Improved purification of beta-lactoglobulin from acid whey by means of ceramic hydroxyapatite chromatography with sodium fluoride as a displacer

The successful separation of beta-lactoglobulin from other bovine whey proteins was performed by ceramic hydroxyapatite chromatography with a fluoride ion gradient in phosphate buffer as displacement agent. The method was applied to acid whey originating from milk of healthy as well as of mastitic cows. beta-Lactoglobulin was completely eluted in one peak at a fluoride concentration of about 0.6 mol/l. The purity of beta-lactoglobulin in this fraction was at least 96% if whey from healthy milk was processed. Co-eluted contaminants are traces of immunoglobulin G, serum albumin and lactoferrin. In case of mastitic whey the proportion of beta-lactoglobulin is diminished as the amounts of immunglobulin G, serum albumin and lactoferrin are increased within this fraction. Size exclusion chromatography on Superdex 75 pg effectively removed contaminants resulting in a purity for beta-lactoglobulin from normal whey of approximately 99%. The yield of beta-lactoglobulin from physiological whey was 50-55% referring to the fraction highly enriched with beta-lactoglobulin by hydroxyapatite chromatography. In case of mastitic milk the higher amounts of contaminants were also removed successfully by size exclusion chromatography.     

Activation of Plasmin in Mastitic Milk

Milk and whey samples from healthy and inflamed udder quarters of 10 Ayrshire cows were analyzed for proteolytic activity using radial caseolysis procedures, a fluorogenic coumaryl peptide substrate, and casein agarose zymography. Free lysosomal enzyme activity (N–acetyl–beta–D–glucosaminidase) was used as the criterion for inflammation. All mastitic milk samples had proteolytic activity, tentatively identified as plasmin (comigration at Mr 83 000 and characteristic fragmentation). The plasmin activities in mastitic milk were on average 2.9 μg/ml (range 0.5–12.5) as measured by radial caseolysis. Milk or whey specimens from healthy quarters were all negative except 1 in which an activity of 0.1 μg/ml was found in both specimens. The caseolytic activities were totally inhibited by 50 KITJ/ml of aprotinin, a serine proteinase inhibitor from bovine lung. No free plasminogen activator (PA) activity was found in any of the samples. Howewer, according to zymographic analyses PA molecules corresponding to urokinase were found in healthy and especially in mastitic specimens. Analysis of plasmin may provide an alternative means of screening for mastitic milk samples.     

Pseudocompact-Type Growth and Conversion of Growth Types of Strains of Staphylococcus aureus In Vitro and In Vivo

Some strains of Staphylococcus aureus grew as compact colonies in Brain Heart Infusion-serum-soft agar but as diffuse colonies in a modified Staphylococcus 110-serum-soft agar. These strains were designated "pseudocompact." Strains showing compact-type colonial morphology in both media were designated "compact," whereas strains showing diffuse-type growth in both media were designated "diffuse." It was observed that the most recently isolated strains of S. aureus were of the pseudocompact type, whereas most stock culture strains tested were of the compact type. Using cultures recently isolated from clinical material, it was shown that pseudocompact strains convert to compact-type growth after prolonged incubation. Interconversion of compact, diffuse, and pseudocompact growth forms could be induced in vitro by appropriate cultural conditions, and conversion of growth type was also observed in vivo. Femoral abscesses produced in mice by four different compact-type strains showed conversion to diffuse or pseudocompact-type growth during the course of the infection.     

Differential protein composition of bovine whey: a comparison of whey from healthy animals and from those with clinical mastitis

During clinical mastitis in dairy cows, the quantity of milk produced decreases and the composition of the milk is altered. As the severity of inflammation associated with the disease increases, the chemical composition of milk approaches that of blood as a consequence of increased permeability of the blood mammary barrier, or de novo intramammary synthesis, as has been suggested for mammary associated serum amyloid A3. A better understanding of these events may provide new approaches for the diagnosis and treatment of mastitis. The objective of this study was to document the changes in the protein composition of milk during clinical mastitis using a proteomic approach, with the objective of identifying new diagnostic markers of mastitis. Whey from dairy cows with clinical mastitis was compared to whey from healthy animals by two-dimensional gel electrophoresis (2-DE) with colloidal Coomassie staining and matrix-assisted desorption/ionization mass spectrometry. Increases in the concentrations of proteins of blood serum origin, including serotransferrin and albumin, were identified in mastitic whey compared to normal whey, while concentrations of the major whey proteins alpha-lactalbumin and beta-lactoglobulin were reduced in mastitic whey. Mass spectrometry subsequently confirmed the location of albumin, alpha-lactalbumin and beta-lactoglobulin on the 2-DE gels at M(r)/pI of 69 294/5.8, 14 200/4.5 and 19 883/4.9 respectively.     

Mitogenic whey extract stimulates wound repair activity in vitro and promotes healing of rat incisional wounds

The ability of single growth factors to promote healing of normal and compromised wounds has been well described, but wound healing is a process requiring the coordinated action of multiple growth factors. Only the synergistic effect on wound healing of combinations containing at most two individual growth factors has been reported. We sought to assess the ability of a novel milk-derived growth factor-enriched preparation ?mitogenic bovine whey extract (MBWE), which contains six known growth factors, to promote repair processes in organotypic in vitro models and incisional wounds in vivo. MBWE stimulated the contraction of fibroblast-populated collagen lattices in a dose-dependent fashion and promoted the closure of excisional wounds in embryonic day 17 fetal rat skin. Application of MBWE increased incisional wound strength in normal animals on days 3, 5, 7, and 10 and reversed the decrease in wound strength observed following steroid treatment. Wound histology showed increased fibroblast numbers in wounds from normal and steroid-compromised animals. These data suggest the mixture of factors present in bovine milk exerts a direct action on the cells of cutaneous wound repair to enhance both normal and compromised healing.     

Adhesion of Staphylococcus aureus to Bovine Mammary Epithelial Cells Induced by Growth in Milk Whey

Staphylococcus aureus strains isolated from bovine intramammary infection (mastitis) were tested for adhesion to bovine mammary epithelial cells after growth in milk whey or TSB. Bacteria grown in milk whey adhered more efficiently to mammary gland epithelial cells in vitro than the corresponding homologous bacteria grown in TSB. Trypsin treatment of milk whey-grown S. aureus had no effect on their adherence. Whereas, pretreatment with periodate significantly decreased bacterial adherence capacity. Periodate treatment of TSB-grown bacteria had no effect on adhesion to the mammary gland epithelial cells.     

Mastitis Whey — a Good Medium for Bacteria?

Growth of mastitis pathogenic bacteria was measured in bovine whey samples by a turbidometric microtechnique. Whey from mastitis cows supported growth as compared with whey prepared from normal milk. Blood proteins leak into milk during mastitis. A study was undertaken to analyze which molecules from blood would promote bacterial growth in whey Fractions containing hemoglobin showed a distinct growth-promoting effect. An inadequate iron supply is one of the restricting growth factors for bacteria in milk. By utilizing heme-compounds the pathogens can by-pass the effect of antimicrobial iron-binding present in milk in the form of lactoferrin.     

Involvement of SDS-stable higher M(r) forms of bovine normal milk alpha-lactalbumin in inducing intestinal IEC-6 cell death

Monomeric 14-kDa bovine alpha-lactalbumin was purified with a preparation of lower molecular weight whey protein concentrate from Holstein cow normal milk followed by size exclusion chromatography. The protein showed a stimulatory rather than an inhibitory effect on the proliferation of a cultured IEC-6 cell line from the rat small intestine. But incubation in 30% trifluoroethanol/acetate buffer (pH 5.5) at 37 degrees C for 5 d in a slowly rotating test tube rendered it highly cytotoxic with concomitant appearance of SDS-stable 20- and 30-kDa forms of alpha-lactalbumin on electrophoresis. Furthermore, alpha-lactalbumin obtained by a one-step purification procedure by affinity chromatography on an anti-alpha-lactalbumin antibody column from the lower molecular weight whey protein concentrate, which had been found to contain several SDS-stable higher M(r) forms of alpha-lactalbumin, exhibited potent inhibitory activity on IEC-6 cell growth. These results indicate the involvement of SDS-stable higher M(r) forms of bovine normal milk alpha-lactalbumin in inducing cell death on the intestinal IEC-6 cell line.     

Proliferation and differentiation of Abelson virus-infected murine myeloid leukemia cell lines does not involve an autocrine mechanism

Culture in agar of cloned promonocytic leukemia cell lines derived from Abelson virus-infected mice produced colonies of both a compact and diffuse morphology. Diffuse colonies contained fewer cells capable of forming colonies when recultured in agar than did compact colonies. Serial subcloning of cells from diffuse, but not compact, colonies ultimately led to the complete loss of colony-forming cells, i.e., to clonal extinction. The production of both compact and diffuse agar colonies was independent of the cell density of either the static liquid culture from which cells were taken for culture in agar, or the number of cells per agar culture. Furthermore, bioassays of culture supernatants indicated the leukemia cells did not secrete hemopoietic growth factors active on normal hemopoietic cells, transforming growth factors active on adherent cell lines, or factors that influenced the growth of the leukemic cells themselves. Collectively, these data suggest neither growth-factor independent replication nor the spontaneous differentiation of Abelson virus-infected myeloid cells involves autocrine secretion of growth regulators.     

Occurrence and clonal relatedness of sec/tst-gene positive Staphylococcus aureus isolates of quartermilk samples of cows suffering from mastitis

AIMS: To investigate the prevalence of sec/tst-gene positive Staphylococcus aureus in bovine mastitis and to get information about the clonal relatedness of these clinical isolates. METHODS AND RESULTS: A total of 533 Staph. aureus strains isolated from bovine mastitic quartermilk samples at 493 randomized dairy farms in Hessia, Germany, from January 1997 until June 1998 were examined for enterotoxin C (sec) gene and toxic shock syndrome toxin (tst) gene by multiplex polymerase chain reaction. Fifty-three (9.3%) of the strains were sec/tst-gene positive. Phenotypic TSST-1 production was found in all positive strains by reversed passive latex agglutination test. With DNA macrorestriction analysis, sec/tst-gene positive strains were divided into five different macrorestriction types. Type I (10 isolates) and III (40 isolates) were found to be the predominant types in terms of frequency of isolation in the investigated area. These DNA macrorestriction types differed in only two bands in the 500 and 270 bp region. CONCLUSIONS: Closely related Staph. aureus strains seem to be responsible for an unusual large proportion of bovine mastitis cases in geographically widely distinct locations. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first reports about the relatedness of sec/tst-gene positive Staph. aureus clinical isolates from bovine mastitis.     

Lipocalin-type prostaglandin D synthase in milk: a new biomarker for bovine mastitis.

The whey protein pattern of milk from animals affected by mastitic inflammation was resolved by two-dimensional gel electrophoresis (2D-PAGE) and compared to milk from unaffected cows. Inflammation caused the appearance of four spots aligned at a molecular weight level of 26 kDa and over a pH-region of 5.0 to 6.4. The spots excised from 2D gels were treated with chymotrypsin and the resulting peptides analyzed by MALDI-TOF mass spectrometry and RP-HPLC. All four spots yielded highly similar chymotryptic peptide mass fingerprints as well as chromatographic peak patterns. A database search could identify the four spots as isoforms of the bovine prostaglandin D synthase (PGD-S). In one of the isoforms a defined cysteine residue was shown to be oxidized to a sulfonic acid.     

Growth promotion and cell binding ability of bovine lactoferrin to Bifidobacterium longum

Lactoferrin, a major whey protein of human milk, is considered as growth promoter for bifidobacteria, the predominant microorganisms of human intestine. In the present study, in vitro growth promotion and cell binding ability of bovine lactoferrin to several strains of Bifidobacterium longum have been demonstrated. A dose-dependent as well as strain-dependent growth promotion effect by lactoferrin was observed. Cell binding ability of lactoferrin was inspected under an inverted confocal laser scanning microscope by incubation bacterial cells with biotinylated bovine lactoferrin and FITC-conjugated avidin. Fluorescence staining showed bovine lactoferrin binding to all tested strains. A lactoferrin-binding protein with a molecular weight of approximately 67 kDa was also detected in the extracted membrane and cytosolic fraction of each B. longum strain by far-Western blot technique using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin. Based on these results, we suggest that existence of lactoferrin-binding protein could be a common characteristic in bifidobacteria. It can also be hypothesized that lactoferrin-binding protein in bifidobacteria is not only involved in growth stimulation mechanism but also could play different roles.     

Bovine colostrum fraction as a serum substitute for the cultivation of mouse hybridomas

Summary Fractions of bovine colostrum were prepared and their ability to support the growth of mouse-mouse hybridomas in culture was tested. Whey was prepared from defatted colostrum by removal of casein using acid precipitation. An ultrafiltrate was obtained from cleared whey by filtration through membranes with a nominal molecular mass cut-off of 100 000 Da. Colostrum ultrafiltrate contained 1.16 g/l protein, 0.24 g/l immunoglobulin G (IgG) and less than 0.24 EU (endotoxin unit)/ml endotoxins. The effect of defatted colostrum, whey and ultrafiltrate as serum substitutes was examined by cultivation of hybridoma cells in minimal essential medium containing different concentrations of the supplements. Under optimal conditions in ultrafiltrate-supplemented medium, the maximal cell concentration was 35–40% of that obtained using 10% foetal bovine serum, and IgG production per cell was equal to that achieved using serum. In 1% defatted colostrum the maximum hybridoma concentration was about 30% of that in 10% serum, but at higher concentrations hybridoma growth was significantly reduced. The growth-promoting activity of whey was low. The results show that bovine colostrum ultrafiltrate provides a very attractive alternate to serum for production of monoclonal antibodies. Correspondence to: R. Pakkanen     

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

Summary We 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.     

A factor produced by human cells in vitro that changes HeLa cell colonial morphology

Summary Colonies of HeLa cells cultured in media supplemented with human or bovine serum or both can be morphologically described as three types: diffuse, intermediate, and compact, with their modal distribution depending on the serum or sera added to the growth medium. We have observed that for a particular medium or serum system, the percentage of compact colonies remains fairly constant under normal culture conditions, 0.2%, whereas the diffuse and intermediate colonies vary over a much wider range. The presence of certain substances as trypsin, heparin and Darvan in the medium favor the increase of compact colonies at the expense of other types. Furthermore, we have discovered that colonial morphology is influenced by cocultivation of the HeLa cells with human fibroblastlike cells, the compact colonies increasing as the density of the fibroblast element introduced into the mixed cultures is increased. Subsequent investigation revealed that conditioned medium from confluent fibroblast and HeLa cell cultures contained a factor(s), that significantly increased the percentage of compact colonies. The factor is nondialyzable, heat-stable and can be neutralized by serum. Recorded in this presentation are preliminary observations on the kinetics of colony formation and the interaction among the three HeLa cell colony types, the diffuse, the intermediate, and the compact. The factor's effect on HeLa cell colonial morphology is time dependent and rapidly reversed if the factor(s) is removed and fresh medium added.     

Effects of fibronectin on the compact colony formation in staphylococci

Fifty-two unencapsulated strains of Staphylococcus aureus, including strains of Wood 46 and Cowan I, formed compact colonies in fibronectin -soft agar. However, 20 encapsulated strains of Staphylococcus aureus and 50 strains of Staphylococcus epidermidis showed diffuse growth in the medium. The results suggest that another possible cellular factor, other than protein A, is involved in the binding of the cell surface with fibronectin and that it would be one of factors in forming compact colonies in serum-soft agar.     

Studies on Protein in Abnormal Milk

Normal milk and various abnormal milks, i.e., mastitic milk, Alcohol test positive milk, drying off-secretions and colostrum were investigated by polarography, based upon the so-called Brdicka’s filtrate reaction. Mastitic milk including subclinical mastitic milk, drying-off secretions and colostrum gave the higher protein double wave than that of normal milk. However, any relation was not observed between alcohol sensitivity and wave height. Polarographic filtrate reaction is not specific for mastitic milk but for lactation stage or healthy conditions of cow.     

Factors influencing the survival and multiplication of bacteriophages in calves and in their environment

Seven phages were fairly susceptible in vitro to the lethal effect of acidified whey, more so than the enteropathogenic Escherichia coli strains on which they were active. The low acidity that prevailed in the abomasum contents of calves shortly after a milk feed had little harmful effect on orally administered organisms of these phages; they flooded into the small intestine. The high acidity that prevailed later was lethal to orally administered phage organisms; few entered the small intestine. The lethal effect could be counteracted by giving CaCO3 in the feed. Low concentrations of phage-neutralizing antibodies were found in some serum samples from human beings, cattle and pigs. Antibodies to one of the seven phages were common in the human samples and antibodies to another, phage B44/1, were common in the cattle and pig samples and in bovine colostrum. Phage B44/1 antibodies in a sample of colostral whey were destroyed at pH 3.25 or less. Giving colostrum containing phage B44/1 antibodies with CaCO3 to a calf greatly reduced the numbers of orally administered phage B44/1 organisms in its alimentary tract. Antibodies to another phage were induced in the serum of a calf suffering from E. coli diarrhoea by treating it with that phage. The phages were as susceptible as the E. coli strains to the lethal action of formaldehyde and sodium hypochlorite. In contrast to the E. coli strains, they were almost completely resistant to phenol and chloroxylenol. The in vitro virulence of 21 phages varied according to the temperature at which tests were performed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions among thermophilic lactobacilli during growth in cheese whey

The starters used in the Parmesan cheese technology are whey-cultured, mixed strain cultures of thermophilic rod lactic acid bacteria. In mixed strain cultures, interactions among different strains can result in both stimulatory or inhibitory effects on microbial growth rate and metabolic activity. These effects, studied by the use of conductance, were observed for individual strains of thermophilic lactobacilli, which were grown in whey and in the presence of cell-free supernatant fluids from whey cultures of other strains. The importance of whey as a growth medium on the microbial composition of the whey cultures is discussed.     

Antibacterial Effect of Bovine Lactoferrin Against Udder Pathogens

The antibacterial effect of lactoferrin (Lf) was tested on isolates of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and coagulase-negative staphylococci (CNS) as well as on Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae), originally isolated from bovine mastitis. Concentrations of Lf used were 0.67 mg/ml, 1.67 mg/ml, and 2.67 mg/ml. Growth of udder pathogens was monitored by turbidometry either in broth culture or in whey prepared from normal milk. We focused on 3 different growth variables: lag time, slope, and maximum absorbance of bacterial growth curves. Growth inhibition was seen in the broth but hardly at all in whey. The isolates of E. coli and CNS did not grow sufficiently well in whey to draw any conclusions. The most effective inhibitory activity of Lf was seen against E. coli and P. aeruginosa. All 5 E. coli isolates had similar growth patterns. Inhibition of growth by Lf was concentration-dependent. The concentration of 0.67 mg/ml in broth and whey was generally too low for a significant inhibitory effect.