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WO2021097543A1 - Procédé de production d'hydolysat de protéines lactées hypoallergénique ou non allergénique, hydrolysat de protéines lactées hypoallergénique ou non allergénique, produit alimentaire et utilisation de l'hydrolysat de protéines lactées hypoallergénique ou non allergénique - Google Patents

Procédé de production d'hydolysat de protéines lactées hypoallergénique ou non allergénique, hydrolysat de protéines lactées hypoallergénique ou non allergénique, produit alimentaire et utilisation de l'hydrolysat de protéines lactées hypoallergénique ou non allergénique Download PDF

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Publication number
WO2021097543A1
WO2021097543A1 PCT/BR2020/050337 BR2020050337W WO2021097543A1 WO 2021097543 A1 WO2021097543 A1 WO 2021097543A1 BR 2020050337 W BR2020050337 W BR 2020050337W WO 2021097543 A1 WO2021097543 A1 WO 2021097543A1
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process according
hypoallergenic
fact
hydrolysis
milk protein
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English (en)
Portuguese (pt)
Inventor
Jadyr MENDES DE OLIVEIRA
Eloisa Cristina CARMIGNOLA
Giovana SAPIENSA MORAIS DIZ
Jéssica SACCHI CASTILHO
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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 OR TREATMENT THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/20—Dietetic milk products not covered by groups A23C9/12 - A23C9/18
    • 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
    • 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
    • A23J3/347—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of proteins from microorganisms or unicellular algae

Definitions

  • the present invention relates to a process for the production of a hypoallergenic or non-allergenic milk protein hydrolyzate, as well as to a food product comprising the milk protein hydrolyzate obtained from the process of the present invention and its use for the manufacture of a hypoallergenic or non-allergenic infant formula.
  • Dairy protein hydrolysates are commonly used by the food industry in the manufacture of products such as infant formulas and food supplements. Especially in infant formulas used as a substitute or supplement to breast milk, milk protein hydrolysates must be hypoallergenic and non-allergenic when directed at children at risk of developing allergies.
  • cow's milk proteins are different from the proteins present in breast milk and may constitute allergens for humans.
  • the main recognized allergens in cow's milk are b-lactoglobulin (bLG), a-lactoglobulin (aLA) and bovine serum albumin (BSA).
  • bLG is used as a marker to point out this allergenicity.
  • Whey proteins and / or casein are generally used as protein sources in infant formulas. To reduce the allergenicide, these proteins are hydrolyzed by enzymes to be reduced to smaller size peptides.
  • the hypoallergenic and non-allergenic formulas currently on the market are composed of whey protein hydrolysates and aim to prevent allergies. They also include other ingredients, such as animal oils, vegetables, starch, malto- dextrin, lactose and sucrose. These protein hydrolysates can also be incorporated into dairy beverages for adults or food supplements to improve digestibility.
  • EP 2 436 389 describes a composition comprising a milk protein hydrolyzate obtained from the treatment of a solution of whey proteins, casein or a mixture of these with endo- peptidases similar to trypsin and chymotrypsin, but of micro bian origin.
  • the objective of the present invention is to provide a production process of an improved milk protein hydrolyzate, with fewer steps, faster and more economical, and whose final product has reduced allergenicity and low bitterness.
  • the process of the present invention provides a milk protein hydrolyzate from a fresh dairy substrate, preferably fluid, concentrated or reconstituted whole milk, using proteolytic enzymes.
  • the hydrolyzate thus obtained maintains all the amino acids and other essential nutrients available in milk.
  • the process of the present invention is energy-saving, and because it uses fresh dairy substrates as a starting material, it also has a lower cost compared to state-of-the-art processes, which makes the final product cheaper and economically viable for the consumer. , while maintaining adequate sensory characteristics.
  • a first objective is to provide a process for the production process of a hypoallergenic or non-allergenic milk protein hydrolyzate.
  • a second objective is to provide hydrolyzate of hypoallergenic or non-allergenic milk proteins.
  • the first objective is achieved from the description of a production process of a hypoallergenic or non-allergenic milk protein hydrolyzate comprising the steps of (i) hydrolysis of a dairy substrate with at least one neutral-alkaline proteolytic enzyme and (ii) hydrolysis of the mixture obtained in step (i) with at least one medium alkaline enzyme.
  • the second objective is achieved by the description of a hydrolyzate of hypoallergenic or non-allergenic milk proteins produced from the said process.
  • the third objective is achieved through the food product comprising the hydrolyzate of hypoallergenic or non-allergenic milk proteins produced from the said process.
  • the fourth objective concerns the use of the milk protein hydrolyzate obtained from the process described for the manufacture of an infant formula or hypoallergenic or non-allergenic food supplement.
  • Figure 1 shows the flow chart of a production process of a state-of-the-art milk protein hydrolyzate. The steps are defined as follows:
  • Figure 2 shows the flowchart of the process for producing a milk protein hydrolyzate of the present invention. The steps are defined as follows:
  • Figure 3 represents an average global impression of the test of acceptance between the product developed by Prozyn and the product produced according to the methodology indicated in the state of the art (EP 2 436 389).
  • Figure 4 represents the result of the comparative test of preference between the product developed by Prozyn and the product produced according to the methodology indicated in the state of the art (EP 2436389).
  • fluid milk means milk that has been sanitized, cooled and kept at 5 ° C, optionally subjected to thermalization (preheating), pasteurization and / or standardization (standardization) of the fatty matter , to be processed and that is not destined directly to the final consumer.
  • concentrated milk is understood to mean milk deprived of part of its water, pasteurized, standardized or not in relation to fat.
  • reconstituted milk means the product resulting from the dissolution of powdered or concentrated milk in water, with or without the addition of milk fat, until it reaches the content fixed for the respective type, homogenized or it is not pasteurized.
  • the present invention relates to a process for the production of a new hypoallergenic or non-allergenic milk protein hydrolyzate.
  • the hydrolysis of milk proteins occurs by the use of enzymes.
  • enzymes can be classified by the type of reaction they catalyze: oxidoreductases, transferases, hydrolases, lyases, isomerases, bonds and esterases.
  • Oxidoreductases are enzymes that catalyze redox reactions. Transferases act in group transfer reactions from one compound to another. Hydrolases act on the hydrolysis of various covalent bonds, while lyases modify the substrate, separating or removing groups from the substrate molecule. Isomerases catalyze isomerization reactions and ligases catalyze bonding reactions between two molecules, from the degradation of ATP molecules. Finally, esterases are involved in the hydrolysis of esters, producing acids and alcohol. [0036] Proteases belong to the class of hydrolases and subclass of peptide-hydrolases or peptidases, being classified as EC 3.4. [0037] Proteases can also act in a specific way, that is, hydrolyzing bonds between specific amino acids, or in a non-specific way, hydrolyzing any and all bonds in the peptide chain.
  • proteases are divided into two main groups according to their mechanism of action: exopeptidases (EC 3.4.11 -19) and endopeptidases (EC 3.4.21-99).
  • Exopeptidases act by the C- or N-terminal of the peptide chain.
  • the exopeptidases that act on the N-terminal region of the protein are called aminopeptidases, while those that act on the C-terminal region of the protein are called carboxypeptidases and are divided according to the nature of their catalytic site into serine-carboxypeptidases, metallo- car boxipeptidases and cysteine carboxypeptidases.
  • endopeptidases act on internal bonds of the peptide chain and are divided into four groups, according to the nature of their catalytic site, namely: serine proteases (EC 3.4.21), cysteine proteases (EC 3.4 .22), aspartic or carboxyl proteases (EC 3.4.23) and metalloproteases (EC 3.4.24).
  • proteases Another important classification of proteases occurs by defining the pH range of action. According to this criterion, proteases can be neutral, acidic or alkaline.
  • Acid proteases mainly include aspartic proteases, with better activity in the pH range between 2.0 and 6.0.
  • Neutral proteins which act at pH values between 6.0 and 8.0, are mainly represented by cysteine proteases, metallo proteases and some serine proteases, while alkaline proteases have better activity in the range pH 8.0 to 13.0 and are represented mainly by serine proteases.
  • Neutral-alkaline enzymes are those capable of acting with greater activity in a pH range that covers values of neutral enzymes and alkaline enzymes.
  • high alkaline enzymes for example, act in their optimum form in pH ranges closer to the limit of the alkaline range.
  • the enzymes used in the process of the present invention are proteases, a type of protein naturally found in living beings, catalyzing metabolic reactions essential for the proper functioning of the organism. Because of this, they can be extracted from several sources: animals, vegetables and microorganisms.
  • the main proteases extracted from animal tissues are pepsin, renin, trypsin and chymotrypsin. Already from plant sources, bromelain, papain and ficine are usually extracted. [0046]
  • the extraction of these enzymes from animal and plant sources requires a process with control of various parameters, such as climatic conditions, and, because of this, their availability is lower and the cost can be higher.
  • An alternative to circumvent these problems is the use of enzymes produced by microorganisms, which are obtained, in general, more quickly, simply and with low cost and higher yield.
  • the main source of neutral fungal proteases is Aspergillus oryzae, a filamentous fungus, known for its potential to produce several enzymes, such as proteases, amylases, beta-galactosidase, lipases, cellulase, glutaminase and metallo-peptidase .
  • the process of producing a new hypoallergenic or non-allergenic milk protein hydrolyzate comprises the steps of: hydrolysis of a dairy substrate with at least one neutral-alkaline proteolytic enzyme; and hydrolysis of the mixture obtained in step (i) with at least one proteolytic enzyme of medium alkalinity.
  • the dairy substrate used in the process of this invention is fluid, concentrated or reconstituted milk, preferably pasteurized whole milk.
  • said substrate is subjected to a double enzymatic hydrolysis, carried out by the addition of one or more neuro-alkaline proteolytic enzymes, and one or more medium alkaline proteolytic enzymes, which lead to the breakdown of milk proteins into peptides and amino acids and, thus, the reduction of allergenicity of the final product obtained.
  • the proteases used in the process of the present invention come from microorganisms, in particular, bacteria and fungi.
  • the proteases used in the process of the present invention are derived from Aspergillus melleus, Escherichia coli, Saccharomyces cerevisiae, Streptomyces lividans, Pseudomonas aeruginoza and Aspergillus oryzae.
  • At least one neutral-alkaline proteolytic enzyme from step (i) of the process is a non-specific serine protease that has an endo-exo mechanism of action.
  • said protease is derived from a microorganism selected from Aspergillus melleus, Escherichia coli, Saccharomyces cerevisiae, Streptomyces lividans and Pseudomonas aeruginasa.
  • the serine protease is derived from Asper gillus melleus.
  • At least one neutral-alkaline proteolytic enzyme from step (i) of the process of the present invention is selected from the group comprising subtilisin, trypsin, leucine-endopeptidase, glutamine-peptidase, among others.
  • step (i) of the process of the present invention is carried out at a pH of 3 to 10, preferably 4 to 8, more preferably 6 to 7 and more preferably still 6.5 to 7, where the enzyme activity of serine protease is optimal.
  • the hydrolysis of step (i) of the process of the present invention is carried out at a temperature between 30 ° C and 65 ° C, preferably between 35 ° C and 50 ° C and more preferably between 40 ° C and 45 ° C, where the enzyme activity of serine protease is optimal.
  • the neutralized proteolytic enzyme alkaline of the present invention is added at 9,000 U to 9,000,000 U per liter of reaction mixture, preferably 100,000 U to 6,000,000 U per liter of reaction mixture and more preferably 450,000 U to 3,000,000 U per liter of the reaction mixture reaction.
  • the enzyme activity can be evaluated using the Folin Method, carried out at pH 8.0, in which a unit of activity is defined as the amount of protease that produces amino acids equivalent to 1 pg of Tyrosine in 1 min, according to defined process conditions.
  • At least one medium-alkaline proteolytic enzyme from step (ii) of the process has an exo mechanism of action.
  • said proteolytic enzyme of medium alkalinity is an aminopeptidase derived from fungi of the genus Aspergillus, preferably derived from Aspergillus oryzae.
  • At least one medium alkaline proteolytic enzyme from step (ii) of the process of the present invention is selected from the group comprising glutamine-aminopeptidase, leucine-aminopeptidase, proline-aminopeptidase, among others.
  • step (ii) of the process of the present invention is carried out at pH 5 to 9, preferably 5.5 to 9, more preferably 6 to 8 and most preferably 6.5 to 7, in which the enzymatic activity of aminopeptidase is optimal.
  • step (ii) of the process of the present invention is carried out at a temperature of 30 ° C and 70 ° C, preferably between 35 ° C and 60 ° C and more preferably between 40 ° C and 45 ° C, where the enzymatic activity of aminopeptidase is optimal.
  • the aminopeptidase of the present invention is added at 10 U to 5,000 U per liter of reaction mixture, preferably 30 U to 2,400 U per liter of reaction mixture and more preferably 50 U to 1. 200 U per liter of the reaction mixture.
  • the total hydrolysis time, sum of the steps (i) and (ii) of the production process of a protein hydrolyzate in the hypoallergenic or non-allergenic dairy products of the present invention it is between 1 and 24 hours, preferably between 1 and 9 hours and more preferably between 1 and 7 hours.
  • the hydrolysis of step (i) of the present invention is between 0.5 and 10 hours, preferably between 0.5 and 6 hours and more preferably between 0.5 and 4 hours; while the hydrolysis of step (ii) of the present invention is between 0.5 and 14 hours, preferably between 0.5 and 9 hours and more preferably between 0.5 and 6.5 hours.
  • the enzyme activity can be assessed by hydrolysis of L-leucine-p-nitroaniline, carried out at pH 7.0 and 40 ° C, in which a unit of activity is defined as the amount of protease that produces the equivalent of 1pmol p-nitroaniline in 1 min, according to defined process conditions.
  • the present process comprises a phosphate addition step.
  • phosphate is added to the dairy substrate at 0.01% to 5% by weight, preferably 0.03% to 3% by weight and more preferably 0.05% to 1% by weight, in relation to the total weight of the reaction mixture.
  • the present process also comprises a drying step of the obtained milk protein hydrolyzate.
  • the drying step of the hydrolyzate is carried out with the use of a spray dryer.
  • the present process further comprises a step of mixing the hydrolyzate with syrup and other ingredients.
  • the present process also comprises a heat treatment stage for the inactivation of proteases, lactose hydrolysis with lactase, sterilization of the hydrolyzate, among others.
  • a hypoallergenic or non-allergenic milk protein hydrolyzate obtained from the process described above is also described.
  • said hydrolyzate has partially or extensively hydrolyzed dairy proteins.
  • a food product that comprises a hydrolyzate of milk proteins obtained from the process defined here, such as an infant formula or a food supplement.
  • this product also comprises carbohydrates, minerals, vitamins, fibers, prebiotics, among others.
  • the allergenicity of the dairy protein hydrolysates obtained from the process of the present invention was evaluated using the Free Amino Nitrogen (FAN - Free Amino Nitrogen) Determination method.
  • the FAN compounds are produced during proteolytic hydrolysis, a process in which the proteins that make up milk are cleaved into smaller peptides.
  • the applied enzymes work by breaking moderate to short peptide chains, promoting the production of ANA.
  • the method therefore, refers to the determination of nitrogen present in small amino acids and peptides.
  • the greater the number of hydrolyzed proteins the greater the number of peptides released and, consequently, the greater the product's ANA.
  • the milk protein hydrolysates produced according to Example 1 were diluted 500 times in deionized water and heated in the presence of a solution of Ninhydrin (5.0 g of disodium hydrogen phosphate dihydrate, 6.0 g of monobasic potassium phosphate, 0.5 g of ninhydrin, 0.3 g of fructose and 100 ml of deionized water ) at pH 6.6 to 6.8.
  • the dilution solution 2.0 g of potassium iodate, 400 ml_ of 95% v / v ethanol and 600 ml_ of water
  • the intensity of the color produced (purple) was measured at 570 nm, using a spectrophotometer.
  • FAN Free Amino Nitrogen (free amino nitrogen)
  • Comparative Example E corresponds to a commercial reference product (Danone's Pregomin Pepti) that falls into the category of extensively hydrolyzed product, where at least 95% of its proteins are reduced to peptides with a maximum size of 1000 Daltons.
  • Table 1 shows the averages of the results obtained considering the triplicates.
  • the allergenicity of the dairy protein hydrolysates obtained from the process of the present invention was also evaluated by the ELI SA Competitive test using the commercial kit RIDASCREEN® b-Lactoglobulin (Ref. R4901), according to the manufacturer's instructions.
  • the Competitive ELISA test consists of the quantification of this protein by means of an enzymatic immunoassay for the quantitative analysis of bLG in the samples of milk protein hydrolysates produced according to Example 1 and Comparative Examples E (Danone's Pregomin Punk), F (Danone's Aptamil) and G (Nestlé's NAN).
  • Comparative Examples F and G fall into the category of partially hydrolyzed products, where at least 60% of their proteins are reduced to peptides with a maximum size of 1000 Daltons.
  • sensory analysis is defined as a discipline used to evoke, measure, analyze and interpret the characteristics of food and materials as perceived by the senses: of sight, smell, taste , touch and hearing.
  • the panel of evaluators can be formed by specialists, trained or untrained people - changing only the necessary number of evaluators for each one.
  • the test site should be around 22 ° C, have individual booths, with white or neutral colored walls, with space to accommodate the evaluator and samples.
  • the lighting must be with natural light or natural fluorescent, where colored lamps can be used as a resource for mas- certain samples.
  • the place and the cabins must be isolated from noise and busy places. Always away from odors that may interfere with the analysis.
  • the preparation must be done before and away from the evaluators, and the evaluation time must be two hours before or after meals, as the presence or absence of appetite can interfere with the result (Monteiro, 1984; Teixeira et al, 1987; Moraes , 1988; Pedrero and Pangborn, 1989; Anzaldáua-Morales, 1994).
  • the tests performed were quantitative affective, which consist of the evaluator's subjective manifestation about the product tested. In order to verify the degree of satisfaction and overall acceptability of a product, identify relevant sensory attributes, the probability of acquiring the tested product and measure specific responses to specific sensory attributes. The minimum number of evaluators required for this type of test is 30, where untrained evaluators are allowed and even indicated (Teixeira et al, 1987; Moraes, 1988; Pedrero and Pangborn, 1989; Anzaldáua-Morales, 1994).
  • the acceptance test was carried out, which is used in order to know how much a product is accepted by consumers. Acceptance varies according to the evaluator's living standards and cultural background (Teixeira et al, 1987; Moraes, 1988).
  • the test was carried out in two stages, with 31 evaluators, the first stage was an acceptance test. It was carried out through the distribution of coded samples and the taster was asked to evaluate the sample according to the hedonic scale (7- I liked it a lot; 6- I liked it a lot; 5- I liked it moderately; 4- I neither liked it nor disliked it; 3 - disliked moderately; 2- disliked a lot; 1- disliked) for the attributes: Aroma, Appearance and Flavor. From these results an average global impression of these products was calculated, as shown in the graph in figure 3.
  • the second step was through a preference test, which consists of a direct comparison of a sample under development with an already consolidated product or when comparing a product with its competitors. Where the choice of a product is forced, with no indication of why the preference is. It was done through the paired comparison test, where within two coded samples, the taster is asked to evaluate them and indicate the sample of his preference. The results are shown in figure 4.

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Abstract

La présente invention concerne un procédé de production d'un hydrolysat de protéines lactées hypoallergénique ou non allergénique, ainsi qu'un hydrolysat de protéines lactées obtenu au moyen de ce procédé, un produit alimentaire comprenant cet hydrolysat de protéines lactées obtenu au moyen du procédé de la présente invention, et son utilisation pour la fabrication d'une formule pour enfant ou d'un complément alimentaire hypoallergénique ou non allergénique.
PCT/BR2020/050337 2019-11-22 2020-08-24 Procédé de production d'hydolysat de protéines lactées hypoallergénique ou non allergénique, hydrolysat de protéines lactées hypoallergénique ou non allergénique, produit alimentaire et utilisation de l'hydrolysat de protéines lactées hypoallergénique ou non allergénique Ceased WO2021097543A1 (fr)

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BR102019024709-6A BR102019024709A2 (pt) 2019-11-22 2019-11-22 processo de produção de hidrolisado de proteínas lácteas hipoalergênico ou não alergênico, hidroli-sado de proteínas lácteas hipoalergênico ou não alergênico, produto alimentício e uso do hidrolisado de proteínas lácteas hipoalergênico ou não alergênico
BRBR1020190247096 2019-11-22

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996013174A1 (fr) * 1994-10-26 1996-05-09 Novo Nordisk A/S Procede de fabrication d'un hydrolysat de lacto-proteine, hydrolysat de lacto-proteine et son mode d'utilisation
JP2001333794A (ja) * 2000-05-30 2001-12-04 Morinaga Milk Ind Co Ltd 蛋白質加水分解物の製造方法
WO2014060495A1 (fr) * 2012-10-19 2014-04-24 Dsm Ip Assets B.V. Procédé de production d'une préparation pour nourrissons
US20140134293A1 (en) * 2012-11-13 2014-05-15 Dmk Deutsches Milchkontor Gmbh Allergen-free food compositions
BRPI0721123A2 (pt) * 2006-12-20 2014-07-08 Danisco Hidrolisatos de proteína do leite com potencial imunogênico reduzido

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996013174A1 (fr) * 1994-10-26 1996-05-09 Novo Nordisk A/S Procede de fabrication d'un hydrolysat de lacto-proteine, hydrolysat de lacto-proteine et son mode d'utilisation
JP2001333794A (ja) * 2000-05-30 2001-12-04 Morinaga Milk Ind Co Ltd 蛋白質加水分解物の製造方法
BRPI0721123A2 (pt) * 2006-12-20 2014-07-08 Danisco Hidrolisatos de proteína do leite com potencial imunogênico reduzido
WO2014060495A1 (fr) * 2012-10-19 2014-04-24 Dsm Ip Assets B.V. Procédé de production d'une préparation pour nourrissons
US20140134293A1 (en) * 2012-11-13 2014-05-15 Dmk Deutsches Milchkontor Gmbh Allergen-free food compositions

Non-Patent Citations (3)

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Title
PACHECO, M. T. B. ET AL.: "Propriedades funcionais de hidrolisados obtidos a partir de concentrados proteicos de soro de leite", CIENCIA E TECNOLOGIA DE ALIMENTOS, CAMPINAS, vol. 25, no. 2, June 2005 (2005-06-01), pages 333 - 338, XP055826684 *
SOARES, R. D. L. ET AL.: "Perfil peptidico de hidrolisados enzimaticos de leite em pó desnatado", REV. BRAS. CIENC. FARM., SAO PAULO, vol. 40, no. 3, July 2004 (2004-07-01), XP055826686, DOI: https://doi.org/10.1590/S1516-93322004000300011 *
VILLAS BOAS ET AL.: "Alergia alimentar: uma abordagem sobre as proteinas lacteas e os principais tratamentos fisico-quimicos e enzimaticos aplicados para reduzir a antigenicidade: revisao da literatura", J HEALTH SCI INST., vol. 32, no. 3, 2014, pages 308 - 313, XP055826681 *

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