Enzymatic hydrolysis of whey proteins. II. Molecular-weight range

Using high-pressure liquid chromatography we studied the distribution of molecular weights in whey-protein hydrolysates using the following commercially obtained proteases: Alcalasa 0.6 L and Protease 660 L, both bacterial in origin, and PEM 2500 S, of animal origin. In each of the systems, the range of molecular weights in the hydrolysate depended solely on the degree of hydrolysis (DH) achieved. For DH >/= 20, between 65% and 95% of the hydrolysate is made up of peptides with a molecular weight of less than 1,000 Da. (c) 1994 John Wiley & Sons, Inc.     

Enzymatic hydrolysis of whey proteins: I. Kinetic models

We have studied the enzymatic hydrolysis of whey proteins at pH 8 and50 degrees C with two proteases of bacterial origin, MKC Protease 660 L, and one of animal origin, PEM 2500 S. Our results show that a greater degree of hydrolysis is achieved under the same experimental conditions with the bacterial proteases than with the animal one. In our interpretation of the results we propose a mechanism in which the hydrolytic reaction is a zero-order one for the substrate, and the enzyme denaturalizes simultaneously via a second-order kinetic process due to free enzyme attacking enzyme bound to the substrate. Our results also indicate that there is an irreversible serine-protease inhibitor in whey proteins. (c) 1994 John Wiley & Sons, Inc.     

Simple purification of immunoglobulins from whey proteins concentrate

We have developed a new protocol with only two steps for purification of immunoglobulins (Ig) from a protein concentrate of whey. Following this protocol, we have an 80% recovery of immunoglobulins, fairly pure. The purification was achieved by eliminating the BSA, via a strong adsorption on DEAE-agarose. Full desoprtion of the other serum proteins could be achieved without contamination with BSA. Thus, a protein solution containing only Ig and very small proteins (e.g., beta-lactoglobulins and alpha-lactalbumin) was obtained. Offering this protein mixture to a lowly activated aminated support, only Ig adsorbed on the support. It has been shown that BSA is able to interact with other proteins (including Ig and lactalbumins). This ability to form complexes with other proteins prevented the success of the direct adsorption of Ig on this mildly activated support, even although Ig should be the largest protein presented in dairy whey.     

Complex coacervation of whey proteins and gum arabic

Mixtures of gum arabic and whey protein (whey protein isolate, WP) form an electrostatic complex in a specific pH range. Three phase boundaries (pH(c), pHphi(1), pHphi(2)) have been determined using an original titration method, newly applied to complex coacervation. It consists of monitoring the turbidity and light scattering intensity under slow acidification in situ with glucono-delta-lactone. Furthermore, the particle size could also be measured in parallel by dynamic light scattering. When the pH is lowered, whey proteins and gum arabic first form soluble complexes. This boundary is designated as pH(c). When the interaction is stronger (at lower pH), phase separation takes place (at pHphi(1)). Finally, at pHphi(2) complexation was suppressed by the charge reduction of the gum arabic. The major constituent of the whey protein preparation used was beta-lactoglobulin (beta-lg), and it was shown that beta-lg was indeed the main complex-forming protein. Moreover, an increase of the ionic strength shifted the pH boundaries to lower pH values, which was summarized in a state diagram. The experimental pH(c) values were compared to a newly developed theory for polyelectrolyte adsorption on heterogeneous surfaces. Finally, the influence of the total biopolymer concentration (0-20% w/w) was represented in a phase diagram. For concentrations below 12%, the results are consistent with the theory on complex coacervation developed by Overbeek and Voorn. However, for concentrations above 12%, phase diagrams surprisingly revealed a "metastable" region delimited by a percolation line. Overall, a strong similarity is seen between the behavior of this system and a colloidal gas-liquid phase separation.     

Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers

In this study we describe the design, preparation and testing of superparamagnetic anion-exchangers, and their use together with cation-exchangers in the fractionation of bovine whey proteins as a model study for high-gradient magnetic fishing. Adsorbents prepared by attachment of trimethyl amine to particles activated in sequential reactions with allyl bromide and N-bromosuccinimide yielded a maximum bovine serum albumin binding capacity of 156 mg g(-1) combined with a dissociation constant of 0.60 microM, whereas ion-exchangers created by linking polyethylene imine through superficial aldehydes bound up to 337 mg g(-1) with a dissociation constant of 0.042 microM. The latter anion-exchanger was selected for studies of whey protein fractionation. In these, crude bovine whey was treated with a superparamagnetic cation-exchanger to adsorb basic protein species, and the supernatant arising from this treatment was then contacted with the anion-exchanger. For both adsorbent classes of ion-exchanger, desorption selectivity was subsequently studied by sequentially increasing the concentration of NaCl in the elution buffer. In the initial cation-exchange step quantitative removal of lactoferrin (LF) and lactoperoxidase (LPO) was achieved with some simultaneous binding of immunoglobulins (Ig). The immunoglobulins were separated from the other two proteins by desorbing with a low concentration of NaCl (< or = 0.4 M), whereas lactoferrin and lactoperoxidase were co-eluted in significantly purer form, e.g. lactoperoxidase was purified 28-fold over the starting material, when the NaCl concentration was increased to 0.4-1 M. The anion-exchanger adsorbed beta-lactoglobulin (beta-LG) selectively allowing separation from the remaining protein.     

Reducer driven baric denaturation and oligomerisation of whey proteins

The influence of high pressure on alpha-lactalbumin (ALA)/beta-lactoglobulin (BLG) mixtures of various compositions was studied at pH 8.5 by gel-permeation chromatography and sodium dodecyl sulphate (SDS) gel electrophoresis without 2-mercaptoethanol. High-molecular protein disulfide oligomers formed after denaturation by the pressure of 10 kbar (1000 MPa) if the weight fraction of BLG (W(BLG)(0)) in the protein mixture exceeded 0.2. The maximum yield of these oligomers of order 80-85% is observed at W(BLG)(0)>/=0.4. Conversions of both proteins in the oligomers are roughly the same. The estimates of the oligomerisation yield obtained by the gel-permeation chromatography and SDS gel electrophoresis agree well. This indicates that the formation of intermolecular disulfide bonds is necessary for the oligomerisation. Thus, the oligomerisation of pressure denatured ALA and BLG is driven by the thiol<-->disulfide exchange rendered possible by the vigorous baric denaturation and the exposure of the free thiol group of BLG, which acts as an initiator of disulfide bridges scrambling.     

Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk.

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin.     

New genetic variants identified in donkey's milk whey proteins

Novel genetic variants for donkey milk lysozyme and -lactoglobulins I and II have been identified by the combined use of peptide mass mapping and sequencing by tandem mass spectrometry in association with database searching. The novel donkey lysozyme variant designated as lysozyme B (Mr 14,631 Da) differed in three amino acid exchanges, N⁴⁹ D, Y⁵² S, and S⁶¹ N, from the previously published sequence. Three novel genetic variants for donkey -lactoglobulins were identified. One of them is a type -lactoglobulin I with three amino acid exchanges at E³⁶ S, S⁹⁷ T, and V¹⁵⁰ I (-lactoglobulin I B, Mr 18,510 Da). The two others are type -lactoglobulins II with two amino acid exchanges at C¹¹⁰ P and M¹¹⁸ T (-lactoglobulin II B, Mr 18,227 Da) and with three amino acid exchanges at D⁹⁶ E, C¹¹⁰ P, and M¹¹⁸ T (-lactoglobulin II C, Mr 18,241 Da). All these primary structures are closely related to those of homologous proteins in horse milk (percent identity >96%).     

Some monotreme milk "whey" and blood proteins

1. Electrophoretic studies are made of mature phase milk "whey" proteins and blood serum proteins of echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus). The echidna milk bands are designated A-M, those of platypus A-G. Some of the proteins are isolated and characterized. 2. Echidna band A protein has some similarity to high cystine "whey" proteins. Band E protein (apparent Mr 21,000) may be a beta-lactoglobulin-like protein. Band M is lysozyme. Band C is serum albumin. Bands G-K are transferrins. 3. Platypus milk bands A, C, D, F and G are isolated. Bands F and G are transferrins. 4. Lactose synthase and lytic activities are examined.     

Fractionation of whey proteins with a hexapeptide ligand affinity resin

The isolation of individual proteins from whey would allow production of more consistent and reliable products by the food industry and possibly would also increase their use in the pharmaceutical industry. -Lactalbumin is the second most prevalent protein in bovine milk whey and has many uses including serving as an excellent protein source in infant formulas, power drinks and other beverages that require soluble, nutritional protein. In this study, we describe two methods for production of -lactalbumin from whey protein isolate using bioselective adsorption. The use of a peptide ligand (WHWRKR) attached to a resin allowed production of an -lactalbumin-rich fraction with a purity of 90.6% and a recovery of 47.9%, while also producing other fractions of commercial interest. The combined use of an amino resin followed by the WHWRKR resin produce a highly purified -lactalbumin (100%) with a yield of 35.2%.     

Fractionation and recovery of whey proteins by hydrophobic interaction chromatography

A method for the recovery and fractionation of whey proteins from a whey protein concentrate (80%, w/w) by hydrophobic interaction chromatography is proposed. Standard proteins and WPC 80 dissolved in phosphate buffer with ammonium sulfate 1 M were loaded in a HiPrep Octyl Sepharose FF column coupled to a fast protein liquid chromatography (FPLC) system and eluted by decreasing the ionic strength of the buffer using a salt gradient. The results showed that the most hydrophobic protein from whey is α-lactalbumin and the less hydrophobic is lactoferrin. It was possible to recover 45.2% of β-lactoglobulin using the HiPrep Octyl Sepharose FF column from the whey protein concentrate mixture with 99.6% purity on total protein basis.     

Fractionation of some bovine whey proteins by recycling gel filtration on a large scale.

Fractionation of bovine whey concentrate was performed by gel filtration on Sephadex G-75 both on a laboratory scale and on a large scale. By a recycling procedure and improved separation was obtained and the whey proteins were resolved into four fractions in the weight ratio 3:12:1:4. The fractions were analysed by polyacrylamide gel (PAG) electrophoresis and the apparent molecular weights were determined by thin layer gel chromatography (TLG) and by sodium dodecylsulfate (SDS) gel electrophoresis.