Fast detection of tyramine-producing Lactic Acid Bacteria

Kurzfassung

A Spanish public research institution has developed a fast, sensitive and easy method to detect, in any point of the food chain, tyramine-producing strains. Lactic Acid Bacteria are essential in the dairy industry as starters; nevertheless, the metabolic activity of some strains can produce toxic substances known as biogenic amines and tyramine is the most frequent and concentrated in cheeses. A biotechnological company is required to license or to collaborate to reach a kit for detection.

Details

Biogenic amines are organic bases of low molecular weight that are formed via decarboxylation of their corresponding amino acids through the action of enzymes produced by microorganisms present in the food. Producer strains can appear as contaminants of fermented foods but they can also form part of starter cultures. Ingestion of foods with high levels of biogenic amines causes serious upheavals, that could even jeopardizes the life of the consumer, specially in those people with deficiencies in the intestinal amino-oxidase activity, the detoxification enzyme. Furthermore, biogenic amines are precursors of well-known carcinogenics as nitrosamines. AB production is more related to strain than species. It is therefore very important to determine which strains produce these undesirable compounds, so that these do not form part of starter cultures. Although several qualitative and quantitative methods have been developed to determine Biogenic amines production, consumer demand for better and healthier foods has increased interest in the development of rapid and sensitive methods for detecting biogenic amine producers in food.
The present invention allows fast and sensitive identification of tyramine producing Lactic Acid Bacteria by PCR (Polymerase Chain Reaction), using specific tdcA primers. The gene tdcA encodes tyrosine decarboxylase, the enzyme that catalyses the synthesis of tyramine. The comparison of the tdcA sequence from Lactococcus lactis IPLA10000 with those held in databases, led to the design of specific oligonucleotides that amplified an internal fragment of the tdcA gene of different genera, allowing the identification of tyramine-producing Lactic Acid Bacteria. Since it is unnecessary to purify the DNA of the strains to be tested (an isolated colony can be used as a template) the proposed method is fast and easy. Moreover, the proposed PCR technique offers the possibility of detecting potential tyramine-producing strains in milk, at any point in milk fermentation, and in final products such as cheese.

Innovative Aspects:
At the present time, two types of detection methods are being used for biogenic amines detection:
- Microbiological. They are based on the growth of microorganisms in differential media with a pH indicator and the decarboxylation substrate amino acid that gives rise to the corresponding biogenic amines. Their main disadvantages are the difficulty for some biogenic amines producing Lactic Acid Bacteria to grow in these media, and the long periods of incubation (72 hours).
- Chromatographic techniques (HPLC). In this case it is necessary to consider that production of these compounds is induced by environmental conditions, such as the presence of the substrate amino acid or the pH. For this reason, a negative result cannot guarantee the security of the strain since in different conditions it could produce AB that would be accumulated in the food or corresponding drink. Furthermore, the detection is realised at the end of the production process, making it impossible to avoid the loss of the product and the subsequent negative economic impact.

The proposed procedure, besides resolving these drawbacks, has the following advantages:·
- Specificity, since the oligonucleotide starters used in the PCR are specific for tdcA, the gene responsible for tyramine synthesis.
- Simplicity. In general, PCR is a very simple technique and in this case, it has been so optimised that it is not even necessary to isolate the genetic material to use as template. An isolated colony or even milk, in any point of fermentation, can be used in the reaction.
- Sensitivity. Lactic Acid Bacteria produce HPLC (High Pressure Liquid Chromatography)-detectable tyramine concentrations when present at densities of 107 cfu ml-1 or greater, but this PCR method is much more sensitive, allowing tyramine-producing strains to be detected at 104 cfu ml-1.
- Speed. In less than 4 hours it can be known if a strain is a tyramine potential producer or if a tyramine producing strain is present in a food sample. The food companies must guarantee the sanitary quality of their products, and therefore they must avoid the presence of biogenic amines in foods. Moreover, the consumers demand greater sanitary control of the foods that they consume. For this reason, the better control mechanisms they apply, and the more competitive they are. The developed method has advantages with respect the conventional ones, such as greater specificity and sensitivity, which contributes to greater security of produced foods, and therefore greater competitiveness. On the other hand, it is easier, making highly qualified personnel unnecessary for his application, and faster, something that in the industry always reduce manufacturing costs.