The aim of this programme is the development and the optimization of a deoxyribonucleic acid (DNA) probe method for the detection of C. tyrobutyricum in milk.
Grass silage is the source of contamination of milk by Clostridium tyrobutyricum. The bacterium is responsible for butyric acid fermentation which leads to severe gas formation during cheese manufacturing (late blowing of cheese) and to abnormal cheese flavour.
Research was carried out with respect to the development and optimization of a deoxyribonucleic acid (DNA) probe method for the detection of C. tyrobutyricum in milk.
Initially 2 DNA probes specific of C. tyrobutyricum were determined. Research then developed to involve nonradioactive labelling of the probe, sample pretreatment and filtration, spore germination and colony growth, bacterial lysis and contaminated milk storage conditions.
To increase the hybridization signal, different probes containing more than 1 label per molecule were produced. Poly-dU-biotin and poly-dU-digoxigenin tailed oligodeoxyribonucleotides, and double stranded DNA fragment biotin or digoxigenin labelled by polymerase chain reaction (PCR) amplification were tested. The best results were obtained with the digoxigenin labelled probe obtained by PCR amplification of the V2 region of 16s ribosomal ribonucleic acid (rRNA).
The repeatability of the filtration procedure was assessed. Its effectiveness on milk samples of Dutch origin was verified. The positive effects of germination activators on recovery was cofirmed. Addition of oxyrase to establish anaerobic conditions did not improve the process.
For the constitution of a bank of contaminated milk samples, different storage methods were evaluated. The best and more convenient method was to filter the milk on the cellulose ester membranes and to store these membranes on agar at 4 C.
The first attempts to enumerate C. tyrobutyricum in artifically contaminated milk samples using the DNA probe method showed that it was necessary to improve the signal to noise ratio and to shorten the procedure.
Grass silage is the source of contamination of milk by Clostridium tyrobutyricum (1E4 to 1E8 spores per g). The bacterium is responsible for butyric acid fermentation which leads to severe gas formation during cheese manufacturing (late blowing of cheese) and to abnormal cheese flavour. This affects several types of cheese like Gouda, Edam, Emmental and Gruyere. For years the outgrowth of C. tyrobutyricum was fought in an effective way by the addition of small amounts of sodium nitrate to the milk. However the presence of spores became a serious problem again due to changes in the way of making grass silage and to new regulations limiting the use of sodium nitrate.
It is therefore necessary to discard highly contaminated milk batches and to look for contamination sources in the farms. But the microbiological enumeration methods are slow (4 to 6 days) and they are most probable number methods. They require highly experienced technicians. In addition the specificity for C. tyrobutyricum is far from perfect. There is a demand for a highly specific and rapid enumeration method.
The following experimental scheme is being used as a guideline all over the programme:
1. heat treatment and filtration of the sample;
2. germination of C. tyrobutyricum on specific medium;
3. cellular lysis and DNA fixation to the filter;
4. hybridization with a nonradioactively labelled DNA probe directed to the 16s ribosomal ribonucleic acid (rRNA).
This DNA probe technology will be compared to the traditional methods of detection of Clostridium species on samples obtained from the dairy industries. This comparison will permit an evaluation of this approach in terms of sensitivity, specificity, absence of false positive and reproducibility. This protocol will not only be adapted for the detection of C. tyrobutyricum in the milk before its transformation into cheese, but it will also be used for the determination of all sources of contamination in the farms, an unpractical task today.
Funding SchemeCSC - Cost-sharing contracts
6710 BA Ede