阅读说明：本技术 特殊医学用途婴儿乳蛋白部分水解配方食品 (Infant milk protein partial hydrolysis formula food for special medical use ) 是由 徐庆利 刘宝华 杜玲玲 于 2021-07-22 设计创作，主要内容包括：本申请提供了一种特殊医学用途婴儿乳蛋白部分水解配方食品,其包括由碱性蛋白酶或胰蛋白酶水解乳清蛋白制备的水解乳清蛋白。本申请的配方食品在适度水解保持良好风味的同时有效降低的其β-乳球蛋白致敏性。(The present application provides a partially hydrolysed infant milk protein formula for particular medical use comprising hydrolysed whey protein prepared by hydrolysing whey protein with an alkaline protease or trypsin. The formula food of the application can effectively reduce the beta-lactoglobulin sensitization of the formula food while maintaining good flavor through moderate hydrolysis.)

1. Infant milk protein partially hydrolysed formula for special medical use, characterised in that it comprises hydrolysed whey protein; the hydrolyzed whey protein is prepared by hydrolyzing whey protein by alkaline protease or compound flavor protease.

2. The food product of claim 1, wherein the ingredients in the formula are: 69.9 percent of vegetable fat powder C, 24 percent of vegetable fat powder E, 2 percent of fructo-oligosaccharide, 1.1 percent of choline, 0.7 percent of potassium chloride, 0.5 percent of calcium hydrophosphate, 0.5 percent of mineral substance, 0.3 percent of inositol, 0.3 percent of tricalcium phosphate, 0.3 percent of vitamin, 0.2 percent of sodium chloride, 0.1 percent of calcium carbonate, 0.05 percent of L-carnitine and 0.05 percent of nucleotide; wherein the vegetable fat powder C is: 45% of lactose, 26% of hydrolyzed whey protein, 26.6% of compound vegetable oil, 1.4% of calcium hydrophosphate, 0.7% of mono-diglycerol fatty acid ester, 0.2% of L-sodium ascorbate, 0.033% of ascorbyl palmitate, 0.033% of phospholipid and 0.034% of mixed tocopherol concentrate; wherein the vegetable fat powder E is: 26.8% of solid starch sugar, 25% of anhydrous cream, 23% of hydrolyzed whey protein, 19.2% of maltodextrin, 5% of compound vegetable oil, 0.71% of mono-diglycerol fatty acid ester, 0.21% of L-sodium ascorbate, 0.03% of ascorbyl palmitate, 0.03% of phospholipid and 0.02% of mixed tocopherol concentrate.

3. A food product according to claim 1 or 2, wherein the hydrolyzed whey protein is prepared by a process comprising enzymatic hydrolysis of whey protein with an alkaline protease at an enzyme/substrate mass ratio of 7%.

4. A food product according to claim 3 wherein 0.1% by mass of whey protein is added during enzymatic hydrolysis of the whey protein.

5. The food product of claim 4, wherein the enzymatic conditions are: pH 8.5, temperature 50 ℃ for 4 hours.

6. A method for producing a hydrolyzed whey protein, characterized in that a whey protein is enzymatically hydrolyzed with an alkaline protease at an enzyme/substrate mass ratio of 7%.

7. A process according to claim 6 wherein 0.1% by weight of whey protein is added to the whey protein during enzymatic hydrolysis.

8. The method of claim 7, wherein the enzymatic conditions are: pH 8.5, temperature 50 ℃ for 4 hours.

9. Use of a hydrolysed whey protein prepared by a process according to any one of claims 6 to 8 in the preparation of a hypoallergenic food product.

10. Use according to claim 9, wherein the food product is an infant milk powder or a formula.

Technical Field

The present application belongs to the field of food products, in particular infant formula, and specifically provides an infant milk protein partial hydrolysis formula for special medical use, which comprises hydrolyzed whey protein prepared by hydrolyzing whey protein with alkaline protease or trypsin.

Background

Cow milk is a main food protein source for replacing breast milk of infants, but is one of the most common food allergens (the cow milk allergy rate of preschool children can reach about 10%, and the cow milk allergy rate of preschool children can still reach about 3% in the adult stage), and the cow milk product or the substitute formula food for preventing allergy is prepared, so that the cow milk product or the substitute formula food has great significance for the health of cow milk allergy people, especially infant cow milk allergy people. Beta-lactoglobulin accounts for about 50% of the total amount of whey protein and is the main functional active component of whey protein. More than 80% of people with cow milk allergy belong to the allergy to beta-lactoglobulin, and the probability of cow milk allergy occurrence can be effectively reduced by eliminating the beta-lactoglobulin or the allergen epitope thereof.

The existing method for eliminating beta-lactoglobulin or allergen epitope thereof mainly comprises heat processing, high-pressure treatment, fermentation, radiation treatment, enzymolysis treatment and the like. Because the problems of difficult overcoming of the actual effect and safety of the heat processing, the high pressure treatment, the fermentation and the radiation treatment exist, the enzymolysis treatment is still the mainstream method for producing the hypoallergenic dairy product/substitute formula food.

On one hand, the beta-lactoglobulin has the characteristic of resisting protease hydrolysis due to the compact space folding structure of the beta-lactoglobulin; on the other hand, increasing the degree of hydrolysis (e.g. extensively hydrolyzed milk powder/whey protein or casein) can reduce allergenicity, but is generally accompanied by an increase in cost and a decrease in flavor; thus, controlling the hydrolysis process/degree is a key step in the preparation of hypoallergenic dairy/alternative formulas, and it is necessary to investigate hydrolysis patterns that further reduce allergenicity at lower hydrolysis levels (meaning better mouthfeel and lower cost).

Disclosure of Invention

The applicant researches the types of enzymes used for hydrolysis, the enzymolysis conditions and the additives added in the enzymolysis, and finds that the alkaline protease added with a proper amount of sodium pyrophosphate can effectively reduce the allergenicity under a proper hydrolysis degree and flavor by enzymolysis of the whey protein, so that the flavor and the low allergenicity are well balanced, and a good raw material is provided for formula food or milk powder.

Infant milk protein partially hydrolysed formula for special medical use, characterised in that it comprises hydrolysed whey protein; the hydrolyzed whey protein is prepared by hydrolyzing whey protein by alkaline protease or compound flavor protease.

Further, the ingredients in the formula food are as follows: 69.9 percent of vegetable fat powder C, 24 percent of vegetable fat powder E, 2 percent of fructo-oligosaccharide, 1.1 percent of choline, 0.7 percent of potassium chloride, 0.5 percent of calcium hydrophosphate, 0.5 percent of mineral substance, 0.3 percent of inositol, 0.3 percent of tricalcium phosphate, 0.3 percent of vitamin, 0.2 percent of sodium chloride, 0.1 percent of calcium carbonate, 0.05 percent of L-carnitine and 0.05 percent of nucleotide;

wherein the vegetable fat powder C is: 45% of lactose, 26% of hydrolyzed whey protein, 26.6% of compound vegetable oil, 1.4% of calcium hydrophosphate, 0.7% of mono-diglycerol fatty acid ester, 0.2% of L-sodium ascorbate, 0.033% of ascorbyl palmitate, 0.033% of phospholipid and 0.034% of mixed tocopherol concentrate;

the vegetable fat powder E is: 26.8% of solid starch sugar, 25% of anhydrous cream, 23% of hydrolyzed whey protein, 19.2% of maltodextrin, 5% of compound vegetable oil, 0.71% of mono-diglycerol fatty acid ester, 0.21% of L-sodium ascorbate, 0.03% of ascorbyl palmitate, 0.03% of phospholipid and 0.02% of mixed tocopherol concentrate;

further, the hydrolyzed whey protein preparation method comprises enzymolysis of whey protein with alkaline protease at an enzyme/substrate mass ratio of 7%

Further, sodium pyrophosphate with the mass of 0.1% of whey protein is added in the process of carrying out enzymolysis on the whey protein.

Further, the enzymolysis conditions are as follows: pH 8.5, temperature 50 ℃, time 4 hours:

in another aspect, the present application provides a method for producing hydrolyzed whey protein, characterized in that the whey protein is enzymatically hydrolyzed with an alkaline protease at an enzyme/substrate mass ratio of 7%.

Further, sodium pyrophosphate with the mass of 0.1% of whey protein is added in the process of carrying out enzymolysis on the whey protein.

Further, the enzymolysis conditions are as follows: pH 8.5, temperature 50 ℃, time 4 hours;

in another aspect, the present application provides the use of the above hydrolyzed whey protein in the preparation of hypoallergenic food products.

Further, the food is infant milk powder or formula food.

Detailed Description

Primary reagents, instruments and methods

The whey protein is self-made by the applicant from an own milk source, and the same batch of products are used in the experimental process;

alkaline protease, complex flavourzyme: novacin;

papain: dong Heng Hua dao;

sodium pyrophosphate: sincere biotechnology limited;

KU812 (human peripheral blood basophilic leukemia cells): purchased from EK-bioscience;

beta-HEX ELISA detection kit: rui Xin biology;

liquid chromatography-mass spectrometry instrument: shimadzu LCMS-2020;

other reagents and ingredients are conventional domestic or imported varieties which meet the relevant regulations.

The enzymolysis method comprises the following steps:

preparing 10g/L whey protein solution with 5mM required PBS, adding required enzyme and sodium pyrophosphate, performing enzymolysis in a constant temperature water bath for a preset time (stirring at low speed), inactivating enzyme at 100 ℃ for 10 minutes, and then measuring or storing in a refrigerator freezing chamber for later use.

And (3) determining the degree of hydrolysis:

OPA (ortho-phthalaldehyde) method, in which 400. mu.l of a sample or a serine control solution (0.1g/L) was added to 3mLOPA reagent, reacted for 2 minutes, and then measured for absorbance at 340nm and calculated (detailed reagents and procedures refer to CHURCH F C et al, Spectrophotometric assay Using o-chromatographic for Determination of protein in Milk and Isolated Milk Proteins, Journal of Dairy Science, 1983, vol.66, stage 6);

in addition, the molecular weight distribution of the hydrolyzed whey protein fragments was determined by SDS-PAGE electrophoresis.

Determination of level of sensitized cells:

serum from patients diagnosed with allergy to bovine milk beta-lactoglobulin (specific IgE >0.8KUA/L) was obtained by a cooperative medical facility and the serum pool used for the test was a mixture of the sera of 25 patients.

Recovering and passaging the purchased cryopreserved KU812 cells; adding 2% cow milk beta-lactoglobulin into the culture medium to confirm serum pool serum (blank group is sterile buffer solution) of an allergic patient, and then incubating for 12 hours to enable cells to be in a sensitized state; adding a sample with the protein concentration of 10mg/mL (a negative control group is sterile buffer solution) with the volume of 1% of the culture medium, and incubating for 6 hours; after Triton X-100 lysis, β -HEX was detected using the above kit and the β -HEX release rate was calculated: β -HEX release (100%) × (sample group OD value-negative group OD value)/(positive group OD value-negative group OD value) × 100%.

HPLC-MS detection

Taking hydrolyzed whey protein, diluting properly and then injecting a sample; mobile phase 1: 0.1% aqueous formic acid, mobile phase 2: 0.1% formic acid acetonitrile solution, and the mobile phase 2 is 0-100% in 0-60 minutes; electrospray positive ion mode, collision voltage 28 ev; the PEAKS software analyzes the Swiss-Prot database.

Example 1 preparation of hydrolysed whey protein

Based on previous research experience and data of hydrolysis degree, time, bitterness value, flavor evaluation and the like in practical hydrolysis experiments (which are not shown here for reasons of space), three single-enzyme enzymolysis methods of alkaline protease, compound flavor protease and papain are selected from various available proteases and combinations thereof, and suitable enzymolysis conditions are further determined:

alkaline protease: 7% enzyme/substrate mass ratio, pH 8.5, temperature 50 ℃, time 4 hours;

compound flavor protease: 9% enzyme/substrate mass ratio, pH 7, temperature 50 ℃ for 4 hours;

papain: 6% enzyme/substrate mass ratio, pH 7, temperature 55 ℃, time 4 hours.

The following samples were prepared using the method described above:

unhydrolyzed whey protein: whey protein sample without any hydrolysis, heat treatment, pressure treatment, irradiation treatment;

hydrolyzed whey protein 1: carrying out enzymolysis by alkaline protease under the conditions;

hydrolyzed whey protein 2: carrying out enzymolysis on the compound flavor protease under the conditions;

hydrolyzed whey protein 3: carrying out enzymolysis on the papain under the conditions;

hydrolyzed whey protein 4: adding sodium pyrophosphate alkaline protease accounting for 0.1 percent of the weight of the whey protein under the above conditions for enzymolysis;

hydrolyzed whey protein 5: adding sodium pyrophosphate compound flavourzyme with the mass of 0.1 percent of whey protein under the above conditions for enzymolysis;

hydrolyzed whey protein 6: adding 0.1% of sodium pyrophosphate papain of the mass of the whey protein for enzymolysis under the conditions;

hydrolyzed whey protein 7: adding sodium pyrophosphate alkaline protease accounting for 0.05 percent of the weight of the whey protein under the above conditions for enzymolysis;

hydrolyzed whey protein 8: adding sodium pyrophosphate alkaline protease accounting for 0.5 percent of the weight of the whey protein under the above conditions for enzymolysis;

example 2 basic Properties of hydrolysed whey proteins

Determination of the degree of hydrolysis:

the hydrolysis of the hydrolyzed whey proteins 1 to 8, as measured by the OPA method and observed by SDS electrophoresis, is shown in the following table:

table 1 hydrolysis of various hydrolyzed whey proteins:

the result shows that the enzymolysis effect of the alkaline protease and the compound flavor protease is better than that of the papain, and the size of the hydrolyzed fragment is obviously different from that of deep hydrolysis (generally, the 3kDa fragment is more than 90%). The taste and flavor of the formula (specific formula is shown in the following examples) prepared by the hydrolyzed whey protein 4 are higher than those of the deeply hydrolyzed formula (multiple protease complex enzymes are hydrolyzed for a long time) prepared by the applicant and certain brand of deeply hydrolyzed milk powder sold in the market (specific scheme and data are not shown for reasons of space) according to the evaluation of scores, and the general understanding of the difference between the partially hydrolyzed milk powder and the deeply hydrolyzed milk powder is also met.

Example 3 sensitization assay and residual peptide short assay

The method is used for detecting the release rate of the beta-HEX of the unhydrolyzed whey protein, the hydrolyzed whey protein 1-8 and the commercially available deeply hydrolyzed milk powder, and the results are as follows:

TABLE 2 beta-HEX Release rates for various hydrolyzed whey proteins

Sample (I)	beta-HEX Release Rate (%)
		Unhydrolyzed whey protein	44.51
Hydrolyzed whey protein 1	29.22
		Hydrolyzed whey protein 2	28.75
Hydrolyzed whey protein 3	37.41
		Hydrolyzed whey protein 4	20.34
Hydrolyzed whey protein 5	27.69
		Hydrolyzed whey protein 6	36.71
Hydrolyzed whey protein 7	22.45
		Hydrolyzed whey protein 8	23.96
Commercially available milk powder with certain degree of deep hydrolysis	19.85

In combination with the results of example 2, it can be seen that the addition of a suitable amount of sodium pyrophosphate to complex the metal components in whey protein or to effect emulsification can change the protein conformation (presumably to be verified) so that certain sensitization key sites are exposed and then hydrolyzed by alkaline protease, probably because of the exposed sites, the combination of compound flavourzyme and sodium pyrophosphate cannot achieve the effect similar to that of alkaline protease. The release rate of beta-HEX of the hydrolyzed whey protein 4 is close to the level of certain deeply hydrolyzed milk powder sold on the market (after the milk powder is prepared according to experience, the sensitization is slightly increased by 2-3 percent, but the amplitude is not too large), but the hydrolyzed whey protein 4 is obviously superior to deeply hydrolyzed products in flavor and manufacturing cost.

Further detecting residual peptide segments in the hydrolyzed whey proteins 1 and 4 by the HPLC-MS method, and finding residual peptide segments of 1-9, 34-40, 42-60, 73-82, 93-101, 87-94 and 140-149 of the beta-lactoglobulin in the hydrolyzed whey protein 1, wherein the residual peptide segments respectively have corresponding allergic epitopes; whereas no 34-40 peptide fragment was detected in hydrolysed whey protein 4 (corresponding to IgE epitopes 21-40, Fritsche R. et al, IgE-mediated rat cell triggering with typing and synthetic peptides of bone beta-lactose, Int Arch Allergy Immunol, 2005, Vol. 138, No. 4).

Example 4 preparation of actual products of formula food

The hydrolyzed whey protein 4 is actually prepared into formula food, and the specific formula is as follows:

69.9 percent of vegetable fat powder C, 24 percent of vegetable fat powder E, 2 percent of fructo-oligosaccharide, 1.1 percent of choline, 0.7 percent of potassium chloride, 0.5 percent of calcium hydrophosphate, 0.5 percent of mineral substance, 0.3 percent of inositol, 0.3 percent of tricalcium phosphate, 0.3 percent of vitamin, 0.2 percent of sodium chloride, 0.1 percent of calcium carbonate, 0.05 percent of L-carnitine and 0.05 percent of nucleotide;

wherein the vegetable fat powder C is: 45% of lactose, 26% of hydrolyzed whey protein, 26.6% of compound vegetable oil, 1.4% of calcium hydrophosphate, 0.7% of mono-diglycerol fatty acid ester, 0.2% of L-sodium ascorbate, 0.033% of ascorbyl palmitate, 0.033% of phospholipid and 0.034% of mixed tocopherol concentrate;

the vegetable fat powder E is: 26.8% of solid starch sugar, 25% of anhydrous cream, 23% of hydrolyzed whey protein, 19.2% of maltodextrin, 5% of compound vegetable oil, 0.71% of mono-diglycerol fatty acid ester, 0.21% of L-sodium ascorbate, 0.03% of ascorbyl palmitate, 0.03% of phospholipid and 0.02% of mixed tocopherol concentrate

The minerals comprise copper sulfate, magnesium sulfate, ferrous sulfate, zinc sulfate, manganese sulfate, potassium iodate, and sodium selenite;

vitamins include retinyl acetate, cholecalciferol, dl-alpha-tocopherol acetate, menadione, thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, cyanocobalamin, nicotinamide, folic acid, D-calcium pantothenate, sodium L-ascorbate, and D-biotin;

the composite vegetable oil comprises sunflower seed oil, soybean oil, low erucic acid rapeseed oil, coconut oil and corn oil.

Through evaluation and trial, the formula food has good taste, effectively avoids the allergy of infants, and is superior to the existing partial hydrolysis products.