Analytical strategies for the determination of biogenic amines in dairy products

Biogenic amines (BA) are mainly produced by the decarboxylation of amino acids by enzymes from microorganisms that emerge during food fermentation or due to incorrectly applied preservation processes. The presence of these compounds in food can lead to a series of negative effects on human health. To prevent the ingestion of high amounts of BA, their concentration in certain foods needs to be controlled. Although maximum legal levels have not yet been established for dairy products, potential adverse effects have given rise to a substantial number of analytical and microbiological studies: they report concentrations ranging from a few mg/kg to several g/kg. This article provides an overview of the analytical methods for the determination of biogenic amines in dairy products, with particular focus on the most recent and/or most promising advances in this field. We not only provide a summary of analytical techniques but also list the required sample pretreatments. Since high performance liquid chromatography with derivatization is the most widely used method, we describe it in greater detail, including a comparison of derivatizing agents. Further alternative techniques for the determination of BA are likewise described. The use of biosensors for BA in dairy products is emerging, and current results are promising; this paper thus also features a section on the subject. This review can serve as a helpful guideline for choosing the best option to determine BA in dairy products, especially for beginners in the field

Identification by means of molecular tools of the microbiota responsible for the formation of histamine accumulated in commercial cheeses in Spain

Histamine intoxication is an important food safety and public health concern. Ripened cheeses are the most common dairy products in which histamine can accumulate. Histamine is formed by the microbiota present in cheese by decarboxylation of histidine, due to the action of the histidine decarboxylase. This study's objective was to identify the responsible for the formation of histamine accumulated in commercial cheeses. The content of histamine of 39 different types of cheeses marketed in Spain, of varying milk origin, was assessed. About one third of the cheeses analysed contained more than 200 mg/kg histamine; two cheeses exceeded 500 mg/kg histamine, the consumption of such cheeses can be harmful or even toxic for consumers. The five cheeses with the highest histamine concentrations were selected for in-depth molecular analysis. Firstly, bacterial and yeast isolates were obtained, and then the total genetic material from the cheeses was analysed, in order to verify the putative presence of the hdc histidine decarboxylase gene. In order to identify the histamine producing microorganisms, the nucleotide sequences of the histidine decarboxylase genes from the cheeses were amplified, and subjected them to Sanger sequencing. In four of the five selected cheeses, the main histamine producer was identified as Lentilactobacillus parabuchneri, whereas in the remaining cheese it was Tetragenococcus halophilus. The hdc gene was located in an unstable plasmid, only present in that cheese sample. Since all histamine producing microorganisms identified in this study are not part of the species used in cheese starter cultures, an improvement of hygienic manufacturing practices and/or thermal treatments for microbial inactivation in milk may be considered to prevent histamine accumulation in cheeses during ripening

Potential of histamine-degrading microorganisms and diamine oxidase (DAO) for the reduction of histamine accumulation along the cheese ripening process

Lentilactobacillus parabuchneri is the main bacteria responsible for the accumulation of histamine in cheese. The goal of this study was to assess the efficiency of potential histamine-degrading microbial strains or, alternatively, the action of the diamine oxidase (DAO) enzyme in the reduction of histamine accumulation along the ripening process in cheese. A total of 8 cheese variants of cow milk cheese were manufactured, all of them containing L. parabuchneri Deutsche Sammlung von Mikroorganismen 5987 (except for the negative control cheese variant) along with histamine–degrading strains (Lacticaseibacillus casei 4a and 18b; Lactobacillus delbrueckii subsp. bulgaricus Colección Española de Cultivos Tipo (CECT) 4005 and Streptococcus salivarius subsp. thermophilus CECT 7207; two commercial yogurt starter cultures; or Debaryomyces hansenii), or DAO enzyme, tested in each cheese variant. Histamine was quantified along 100 days of cheese ripening. All the degrading measures tested significantly reduced the concentration of histamine. The highest degree of degradation was observed in the cheese variant containing D. hansenii, where the histamine content decreased up to 45.32 %. Cheese variants with L. casei, or L. bulgaricus and S. thermophilus strains, also decreased in terms of histamine content by 43.05 % and 42.31 %, respectively. No significant physicochemical changes (weight, pH, water activity, color, or texture) were observed as a consequence of the addition of potential histamine-degrading adjunct cultures or DAO in cheeses. However, the addition of histamine-degrading microorganisms was associated with a particular, not unpleasant aroma. Altogether, these results suggest that the use of certain histamine-degrading microorganisms could be proposed as a suitable measure in order to decrease the amount of histamine accumulated in cheeses. © 2022 The Author

The significance of cheese sampling in the determination of histamine concentration: Distribution pattern of histamine in ripened cheeses

Cheeses are becoming a major safety and public health concern: cheeses available in supermarkets occasionally contain high histamine concentrations that can have negative effects on consumer health. In this study, we have attempted to assess the histamine distribution pattern in ripened cheeses, with the purpose of establishing a correct cheese sampling strategy for the quantification of histamine. To this aim, histamine was determined in four distinct areas of twelve long-ripened hard cheeses: the external and internal rind, along with the outer and inner core of the wedge. The concentrations measured were remarkably different: histamine accumulated in the central core, whereas the lowest amount was found in the peripheral rind. To explain this heterogenous distribution, histamine producers were determined in the four areas by identifying the hdc sequences obtained from cheese samples. Non-starter bacteria were identified as main histamine producers; however, these microbiota were homogeneously distributed throughout the wedge. Nevertheless, the analysis of psychochemical properties of the different areas revealed an observable trend: histamine tended to accumulate in the saltier, more humid, and less oxidized areas in a wedge. Overall, this study highlights the significance of a correct sampling strategy when histamine is quantified in cheese

Protein S-Bacillithiolation Functions in Thiol Protection and Redox Regulation of the Glyceraldehyde-3-Phosphate Dehydrogenase Gap in Staphylococcus aureus Under Hypochlorite Stress

Aims: Bacillithiol (BSH) is the major low-molecular-weight thiol of the human pathogen Staphylococcus aureus. In this study, we used OxICAT and Voronoi redox treemaps to quantify hypochlorite-sensitive protein thiols in S. aureus USA300 and analyzed the role of BSH in protein S-bacillithiolation.  Results: The OxICAT analyses enabled the quantification of 228 Cys residues in the redox proteome of S. aureus USA300. Hypochlorite stress resulted in >10% increased oxidation of 58 Cys residues (25.4%) in the thiol redox proteome. Among the highly oxidized sodium hypochlorite (NaOCl)-sensitive proteins are five S-bacillithiolated proteins (Gap, AldA, GuaB, RpmJ, and PpaC). The glyceraldehyde-3-phosphate (G3P) dehydrogenase Gap represents the most abundant S-bacillithiolated protein contributing 4% to the total Cys proteome. The active site Cys151 of Gap was very sensitive to overoxidation and irreversible inactivation by hydrogen peroxide (H2O2) or NaOCl in vitro. Treatment with H2O2 or NaOCl in the presence of BSH resulted in reversible Gap inactivation due to S-bacillithiolation, which could be regenerated by the bacilliredoxin Brx (SAUSA300_1321) in vitro. Molecular docking was used to model the S-bacillithiolated Gap active site, suggesting that formation of the BSH mixed disulfide does not require major structural changes.  Conclusion and Innovation: Using OxICAT analyses, we identified 58 novel NaOCl-sensitive proteins in the pathogen S. aureus that could play protective roles against the host immune defense and include the glycolytic Gap as major target for S-bacillithiolation. S-bacillithiolation of Gap did not require structural changes, but efficiently functions in redox regulation and protection of the active site against irreversible overoxidation in S. aureus. Antioxid. Redox Signal. 28, 410–430

Occurrence of mutations impairing sigma factor B (SigB) function upon inactivation of Listeria monocytogenes genes encoding surface proteins.

Bacteria of the genus Listeria contain the largest family of LPXTG surface proteins covalently anchored to the peptidoglycan. The extent at which these proteins may function or be regulated cooperatively is at present unknown. Because of their unique cellular location, we reasoned that distinct LPXTG proteins could act as elements contributing to cell wall homeostasis or influencing the stability of other surface proteins bound to peptidoglycan. To test this hypothesis, we analysed by proteomics mutants of the intracellular pathogen L. monocytogenes lacking distinct LPXTG proteins implicated in pathogen-host interactions as InlA, InlF, InlG, InlH, InlJ, LapB and Vip. Changes in the cell wall proteome were found in inlG and vip mutants, which exhibited reduced levels of the LPXTG proteins InlH, Lmo0610, Lmo0880 and Lmo2085, all regulated by the stress-related sigma factor SigB. The ultimate basis of this alteration was uncovered by genome sequencing, which revealed that these inlG and vip mutants carried loss-of-function mutations in the rsbS, rsbU and rsbV genes encoding regulatory proteins that control SigB activity. Attempts to recapitulate this negative selection of SigB in large series of new inlG or vip mutants constructed for this purpose were however unsuccessful. These results indicate that inadvertent secondary mutations affecting SigB functionality can randomly arise in L. monocytogenes when using widely used genetic procedures or during sub-culturing. Testing of SigB activity could be therefore valuable when manipulating genetically L. monocytogenes prior to any subsequent phenotypic analysis. This test may be even more justified when generating deletions affecting cell envelope components.Fil: Quereda, Juan J.. Consejo Superior de Investigaciones Cientificas. Centro Nacional de Biotecnologia; EspañaFil: Graciela Pucciarelli, M.. Consejo Superior de Investigaciones Cientificas. Centro Nacional de Biotecnologia; España. Universidad Autónoma de Madrid. Departamento de Biología Molecular. Centro de Biología Molecular "Severo Ochoa"; EspañaFil: Botello Morte, Laura. Consejo Superior de Investigaciones Cientificas. Centro Nacional de Biotecnologia; EspañaFil: Calvo, Enrique. Centro Nacional Investigaciones Cardiovasculares; EspañaFil: Carvalho, Filipe. Universidade do Porto. Instituto de Biologia Molecular e Celular. Group of Molecular Microbiology; PortugalFil: Bouchier, Christiane. Institut Pasteur. Département Génomes et Génétique. Plate-forme PF1 Génomique; FranciaFil: Vieira, Ana. Universidade do Porto. Instituto de Biologia Molecular e Celular. Group of Molecular Microbiology; PortugalFil: Mariscotti, Javier Fernando. Consejo Superior de Investigaciones Cientificas. Centro Nacional de Biotecnologia; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Chakraborty, Trinad. Justus-Liebig-University. Institute of Medical Microbiology; AlemaniaFil: Cossart, Pascale. Institut National de la Santé et de la Recherche Médicale. Unité des Interactions Bactéries-Cellules; Francia. Instituto Pasteur; Francia. Centre de Recherche de Nantes. Institut National de la Recherche Agronomique; FranciaFil: Hain, Torsten. Justus-Liebig-University. Institute of Medical Microbiology; AlemaniaFil: Cabanes, Didier. Universidade do Porto. Instituto de Biologia Molecular e Celular. Group of Molecular Microbiology; EspañaFil: Garcia del Portillo, Francisco. Consejo Superior de Investigaciones Cientificas. Centro Nacional de Biotecnologia; Españ

Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA from Anabaena

Aims: The ferric uptake regulator (Fur) is the main transcriptional regulator of genes involved in iron homeostasis in most prokaryotes. FurA from Anabaena sp. PCC 7120 contains five cysteine residues, four of them arranged in two redox-active CXXC motifs. The protein needs not only metal but also reducing conditions to remain fully active in vitro. Through a mutational study of the cysteine residues present in FurA, we have investigated their involvement in metal and DNA binding. Results: Residue C(101) that belongs to a conserved CXXC motif plays an essential role in both metal and DNA binding activities in vitro. Substitution of C(101) by serine impairs DNA and metal binding abilities of FurA. Isothermal titration calorimetry measurements show that the redox state of C(101) is responsible for the protein ability to coordinate the metal corepressor. Moreover, the redox state of C(101) varies with the presence or absence of C(104) or C(133), suggesting that the environments of these cysteines are mutually interdependent. Innovation: We propose that C(101) is part of a thiol/disulfide redox switch that determines FurA ability to bind the metal corepressor. Conclusion: This mechanism supports a novel feature of a Fur protein that emerges as a regulator, which connects the response to changes in the intracellular redox state and iron management in cyanobacteria. Antioxid. Redox Signal. 24, 173–185