DESCRIPTION

BA are organic bases of low molecular weight that are formed via descarboxylation 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 AB causes serious upheavals, that could even jeopardizes the life of the consumer, specially in those people with deficiencies in the intestinal amino-oxidasa activity, the detoxification enzyme. Furthermore, AB 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 BA production, consumer demand for better and healthier foods has increased interest in the development of rapid and sensitive methods for detecting BA producers in food.

The present invention allows fast and sensitive identification of tyramine producing BAL by PCR, using specific tdcA primers. The gene tdcA encodes tyrosine descarboxilase, the enzyme that catalyzes the synthesis of tyramine. The comparison of the tdcA sequence from Lc. 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 LAB. 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 AB detection:

· Microbiological. They are based on the growth of the microorganisms in differential media with a pH indicator and the descarboxilation substrate amino acid that gives rise to the corresponding AB. Their main disadvantages are the difficulty of some AB producing BAL 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, as the presence of the substrate amino acid or the pH. For this reason, a negative result can not 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 realized at the end of the productive process being impossible to avoid the loss of the product and the consequent negative economic impact.

The proposed procedure, besides to resolve these inconvenients, have the following advantages:

· Specificity, since the oligonucleotide starters used in the PCR are specific for tdcA, the gene responsible of the tyramine synthesis.

· Simplicity. In general, PCR is a very simple technique and in this case, it has been so optimized, that 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. LAB produce HPLC-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 can be known if a strain is a tyramine potential producer or if a tyramine producing strain is present in a food sample.

COMPETITIVE ADVANTAGES

The food companies must guarantee the sanitary quality of their products and therefore they must avoid the presence of AB in foods. Moreover, the consumers demands a greater sanitary control of the foods that consume. For this reason, the better control mechanisms they apply, the more competitive they are. The developed method has advantages respect the conventional ones, such as greater specificity and sensitivity, which contributes to a greater security of produced foods and therefore a greater competitiveness. On the other hand, it is easier, making unnecessary highly qualified personnel for his application, and faster, something that in the industry always reduce manufacturing cost.

KEYWORDS

Lactic acid bacteria, biogenic amines, tyramine, tyrosine decarboxylase gene, PCR detection

PATENT

P200302572 2003-11-04

CONTACT

Estrella Maroto 
email: e.maroto@orgc.csic.es
phone: +34 91 585 52 51

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