Extensively Hydrolysed Protein Hypoallergenic Infant Formula for the nutritional treatment of infants with CMA

Author: Angel Gil, Full Professor Department of Biochemistry and Molecular Biology, Institute of Nutrition and Food Technology, Centre of Biomedical Research, University of Granada, Granada, Spain

Cow’s milk protein allergy

Food allergies have dramatically increased over the last decades, those mediated by IgE affecting about 4-8% of children, namely infants and toddlers.  Among food allergies, cow’s milk protein allergy (CMPA) is one of the most frequent food allergies, as milk and dairy products are usual components of the diet.  CMPA is defined by the occurrence of clinical symptoms related to the abnormal immune response of the host after ingestion of these CM proteins. CMPA seems to peak in the first year of life, with a prevalence of 2% - 3% in the infant population. Most infants with CMPA develop symptoms in the first year of life and approximately 85% become clinically tolerant by the third year of life. Adverse reactions to cow’s milk protein include true allergy (CMPA) and intolerance (CMPI). The former is mainly mediated by IgE response whereas the latter is largely due to depending-cell immune responses.  CMPA symptoms usually appear immediately or within two hours after the intake, while in non-IgE-mediated CMPA, symptoms may take a long time to appear (48 h to even 1 week following ingestion). Both usually lead to cutaneous (atopic dermatitis, erythema and angioedema), respiratory (sibilance, bronchospasm, rhinitis, coughing and asthma) and gastrointestinal (vomiting, diarrhoea, colic, constipation and gastroesophagic reflux) symptoms. The treatment for CMPA in infants involves the complete elimination of CMP from the diet and adequate replacement with a hypoallergenic formula.

Extensively hydrolysed milk formulas for the treatment of cow’s milk protein allergy in infancy and childhood

Human breast milk feeding is the gold standard for infants at least from birth up to six months. Only in cases where breast-feeding is not possible, an infant formula specially designed for the nutritional treatment of infants can be used. However, artificial formulas based on cow's milk proteins are widely used, mainly at weaning, despite the fact that early introduction of formula can lead to an allergic reaction against those proteins as well as other foods, especially in infants with a history of familial atopy. Hence, alternative formulas have been developed for babies with proven or suspected CMPA. Although formulas based on soybean proteins and goat and other ruminant milks are among such alternatives, they can produce several gastrointestinal reactions and even characteristic CMPA symptoms.  Extensively hydrolysed protein formulas (eHF) which are less antigenic should be used for infants at risk, CMPA infants and those showing clinical signs of intolerance to milk dietary proteins.

The only means of obtaining true hypoallergenic formulas is to sufficiently modify the structure of the native protein in such a way that the quantity of antigenic material is drastically reduced. This can be achieved by heat treatment or hydrolysis. However, heat treatment by itself is unable to adequately reduce the potential antigenic material and may produce new epitopes that are not present in the native protein. The enzymatic hydrolysis of protein is the best way to reduce its antigenicity. Acid or alkaline hydrolysis induces severe amino acid degradation, decreasing nutritional value. On the other hand, enzymatic proteolysis by selected endo- and exopeptidases is gentler, preserving the nutritional qualities of the amino acids and peptides produced. The drawbacks to enzymatic hydrolysis are the production of a large amount of free amino acids, a bitter taste and an increase in the formula osmolarity; nevertheless, these problems can be overcome by means of an adequate control of the factors involved in the processing (type and amount of enzymes, temperature of hydrolysis, pH, etc.).

Protein hydrolysates included in hypoallergenic formulas for infants with CMPA should have the following characteristics: a high content of di- and tripeptides, which are quickly absorbed by the gut as peptides, relatively small free amino acid content, a limited bitterness, a high nutritional value and, of course, a very low antigenicity. Although it is very difficult to set an upper limit for the size of residual peptides below which hypoantigenicity could be guaranteed, peptides of 2-10 amino acids will probably have a highly reduced residual antigenicity and thus be potentially hypoallergenic. To ensure the hypoallergenicity of the protein hydrolysates used in formulas, it is necessary to check that the immunoreactive protein concentration is less than 1/105- 1/106 of the protein content normally present in the starting materials. It is also necessary to ensure, using animal models, that the oral administration of the formula does not induce sensitization to major milk proteins, to other proteins present in the starting materials used or to residual peptides, including anaphylactic shock or the development of antibodies detectable by passive cutaneous anaphylaxis tests.

A new extensively casein and whey milk protein hydrolysed formula with proven safety, tolerance and efficacy for the treatment of infants with cow’s milk allergy

We have recently reported the results of a study in which a new eHF was designed and clinically evaluated (Matencio E., Maldonado J., Olza J., Mesa M.D., Romero F., Ros G., Abellan P., Gil A. (2016) A Hypoallergenic Infant Formula Comprising Extensively Hydrolysed Protein for the Nutritional Treatment of Infants with Cow’s Milk Allergy: Safety, Tolerance and Efficacy. J Hum Nutr Food Sci 4(3): 1090). The new eHF is based on a mixture of casein and whey protein hydrolysates to mimic the amino acid composition of human milk. One additional reason for that design was to decrease the amount of peptides from casein protein, which are known for their strong bitter taste. The eHF also contained, in addition to the extensively hydrolysates as protein source, lactose and maltodextrin as carbohydrate sources, vegetable fats and added vitamins, minerals, docosahexaenoic acid (DHA) and arachidonic acid (AA) and nucleotides to mimic human milk composition.

The quality of the protein was determined by the Thomas-Mitchell technique and the indices employed for estimating the protein quality were true digestibility, net protein utilisation (NPU), biological value (BV) and protein efficiency ratio (PER). The protein hydrolysates had a BV higher than 73% and a PER of about 3.6, which indicates their adequacy for supporting adequate growth and development of the infant.

The low antigenicity of this eHF was validated by means of in vitro and in vivo studies and subsequently in a clinical trial. The molecular distribution of peptides was assessed by high-performance liquid chromatography and the potential antigenicity of the native proteins (whey protein and casein) and their hydrolysates was measured by a competitive inhibition ELISA. 

The tested formula based on extensively hydrolysed casein (20%) and extensively hydrolysed whey proteins (80%), exhibited a molecular weight distribution in which 26% of the peptides had between 1000 to 3000 Da and 64% had a molecular weight of less than 1000 Da. The mean molecular weight was 989 Da (peptides containing on average 7 amino acids) and the highest was less than 2150 Da (peptides lower than 15 amino acids).

In addition, in vitro studies and an Active Systemic Anaphylaxis Test (ASA test) were used to determine the antigenicity of the new eHF, and a prospective clinical trial was performed to evaluate the safety and tolerance of this formula for use in infants with proven CMPA. The safety and clinical tolerance was evaluated by means of an oral food challenge provocation test (OFC).

The clinical trial was a prospective, open, multicentre study conducted in different hospitals in Spain [(Hospital Virgen de las Nieves (Granada), Hospital Vall D’Hebrón (Barcelona); Hospital Infanta Sofía and Hospital Gregorio Marañón (Madrid)]. This study was performed according to Good Clinical Practice guidelines and conforms to the Helsinki Declaration. The inclusion criteria were: Infants diagnosed with CMPA based on clinical history of IgE-mediated CMPA, acute cutaneous symptoms (erythema, angioedema), which seemed to be related to the intake of cow’s milk, a positive test in IgE antibodies specific to cow’s milk and or milk protein (skin prick test (SPT) and/or measure-specific IgE antibodies) and an OFC test, except when the challenge was contraindicated in accordance with current paediatric allergy guidelines. Forty seven infants (26 boys, 21 girls; age 4.6 ± 1.8 months (mean ± SD), who had been previously diagnosed with CMPA, were selected for the study. Follow up visits were carried out 1 month, 2 months and 3 months after enrolment. At each visit, the paediatricians drew up a general clinical history and evaluated the tolerance to the study formula and any potential adverse events.

At the baseline, CMPA was confirmed in all the patients according to the diagnosis criteria. 79% of patients had a positive skin prick test result to some of the milk proteins. An OFC test with CMP was carried out in 37 infants (79%), while ten (21%) were not presented with cow’s milk because provocation was contraindicated. Approximately 81% of the infants showed cutaneous eruption, 30% tract symptoms such as vomiting, 21% pruritus, 17% erythema and 11% angioedema, whereas anaphylaxis, abdominal pain, colic or thrive failure were less common (2%). 

The new eHF was tolerated by 46 of the 47 infants, a tolerance level of 98% (95% CI: 94-100%). This means that more than 90% of the infants tolerated the formula, thus complying with the requirements established by various scientific bodies such as AAP, ESPGHAN and ESPACI. In addition, at the end of the follow up, no serious adverse event related to the study formula had been recorded.

Observational and interventional studies have shown that infants with allergies usually suffer impaired growth compared to healthy infants, so that hypoallergenic formulas should ensure normal development. In this regard, the new eHF led to a positive z-score of weight for age close to the median of healthy infants, meaning there was no risk of under nutrition.

In conclusion, the new designed eHF was well tolerated by infants with CMPA. According to the results obtained in the safety, tolerance and nutritional validation, this eHF formula complies with the requirements established by the scientific bodies (AAP, ESPGHAN) and it is useful for the nutritional treatment of infants with proven CMPA.