Biosensor assay for determination of haptoglobin in bovine milk.

Despite more than 30 years of research into mastitis diagnostics, there are few alternatives to the somatic cell count (SCC) in practical use for identification of cows with subclinical mastitis. Mastitis is not only an animal welfare problem, but also affects the yield, composition and technological properties of milk. Hence, dairy cooperatives give farmers a premium quality payment to encourage low SCC although there is no clear scientific data defining the level of SCC in bulk tank milk that is associated with additional benefits in terms of milk quality. Recent research on alternative markers for inflammatory reactions in the lactating cow, e.g. in mastitis, includes investigations of the acute phase protein, haptoglobin (Hp). So far, the content of Hp in milk has mainly been studied in relation to mastitis diagnostics, with little attention given to its importance for milk composition and technological properties. At present, Hp in milk is measured using ELISA, but this technique is not suitable for routine large-scale analysis. In recent years, optical biosensor technology has been used for automated and rapid quantitative analysis of different components in milk, but so far not for analysis of acute phase proteins. The aim of the present study was to develop a rapid and sensitive biosensor method to determine Hp in milk. An affinity sensor assay based on the interaction between Hp and haemoglobin was developed using surface plasmon resonance (SPR) biosensor technology. The assay was used to analyse Hp in composite milk samples from cows without any clinical signs of mastitis and quarter milk samples with a weak to strong reaction in the California Mastitis Test (CMT). A commercial ELISA for determination of Hp in milk was used for comparison. The limit of detection (LOD) of the biosensor assay was determined as 1.1 mg/l. Within-assay and between-day variations were determined both with bulk tank milk spiked with human Hp and with composite milk samples containing bovine Hp. Coefficients of variation varied between 3.6 and 8.6% at concentrations between 4.0 and 12 mg/l, respectively. Agreement between the results obtained by the biosensor assay and the ELISA was satisfactory; however, the results obtained by the biosensor were generally lower than the results obtained by the ELISA. Possible explanations for this observation are discussed.

Evaluation of cooling strategies for pumping of milk - impact of fatty acid composition on free fatty acid levels.

Cooling strategies for pumping of raw milk were evaluated. Milk was pumped for 450 s at 31 degrees C, or pumped after cooling to 4 degrees C and subsequently subjected to various incubation times. Two types of milk were used; i.e. milk from cows fed a diet high in saturated fat supplements resulting in significantly larger milk fat globules than the other type of milk which comes from cows fed a low-fat diet that stimulates high de novo fat synthesis. The content of liquid fat was determined by low-field 1H NMR, which showed that milk from cows given the saturated fat diet also contained less liquid fat at both 4 degrees and 31 degrees C than the other type of milk. This can be ascribed to the differences in the fatty acid composition of the milk as a result of the fatty acid composition of the diets. After pumping of the milk at 31 degrees C, measurement of fat globule size distribution revealed a significant coalescence of milk fat globules in the milk obtained from the saturated fat diet due to pumping. Pumping at 4 degrees C or pumping the other type of milk did not result in coalescence of milk fat globules. Formation of free fatty acids increased significantly in both types of milk by pumping at 31 degrees C. Cooling the milk to 4 degrees C immediately before pumping inhibited an increased content of free fatty acids. However, when the milk was incubated at 4 degrees C for 60 min after cooling and then subjected to pumping, a significant increase in the formation of free fatty acids was observed in both types of milk. It is suggested that this increase in free fatty acids is caused by transition of polymorphic crystal forms or higher level of attached lipoprotein lipases to the milk fat globule before pumping.

Affinity and rate constants for interactions of bovine folate-binding protein and folate derivatives determined by optical biosensor technology. Effect of stereoselectivity.

The interactions between bovine folate-binding protein (FBP) and different folate derivatives in pure diastereoisomeric forms were studied at pH 7.4 by a surface plasmon resonance technology (Biacore). The results show that folic acid had the most rapid association rate (k(a) = 1.0 x 10(6) M(-)(1) s(-)(1)), whereas (6S)-5-HCO-5,6,7,8-tetrahydrofolic acid had the most rapid dissociation rate (k(d) = 3.2 x l0(-)(3) s(-)(1)). The equilibrium dissociation constant (K(D)), calculated from the quotient of k(d)/k(a), showed that the two forms of folates not occurring in nature, that is, folic acid and (6R)-5-CH(3)-5,6,7,8-tetrahydrofolic acid, had the highest affinities for FBP, 20 and 160 pmol/L, respectively. The results thus show that there were great differences in the interactions between folate-binding protein and the major forms of folate derivatives. The nutritional implications of these differences are discussed.

Comparison of Biosensor, Microbiological, Immunochemical, and Physical Methods for Detection of Sulfamethazine Residues in Raw Milk.

A biosensor assay based on biospecific interaction analysis (BIA) was compared with already existing methods for detection of sulphamethazine (SMZ) residues in milk. Microbial inhibitor and receptor assays, an enzyme-linked immunosorbent assay (ELISA), high-pressure liquid chromatography (HPLC), and BIA were used to analyze milk samples from SMZ-treated cows. The results of the commercially available tests (Delvotest SP Special, BR-test Blue Star, Charm II test) were in agreement with the claimed sensitivity of the respective assays. The agreement between the quantitative methods (ELISA, HPLC, BIA) varied. The microbial inhibitor assays and BIA were also used to screen 330 tanker milk samples, All samples were negative in the inhibitor tests, whereas the BIA indicated the occurrence of less than 0.9 µg of SMZ per kg of milk in 5 samples and 1.5 ± 0.6 µg/kg in one sample, HPLC indicated the presence of SMZ in the latter sample, although the concentration was below the detection limit of the method. The advantages offered by the BIA: no sample preparation, high sensitivity, and rapid, fully automated analysis in real time make the technology an interesting alternative to existing screening methods within future food-quality control systems.

Liquid Chromatography Verification of Tetracycline Residues in Milk and Influence of Milk Fat Lipolysis on the Detection of Antibiotic Residues by Microbial Assays and the Charm II Test.

Farm milk samples, which were positive in the routine assay for inhibitory substances detected by the Arla microtest but negative in the Delvotest SP, were used in the study. All samples analyzed gave positive results in the determination of tetracyclines and macrolides by the Charm II microbial receptor tests. To confirm the presence of tetracyclines, liquid chromatography and the Charm II immuno-receptor test were used. All samples, except one, showed negative results in the analyses by both these methods. This suggested that some other factor(s) was causing the observed positive results for tetracyclines and macrolides. Circumstantial evidence indicated that free fatty acids (FFA) may be a causative factor. Consequently, the influence of FFA on the Arla microtest, Valio T101, Delvotest SP, and the tetracycline and macrolide determinations by the Charm II test was investigated. Lipolysis of milk fat was induced by the addition of human blood serum, followed by storage for 24 and 48 h at 4°C. Samples were found to be inhibitory in the Arla microtest and the Valio T101 test at FFA levels of 4-5 mM, whereas the results of the Delvotest SP remained negative. The Charm II counts for tetracyclines and macrolides as determined by the microbial receptor tests were also highly correlated with FFA. It was concluded that lipolysis of milk fat may cause positive results in the microbial nonagar assays and interfere in the confirmation by the Charm II test.

Charm Test II for Confirmation of Inhibitory Substances Detected by Different Microbial Assays in Herd Milk.

In Sweden, milk delivered to dairies is assayed with either the Arla microtest or the Delvo test P for the presence of inhibitory substances. A positive result has to be verified with the Delvo test P to be considered positive. In the present investigation, Charm test II was used for confirmation analysis of approximately 300 milk samples positive in the routine assays. The samples were analyzed for the presence of beta-lactams, tetracyclines, and aminoglycosides. In addition, the samples were analyzed with the Valio T 101 test. Approximately 90% of the samples, which were positive in the verification with Delvo test P, were positive for beta-lactams in the Charm test II. Of the samples, which were found positive by the Arla microtest, 37% were negative in the verification with Delvo test P. The majority of these samples were found to be positive for tetracyclines in Charm test II. The T 101 test showed a different pattern of sensitivity compared to the Delvo test P and Arla microtest. Of the samples which were positive in the verification with Delvo test P, only 68% were positive in the T 101 test, while 49% of the samples which were positive in the Arla microtest but negative in the verification with Delvo test P were positive in the T 101 test. Most of the latter samples, i.e., 95%, were positive for tetracyclines in the Charm test II. It can be concluded that the microbial assays used have quite different patterns of sensitivity toward inhibitory substances in milk and that confirmation with Charm test II gives a high degree of clarification of antibiotic substances present.

Antibacterial Effect of the Lactoperoxidase/Thiocyanate/Hydrogen Peroxide System Against Strains of Campylobacter Isolated from Poultry.

The antibacterial effect of the lactoperoxidase/thiocyanate/hydrogen peroxide system (lactoperoxidase system) was tested against strains of Campylobacter jejuni and Campylobacter coli isolated from poultry. The effect was studied at different pH-values and temperatures in UHT-milk (control); UHT-milk containing lactoperoxidase, sodiumthiocyanate and hydrogen peroxide (lactoperoxidase system); UHT-milk containing lactoperoxidase, sodiumthiocyanate, hydrogen peroxide and sodiumpyrosulfite (inactivated lactoperoxidase system). The lactoperoxidase system had a strong bactericidal effect against C. jejuni and C. coli . The bactericidal effect was more rapid at 37°C compared to 20°C. The effect of lactoperoxidase system decreased with decreasing pH-values. The fastest reduction in viable numbers was obtained at pH 6.6 and 37°C.