Beatriz del Rio

Factors influencing biogenic amines accumulation in dairy products

Fermented foods are among the food products more often complained of having caused episodes of biogenic amines (BA) poisoning. Concerning milk-based fermented foods, cheese is the main product likely to contain potentially harmful levels of BA, specially tyramine, histamine, and putrescine. Prompted by the increasing awareness of the risks related to dietary uptake of high biogenic amine loads, in this review we report all those elaboration and processing technological aspects affecting BA biosynthesis and accumulation in dairy foods. Improved knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in milk products. Synthesis of BA is possible only when three conditions converge: (i) availability of the substrate amino acids; (ii) presence of microorganisms with the appropriate catabolic pathway activated; and (iii) environmental conditions favorable to the decarboxylation activity. These conditions depend on several factors such as milk treatment (pasteurization), use of starter cultures, NaCl concentration, time, and temperature of ripening and preservation, pH, temperature, or post-ripening technological processes, which will be discussed in this chapter.

HPLC quantification of biogenic amines in cheeses: correlation with PCR-detection of tyramine-producing microorganisms.

The consumption of food and beverages containing high amounts of biogenic amines (BA) can have toxicological effects. BA found in foods and beverages are synthesized by the microbial decarboxylation of certain amino acids. This paper reports the concentrations of BAs in a number of commercial cheeses, as determined by HPLC. The cheeses studied were made from raw and pasteurized milk of different origin, and were subjected to different ripening periods. BA concentrations were lower in short ripening period than in long ripening period cheeses, and higher in cheeses made from raw milk than in those made from pasteurized milk. The highest BA concentrations were recorded in blue cheeses made from raw milk. Tyramine was the most commonly recorded and abundant BA. The presence of tyramine-producing bacteria was determined by PCR, and a good correlation obtained between the results of this method and tyramine detection by HPLC. These methods could be used to complement one another in the detection and quantification of tyramine in cheese prevention of tyramine accumulation in cheese.

Detection and Characterization of Streptococcus thermophilus Bacteriophages by Use of the Antireceptor Gene Sequence

ABSTRACT In the dairy industry, the characterization of Streptococcus thermophilus phage types is very important for the selection and use of efficient starter cultures. The aim of this study was to develop a characterization system useful in phage control programs in dairy plants. A comparative study of phages of different origins was initially performed based on their morphology, DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and lifestyles and on the presence of a highly conserved DNA fragment of the replication module. However, these traditional criteria were of limited industrial value, mainly because there appeared to be no correlation between these variables and host ranges. We therefore developed a PCR method to amplify VR2, a variable region of the antireceptor gene, which allowed rapid detection of S. thermophilus phages and classification of these phages. This method has a significant advantage over other grouping criteria since our results suggest that there is a correlation between typing profiles and host ranges. This association could be valuable for the dairy industry by allowing a rational starter rotation system to be established and by helping in the selection of more suitable starter culture resistance mechanisms. The method described here is also a useful tool for phage detection, since specific PCR amplification was possible when phage-contaminated milk was used as a template (detection limit, 105 PFU ml−1).

Comparative analysis of the in vitro cytotoxicity of the dietary biogenic amines tyramine and histamine

Tyramine and histamine, the most toxic biogenic amines (BA), are often found in high concentrations in certain foods. Prompted by the limited knowledge of BA toxicity, and increasing awareness of the risks associated with high intakes of dietary BA, the in vitro cytotoxicity of tyramine and histamine was investigated. Tyramine and histamine were toxic for HT29 intestinal cell cultures at concentrations commonly found in BA-rich food, as determined by real-time cell analysis. Surprisingly, tyramine had a stronger and more rapid cytotoxic effect than histamine. Their mode of action was also different, while tyramine caused cell necrosis, histamine induced apoptosis. To avoid health risks, the BA content of foods should be reduced and legal limits established for tyramine.

Multiplex Fast Real-Time PCR for Quantitative Detection and Identification of cos- and pac-Type Streptococcus thermophilus Bacteriophages

ABSTRACT The fermentation of milk by Streptococcus thermophilus is a widespread industrial process that is susceptible to bacteriophage attack. In this work, a preventive fast real-time PCR method for the detection, quantification, and identification of types of S. thermophilus phages in 30 min is described.

The putrescine biosynthesis pathway in Lactococcus lactis is transcriptionally regulated by carbon catabolic repression, mediated by CcpA.

Lactococcus lactis is the lactic acid bacterium most widely used by the dairy industry as a starter for the manufacture of fermented products such as cheese and buttermilk. However, some strains produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The proteins involved in this pathway, including those necessary for agmatine uptake and conversion into putrescine, are encoded by the aguB, aguD, aguA and aguC genes, which together form an operon. This paper reports the mechanism of regulation of putrescine biosynthesis in L. lactis. It is shown that the aguBDAC operon, which contains a cre site at the promoter of aguB (the first gene of the operon), is transcriptionally regulated by carbon catabolic repression (CCR) mediated by the catabolite control protein CcpA.

Impact of HLA mismatch direction on outcomes after umbilical cord blood transplantation for hematological malignant disorders: a retrospective Eurocord-EBMT analysis

HLA matching is a critical determinant of outcomes for patients who have undergone umbilical cord blood transplantation (UCBT). Data have been published on the importance of donor/recipient HLA mismatch direction on UCBT outcomes. HLA mismatch in the graft-versus-host (GVH) direction is defined as a donor homozygous at an HLA locus, while the recipient shares one HLA Ag with the donor. HLA mismatch in the host-versus-graft (HVG) direction is defined as a recipient homozygous with the donor sharing one HLA Ag. In our study we focused on confirming, using an independent population, whether transplantation outcomes would be different when HLA mismatch direction was considered. We analyzed 1565 patients who received a single-unit UCBT for malignant disease. Median age was 15 years and 72% of patients were transplanted for leukemia. In multivariate analysis, using the 5/6 HLA-matched population as reference, one or two HLA mismatches in the GVH or HVG direction were not associated with non-relapse related mortality and survival. On the basis of our results, there is no evidence to support a change in the current practice for cord blood unit selection.

The Relationship among Tyrosine Decarboxylase and Agmatine Deiminase Pathways in Enterococcus faecalis

Enterococci are considered mainly responsible for the undesirable accumulation of the biogenic amines tyramine and putrescine in cheeses. The biosynthesis of tyramine and putrescine has been described as a species trait in Enterococcus faecalis. Tyramine is formed by the decarboxylation of the amino acid tyrosine, by the tyrosine decarboxylase (TDC) route encoded in the tdc cluster. Putrescine is formed from agmatine by the agmatine deiminase (AGDI) pathway encoded in the agdi cluster. These biosynthesis routes have been independently studied, tyrosine and agmatine transcriptionally regulate the tdc and agdi clusters. The objective of the present work is to study the possible co-regulation among TDC and AGDI pathways in E. faecalis. In the presence of agmatine, a positive correlation between putrescine biosynthesis and the tyrosine concentration was found. Transcriptome studies showed that tyrosine induces the transcription of putrescine biosynthesis genes and up-regulates pathways involved in cell growth. The tyrosine modulation over AGDI route was not observed in the mutant Δtdc strain. Fluorescence analyses using gfp as reporter protein revealed PaguB (the promoter of agdi catabolic genes) was induced by tyrosine in the wild-type but not in the mutant strain, confirming that tdc cluster was involved in the tyrosine induction of putrescine biosynthesis. This study also suggests that AguR (the transcriptional regulator of agdi) was implicated in interaction among the two clusters.

The dietary biogenic amines tyramine and histamine show synergistic toxicity towards intestinal cells in culture

Tyramine and histamine are the biogenic amines (BA) most commonly found at high concentrations in food; they may even appear together at toxic concentrations. The present work examines, via real-time cell analysis, whether histamine and tyramine show synergistic toxicity towards intestinal cell cultures. Employing a constant equipotency ratio, their interaction was examined via the combination index (CI) method of Chou & Talalay. Co-treatment with tyramine and histamine was associated with a stronger cytotoxic effect than was treatment with either BA or on its own. Indeed, a synergistic interaction (CI<1) was observed in the range of concentrations found in foods. The results also show that histamine, at concentrations below the legal limit, increases the cytotoxicity of tyramine at concentrations frequently reached in some foods. The synergistic cytotoxicity of tyramine and histamine should be taken into account when establishing legal limits designed to ensure consumer safety.

Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase

Prolyl endopeptidases (PEP) (EC 3.4.21.26), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in the future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients.