Lactalbumin hydrolysate

There are a number of media available which are not based on a detailed investigation of growth requirements, but rather include crude mixtures of nutrients added to promote cell growth. These include lactalbumin hydrolysate (Appendix 1 Table 9) or yeast extract (Appendix 4) to provide an inexpensive source of amino acids or vitamins. Thus Melnick s monkey kidney media A and B (Melnick, 1955) contain lactalbumin hydrolysate and calf serum in Hanks and Earle s BSS, respectively. Chick embryo extract and tryptose phosphate broth (Appendix 1, Tables 11 and 12) are also used occasionally and their use is referred to where appropriate throughout the book. Mitsuhashi and Maramorosch mosquito cell medium contains lactalbumin hydrolysate, yeast extract and foetal calf serum in a specially developed saline (Mitsuhashi and Maramorosch, 1964 Singh, 1967). 

The high levels of amino acids found in haemolymph may be supplied from a 10% lactalbumin hydrolysate supplemented with non-essential amino acids. Few studies have been done on the amino acid requirements of cultured insect cells and, although some media (Grace, 1962) do specify an amino acid mixture, the presence of additives, such as whole egg ultrafiltrate, makes the significance of specific amino acid concentrations doubtful. 

Subculture Don-C cells every 24 h in McCoy s 5a medium (Appendix 1) containing 20% foetal calf serum and 0.08% lactalbumin hydrolysate, seeding cells at 1.2 X 105/ml(3 X 104 cells/cm2). 

Lactalbumin hydrolysate purchased as a dry powder from Nutritional Biochemicals (U.S.A.) is kept in brown sealed bottles. It is dissolved to 5% in Hanks BSS and autoclaved at 115°C for 10 min when it may be kept at room temperature for a month or more. If kept frozen a precipitate forms which will redissolve on heating in a boiling water bath. For use it is diluted tenfold to 0.5% with growth medium. 

Growth is achieved at 27°C in TC-lOO medium (Gibco BRL) with 10% heat-inactivated FBS or IPL-41 basal medium (J.R. Scientific, CA) with 2% FBS plus 3.3 g each of yeastolate and lactalbumin hydrolysate (Difco), suspension cultures being supplemented with 1-2 g H pluronic F-68 (BASF Corp., NJ). Sf9 cells can also be cultivated in serum-free media such as Excell-400 (J.R. Scientific, CA) and SF900. 

Originally cultures were established in Earle s BSS containing 0.5% lactalbumin hydrolysate, 0.1% yeast extract, 0.1% polyvinylpyrrolidone and 2-5% FBS but they can be maintained successfully in Eagle s MEM (EBSS) with added 7.5% bovine serum and 2.5% FBS or Medium 199 supplemented with 5% FBS. Vero cells are adaptable to batch and continuous perfusion culture. 

The cytotoxicity of distamycin derivatives was estimated on the basis of the morphological modifications induced in HeLa cell cultures, after incubation for 40 h in Hanks saline solution + 0.5 % lactalbumin hydrolysate + 5 % calf serum (HLS). Assay on vaccinia virus Cultures of HeLa cells (grown in HLS medium) or mouseembryo cells (grown in HLS medium plus 0.1 % yeastolate) infected with vaccinia virus (Strain WR/ATCC) were used. Preliminary assays were made according to Herrmann et al.° Subsequent studies were carried out by assessing the inhibition of plaque formation (ECP) as well as the inhibition of infectious virus production in test tube cultures treated with the compounds for 40 h after the absorption of the virus. 

Hernandez-Ledesma, B., D avalos, A., Bartolom e, B., and Amigo, L. (2005). Preparation of antioxidant enzymatic hydrolysates from a-lactalbumin and p-lactoglobulin. Identification of active peptides by HPLC-MSMS. ]. Agric. Food Chem. 53,588-593. 

One of the more spectacular examples of the development of novel interactions for nanostructuring of food systems is the self-assembly of partially hydrolysed molecules of a-lactalbumin at neutral pH in the presence of appropriate cations (Ca2+, Mn2+, Zn2+, Cu2+ or Al3+). These ordered nanostructures possess enhanced functionality for thickening, gelation and encapsulation, as compared to the individual protein molecules or their disordered aggregates. The molecules assemble into rather stiff nanotubes with a cavity diameter of 16 nm and a length of a few micrometres (Figure 1.1). The specific ion size and its preferred ligand coordination number seem to play a key mechanistic role. But hydrolysis is needed to make the a-lactalbumin prone to self-assembly. 

Figure 1.1 Schematic representation of self-assembly of partially hydrolysed a-lactalbumin into nanotubes in the presence of Ca2+. The image at the extreme right show s an electron micrograph of the nanotubes (75 mM Tris buffer, pH = 7.5, 2 mole Ca2+ per mole a-lactalbumin prepared by negative staining with 3% uranyl acetate for 1 min). Reproduced from Graveland-Bikker and de Kruif (2006) with permission.

In contrast, the whey proteins are relatively small globular proteins. a-Lactalbumin represents about 20 % of the protein content of bovine whey (3.5 % of total bovine milk protein), and it is the principal protein in human milk (Brew and Grobler, 1992). Nanotube assembly has been discovered in some solutions containing a hydrolysed derivative of this protein. And it appears that the a-lactalbumin nanotube is unique in the sense that it is the only artificial nanotube that has so far been made from a food protein (Graveland-Bikker et al., 2004 Graveland-Bikker and de Kruif, 2006). As for p-lactoglobulin, it has the capacity under certain specific conditions to form nano-fibres in aqueous media (as can various other globular food proteins, such as ovalbumin, soy proteins, and bovine serum albumin) (van der Linden, 2006 Nicolai, 2007). 

Graveland-Bikker, J. F. (2005). Self-assembly of hydrolysed a-lactalbumin into nanotubes. PhD Thesis, Utrecht University, the Netherlands. 

Ipsen, R., Otte, J., Qvist, K.B. (2001). Molecular self-assembly of partially hydrolysed alpha-lactalbumin resulting in strong gels with a novel microstructure. Journal of Dairy Research, 68, 277-286. 

In a recent work (Sitohy et al, 2001b) 100% methyl-, 59% ethyl- and 56% propyl-esters of (3-casein, and 52% methyl-, 36% ethyl- and 25% propyl-esters of a-lactalbumin were prepared. The degree of pepsinolysis (% DH) was enhanced considerably after esterification. Methyl esters of both proteins yielded the highest levels of DH. Compared to SDS-PAGE of peptic hydrolysates of native proteins, those of esterified (3-caseins demonstrated the... 

Pihlanto-Leppala, A., Marnila, P., Hubert, L., Rokka, T., Korhonen, H., and Karp, M. 1999. The effect of alpha-lactalbumin and beta-lactoglobulin hydrolysates on the metabolic activity of Escherichia coli JM103. J. Appl. Microbiol. 87, 540-545. 

The peptide map of a trypsin hydrolysate of hen egg white lysozyme oxidized at the S—S bonds was obtained and characterized. A comparative study of it with the map of the same hydrolysate of lysozyme reduced at the S—S bonds was conducted.Comparison of the (structural) signal regions of hen egg white lysozyme and of ovine a-lactalbumin have been reported. 

Milk was probably the first proteinaceous cosmetic ingredient used. Milk proteins are composed of about 80% casein, an acidic phosphoprotein present as colloidal dispersion, and a soluble protein fraction composed of lactalbumine and lactoglubulins, which are found in milk serum after acid precipitation of casein. These three protein substances are useful as cosmetic ingredients as partial hydrolysates (casein, lactalbumin) or in native form (lactoglobulin). 

Ipsen R, Otte J (2007) Self-assembly of partially hydrolysed alpha-lactalbumin. Biotechnol Adv 25 602-605... 

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