Identification of a Tyrosine Decarboxylase Gene ( tdcA ) in Streptococcus thermophilus 1TT45 and Analysis of Its Expression and Tyramine Production in Milk

Abstract

In this study, a tyrosine decarboxylase gene (tdcA) was identified in 1 among 83 Streptococcus thermophilus strains tested. Its sequence, nearly identical to that of a tdcA of Lactobacillus curvatus, indicated a horizontal gene transfer event. Transcription in milk and the formation of critical levels of tyramine were observed in the presence of tyrosine.Biogenic amine (BA) formation in fermented food has recently been included by the European Food Safety Authority (EFSA) among the safety concerns requiring evaluation. Among these compounds, tyramine is the most common in ripened cheeses and fermented sausages, where it usually accumulates during ripening and can be present in toxicologically relevant levels (Ͼ100 mg kg Ϫ1 ) (4). The symptoms of tyramine poisoning, also known as "cheese reaction," are hypertension due to peripheral vasoconstriction caused by the release of noradrenaline, pupil dilation, and increased respiration frequency and blood sugar level (22).Tyramine formation is the result of bacterial decarboxylation of tyrosine that is carried out by several microorganisms, including strains of lactic acid bacteria (LAB) belonging to different genera and species (7, 23). Among LAB, Streptococcus thermophilus is of major importance for the dairy industry, where it is commonly used in starter cultures for the manufacture of yogurt, fermented milks, and many varieties of cheese. Recently, a few strains of dairy origin assigned to the species S. thermophilus have been described for their tyraminogenic activity by both physiological assays (15) and consensus PCR on tyrosine decarboxylase genes (tdcA genes) (3). However, the identity of the genetic determinant involved was not unveiled.The aims of this study were (i) to screen the tyramine production of S. thermophilus in synthetic medium, (ii) to identify the gene responsible for this activity, (iii) to quantify its expression by quantitative real-time reverse transcription-PCR (RT-qPCR), and (iv) to monitor the levels of tyramine production in the presence of physicochemical parameters common to a cheese-making process in order to obtain indications on how to limit the formation of this BA.Screening of S. thermophilus strains for tyramine production. Eighty-three S. thermophilus dairy isolates, identified and genotypically characterized in previous studies (12, 20), were investigated. All strains were subcultured in LM17 medium, i.e., M17 medium (Fluka, Italy) supplemented with 0.5% (wt/vol) lactose, at 37°C for 24 h. Initially, the strains were screened for tyramine production according to the method of Bover-Cid and Holzapfel (2). Only the strain S. thermophilus 1TT45 (16S rRNA gene sequence has EMBL/GenBank/DDBJ accession no. FR725449), isolated from Taleggio cheese, gave a positive reaction in plates. Consensus tdcA-targeted PCR with the degenerate primers DEC5/DEC3 (23) on genomic DNA extracted with the method of Rossi et al. (21) from 2-ml cultures generated an amplicon of the expected size of close to 336 bp only from this strain (data not shown). T...

Quantitative Analysis of Histidine Decarboxylase Gene (hdcA) Transcription and Histamine Production by Streptococcus thermophilus PRI60 under Conditions Relevant to Cheese Making

“…This was chosen as a reference gene among those reported to be stably expressed in bacteria (23). The stability of expression of rpoA in S. thermophilus PRI60 was experimentally confirmed as described in a previous paper (13), with a maximum intertreatment variation of the transcript 2 ⌬CT ϭ 2.0. An RT-qPCR test was assessed for each target gene using the primer pairs STHDEC-F/STHDEC-R and RPOST-F/RPOST-R (Table 1), specific for hdcA and rpoA, respectively.…”

“…RNA extraction from skim milk cultures and reverse transcription for the RT-qPCR tests were carried out as previously described (13). Duplicate RT-qPCR trials were carried out amplifying hdcA and rpoA in separate sets of reactions on the same cDNAs.…”

Growth of Aerobic Ripening Bacteria at the Cheese Surface Is Limited by the Availability of Iron

“…The present study is also an example of gene expression measurements in cheese by real-time reverse transcription-PCR. Such analyses are increasingly used for a better understanding of the physiology of microorganisms and their expression of beneficial or undesirable properties during cheese production (1,12,13,15,20,24,29,31,33,34). …”

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

“…RNA extraction methods involving prior separation of the cells from cheeses and other dairy products have been used in several studies (Randazzo et al, 2002;Bleve et al, 2003;Sanchez et al, 2006;Bogovic Matijasic et al, 2007;Smeianov et al, 2007;Makhzami et al, 2008;Rantsiou et al, 2008a;Rantsiou et al, 2008b;Ulvé et al, 2008;Duquenne et al, 2010;Falentin et al, 2010;Cretenet et al, 2011;La Gioia et al, 2011;Masoud et al, 2011;Taïbi et al, 2011). The recovery of microbial cells is done following similar protocols than for DNA extraction methods (see above).…”