CN113519687B - Infant milk protein partial hydrolysis formula food for special medical use - Google Patents

Infant milk protein partial hydrolysis formula food for special medical use Download PDF

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CN113519687B
CN113519687B CN202110831539.1A CN202110831539A CN113519687B CN 113519687 B CN113519687 B CN 113519687B CN 202110831539 A CN202110831539 A CN 202110831539A CN 113519687 B CN113519687 B CN 113519687B
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whey protein
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alkaline protease
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CN113519687A (en
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徐庆利
刘宝华
杜玲玲
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Qingdao Shengtong Nutritional Food Co ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/04—Animal proteins
    • A23J3/08—Dairy proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/1522—Inorganic additives, e.g. minerals, trace elements; Chlorination or fluoridation of milk; Organic salts or complexes of metals other than natrium or kalium; Calcium enrichment of milk
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/1526—Amino acids; Peptides; Protein hydrolysates; Nucleic acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/156—Flavoured milk preparations ; Addition of fruits, vegetables, sugars, sugar alcohols or sweeteners
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
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    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
    • A23J3/343—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of dairy proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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Abstract

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.

Description

Infant milk protein partial hydrolysis formula food for special medical use
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's milk is the main food protein source for infant to replace breast milk, but it is one of the most common food allergens (cow's milk allergy rate of preschool children can reach about 10%, and it still has about 3% in adult stage), and the preparation of cow's milk products or substitute formula foods for preventing allergy is very significant for cow's milk allergy people, especially for the health of infant cow's 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 allergy to beta-lactoglobulin, and the probability of cow milk allergy occurrence can be effectively reduced by eliminating beta-lactoglobulin or allergen epitopes 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 thermal 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 products/substitute formula foods.
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 plant 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 whey protein enzymolysis process.
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 mass spectrometer: 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 (0.1 g/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 >0.8 KUA/L) was obtained by a cooperative medical facility and the serum pool used for the test was a mixture of the sera from 25 patients.
Recovering and passaging the purchased cryopreserved KU812 cells; adding 2% cow milk beta-lactoglobulin serum (blank group is sterile buffer solution) into the culture medium, and incubating for 12 hours to enable the 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, beta-HEX was detected using the above kit and the beta-HEX release rate was calculated: β -HEX release rate (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 28ev; 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 ℃ for 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 flavourzyme 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 in the mass of whey protein under the conditions for enzymolysis;
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 0.5% of sodium pyrophosphate alkaline protease in the amount of 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:
Figure BDA0003175619250000041
Figure BDA0003175619250000051
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(s) beta-HEX Release Rate (%)
Unhydrolyzed whey protein 44.51
Hydrolysed whey protein 1 29.22
Hydrolyzed whey protein 2 28.75
Hydrolyzed whey protein 3 37.41
Hydrolyzed whey protein 4 20.34
Hydrolysed 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 the beta-HEX of the hydrolyzed whey protein 4 is close to the level of certain deeply hydrolyzed milk powder sold in 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 map 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 sodium L-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.

Claims (4)

1. Infant milk protein partial hydrolysis formula for special medical use, characterized in that the formula comprises the following components: 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 comprises the following components: 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 plant fat powder E comprises the following components: 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 hydrolyzed whey protein is prepared by hydrolyzing whey protein with alkaline protease, and the preparation method of the hydrolyzed whey protein comprises adding alkaline protease with an enzyme/substrate mass ratio of 7% and sodium pyrophosphate with a whey protein mass ratio of 0.1% to carry out enzymolysis on the whey protein.
2. The food product of claim 1, wherein the enzymatic conditions during the preparation of the hydrolyzed whey protein are: pH 8.5, temperature 50 ℃ for 4 hours.
3. A preparation method of hydrolyzed whey protein is characterized in that alkaline protease with an enzyme/substrate mass ratio of 7% and sodium pyrophosphate with a whey protein mass ratio of 0.1% are added in the preparation method to carry out enzymolysis on the whey protein.
4. The method of claim 3, wherein the enzymatic conditions are: pH 8.5, temperature 50 ℃ for 4 hours.
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