate; then mixing the concentrate with the aseptically concentrated active whey rich in lactoferrin obtained in step (4), concentrating and spray-drying to obtain an infant formula milk powder with high active immune protein.

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

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

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

5. The method according to claim 1, characterized in that: In step (3), desalination is performed by filtering with a 0.05-0.15 μm microfiltration membrane, and deionized water is used as a secondary filtration solution; the amount of the secondary filtration solution used is 5-10 times the volume of the retentate, and the end point of the desalination operation is determined by "the conductivity value is reduced to the normal retentate conductivity range", and the protein concentration is determined by the Kjeldahl method to determine the total protein concentration in the retentate.

6. The method according to claim 1, characterized in that The ultrafiltration membrane used in step (4) has a molecular weight cutoff of 9-11 kDa, a concentration multiple of 5-10 times, and an ultrafiltration temperature not exceeding 45°C.

7. The method according to claim 1, characterized in that The spray drying in step (6) is low-temperature and low-pressure spray drying, specifically: the material inlet temperature is not higher than 80°C, and the material outlet temperature is adjusted to 52-55°C by flow regulation.

8. Infant formula milk powder containing highly active immune protein prepared by the method according to any one of claims 1 to 7.

9. Application of the infant formula milk powder containing the highly active immune protein according to claim 8 in the food field.

10. A method for increasing the active protein content in infant formula milk powder, characterized in that: Infant formula milk powder using the highly active immune protein described in claim 8.

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