Buffalo cheese whey proteins, identification of a 24 kda protein and characterization of their hydrolysates: in vitro gastrointestinal digestion

Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion, several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

SDS-PAGE patterns for the H1 and H2 hydrolysates.

<p>In the both figures the Lane 1: molecular mass standards; Lane 2: treated bovine milk whey; Lane 3: treated buffalo milk whey. The lanes 4–12 are showing hydrolysates produced using the M1 method (Fig 5a) with incubation between 0–180 min, whereas the lanes 4–16 are showing hydrolysates produced using the M2 method (Fig 5b) with incubation between 0–1440 min.</p

Protein, lactose and fat concentrations of the <i>in natura</i> and treated buffalo milk whey.

<p>*reduction of lactose</p><p>**dialyzed, defatted and centrifuged</p><p>Protein, lactose and fat concentrations of the <i>in natura</i> and treated buffalo milk whey.</p

Relative concentration (nmol. L^-1) of the amino acids from buffalo milk whey before and after dialyzability.

<p>1 Hydrolysis with pepsin, trypsin, chymotrypsin and carboxypeptidase-A</p><p>2 Total of released amino acids by dialyzability (Dext + Dint) NH = non-hydrolyzed; H1 = low degree of hydrolysis; H2 = high degree of hydrolysis; GD = gastric digest; Dext = external intestinal digest, samples collected inside of the membrane; Dint = internal intestinal digest, samples collected outside of the membrane</p><p>3 Essential amino acids</p><p>4 Non-essential amino acids</p><p>Relative concentration (nmol. L^-1) of the amino acids from buffalo milk whey before and after dialyzability.</p

Electrophoretic profile of the buffalo cheese whey.

<p>a) Ten percent PAGE silver stained protein profile of treated bovine (1) and buffalo (2) whey. b) Twelve percent SDS-PAGE of the bovine and buffalo milk whey proteins. (1) Molar mass standards; (2) bovine milk whey; (3) buffalo milk whey. Ig: immunoglobulin; BSA: bovine serum albumin; α-La: alpha-lactalbumin; β-Lg: beta-lactoglobulin.</p

Determination of the hydrolysis degree for buffalo cheese whey hydrolysates.

<p>The values of DH were obtained by TNBS and OPA methods for hydrolysis using the M1 and M2 methods.</p

The HPLC chromatographic of the buffalo cheese whey hydrolysates.

<p>a) non-hydrolyzed (NH), b) with a medium degree of hydrolysis (H1), and c) with a high degree of hydrolysis (H2). Reverse phase chromatography using column Kromasil C18 (250 x 4.6 mm) Φ = 5 μm, 300 Ǻ porosity with a 5–95% linear gradient (solvent A: water with 0.045% TFA and solvent B: acetonitrile containing 0.036% TFA, 30 min), a flow rate of 1.0 mL min^-1 and with detection at 220 nm.</p

Amino acid sequence for β-Lg from <i>Bos taurus</i> (gi/229460).

<p>The tryptic peptides obtained for 24 kDa protein from buffalo cheese way were identical to the red sequence. It was obtained 37% of coverage.</p