Geoffrey O. Regester

Whey proteins as functional food ingredients

Putative anti-cancer activity of whey proteins has been investigated in an animal model to evaluate their potential role in disease prevention, and to contribute to a basis for their inclusion as ingredients in functional foods. Animal feeding trials have compared the efficacy of dietary whey proteins in retarding chemically induced colon cancer in a rat model of the disease. Dairy proteins, in particular whey protein, were found to be efficacious in retardation of colon cancer in young rats compared with other dietary proteins (meat, soy). The influence of dietary whey protein on development of colon cancer in mature rats has also been examined. Results similar to those with younger animals have been demonstrated, a finding that suggests age does not significantly alter the outcome. Efficacy of whey protein fractions has also been assessed. Preliminary results suggest that diets supplemented with lactoferrin or with β-lactoglobulin enhance protection against the development of putative tumor precursors (aberrant crypts) in the hind gut wall. The mechanism behind the apparent anti-cancer activity of dietary whey proteins in these studies may be related to their sulfur amino acid content, for which there is a high requirement in the rat, and hypothesised role in protecting DNA in methylated form. In a parallel study, a number of potential functional foods containing whey protein (flavored milk, pasta, ice cream, dessert pudding, muesli, and savory dip) have been developed in preparation for human clinical trials. The foods containing whey protein were generally highly acceptable in taste trials. These products are expected to be suitable as delivery vehicles for dietary whey protein in studies aimed at substantiating the human health benefits of this protein source.

Effects of purified bovine whey factors on cellular immune functions in ruminants

The immunomodulatory properties of bovine milk and whey have long been documented. The recent advance of whey protein fractionation technology has now allowed us to study the immunobiological properties of some highly purified components of whey, with a view to exploiting their possible industrial and biomedical applications. The effects of fractionated bovine whey proteins on cellular immune responses were therefore examined using a panel of in vitro assays. Both lactoferrin (LF) and lactoperoxidase (LP) were found to inhibit proliferation and interferon-gamma (IFN-gamma) production of ovine blood lymphocytes in response to mitogenic stimulation. However, their effects in a combined fraction or in whey protein concentrate (WPC) were either diminished or eliminated. LF and LP had no effect on lipopolysaccharide (LPS)-induced ovine blood lymphocyte proliferation, production of interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF alpha) by ovine bronchoalveolar lavage (BAL) macrophages, major histocompatibility complex (MHC) Class II antigen expression by ovine BAL macrophages and bovine natural killer (NK) cell activity. However, alpha-lactalbumin (alpha LA) exhibited an enhancing effect on IL-1 beta production. It is noteworthy that as bovine whey fractions become progressively more purified, their modulatory effects on the immune response also become more clear-cut. The effects of LF, LP and alpha LA may be eliminated by their combination in whey or by other minor components of whey. Further investigation of industrial applications for whey proteins of high purity is warranted.

Milk-derived growth factors as serum supplements for the growth of fibroblast and epithelial cells

SummaryWe have investigated the response of several epithelial and fibroblastic cells to a mitogenic extract of bovine milk. Cation exchange chromatography was used to produce a mitogen-rich fraction from an industrial whey source that, although comprising only 0.5% of total whey protein, contained the bulk of the growth factor activity. This fraction was a source of potent growth promoting activity for all mesodermal-derived cells tested, including human skin and embryonic lung fibroblasts, Balb/c 3T3 fibroblasts, and rat L6 myoblasts. Maximal growth of all these cell types exceeded that observed in 10% fetal bovine serum. Feline kidney and baby hamster fibroblasts and Chinese hamster ovary cells were less responsive, achieving a maximal growth response of 50–75% that observed in 10% fetal bovine serum. Maximal growth achieved in whey-extract-supplemented cultures of Balb/c 3T3 and human skin fibroblasts, and L6 myoblast cultures exceeded that seen in response to recombinant acidic or basic fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, or epidermal growth factor. Importantly, addition of low concentrations of fetal bovine serum to the whey-derived mitogenic fraction produced an additive response. However, concentrated milk-derived factors were found to be inhibitory to the growth of all epithelial lines tested, including rat intestinal epithelial cells, canine kidney epithelial cells, and mink lung cells. It is concluded that industrial whey extracted in this form constitutes an important source of potent growth-promoting agents for the supplementation of mesodermal-derived cell cultures.

Influence of whey and purified whey proteins on neutrophil functions in sheep

The effects of ruminant whey and its purified fractions on neutrophil chemotaxis and superoxide production in sheep were studied. Both colostral whey and milk whey were found to inhibit chemotaxis regardless of whether they were autologous or homologous, but the inhibitory effects were abolished by washing neutrophils with culture medium before their use in the chemotaxis assay. Colostral whey and milk whey also inhibited the chemotactic activity of zymosan-activated serum. Whey fractions of various degrees of purity such as lactoferrin, lactoperoxidase, lactoferrin-lactoperoxidase, alpha-lactalbumin, bovine serum albumin and whey protein concentrate were then studied. While none of these proteins showed any effects on chemotaxis, lactoferrin-lactoperoxidase and whey protein concentrate were found to have an enhancing effect on superoxide production in a dose-dependent manner. Our results provide information on the modulatory role of ruminant milk proteins in inflammatory responses and warrant future investigation.

Immune modulation in suckling rat pups by a growth factor extract derived from milk whey.

Oral tolerance to foreign enteral antigens is not fully developed in early neonatal life. Epidemiological evidence supports a role for maternal milk in the development of immune responses, including oral tolerance. Formula fed infants have an increased susceptibility to food allergy and the later development of autoimmune disease. This may relate to the lack in infant formula of growth factors found in maternal milk. Bovine milk contains proteins, growth factors and cytokines. Various studies have outlined the immune modulating potential of bovine milk-derived products. Fractionated whey extracts have therapeutic potential in disease states where there is an excessive inflammatory reaction, and disease preventive potential for infants who are not breast-fed. We have shown that daily oral administration of a growth factor-enriched fraction from milk whey to naturally suckling rat pups between days 4-9 postnatal can down-regulate immune activation to a specific orally administered food antigen, ovalbumin, assessed by lymphocyte proliferation. In addition, non-specific down-regulation in the intestine was observed as assessed by the expression of MHC I. Treatment of rat pups with whey extract at the time of oral sensitisation to ovalbumin also resulted in an increased secretion of TGF-beta into the culture supernatant of spleen cells incubated with specific antigen. TGF-beta is an immuno-down-regulatory cytokine involved in tolerance induction. Immune modulation by extracts derived from milk whey could be of potential benefit for formula-fed and pre-term infants in reducing susceptibility to inappropriate activation to food antigens.

New therapeutics from a dairy byproduct—Cheese whey

An efficient process has been developed for extraction of growth factors, antimicrobial agents, and minor whey proteins from bovine cheese whey. The whey‐derived extract stimulates growth of mesodermal cell lines in culture but inhibits proliferation of epithelial‐derived cells. Known growth factors characterised in the material do not account for its complete biological activity in cultured fibroblasts, suggesting the presence of unidentified cell mitogens. The protein extract is being investigated for use in prevention and repair of gastrointestinal injury and surface wounds. In preclinical studies with experimental animal models, the protein extract has been shown to reduce damage in the small bowel and oral mucosa associated with ablative cytotoxic drug regimens, as well as to promote healing of incisional and excisional surface wounds. Human trials of the whey extract incorporating novel protein delivery systems are now under way to assess its safety and efficacy. If successful, the whey‐based extract will offer a valuable source of natural cell mitogens with widespread clinical application in preventing tissue damage and stimulating wound repair. Drug Dev. Res. 46:286–291, 1999.

Immunomodulatory activities of whey fractions in efferent prefemoral lymph of sheep.

Studies on the immunomodulatory activities of ruminant milk and colostral whey fractions were undertaken. By comparing with boiled colostral whey in a preliminary experiment, a putative heat-labile immunostimulatory factor for antibody responses was found to be present in ovine colostral whey. Studies were then undertaken in sheep in which the efferent prefemoral lymphatic ducts were cannulated bilaterally, and immune responses in the node were measured following subcutaneous injection in the flank fold of whey protein preparations of various purities. A significant sustained decline of efferent lymphocyte output was observed following injection with autologous crude milk whey or colostral whey preparations, but no changes were observed in interferon-gamma levels in lymph plasma. Two bovine milk whey fractions (lactoperoxidase and lactoferrin) of high purity were compared in bilaterally cannulated sheep. A transient decline over the first 6 h was seen in the efferent lymphocyte output and lymph flow rate after injection of both fractions. A significant difference was seen between the two fractions in interferon-gamma levels in lymph at 6 h after injection. However, no significant changes in the proportion of the various efferent lymphocyte phenotypes were seen following either treatment. Whereas both fractions showed a significant inhibitory effect in a dose-dependent manner on the proliferative response of T lymphocytes, but not B lymphocytes, to mitogenic stimulation in vitro, no similar changes were seen following in vivo stimulation with these two fractions.

Comparison of immunomodulatory properties of purified lactoferrin, lactoperoxidase and β-casein in sheep

Bovine milk contains a variety of proteins and peptides that are biologically active (Ogra & Ogra, 1978; Duncan & McArthur, 1981; Newby et al . 1982; Juto, 1985; Stoeck et al . 1989; Mincheva-Nilsson et al . 1990; Watson, 1990; Barta et al . 1991; Politis et al . 1991; Fiat et al . 1993). Our laboratory has a long-term interest in some purified milk proteins, particularly lactoferrin (LF), lactoperoxidase (LP) and β-casein (β-CN), which have been shown to be immunologically significant. Some of our recent studies on these bovine milk proteins, particularly β-CN, indicated that their in vitro immunological effects did not always parallel their in vivo activities (Wong et al . 1996 a , b ; 1997 a , b ). This study was designed to investigate and compare the capacity of these purified bovine milk proteins to modulate a range of components that are vital to in vivo immune responses in sheep, with a view to providing further information on their potential in biomedical applications. To achieve this objective, a sensitive lymphatic cannulation model was employed that allows in vivo immune components and their functions to be measured in lymph collected under physiological conditions.

A Potent Growth Factor Supplement For Cell Culture Purified From Whey

We developed a single-step cation-exchange process for enriching growth factors present in cheese whey. Whey-derived growth factor extract (WGFE) contained only 0.5% whey protein, but all the growth factor activity. It stimulated the growth of mesodermal-derived cells and inhibited epithelial cells. Maximum growth of fibroblasts exceeded the response to serum, whereas other lines, such as CHO cells and L6 myoblasts, required the presence of low serum concentrations (1% v/v) to achieve similar growth rates. Specific assays detected IGF-I and -II, FGF-1 and -2, PDGF, and TGF-s1 and -s2 at concentrations that partially account for the growth-promoting activity. A combination of these growth factors could not reproduce the maximum effect of WGFE, suggesting the presence of other growth factors. Membrane Ultrafiltration of WGFE partitioned and concentrated growth factors, producing fractions enriched in IGF or TGF-s peptides, with enhancement of potency by up to 30-fold. WGFE as a source of growth factors, has application as a serum supplement or extender, particularly for fibroblasts and other mesodermal-derived commercially important cell lines.