Technological possibilities of changing immunoreactive properties of cow
milk protein
Barbara Wróblewska, Lucjan Jędrychowski
Institute of Animal Reproduction and Food Research of Polish Academy of Sciences,
Department of Food Enzymes and Allergens,
Tuwima str 10, 10-747 Olsztyn, Poland
e-mail: basia@pan.olsztyn.pl
Introduction
Food allergy is today recognized as an immune-mediated state of hypersensitivity that
results from an exposure to an allergen, usually a protein of food origin, sometimes also
defined as type I allergy, an immunoglobulin E (IgE) – mediated immunologic reaction to
specific food [Hayakawa et al., 1999]. The development of food allergy and intolerance
depends of the following factors: genetic allergy predisposition, early ‘foregin’ food protein
exposure (time, dose, frequency), allergen uptake and handling. Adverse reaction to food,
mainly cow’s milk protein, are most common in the first years of life [Halken, 1997].
Breast feeding is undoubtfuly the ideal kind of nutrition for young infants. It is
generaly known that their properties can supports optimal growth of children and .enhances
immune functions as induces oral tolerance and enhances IgA production. Mother milk
usually is hypoallergenic and establishes bifidogenic gut flora. Preventive effect of breast
feeding on the development of atopic dermatitis may be enhanced by maternal avoidance of
potential food allergen (milk, egg, fish) during the lactation period [Halken, 1997].
When breastmilk is insufficient or lacking, a substitute formula is nedded.
Thermal processing
Thermal processes are the most common and popular as well as the oldest methods
for treatment of raw food materials. They are able to make food proteins more edible, by
improvement of intrinsic digestibility and to modify their functional properties. [Korhonen et
al., 1998]. Heat treatment causes Maillard’s reaction (non-enzymatic browning) as a result of
bound between lysine residues of proteins and carbohydrates
Heat denaturation can change antigenic and allergenic properties of the proteins.
Within the milk proteins - la and - lg are more sensitive to thermal treatment than casein
which is more stable. During immunological reaction the antibodies recognize and interact
with allergenic proteins through epitopes, coming from a few aminoacids, on the molecule.
There are two types of epitopes, linear and conformational [Davis, 1998]. Usually heating
destroys most conformational epitopes by unfolding native proteins, leaving only the linear
epitopes, which are is mainly involved into the allergenic problems. Together with an increase
in the severity of process conditions (time, temperature), the changes of immunoreactivity
properties were noticed. Thermal processes are necessary for preparing raw material (e.g.
milk) as a base to transform it into hypoallergenic food and should therefore be combined
with other processes like e.g. enzymatic hydrolysis.
Enzymatic modification
Tradition of protein enzymatic modification dates back to the 1940s. This process was
used for making functional properties of protein and for clinical nutrition. The bitter taste of
the obtained products was the main reason of small popularity of proteolysis as a
technological process. Nowadays enzymatic hydrolysis is an essential type of milk protein
treatment to produce hypoallergenic formulas in the industrial scale. The inquires into the
optimal baby formula for allergenic patients are still continued. Residual immunoreactivity
measured by the ELISA method of -la and -lg remaining after proteolytic reaction was
various. In our own studies the most effective was rennin, which reduced immunoreactivity of
-la to 3.99% after 1 hour of hydrolysis. The other enzymes did not react so quickly and after
1 h the results obtained for -la were higher, e.g. for Alcalase, trypsin and pepsin we noticed
98.59; 95.13; 75.81%, respectively. Hydrolysis continued for 24 h showed that alcalase could
reduce immunoreactivity of -la to 0.62% compared to the results reported for raw milk
[Jędrychowski et al., 2000]. 
According to Van Berenstein [1994], the minimum molecular weight required for
peptides obtained from whey to obtain a response was between 3,000 and 5,000 Da. Ena
[1995] showed in his study that peptides with molecular weight below 3,400 Da did not
provoke allergic reactions. The residual antigenicity depended mostly on the enzyme action
specificity and not on hydrolysis degree or molecular distribution of molecular weights of the
compounds formed. Van Hoeyveld et al. [1998] reported that peptides with molecular weight
above 2,600 Da provoke positive skin response and inhibit IgE binding. Peptides below
1,400 Da do not provoke positive skin response although they are still potent to partly inhibit
IgE binding with hydrolysate. The authors concluded that the minimum molecular weight of
antigen which can react with IgE in vitro is within the range of 970-1,400 Da. Such
observations are significant in the case of patients in whom IgE is a mediator. In the case of
infants, allergies are caused by T cells reaction to peptides.
In their publication on hypoallergenic formulas, Giampietro et al. [2001] stressed that
none of the so far prepared and available formulas for allergic infants is completely safe.
When recommending such formulas, the American Academy of Paediatrics should at the
same time be responsible for carrying out tests with double blind placebo controled food
challenge (DBPDFD) samples in which tolerance is confirmed in at least 90% of infants with
formerly diagnosed milk allergy. Such point of view is represented also by the European
organisations such as European Society for Paediaric Allergology and Clinical Immunology
(ESPACI), Committee on Hypoallergenic Formulas and the European Society for Pediatric
Gastroenterology, Hepatology and Nutrition (ESPGHAN), and Committee on Nutrition [Høst
et al., 1999].
Further studies on the presence of the remaining immunogenic epitopes in the so
called hypoallergenic protein hydrolysates are necessary.
Chemical modification
Chemical modifications such as: acetylation, succinylation and phosphorylation are
useful tools in changing functional properties of food. The ELISA method results of our
studies showed that acetylation and succinylation were a useful method for modification of
immunoreactive properties of whey allergen. Addition of acetic anhydride to milk protein
caused the reduction of -la and -lg immunoreactivity to 0.73% and 13.02%, respectively.
The same addition of succinyl anhydride was less effective, and residual immunoreactivity of
-la and -lg was 19.83 and 14.24 % respectively. Conjugation of whey proteins with
polyethylene glycol (PEG) were applied to reduce immunoreactive properties of -la and lg [Wróblewska & Jędrychowski, 2002]. Polyethylene glycol (PEG) is a non-toxic and nonimmunogenic compound. Currently, over 40 proteins have been modified with the use of
either polyethylene glycol or its derivatives. The conditions of conjugation reaction were
mild (4˚C) and any conformation changes within the protein molecule were not expected. The
reduction of the residual immunoreactivity of -la and -lg depended on the number of
polymer chains bound to the protein surface. It is suspected that the decrease of protein
immunoreactivity could have been caused by covering the epitope area i.e. places on the
protein surface that have the ability to bind antibodies of -la and -lg Protein modification
with PEG holds our hope for obtaining non-immunogenic and non-toxic compound forms for
people with allergies.
Modification of milk protein during lactic acid fermentation
The fermentation process involves natural and/or added microorganisms which during
their growth hydrolyse sugars and proteins available in their surrounding medium. During
such a process peptides with different amino acid sequencies and single amino acid are
formed [Korhonen, 1998]. The effect of microorganisms cultures on human organisms is well
documented. Lately a high interest concerning probiotics and their immunomodulative
properties has been noticed. In our experiment there were used about 400 different strains and
cultures of meso and thermophilic lactic acid bacteria. Generally the results showed that
residual immunoreactivity of -la and -lg was reduced sometimes by over 99% as compared
to raw milk. The PRICK test was carried out in Chair and Clinical of Allergy and Internal
Diseases at Bydgoszcz, Poland by interdermal injecting of 0.1 ml of whey samples of which
immunoreactivity was reduced the most for both estimated proteins. In vivo allergenicity test
allow to observe only slight attenuation of the immediate reaction to the provocation test.
Nevertheless fermented products have slightly sour taste and pleasant aroma, what is very
significant for potential consumers [Jędrychowski & Wróblewska, 1999].
Final remarks
Cow milk protein allergy is very important health problem, especially for small
children. The best solution in food allergy management is avoidance of potential allergens,
elimination diets and infant formula substitutes. There are some posibilities to decrease the
whey milk protein immunoreactivity (-la and -lg) by thermal processes, enzymatic
hydrolysis and chemical modifications. Conjugation of whey protiens with PEG seems to be
quite interesting solution. The most promising kind of modification is lactic acid
fermentation. The obtained products were characterized by good properties, nice aroma and
slightly acidic taste very important for potential consumers.
References
1. Davis P.J., Williams S.C., Protein modification by thermal processing. Allergy 1998: 53
Suppl 46: 102 – 105.
2. Ena J.M., van Berestaijn E.C.H., Robben A.J.P.M., Schmidt D.G.,: Whey protein
antigenicity by fungal proteinases and a pepsin/pancreatin combination. J. Food Sci. 1995,
60, 104-116.
3. Giampietro P.G., Kjellman N.I.M., Oldaeus G., Wouters-Wesseling W., Businco L.
Hypoallergenicity of an extensively hydrolyzed whey formula. Pediatric Allergy and
Immunology, 2001,12 (2): 83-86.
4. Halken S., Prevention of food allergy. Environmental Toxicology and Pharmacology 4
(1997), 149-156.
5. Hayakawa K., Linko Y.-Y., Linko P., Mechanism and Control of Food Allergy.
Lebensm.-Wiss. u.-Technol., 1999, 32, 1-11.
6. Høst A., Koletzko B., Dreborg S., Muraro A., Wahn U., Aget P., Breson J.-L., Hernel O.,
Lafeber H., Michaelsen K.F., Micheli J.-L,. Rigo J., Weaver L., Heymans H., Strobel S.,
Vandenplas Y.: Dietary products used in infants for treatment and prevention of food
allergy. Joint statement of the European Society for Paediaric Allergology and Clinical
Immunology (ESPACI) Committee on Hypoallergenic Formulas and the European
Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN)
Committee on Nutrition. Arch. Dis. Child, 1999, 81, 80-84.
7. Jędrychowski L., Wróblewska B., Mierzejewska D., Changes in the immunoreactive
properties of cow milk proteins upon enzymatic modification. Pol. J. Food Nutr. Sci.
2000, Vol. 9/50, No 3S, 23-31.
8. Jędrychowski L., Wróblewska B., Reduction of antigenicity of whey proteins by lactic
acid fermentation. Food and Agricultural Immunology 1999, 11, 91-99.
9. Korhonen H., Pihlanto – Leppälä A., Rantamäki P., Tupasella T., Impact of processing on
bioactive proteins and peptides. Trends in Food Science & Technology 1998, 9, 307–319.
10. van Beresteijn E.C., Peeters R.A., Kaper J., Meijer R., Robben A., Schmidt D.: Molecular
mass distribution, immunological properties and nutritive value of whey protein
hydrolysates. J. Food Protect., 1994, 57, 619-62.
11. van Hoeyveld E.M, Escalona-Monge M., de Swert L.F.A, Stevens E.A.M.: Allergenic
and antigenic activity of peptide fragment in whey hydrolysate formula. Clin. Exp.
Allergy, 1998, 28: 1131-1137.
12. Wróblewska B., Jędrychowski L., Effect of conjugation of cow milk whey protein with
polyethylene glycol on changes in their immunorective and allergic properties. Food and
Agricultural Immunol. 2002, 14, 157 – 164.