Study of human allergic milk whey protein from different mammalian species using computational method

Nowadays, safety and quality assessment of food used for human consumption have to consider by its possible contribution to the maintenance or improvement of the consumer's health. Milk is an important food with many nutrients. Cow milk is an important source of energy, protein, vitamins and minerals for the growing child as well as adults. But, numerous cow milk proteins have been implicated in allergic responses and most of these have been shown to contain multiple allergic epitopes. The present study disclosed best alternatives to cow milk, which are not allergic and as good as cow milk in nutritional value. The in silico analysis of casein (alpha s1, alpha s2, beta and kappa) and beta-lactoglobulin, unveils that sheep milk is a more suitable alternate to cow milk for allergic infants and buffalo milk for allergic adult humans.

lactoferrin as well as 4 caseins, have been identified as allergens [10]. The casein fraction is composed of alpha s1, alpha s2, beta and kappa-casein, of which alpha-s1 casein seems to be a major allergen according to .
In developed countries there is increasing interest in goat milk and its derivatives, the quality of which is considered of special importance in the light of current tendencies favoring healthy eating. In particular, the composition of goat milk is said to have certain advantages over that of cow milk, and thus former is preferable for some consumers [15]. In one case Scientific and clinical studies also suggest that infants and children who are sensitive to cow milk based product often thrieve better when goat milk based product is substituted [16]. In order french extensive clinical studies with children allergic to cow milk, the treatment with goat milk produced positive results in 93% of the children and was recommended as a valuable aid in child nutrition because of less allergenicity and better digestibility than cow milk [17]. But in some cases goat milk also proved to be allergenic and intolerant for infants [18]. In some childrens and adult humans,water buffalo milk show more tolerance than cow milk. Repeat skin testing was performed at age 2 years of children and continued to be positive for cow milk but children found to be negative for skin prick tested to water buffalo milk based yogurt [19]. On these bases, selection of suitable milk for consumption is an important target for nutritionist and pediatricians. Therefore, the present study leads to evaluate the suitability of buffalo and sheep milk for nutrition to the people and children allergic to cow milk by study of amino acid composition, secondary structure, CLUSTALW2 analysis and phylogenetic relationship of Cow, Goat, Sheep and Buffalo milk whey proteins.

Methodology: Dataset
Search for some milk whey protein was made in GenBank and EMBL databases. Obtained protein sequences filtration was done to remove any redundant sequences from the population and cross-checked by UniProtKB data base. The sequences are available at NCBI (http://www.ncbi.nlm.nih.gov/protein) with accession no. as given in Table 1 (see supplementary material). The dataset consists of casein protein (α-s1, α-s2, β and κ) and β-lactoglobulin.

Sequence analysis
The ProtParam tool of ExPASy (http://www.expasy.ch/) was used to analyze amino acid and atomic composition [20]. Further isoelectric point, Extinction coefficient and hydropathicitywas observed and compared between four species.Secondary structure predictions was done using SABLE (http://www.sable.cchmc.org) of obtained sequence in dataset.

Multiple Sequence Alignment CLUSTALW2
at European Bioinformatics Institute (www.ebi.ac.uk/tools/msa/clustalw2/) was used for protein multiple sequence alignment (MSA) using standard parameters.

Phylogeny tree construction
The Phylip tool (http://bioweb.pasteur fr/phylogeny/intro.en.html) was used to construct phylogenetic trees from aligned sequences. Computation of distance was done by program Protdist, which constructs distance matrices by a process called "Bootstrapping". A rooted tree was plotted using NJ plot software package [21] and distance matrix was calculated between each selected species [22].

Sequence analysis
Number of amino acid residue in selected milk whey protein in each species given in Table 2 (see supplementary material). These proteins also share considerable similarity in their percentage content of amino acids as given in Table 3-7 (see supplementary material). There was considerable similarity in predicted protein secondary structure of Capra hircus and Ovis aries as shown in Figure 1 (A-T). At the same time there were some similarities as well as some differences in predicted protein secondary structure of Bos Taurus and Bubalus bubalis as shown in Figure 1

Multiple sequence Alignment
The MSA of protein sequences obtained from CLUSTALW2 including the family of candidate protein sequences, show the maximum pairwise similarity in beta-casein group (95%) between goat and sheep. On the other hand minimum pair wise similarity shown in kappa casein (85%) between cow -goat and cow-sheep. The most common and generally more accessible approach to protein function prediction is 'inheritance through homology'-that is, the knowledge that proteins with similar sequences frequently carry out same function [23]. For a majority of proteins it is already posible to predict their approximate function with resonable accuracy based on their evolutionary relationship or sequence similarity to proteins with known functions [24-26]. Since the whey protein from goat and sheep share a great similarity this supports functional similarity between goat and sheep milk. At the same time buffalo shows great similarity with cow at sequence level but some dissimilarities at secondary structure level makes it less allergenic. The reason behind this may be absence of allergenic epitopes but it should be analyzed further in vitro.

Phylogenetic tree construction
The result of phylogenetic tree analysis shown that for casein and beta-lactoglobulin each subgroup is divided in to two sub groups containing two species (Bos Taurus, Bubalus bubalis) in one group and two species (Capra hircus, Ovis aries) in another group is shown in Figure 2 (A-E). Distance matrix for each protein groups in between four different species was calculated as given in Table 8-12 (see Supplementary material). Calculated distance matrix evaluates close relationship between goat-sheep and cow-buffalo.

Conclusion:
In case of cow milk allergic infants, since milk whey proteins of sheep and goat are almost identical , hence sheep milk is suitable and acceptable substitute of goat milk. In cow milk allergic childrens and adult humans buffalo milk is a good substitute of cow milk without any change in nutritional quality.