Identification and detection of Bacillus sporothermodurans spores in 1, 10, and 100 milliliters of raw milk by PCR.

A PCR method was developed to detect spores of Bacillus sporothermodurans in 1, 10, and 100 ml of raw milk. Two primers were derived from a unique sequence after subtractive hybridization of B. sporothermodurans DNA with DNA of MB 397, a not yet identified spore-forming bacterium isolated from raw milk, closely related to B. sporothermodurans. Specific identification was proven on a large collection of Bacillus strains and on strains from relevant taxa. The detection of B. sporothermodurans in raw milk is based on activation, germination, and outgrowth of the spores, followed by PCR identification. Spores from 10 and 100 ml were concentrated by centrifugation after chemical extraction of the milk components. The total test takes 28 h. The detection limits are 9, 0.4, and 0.22 CFU/ml for 1, 10, and 100 ml, respectively.

beta-agonists in animal feed. III: Optimization of the clean-up and the end-determination step.

This study represents the second part of an interlaboratory study intended to develop an official modular Community confirmatory method for the detection of beta-agonists in animal feed. Homogeneous pools of primary extracts were prepared by means of an extraction module based on the conclusions of a previous part of this work. The primary extracts were further processed by four laboratories each using a different clean-up scheme. The final extracts thus obtained were cross-distributed between the same laboratories and measured either by GCMS or HPLC. Two laboratories (B and D) applied separate clean-up schemes for clenbuterol and salbutamol. All clean-up schemes for clenbuterol were found to be compatible with all end-determination steps. In contrast, for salbutamol clean-up method D was found not to be compatible with the end-determination steps applied by laboratories B and C. The results of this study have clearly demonstrated that the clean-up methods for both clenbuterol and salbutamol applied by laboratory B yielded superior recoveries with an acceptable standard deviation. Therefore, in conclusion to this study, the participating laboratories recommend the clean-up schemes applied by laboratory B to serve as part of the official Community confirmatory method.

Beta-agonists in animal feed. II: Optimization of the extraction.

New projects of the European Commission, Measurement and Testing Programme (BCR) were set up in order to develop a modular sample preparation system for the determination of beta-agonists and animal feeds. Three phases are included: an extraction study, a clean-up study and finally a Second Intercomparison. This paper describes the extraction study in which four laboratories were involved. A total of 33 extraction conditions were tested regarding their yield on clenbuterol and salbutamol, their compatibility towards several clean-up and chromatographic end-methods and the influence of undesired coextractives. The conditions differed with respect to five factors: with or without organic solvent, temperature, pH, agitation and centrifugation. Their influence was examined via a ruggedness-test approach. A unique set-up allowed the combination of individual results in a complete factorial design. The addition of an organic solvent was found to be the most important factor. Interactions between factors were also studied. The best combinations of factors regarding the extraction are given. Finally limits for applicability and influence of organic solvents, pH and temperature were evaluated in a fifth laboratory towards enzyme immunoassay as detection method.

Direct detection of Listeria monocytogenes in 25 milliliters of raw milk by a two-step PCR with nested primers.

A method for direct detection of Listeria monocytogenes in 25 ml of raw milk is presented. The detection limit can be situated between 10 and 5 CFU. The detection method is based on chemical extraction of the milk components and PCR amplification with two nested pairs of primers specific for Listeria monocytogenes.

Routine limulus amoebocyte lysate (LAL) test for endotoxin determination in milk using a Toxinometer ET-201.

A rapid method of performing the Limulus amoebocyte lysate (LAL) test in milk is proposed using the Toxinometer ET-201. This instrument measured the increase in turbidity due to the interaction between the endotoxins of the Gram-negative bacteria and the LAL reagent, monitored the ratio Rt of the sequential to the initial transmission at 12 s intervals and quantified endotoxins by determination of the reaction time Tr required to obtain a 5% decrease in Rt. There was a good correlation between the toxinometrically determined endotoxin concentrations and the number of Gram-negative bacteria (SD, 0.18 log(plate count units)), and the repeatability (CV, 6-10%) was high. The assay may be useful for screening raw materials for UHT milk production, as the endotoxin content of the raw material is related to the rest proteinase activity in the UHT milk.

A comparison of the reproductive and growth performances of offspring from broiler breeder males selected for early growth rate using artificial insemination and unselected males kept on deep litter.

Broiler breeder males were selected for early growth rate at 5 weeks of age (average weight + 0.5 SD). The reproductive and growth performances of the offspring of these males using artificial insemination with stored semen was compared with those from non-selected males kept on deep litter under conditions of natural mating. On three different occasions (31, 41 and 51 weeks of age), the eggs of the two treatments were incubated and, although the reproductive performances showed little difference over the entire period, hatchability of eggs set was significantly (p less than 0.01) higher for the field hens on the first occasion. The opposite was true after 50 weeks of age due to a serious decline in fertility in natural mating. At all periods, the selected males produced significantly (p less than 0.01) heavier offspring at six weeks of age.

The response of CO2 exchange rate to photosynthetic photon flux density for several Populus clones under laboratory conditions.

No significant differences were found between four mathematical equations describing the response of CO2 exchange rate to photosynthetic photon flux density in seven poplar clones under laboratory conditions. Choice of an optimal equation for poplar may be based on the contemplated aims. High significant differences (at p<0.001) were found among the clones.

Early body-weight selection of broiler breeder males in relation to reproductive and growth performance of their offspring.

1. The effectiveness of selection of broiler breeder males for body weight at 3 weeks of age on later growth, semen quality and performance of progeny has been tested. 2. Correlation between 3- and 20-week body weights in the breeders was poor but significant. 3. On the basis of 3-week body weight males were divided into heavy birds (mean + 0.5 standard deviation) and all birds. 4. Semen quality was not different between the two groups, but some selection for semen quality was practised within groups. 5. There were no differences in fertility and hatchability of eggs produced from hens artificially inseminated with diluted, stored semen from both groups. 6. There was a small, positive, but non-significant, effect of selection of breeder males on body weight of progeny at 6 weeks of age.

Compounds determining pH in dry sausage.

The relative importance of various compounds determining pH in dry sausage was investigated using 'multiple linear regression' analysis. Analytical data were obtained for the concentrations of water, lactate, ammonia, acetate, α-amino nitrogen and salt related to sausage crude protein, using 55 different brands of dry sausage. The pH (Y) was significantly related to water, ammonia and lactate concentrations as follows: Y = 4·317 - 1·152X(1) + 0·751X(2) where X(1) = log(H(2)O) and X(2) = [log (ammonia)/(lactate) · 100] with (H(2)O) expressed as g/100g crude protein and (lactate) and (ammonia) as mmoles/100 g crude protein. The results suggest that the increase in pH often observed in the later stages of ripening is related to a decrease in electrolyte dissociation and/or an increase in the concentration of buffering proteins as well as to the formation of ammonia.