Mika Tuomola

Influence of chicory roots ( Cichorium intybus L ) on boar taint in entire male and female pigs

Boar taint is an off-flavour of pork caused primarily by a microbial breakdown product, skatole and a testicular steroid, androstenone. As skatole is produced in the large intestine from tryptophan, it is possible that some ‘bioactive’ ingredients could modify protein fermentation and, in the process, diminish boar taint. The aim of this study was to examine the effect of inulin-rich chicory roots (Cichorium intybus L.) on boar taint. In the first of three trials individually penned, entire males and females were given an organic concentrate in which 0·25 of the daily energy intake was replaced with crude chicory roots for 9 or 4 weeks prior to slaughter. In the second trial, entire male pigs were given diets that included, either crude chicory roots, dried chicory roots, or inulin (extracted from chicory roots) for 6 weeks pre-slaughter. In the third trial, intact male pigs were given the dried chicory diet for either 2 or 1 week before slaughter. In all trials the chicory diets were offered on a scale at 0·95 of the Danish recommendation for energy intake, and pig performance was compared with a control group given the organic concentrate at 0·95 of recommended energy intake plus silage ad libitum. In trial 1 an additional control group was offered the organic concentrate at a daily energy intake level of 1·0 of Danish recommendations. The pigs in trials 1, 2, and 3 were slaughtered at an average live weight of 118, 124, and 110kg, respectively, in order to ensure that they had achieved sexual maturity. Overall, skatole concentrations in blood plasma and backfat at slaughter were reduced to almost zero levels by including crude or dried chicory or inulin in the diet. This occurred irrespective of sex and length of feeding period (1 to 9 weeks). In trial 3 a significant effect on blood plasma concentration was observed after 3 days of feeding a diet containing dried chicory. The only significant reduction in plasma androstenone levels was detected in pigs given the crude chicory for a 9 week duration in trial 1. The production and proportion of lean was generally not affected by the addition of either form of chicory to the diets in trials 1 and 2. Therefore, dried chicory may be the most suitable form for commercial use because it: had no initial adverse effects on food intake, consistently reduced skatole without reducing performance, was easy to handle throughout the entire year and is relatively inexpensive.

An 8-hour system for Salmonella detection with immunomagnetic separation and homogeneous time-resolved fluorescence PCR

We describe a system consisting of rapid sample enrichment and homogeneous end-point PCR analysis that enables the detection of Salmonella in various food matrices in 8 h. Sample preparation starts with 6 h enrichment step in supplemented broth, after which Salmonella cells are collected with immunomagnetic particles. The particles are washed and dispensed to ready-to-use PCR reaction vessels, which contain dried assay-specific reagents and an internal amplification control. PCR is performed with a novel instrument platform utilising the sensitive label technology of time-resolved fluorometry. Qualitative assay results are automatically interpreted and available in 45 min after sample addition. The overall accuracy, sensitivity and specificity of the Magda CA Salmonella system were 99.1%, 98.4% and 100.0%, respectively, based on the evaluation of 107 samples (beef, pork, poultry and ready-to-eat meals) artificially contaminated with sub-lethally injured Salmonella cells.

A specificity-enhanced time-resolved fluoroimmunoassay for zeranol employing the dry reagent all-in-one-well principle

A simple dry chemistry time-resolved fluorescence immunoassay (TR-FIA) method was developed for the measurement of zeranol in bovine urine samples. The samples were purified by immunoaffinity chromatography and a specificity-enhanced zeranol antibody was employed in the immunoassay. This resulted in a highly selective method, which had only negligible reactivity with Fusarium spp. toxins. The all-in-one-well dry chemistry concept made the assay very simple to use because all the assay-specific reagents were already present in the reaction wells in dry form. Only the addition of diluted sample extract was required to perform the competitive one-step TR-FIA and the results were available in less than 1 h. The analytical limit of detection (mean + 3s) for the immunoassay was 0.16 ng ml(-1) (n = 12) and the functional limit of detection for the whole method, estimated by the analysis of zeranol-free samples, was 1.3 ng ml(-1) (n = 20). The recovery of zeranol at the level of 2 ng ml(-1) was 99% (n = 18) and the within-assay variation ranged between 4.5 and 9.0%.

Determination of androstenone in pig fat using packed column supercritical fluid chromatography–mass spectrometry

Packed column supercritical fluid chromatography (SFC) in combination with atmospheric pressure chemical ionisation mass spectrometry was applied to the analysis of androstenone in pig fat samples. Liquefied fat samples were dissolved in dichloromethane and analysed directly by SFC without any sample purification. Chromatographic separation was achieved with a density/pressure gradient using pure carbon dioxide as the mobile phase and the analysis resulted in a quantitation limit of 0.25 microg/g with 1 microl injection volume. Good agreement was found between the SFC method and time-resolved fluoroimmunoassay by the analysis of 15 boar back fat samples.

Production and characterisation of monoclonal antibodies against a very small hapten, 3-methylindole.

Monoclonal antibodies were produced against a very small (131.2 Da) hapten, 3-methylindole. Nine derivatives of 3-methylindole were synthesised with spacers ending in a carboxyl group, and coupled to immunogenic carriers and europium chelate labels. Almost all the antigens elicited an antihapten response, but the majority of the mAbs produced strongly recognised the spacer group and did not bind free 3-methylindole. However, specific antibodies were obtained with five immunogens. Specificity could be directed against the pyrrole ring by locating the bridging group to the aromatic moiety of the indole ring system. Any modification in the position 3 of the indole ring strongly hindered mAb binding to the compound, and the cross-reactivity of physiologically important compounds, such as tryptophan and tryptamine, was negligible for all of the mAbs. The developed hapten structures successfully focused antibody recognition to the important sub-determinants in the indole ring system. Similar constructs could also be useful in the development of antibodies against other indolic compounds.

Monitoring androstenone levels in boars by direct immunochemical analysis of serum samples.

The possibility of using blood samples for screening high levels of boar taint steroid androstenone (5α-androst-16-en-3-one) was studied both in living animals at the farm and carcasses at the slaughterhouse. The steroid was measured from boar serum and fat samples with a simple time-resolved fluoroimmunoassay. Fat samples contained androstenone in the range of 90-7500 ng/g (n=214), and 74.8% of the samples exhibited fat androstenone levels above 500 ng/g. Androstenone concentrations in blood samples were measured by direct serum assay and ranged up to 215 ng/ml (n=214). The levels of androstenone were correlated (r=0.78-0.88, P<0.001) between the serum and fat samples obtained at slaughter and serum samples taken at the farm 7-11 days before slaughter. A direct serum analysis seems to give a reliable indication of the androstenone level in fat and it can also be used in the screening of living animals.

Development and validation of dry reagent time-resolved fluoroimmunoassays for zeranol and α -zearalenol to assist in distinguishing zeranol abuse from Fusarium spp. toxin contamination in bovine urine

Zeranol, an oestrogenic growth promoter in food animals, is banned within the European Union (EU). However, commercially available immunoassay kits for zeranol cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the validation of a specificity enhanced, rapid dry reagent time-resolved fluoroimmunoassay (TR-FIA) for zeranol (recovery 99%, limit of detection 1.3 ng ml-1) demonstrating that up to 150 ng ml-1 of Fusarium spp. toxins in urine do not lead to false-positive results. This assay will assist EU Member States to implement Council Directive 96/23/EC, which requires states to monitor for potential abuses of zeranol. A similar TR-FIA for the Fusarium spp. toxin α-zearalenol, using the same sample extract, is also described (recovery 68%, limit of detection 5.6 ng ml-1). Only the addition of diluted sample extract is required to perform these dry-reagent TRFIAs, the results being available within 1h of extract application. The EU-funded project ‘Natural Zeranol’ (FAIR5-CT97-3443) will use these fluoroimmunoassays to screen bovine urine in four Member States to gather data on the seasonality of Fusarium spp. toxin contamination of urine and the incidence of zeranol screening test positives.