A composition suitable for use in infants and young children with milk protein allergy and a method for its preparation

Abstract

The present application relates to the technical field of infant nutrition, in particular to a composition suitable for infants with milk protein allergy and a preparation method thereof. The composition comprises modified deep hydrolyzed milk protein complex, carbohydrate, complex vegetable oil, 2'-fucosyllactose, complex vitamins, complex minerals, nucleotide complex and potassium citrate. The deep hydrolyzed milk protein complex is prepared by the following method: enzymolysis is performed on a mixture of whey protein and casein protein, ultrafiltration is performed to obtain a deep hydrolyzed milk protein solution, deamidation treatment is performed, bovine milk fat globule membrane polar lipid concentrate is added, and shearing, homogenization and drying are performed. The composition has the characteristics of comprehensive nutrition, suitable osmotic pressure and good brewing property, can promote intestinal mucosa repair and immune tolerance establishment, and is suitable for desensitization feeding and long-term nutritional support of infants with milk protein allergy.

Description

Technical Field

[0001] This invention relates to the field of infant nutrition food technology, specifically to a composition suitable for infants with milk protein allergies and its preparation method. Background Technology

[0002] Milk protein allergy is one of the most common food allergies in infants and young children. Statistics show that the global incidence rate among infants aged 0-3 years is 3%-8%, while in my country it is 2%-7.5%, and it has been increasing year by year in recent years. Because infants' intestinal barrier function is not yet fully developed and their immune system is immature, allergenic fragments such as β-lactoglobulin in whey protein and αS1-casein in casein can easily trigger an IgE-mediated immune response, manifesting as allergic symptoms such as skin rashes, vomiting, diarrhea, abdominal distension, and persistent crying. In severe cases, it can also affect the growth and development of infants and young children, and even lead to serious complications such as anaphylactic shock.

[0003] Currently, the mainstream clinical solution for infants with cow's milk protein allergy is extensively hydrolyzed milk protein formula. Its core principle is to use enzymatic hydrolysis to break down large milk protein molecules into smaller peptides and amino acids, reducing protein allergenicity while providing the protein nutrition necessary for infants' growth and development. However, existing extensively hydrolyzed milk protein medical foods still have many key deficiencies in their formulation design, failing to meet the special needs of long-term desensitization feeding for infants.

[0004] 1. Incomplete control of allergens: The extensively hydrolyzed milk proteins used in existing formulas are mostly prepared by single enzymatic hydrolysis, which cannot accurately cut allergenic peptides. This results in the residue of allergenic peptides with a molecular weight >1200Da, and the proportion of low molecular weight peptides (<1200Da) is difficult to reach more than 95%. The residual antigenicity is high, which can easily lead to repeated allergic symptoms and fail to achieve efficient desensitization.

[0005] 2. Imbalance between nutrition and desensitization: Some formulas, in pursuit of low allergenicity, excessively hydrolyze proteins, resulting in the destruction of essential amino acids for infants and young children, such as lysine and methionine, which cannot meet the complete nutritional needs for infant growth and development. At the same time, the fat system is not properly proportioned and lacks functional components suitable for infant intestinal absorption, resulting in low nutrient absorption efficiency.

[0006] 3. Slow establishment of intestinal immune tolerance: Existing formulas only focus on the single goal of "low allergenicity" without taking into account the characteristics of infant gut microbiota, and do not add targeted prebiotics or intestinal mucosal repair ingredients. They cannot regulate the structure of gut microbiota or strengthen the intestinal barrier function, resulting in slow establishment of immune tolerance to milk protein and an allergy recurrence rate of over 30%.

[0007] 4. Physicochemical properties are not suitable for the physiological characteristics of infants and young children: Some formulas have a high osmotic pressure (mostly 320-350 mOsm / L), which is significantly different from the osmotic pressure of breast milk (280-300 mOsm / L), which can easily cause gastrointestinal discomfort such as abdominal distension and diarrhea in infants and young children; at the same time, they have poor reconstitution properties and are prone to sticking to the bottle or clumping, which reduces the acceptance of infants and young children for feeding.

[0008] 5. Safety needs improvement: Some formulas add artificial flavors, colorings, preservatives or excessive thickeners to improve taste and stability, which do not meet the safety requirements for special medical foods for infants and young children. Long-term consumption may have potential effects on the health of infants and young children.

[0009] To address the shortcomings of the existing technologies, this invention develops a low-allergenic, nutritionally complete, gut-friendly, and physiologically appropriate deep-hydrolyzed milk protein special medical food by precisely compounding core protein sources, scientifically matching functional components, and optimizing physicochemical properties. The aim is to provide a better formula option for desensitizing infants with milk protein allergies. Summary of the Invention

[0010] In view of the shortcomings of the prior art, the present invention provides a composition suitable for infants with milk protein allergy and a method for preparing the same.

[0011] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0012] A composition suitable for infants with milk protein allergy comprises the following raw materials: 12-28 parts of modified extensively hydrolyzed milk protein complex, 15-40 parts of maltodextrin, 18-32 parts of compound vegetable oil, 0.5-1.5 parts of 2'-fucosylated lactose, 7-15 parts of pregelatinized starch, 0.2-0.6 parts of compound vitamins, 2-6 parts of compound minerals, 0.02-0.1 parts of nucleotide complex, and 0.02-0.2 parts of potassium citrate; the modified extensively hydrolyzed milk protein complex is prepared by a method including the following steps: enzymatic hydrolysis and ultrafiltration of a mixture of whey protein and casein to obtain extensively hydrolyzed milk protein solution;

[0013] The deeply hydrolyzed milk protein solution is subjected to deamide treatment to obtain a deamided deeply hydrolyzed milk protein solution;

[0014] Bovine fat globule membrane polar lipid concentrate was added to the deamidated deep hydrolyzed milk protein solution, and after shearing, homogenization and drying, a modified deep hydrolyzed milk protein complex was obtained.

[0015] Preferably, the mass ratio of whey protein to casein is (6-8):(4-2).

[0016] Preferably, in the enzymatic hydrolysis step, the enzymes used are trypsin and chymotrypsin, and the mass ratio of trypsin to chymotrypsin is 1:1, and the total amount of enzymes added is 0.5%-2% of the total mass of whey protein and casein; the ultrafiltration step uses an ultrafiltration membrane with a molecular weight cutoff of 1000-1500 Da.

[0017] Preferably, the deamidation treatment uses glutaminase, with an addition amount of 50-150 U / g protein, a reaction temperature of 40-50℃, and a reaction time of 30-60 min.

[0018] Preferably, the bovine fat globule membrane polar lipid concentrate is prepared by a method comprising the following steps:

[0019] Using sweet buttermilk as raw material, the mixture is treated by microfiltration and ultrafiltration, and the ultrafiltration residue is collected, concentrated and dried. The microfiltration uses a microfiltration membrane with a pore size of 0.1-0.5 μm, and the ultrafiltration process is carried out by washing with deionized water.

[0020] Preferably, the polar lipid concentrate of the milk fat globule membrane is prepared as follows: Q1, take sweet buttermilk, a by-product of milk butter production, as raw material, filter it through a 100-mesh sieve, pasteurize it at 70-85℃ for 10-20s, and then cool it to 45℃ for later use.

[0021] Q2. Centrifuge the pretreated buttermilk at 6000-10000g for 10-20 minutes at 2-6℃ to remove the upper free fat and the bottom sediment, and collect the intermediate liquid.

[0022] Q3. The intermediate solution is first microfiltered through a 0.1-0.5 μm microfiltration membrane, with the feed temperature controlled at 40-50℃, transmembrane pressure difference at 0.05-0.2 MPa, and volume concentration factor at 3-6 times, to enrich milk fat globule membrane fragments; then ultrafiltered, and washed with 2-4 times the volume of deionized water, with the ultrafiltration temperature controlled at 40-60℃ and transmembrane pressure difference at 0.1-0.4 MPa; to obtain the ultrafiltration retention solution;

[0023] Q4. Concentrate the ultrafiltration retention solution under vacuum to a solid content of 20-30 wt%, homogenize once at 15-25 MPa, and spray dry to obtain a concentrated product of polar lipids from milk fat globule membranes.

[0024] Maltodextrin: As a major source of carbohydrates, it provides sufficient energy for infants and young children. At the same time, it can regulate the osmotic pressure of the formula, improve the solubility and stability of the product, and avoid gastrointestinal discomfort in infants and young children caused by excessive osmotic pressure.

[0025] Compound vegetable oil: Utilizing a functional compound fat system tailored to the absorption characteristics of infants' intestines, it is specifically composed of 30%-40% OPO structured lipids, 20%-25% medium-chain triglycerides (MCT), and the remainder being a 1:1 blend of sunflower seed oil and palm oil. OPO structured lipids reduce calcium soap formation, promote calcium absorption, and alleviate constipation in infants; MCT has a small molecular weight, allowing for direct absorption by the intestines to provide energy, reducing the digestive burden on infants; sunflower seed oil and palm oil provide unsaturated fatty acids to meet the needs of infant growth and development.

[0026] 2'-Fucose-based lactose (2'-FL): It belongs to the prebiotics derived from human milk. It can specifically proliferate beneficial bacteria such as Bifidobacteria and Lactobacillus in the intestines of infants and young children, inhibit the growth of harmful bacteria, regulate the balance of intestinal microecology, promote the repair of intestinal mucosa, strengthen the intestinal barrier function, and moderately stimulate the body's immune system to produce a low-level response. It helps to regulate the intestinal microecology and improve the intestinal barrier function, thus benefiting the long-term feeding adaptation of infants with milk protein allergy.

[0027] Pregelatinized starch: It has good water solubility and thickening properties, which can significantly improve the product's reconstitution properties, allowing the product to dissolve quickly in warm water (around 37℃) without sticking to the wall or forming clumps, thus improving infants' and young children's acceptance of feeding; at the same time, it can help regulate the osmotic pressure of the formula and enhance product stability.

[0028] Multivitamins: Contain vitamins A, D3, E, K1, B1, B2, B6, B12, niacin, folic acid, pantothenic acid, C, biotin, etc. The content of each component strictly follows the requirements of GB 25596-2010, precisely matching the physiological needs of infants aged 0-3 years, providing comprehensive vitamin support for their growth and development, and avoiding problems such as growth retardation and decreased immunity caused by vitamin deficiency;

[0029] Complex minerals: Contains calcium, iron, zinc, copper, manganese, selenium, potassium, sodium, chlorine, phosphorus, etc. The content of each component meets the requirements of GB25596-2010. Among them, iron is made of ferric pyrophosphate and zinc is made of zinc gluconate. Compared with ordinary minerals, they are more easily absorbed by the intestines of infants and young children, which can effectively prevent iron deficiency anemia, zinc deficiency and other nutritional deficiencies in infants and young children, and meet the needs of infants and young children's bone, nerve and immune system development.

[0030] Nucleotide complex: composed of disodium 5'-cytidine, disodium 5'-uridine, disodium 5'-adenosine, disodium 5'-guanylate, and disodium 5'-inosine in a mass ratio of 2:2:1:1:1. It can repair intestinal mucosal damage, strengthen intestinal barrier function, regulate intestinal microecology in synergistic effect with prebiotics, and promote the development of infant immune system and enhance the body's resistance.

[0031] Potassium citrate: mainly used to adjust the pH of the formula to 6.5-7.0, which is compatible with the acid-base environment of the infant's gastrointestinal tract, reduces gastrointestinal irritation, and lowers the probability of discomfort symptoms such as bloating and diarrhea. It can also help regulate electrolyte balance.

[0032] The compound vegetable oil contains 30-40 wt% OPO structured lipids, 20-25 wt% medium-chain triglycerides, 15-25 wt% sunflower seed oil and 15-25 wt% palm oil, and the mass ratio of sunflower seed oil to palm oil is 0.8-1.2:1.

[0033] The complex vitamins contain vitamin A, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, pantothenic acid, vitamin C, and biotin; the complex minerals contain calcium carbonate, potassium dihydrogen phosphate, magnesium hydrogen phosphate, potassium chloride, sodium chloride, ferric pyrophosphate, zinc gluconate, copper gluconate, manganese gluconate, sodium selenite, and potassium iodide; the nucleotide complex contains disodium 5'-cytidine, disodium 5'-uridine, disodium 5'-adenosine, disodium 5'-guanylate, and disodium 5'-inosine.

[0034] A method for preparing the composition suitable for infants with milk protein allergy includes the following steps:

[0035] (1) The extensively hydrolyzed milk protein complex, maltodextrin, pregelatinized starch, complex minerals, nucleotide complex and potassium citrate are mixed with water to form an aqueous phase;

[0036] (2) Heat the compound vegetable oil to 50-60℃ to form an oil phase;

[0037] (3) Add the oil phase to the aqueous phase and emulsify;

[0038] (4) Homogenize the emulsion;

[0039] (5) The homogenized liquid is sterilized, concentrated, and spray-dried to obtain the base powder;

[0040] (6) The base powder is mixed with 2'-fucosylated lactose and compound vitamins to obtain the composition.

[0041] Preferably, in step (3), the emulsification speed is 4000-5000 r / min and the emulsification time is 5-15 min; in step (4), the homogenization pressure is 15-25 MPa; in step (5), the sterilization temperature is 80-90℃ and the sterilization time is 15-30 seconds; the inlet air temperature of the spray drying is 170-190℃ and the outlet air temperature is 80-85℃.

[0042] The inventors have discovered that while combining whey protein and casein for dual enzymatic hydrolysis followed by fractional ultrafiltration can reduce the molecular weight of large milk proteins and remove some high-molecular-weight sensitizing fragments to a certain extent, desensitization is primarily achieved through peptide chain cleavage and molecular weight sieving. Its ability to regulate the charge distribution, spatial conformation, and interfacial properties of residual small peptides remains limited. After simple enzymatic hydrolysis and ultrafiltration, residual peptides with some antigen recognition ability may still exist in the system, and the exposed hydrophobic groups of some small peptides can easily lead to increased bitterness, insufficient dispersion stability, and poor interfacial compatibility when subsequently combined with fat systems.

[0043] Based on this, after obtaining a deeply hydrolyzed milk protein solution through enzymatic hydrolysis and ultrafiltration, this invention further introduces glutaminase for deamidation treatment, and then combines it with a concentrated polar lipid from bovine fat globule membrane. A modified deeply hydrolyzed milk protein complex is constructed through shearing and homogenization. The inventors believe that deamidation treatment can transform some glutamine side chains, regulate the surface charge distribution and hydrophilic / hydrophobic balance of peptides, weaken the recognition conditions of some residual sensitizing epitopes, and improve the solubility, dispersibility, and system stability of the deeply hydrolyzed milk protein solution. Under shearing and homogenization conditions, the concentrated polar lipid from bovine fat globule membrane and the deamidated milk protein peptides form a more stable composite interface structure, which is beneficial for interfacial embedding and spatial shielding of residual hydrophobic fragments, thereby further reducing the probability of residual antigen exposure and improving compatibility with the composite vegetable oil system and the reconstitution stability after powdering.

[0044] Furthermore, based on the above-mentioned modified deep hydrolyzed milk protein complex, this invention further incorporates 2'-fucosylated lactose, a nucleotide complex, and a complex vegetable oil system containing OPO structured lipids and medium-chain triglycerides. The 2'-fucosylated lactose and nucleotide complex help regulate the intestinal microecology, promote intestinal mucosal repair, and enhance intestinal barrier function, while the complex fat system helps improve fat digestion and absorption efficiency and reduce the digestive burden on the gastrointestinal tract. As a result, the obtained composition is more suitable for long-term nutritional support and desensitization feeding for infants with milk protein allergies.

[0045] The beneficial effects of this invention are:

[0046] 1. This invention provides a composition suitable for infants with milk protein allergies and its preparation method. Firstly, whey protein and casein are compounded, subjected to double enzymatic hydrolysis and fractional ultrafiltration, and further modified by introducing glutaminase deamidation and bovine fat globule membrane polar lipid concentrate to prepare a modified deeply hydrolyzed milk protein complex. Compared with conventional deeply hydrolyzed milk protein prepared solely by enzymatic hydrolysis and ultrafiltration, this invention can further reduce the antigen exposure probability of residual sensitizing peptides, improve the charge distribution, hydrophobicity-affinity balance, and interfacial behavior of small molecule peptides, thereby improving system stability.

[0047] 2. Based on the aforementioned modified deep-hydrolyzed milk protein complex, this invention further incorporates a scientifically formulated system of compound vegetable oils, compound vitamins, compound minerals, and carbohydrates. This reduces the risk of allergies while ensuring nutritional integrity and efficient digestion and absorption. In particular, the introduction of OPO structured lipids and medium-chain triglycerides into the compound vegetable oils reduces calcium soap formation, promotes the absorption of fats and minerals, and alleviates the digestive burden on infants and young children. Simultaneously, the gentle protein modification approach helps retain essential amino acids and avoids nutritional loss due to excessive hydrolysis.

[0048] 3. This invention, by introducing 2'-fucosylated lactose and a nucleotide complex, further endows the composition with excellent intestinal microecological regulation and intestinal barrier repair capabilities. 2'-fucosylated lactose can specifically promote the proliferation of beneficial bacteria such as Bifidobacteria and Lactobacillus, while the nucleotide complex helps intestinal mucosal repair and barrier function enhancement. The synergistic effect of the two is beneficial to accelerate the establishment of intestinal immune tolerance in infants with milk protein allergies and reduce the risk of recurrent allergies. Detailed Implementation

[0049] The invention will be further described in detail below with reference to specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments.

[0050] The raw materials described in this application are partially described; all other raw materials not described are commercially available.

[0051] The maltodextrin was purchased from Shandong Quansheng Chemical Technology Co., Ltd., and is food grade.

[0052] 2'-Fucose-based lactose was purchased from Jiangxi Huihai Biotechnology Co., Ltd., and is food grade.

[0053] The pregelatinized starch was purchased from Yibo Chemical Co., Ltd. in Dacheng County; it was food grade.

[0054] The trypsin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: S141078.

[0055] The chymotrypsin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: C1501338.

[0056] Protein glutaminase was purchased from Zhengzhou Jiuting Chemical Products Co., Ltd., with an enzyme activity of 100,000 / g.

[0057] The commercially available reference product used was Nutricia extensively hydrolyzed milk powder, batch number: TY20235001.

[0058] Example 1

[0059] A composition suitable for infants with milk protein allergy, comprising the following ingredients in parts by weight: 20 parts by weight of modified extensively hydrolyzed milk protein complex, 25 parts by weight of maltodextrin, 24 parts by weight of compound vegetable oil, 0.9 parts by weight of 2'-fucosylated lactose, 10 parts by weight of pregelatinized starch, 0.4 parts by weight of compound vitamins, 3.5 parts by weight of compound minerals, 0.05 parts by weight of nucleotide complex, and 0.08 parts by weight of potassium citrate.

[0060] The compound vegetable oil contains 8.4 parts by weight of OPO structured lipids, 6.0 parts by weight of medium-chain triglycerides, 4.8 parts by weight of sunflower seed oil, and 4.8 parts by weight of palm oil.

[0061] The complex vitamin is composed of vitamin A, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, pantothenic acid, vitamin C, and biotin in a mass ratio of 520:8:12000:70:780:1260:490:3.5:3920:130:5240:178000:25.

[0062] The composite mineral is composed of calcium carbonate, potassium dihydrogen phosphate, magnesium hydrogen phosphate, potassium chloride, sodium chloride, ferric pyrophosphate, zinc gluconate, copper gluconate, manganese gluconate, sodium selenite, and potassium iodide, wherein the mass ratio of each mineral salt is 1070:960:220:390:440:17:29:2.4:1.8:0.05:0.20.

[0063] The nucleotide complex is composed of disodium 5'-cytidine, disodium 5'-uridine, disodium 5'-adenosine, disodium 5'-guanylate, and disodium 5'-inosine in a mass ratio of 2:2:1:1:1.

[0064] The preparation method of the modified deep-hydrolyzed milk protein complex is as follows:

[0065] S1. Mix whey protein and casein at a mass ratio of 7:3, add water at 55°C, and stir to prepare a 15wt% protein mixture. Adjust the pH to 7.4, and add an endonuclease at 45°C and 600 rpm for enzymatic hydrolysis. The endonuclease is trypsin and chymotrypsin, with a mass ratio of trypsin to chymotrypsin of 1:1. The total amount of enzyme added is 1wt% of the total mass of whey protein and casein, and the hydrolysis time is 2 hours. Inactivate the enzyme at 95°C for 15 minutes, and perform fractional ultrafiltration using a 1200 Da ultrafiltration membrane. Control the ultrafiltration pressure at 0.2 MPa and the ultrafiltration temperature at 25°C, and collect the permeate to obtain a deeply hydrolyzed milk protein solution.

[0066] S2. Adjust the pH of the above-mentioned deeply hydrolyzed milk protein solution to 6.7, add 100 U / g protein glutaminase, react at 45℃ for 40 min to inactivate the enzyme, and obtain deamidated deeply hydrolyzed milk protein solution.

[0067] S3. Add bovine fat globule membrane polar lipid concentrate to the above-mentioned deamidated deep hydrolyzed milk protein solution. The amount of bovine fat globule membrane polar lipid concentrate added is 2wt% of the total mass of whey protein and casein. Shear at 6000 rpm for 8 min at 55°C, then homogenize once at 20 MPa, cool and spray dry to obtain the modified deep hydrolyzed milk protein complex.

[0068] The preparation of the polar lipid concentrate of the milk fat globule membrane is as follows: Q1. Take sweet buttermilk, a by-product of milk butter production, as raw material, filter it through a 100-mesh sieve, pasteurize it at 75℃ for 15 seconds, and then cool it to 45℃ for later use.

[0069] Q2. Centrifuge the pretreated buttermilk at 8000g for 15min at 4℃ to remove the upper free fat and the bottom precipitate, and collect the intermediate liquid.

[0070] Q3. The intermediate solution is first microfiltered through a 0.2 μm pore size microfiltration membrane, and the microfiltration retentate is collected after microfiltration. The feed temperature is controlled at 45℃, the transmembrane pressure difference is 0.1 MPa, and the volume concentration factor is 4 times to enrich milk fat globule membrane fragments. Ultrafiltration is then performed, and the solution is washed with 3 times the volume of deionized water. The ultrafiltration temperature is controlled at 50℃ and the transmembrane pressure difference is 0.2 MPa to obtain the ultrafiltration retainer.

[0071] Q4. The ultrafiltration retention solution was concentrated under vacuum to a solid content of 25 wt%, homogenized once at 20 MPa, and spray-dried to obtain a concentrated concentrate of polar lipids from milk fat globule membrane.

[0072] The preparation method of the composition suitable for infants with milk protein allergy is as follows:

[0073] (1) Add the extensively hydrolyzed milk protein complex, maltodextrin, pregelatinized starch, complex minerals, nucleotide complex and potassium citrate to deionized water and stir to form an aqueous phase with a solid content of about 40%;

[0074] (2) The compound vegetable oil is heated to 55°C to form a homogeneous oil phase;

[0075] (3) Under stirring conditions, the oil phase is slowly added to the water phase and emulsified for 10 min at 4500 r / min using a high shear emulsifier;

[0076] (4) The emulsion is fed into a high-pressure homogenizer and homogenized once at 20 MPa;

[0077] (5) After homogenization, the liquid is sterilized at 85°C for 20 seconds, then concentrated to a solid content of 50%, and then spray dried with an inlet air temperature of 180°C and an outlet air temperature of 82°C to obtain the base powder.

[0078] (6) The base powder cooled to no higher than 30°C is added to a mixer along with 2'-fucosylated lactose, compound vitamins and other heat-sensitive components. The mixture is mixed at 1200 r / min for 10 min, passed through an 80-mesh sieve and then packaged with nitrogen to obtain a composition suitable for infants with milk protein allergy.

[0079] Example 2

[0080] The method is basically the same as that in Example 1, except that the amount of the deeply hydrolyzed milk protein complex added is 12 parts by weight, the amount of maltodextrin is increased to 33 parts by weight, and the composition, amount added and preparation method of the other raw materials are the same as those in Example 1.

[0081] Example 3

[0082] The method is basically the same as that in Example 1, except that the amount of the deeply hydrolyzed milk protein complex added is 28 parts by weight, the amount of maltodextrin is reduced to 17 parts by weight, and the composition, amount added and preparation method of the other raw materials are the same as those in Example 1.

[0083] Comparative Example 1

[0084] The method is basically the same as in Example 1, except that the preparation method of the deeply hydrolyzed milk protein complex is as follows: whey protein and casein are mixed at a mass ratio of 7:3, water at 55°C is added, and the mixture is stirred to prepare a 15wt% protein mixture; the pH is adjusted to 7.4, and an endonuclease is added at 45°C and 600 rpm for enzymatic hydrolysis; the endonuclease is trypsin and chymotrypsin, the mass ratio of trypsin to chymotrypsin is 1:1, the total amount of enzyme added is 1wt% of the total mass of whey protein and casein, and the hydrolysis time is 2h; the enzyme is inactivated at 95°C for 15min, and a 1200Da ultrafiltration membrane is used for fractional ultrafiltration treatment, the ultrafiltration pressure is controlled at 0.2MPa and the ultrafiltration temperature at 25°C, the permeate is collected, and the deeply hydrolyzed milk protein complex is obtained.

[0085] Comparative Example 2

[0086] The method is basically the same as in Example 1, except that the preparation method of the deeply hydrolyzed milk protein complex is as follows:

[0087] S1. Mix whey protein and casein at a mass ratio of 7:3, add water at 55°C, and stir to prepare a 15wt% protein mixture. Adjust the pH to 7.4, and add an endonuclease at 45°C and 600 rpm for enzymatic hydrolysis. The endonuclease is trypsin and chymotrypsin, with a mass ratio of trypsin to chymotrypsin of 1:1. The total amount of enzyme added is 1wt% of the total mass of whey protein and casein, and the hydrolysis time is 2 hours. Inactivate the enzyme at 95°C for 15 minutes, and perform fractional ultrafiltration using a 1200 Da ultrafiltration membrane. Control the ultrafiltration pressure at 0.2 MPa and the ultrafiltration temperature at 25°C, and collect the permeate to obtain a deeply hydrolyzed milk protein solution.

[0088] S2. Add bovine fat globule membrane polar lipid concentrate to the above-mentioned deeply hydrolyzed milk protein solution. The amount of bovine fat globule membrane polar lipid concentrate added is 2wt% of the total mass of whey protein and casein. Shear at 6000 rpm for 8 min at 55°C, then homogenize once at 20 MPa, cool and spray dry to obtain the modified deeply hydrolyzed milk protein complex.

[0089] The preparation method of the bovine fat globule membrane polar lipid concentrate is the same as that in Example 1.

[0090] Comparative Example 3

[0091] The method is basically the same as in Example 1, except that the preparation method of the deeply hydrolyzed milk protein complex is as follows: S1, whey protein and casein are mixed at a mass ratio of 7:3, water at 55°C is added, and the mixture is stirred to prepare a 15wt% protein mixture; the pH is adjusted to 7.4, and an endonuclease is added at 45°C and 600 rpm for enzymatic hydrolysis; the endonuclease is trypsin and chymotrypsin, the mass ratio of trypsin to chymotrypsin is 1:1, the total amount of enzyme added is 1wt% of the total mass of whey protein and casein, and the hydrolysis time is 2h; the enzyme is inactivated at 95°C for 15min, and a 1200Da ultrafiltration membrane is used for fractional ultrafiltration treatment, the ultrafiltration pressure is controlled at 0.2MPa and the ultrafiltration temperature at 25°C, and the permeate is collected to obtain the deeply hydrolyzed milk protein solution;

[0092] S2. Adjust the pH of the above extensively hydrolyzed milk protein solution to 6.7, add 100 U / g protein glutaminase, react at 45°C for 40 min to inactivate the enzyme, and obtain the extensively hydrolyzed milk protein complex.

[0093] Comparative Example 4

[0094] The composition is basically the same as that in Example 1, except that: no 2'-fucosylated lactose and nucleotide complex are added to the composition, and the remaining ingredients, amounts, and preparation methods are the same as in Example 1.

[0095] Comparative Example 5

[0096] The method is basically the same as in Example 1, except that: no OPO structured lipids and medium-chain triglycerides are added to the compound vegetable oil, and sunflower seed oil and palm oil are used directly in equal amounts to replace them. The compound vegetable oil contains 12 parts by weight of sunflower seed oil and 12 parts by weight of palm oil.

[0097] Test Example 1

[0098] Physicochemical property test: Weigh 10.00g of each sample and add it to 70.00g of 37℃ deionized water. Stir at 300rpm for 2min using a magnetic stirrer, and then let it stand in a 37℃ water bath for 5min to obtain the reconstitution solution.

[0099] The osmotic pressure of the reconstitution solution was measured using a freezing point osmoremeter; the pH value was measured using a pH meter calibrated with standard buffer solutions of pH 4.00 and pH 7.00; and the moisture content was determined by drying at 105℃ under normal pressure. The measurements were performed in triplicate and the average value was taken.

[0100] The preparation time is defined as the time required from when the sample is added to water until no obvious powder or clumps are visible to the naked eye and the foam on the liquid surface is basically stable. The evaluation criteria for wall adhesion / clumps are: none, meaning there are no obvious deposits on the container wall and the liquid is uniform; slight wall adhesion, meaning a small amount of deposits are visible on the container wall but can be dispersed by slight shaking; present, meaning there are obvious deposits on the container wall or visible clumps in the liquid.

[0101] To evaluate storage stability, each group of samples was packaged in nitrogen-filled aluminum foil composite bags, with the oxygen content inside the packaging controlled to be no higher than 3%. They were then sealed and stored at 25±2℃ and 60±5% relative humidity for 6 months. Appearance was observed at 0, 1, 2, 3, 4, 5, and 6 months, and moisture content, osmotic pressure, and reconstitution properties were measured at 0, 3, and 6 months. If the sample showed no obvious clumping or rancid odor during storage, and the osmotic pressure change did not exceed ±10 mOsm / L, and the reconstitution time change did not exceed 20%, the stability was considered good. If only slight clumping or a slight extension of the reconstitution time occurred, the stability was considered average.

[0102] Three samples were prepared independently for each group, and the test results are expressed as the average value. The results are shown in Table 1.

[0103] Table 1. Results of physicochemical property tests

[0104] Example 1 289 6.78 2.3 18 none good Example 2 284 6.71 2.5 20 none good Example 3 297 6.83 2.4 19 none good Comparative Example 1 308 6.62 2.8 28 Slightly sticking to the wall generally Comparative Example 2 302 6.66 2.7 25 Slightly sticking to the wall generally Comparative Example 3 300 6.68 2.6 24 Slightly sticking to the wall generally Comparative Example 4 292 6.75 2.4 19 none good Comparative Example 5 295 6.73 2.5 22 Slightly sticking to the wall good Commercially available reference materials 326 6.58 2.9 32 have generally

[0105] Test Example 2

[0106] In vitro nutritional digestion and absorption evaluation

[0107] (1) Protein digestibility: Samples from each group were prepared into a protein concentration of 10 mg / mL using equal amounts of protein. 10 mL of the sample solution was taken, pH adjusted to 2.0, and pepsin was added to a final concentration of 2000 U / mL. Digestion was carried out at 37℃ and 100 rpm for 2 h. Subsequently, pH was adjusted to 7.0 with 1 mol / L NaOH, and trypsin and bile salts were added to a final concentration of 100 U / mL and 10 mmol / L, respectively. Digestion continued at 37℃ and 100 rpm for 2 h. After digestion, the reaction was terminated by heating at 95℃ for 10 min. After cooling, the mixture was centrifuged (10000 rpm, 10 min). The supernatant was used to determine the soluble nitrogen content, and the total nitrogen content was determined using the Kjeldahl method. The protein digestibility was calculated using the following formula:

[0108] Protein digestibility (%) = soluble nitrogen content / total nitrogen content × 100%.

[0109] Three parallel samples were set up for each group of samples, and the test was repeated three times. The average value was taken. The results are shown in Table 2.

[0110] (2) Essential amino acid composition retention rate: Approximately 100 mg of each sample was accurately weighed, and 10 mL of 6 mol / L hydrochloric acid was added. After sealing under nitrogen, the mixture was hydrolyzed at 110℃ for 24 h. After cooling, the volume was adjusted, and the mixture was filtered. The contents of essential amino acids such as lysine, methionine, leucine, and valine were determined using an automatic amino acid analyzer. Based on the total amount of the same amino acids in the corresponding raw milk protein, the essential amino acid composition retention rate was calculated using the following formula:

[0111] Essential amino acid composition retention rate (%) = total essential amino acids in the sample / total essential amino acids in the raw milk protein × 100%. Each sample was measured independently three times, and the average value was taken. The results are shown in Table 2.

[0112] (3) Calcium transport rate of Caco-2 cells: Caco-2 cells were purchased from an authoritative cell bank, and cells of passages 20-40 were selected. They were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were then cultured at a rate of 1×10⁶ cells / year. 5 pcs / cm 2 The cells were seeded at a density of 0.4 μm in Transwell chambers, with the medium changed every 2 days, and cultured for 21 days to form a differentiated monolayer; the transepithelial resistance (TEER) value greater than 500 Ω·cm² was used as the criterion for monolayer formation.

[0113] After each group of samples underwent simulated gastrointestinal digestion as described above, the calcium ion concentration was adjusted to the same level. 0.5 mL of the solution was added to the upper chamber, and 1.5 mL of HBSS buffer was added to the lower chamber. The mixture was incubated at 37°C for 2 hours. After incubation, the lower chamber solution was collected, and the calcium content was determined using ICP-OES or atomic absorption spectrometry. The calcium transport rate of Caco-2 cells was calculated using the following formula:

[0114] Calcium transport rate of Caco-2 cells (%) = Calcium content detected in the lower chamber / Total calcium added to the upper chamber × 100%.

[0115] Three parallel holes were set in each group, and the experiment was repeated three times. The average value was taken, and the results are shown in Table 2.

[0116] Table 2 Evaluation of in vitro nutrient digestion and absorption

[0117] Example 1 96.8 98.7 43.8 Example 2 95.9 98.1 41.5 Example 3 95.6 98.0 40.9 Comparative Example 1 91.2 93.6 30.2 Comparative Example 2 92.1 94.8 32.0 Comparative Example 3 92.4 95.0 33.1 Comparative Example 4 94.3 96.2 40.6 Comparative Example 5 93.8 96.0 33.8 Commercially available reference materials 91.9 94.2 28.7

[0118] Test Example 3

[0119] Caco-2 cell scratch healing rate test: (1) Proliferation rate of Bifidobacterium and Lactobacillus: The test bacteria used were Bifidobacterium longum CICC 6195 and Lactobacillus rhamnosus CICC 6158. Bifidobacterium longum was inoculated into Clostridium longum culture medium or suitable Bifidobacterium culture medium, and Lactobacillus rhamnosus was inoculated into MRS culture medium. They were cultured at 37℃ to the logarithmic growth phase and the bacterial concentration was adjusted to about 1×10. 6 CFU / mL. The reconstituted solutions of each group of samples were added to the corresponding basal culture medium according to the set ratio to make the final concentration of the samples 10 mg / mL. After incubation at 37℃ for 24 h, the viable count was determined by plate counting method.

[0120] Using the blank culture medium group as a control, the proliferation rate of Bifidobacterium and Lactobacillus in each group of samples was calculated. The proliferation rate = (number of viable bacteria in the sample group - number of viable bacteria in the blank group) / number of viable bacteria in the blank group × 100%.

[0121] (2) Intestinal mucosal repair rate: A Caco-2 cell scratch repair model was used. Caco-2 cells were cultured into a monolayer and scratched with a sterile pipette tip. Supernatant from each group of samples after simulated digestion was added, and the cells were cultured for 24 hours. The scratch area was photographed and recorded at 0 hours and 24 hours. The scratch healing rate, i.e., the intestinal mucosal repair rate, was calculated using the following formula:

[0122] Intestinal mucosal repair rate (%) = (initial scratch area - scratch area after 24h) / initial scratch area × 100%, the results are shown in Table 3.

[0123] Table 3 Results of gut compatibility assessment

[0124] Example 1 48.6 45.3 92.4 Example 2 42.8 40.7 90.1 Example 3 43.2 41.0 90.5 Comparative Example 1 23.5 16.9 67.2 Comparative Example 2 25.7 18.4 70.3 Comparative Example 3 27.9 19.8 72.1 Comparative Example 4 11.2 10.5 61.4 Comparative Example 5 46.2 43.8 89.5 Commercially available reference materials 15.8 13.6 65.4

[0125] As shown in the table above, the composition prepared by this invention for infants with milk protein allergies has good physicochemical properties and digestibility, as well as good performance in promoting intestinal epithelial cell migration and barrier repair. Example 1 exhibits osmotic pressure, pH, reconstitution properties, and storage stability that are more suitable for infant feeding needs, with high protein digestibility, essential amino acid composition retention, and Caco-2 cell calcium transport rate. It also shows high proliferation rates of Bifidobacteria and Lactobacillus, and good intestinal mucosal repair effects. Examples 2 and 3 only adjusted the amount of extensively hydrolyzed milk protein complex added. Although their overall performance was slightly lower than Example 1, it was still significantly better than the comparative examples. This indicates that the present invention, through the synergistic effect of modified extensively hydrolyzed milk protein complex and functional components, can not only effectively ensure the low allergenicity and nutrient absorption performance of the formula, but also maintain good physicochemical properties and intestinal friendliness within a certain ratio range.

[0126] Comparative Example 1, which only used dual enzymatic hydrolysis and ultrafiltration to prepare a deeply hydrolyzed milk protein complex without further deamidation and blending with milk fat globule membrane polar lipid concentrate, showed significantly lower physicochemical properties, nutritional absorption performance, and intestinal regulation effects compared to Example 1. This indicates that while enzymatic hydrolysis and ultrafiltration alone can reduce some large protein molecules, they have limited control over the surface properties and interfacial behavior of residual small peptides, making it difficult to simultaneously achieve dispersion stability, absorption compatibility, and intestinal friendliness. Comparative Example 2 lacked the deamidation step, and Comparative Example 3 lacked the blending with milk fat globule membrane polar lipid concentrate. Although both were superior to Comparative Example 1 in various indicators, they were still inferior to Example 1. This indicates that both deamidation and blending with milk fat globule membrane polar lipid concentrate have a positive effect on improving system performance, and the effect is even better when they work synergistically. Among them, deamidation is more conducive to improving the hydrophilicity and digestibility of peptides, while milk fat globule membrane polar lipid concentrate is more conducive to improving the interfacial stability and reconstitution performance of the protein-fat system.

[0127] In Comparative Example 4, the removal of 2'-fucosylated lactose and the nucleotide complex did not significantly alter the physicochemical properties and basic nutritional indicators. However, the proliferation rates of Bifidobacteria and Lactobacillus, as well as the intestinal mucosal repair rate, decreased significantly, indicating that the 2'-fucosylated lactose and nucleotide complex are important components for promoting intestinal microecological regulation and intestinal barrier repair. In Comparative Example 5, replacing OPO structured lipids and medium-chain triglycerides with conventional vegetable oils resulted in intestinal flora proliferation and mucosal repair effects similar to those in Example 1. However, the calcium transport rate of Caco-2 cells decreased significantly, and the reconstitution properties deteriorated. This suggests that the complex fat system composed of OPO structured lipids and medium-chain triglycerides primarily helps improve the absorption efficiency of fats and minerals, improve system dispersibility, and reduce the digestive burden.

Claims

1. A composition suitable for use in infants allergic to milk proteins, characterized in that it comprises, The modified extensively hydrolyzed milk protein complex, by weight, comprises the following raw materials: 12-28 parts of modified extensively hydrolyzed milk protein complex, 15-40 parts of maltodextrin, 18-32 parts of compound vegetable oil, 0.5-1.5 parts of 2'-fucosylated lactose, 7-15 parts of pregelatinized starch, 0.2-0.6 parts of compound vitamins, 2-6 parts of compound minerals, 0.02-0.1 parts of nucleotide complex, and 0.02-0.2 parts of potassium citrate; the modified extensively hydrolyzed milk protein complex is prepared by a method comprising the following steps: A mixture of whey protein and casein is enzymatically hydrolyzed and ultrafiltered to obtain a deeply hydrolyzed milk protein solution. The deeply hydrolyzed milk protein solution is subjected to deamide treatment to obtain a deamided deeply hydrolyzed milk protein solution; Bovine fat globule membrane polar lipid concentrate was added to the deamidated deep hydrolyzed milk protein solution, and after shearing, homogenization and drying, a modified deep hydrolyzed milk protein complex was obtained. The amount of the bovine fat globule membrane polar lipid concentrate added is 0.5%-5% of the total mass of whey protein and casein.

2. The composition of claim 1, wherein, The mass ratio of whey protein to casein is (6-8):(4-2).

3. The composition of claim 1, wherein, In the enzymatic hydrolysis step, the enzymes used are trypsin and chymotrypsin, and the mass ratio of trypsin to chymotrypsin is 1:

1. The total amount of enzyme added is 0.5%-2% of the total mass of whey protein and casein. In the ultrafiltration step, an ultrafiltration membrane with a molecular weight cutoff of 1000-1500 Da is used.

4. The composition of claim 1, wherein, The deamidation treatment uses glutaminase at an addition amount of 50-150 U / g protein, a reaction temperature of 40-50℃, and a reaction time of 30-60 min.

5. The composition according to claim 1, characterized in that, The bovine fat globule membrane polar lipid concentrate is prepared by a method comprising the following steps: Using sweet buttermilk as raw material, the mixture is treated by microfiltration and ultrafiltration, and the ultrafiltration residue is collected, concentrated and dried. The microfiltration uses a microfiltration membrane with a pore size of 0.1-0.5 μm, and the ultrafiltration process is carried out by washing with deionized water.

6. The composition according to claim 1, characterized in that, The compound vegetable oil contains 30-40 wt% OPO structured lipids, 20-25 wt% medium-chain triglycerides, 15-25 wt% sunflower seed oil and 15-25 wt% palm oil, and the mass ratio of sunflower seed oil to palm oil is 0.8-1.2:

1.

7. The composition according to claim 1, characterized in that, The complex vitamins contain vitamin A, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, pantothenic acid, vitamin C, and biotin; the complex minerals contain calcium carbonate, potassium dihydrogen phosphate, magnesium hydrogen phosphate, potassium chloride, sodium chloride, ferric pyrophosphate, zinc gluconate, copper gluconate, manganese gluconate, sodium selenite, and potassium iodide; the nucleotide complex contains disodium 5'-cytidine, disodium 5'-uridine, disodium 5'-adenosine, disodium 5'-guanylate, and disodium 5'-inosine.

8. A method for preparing a composition suitable for infants with milk protein allergy as described in any one of claims 1-7, characterized in that, Includes the following steps: (1) The modified deep hydrolyzed milk protein complex, maltodextrin, pregelatinized starch, complex minerals, nucleotide complex and potassium citrate are mixed with water to form an aqueous phase; (2) Heat the compound vegetable oil to 50-60℃ to form an oil phase; (3) Add the oil phase to the aqueous phase and emulsify; (4) Homogenize the emulsion; (5) The homogenized liquid is sterilized, concentrated, and spray-dried to obtain the base powder; (6) The base powder is mixed with 2'-fucosylated lactose and compound vitamins to obtain the composition.

9. The method according to claim 8, characterized in that, In step (3), the emulsification speed is 4000-5000 r / min and the emulsification time is 5-15 min; in step (4), the homogenization pressure is 15-25 MPa; in step (5), the sterilization temperature is 80-90℃ and the sterilization time is 15-30 seconds; the inlet air temperature of the spray drying is 170-190℃ and the outlet air temperature is 80-85℃.