Leona Buňková

The effect of ripening and storage conditions on the distribution of tyramine, putrescine and cadaverine in Edam-cheese.

The aim of the work was to describe the development of selected biogenic amines (histamine, tyramine, putrescine and cadaverine) in 4 layers of Dutch-type cheese (Edam-cheese) depending on 3 ripening/storage regimes during a 98-day period. Biogenic amines were analysed by means of ion-exchange chromatography. A further goal was to identify microbial sources of biogenic amines in the material analysed. Phenotype characterization and repetitive sequence-based PCR fingerprinting were used to identify the isolated bacteria. The highest content of tyramine, putrescine and cadaverine was determined in cheeses stored in a ripening cellar at a temperature of 10 degrees C during the whole observation period. Lower biogenic amines content was determined in samples which were moved into a cold storage device (5 degrees C) after 38 days of storage in a ripening cellar (10 degrees C). The lowest concentrations of biogenic amines were detected in cheeses which were moved into a cold storage device (5 degrees C) after 23 days of storage in a ripening cellar (10 degrees C). During the 98-day period, histamine was not detected in any of the regimes. Within the cheeses analysed, non-starter lactic acid bacteria Lactobacillus curvatus, Lactobacillus casei/paracasei and Lactobacillus plantarum were detected as the main producers of the biogenic amines tested. In starter bacteria Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris the decarboxylase activity tested was not detected.

Formation, Degradation, and Detoxification of Putrescine by Foodborne Bacteria: A Review

Biogenic amines (BAs) represent a considerable toxicological risk in some food products. Putrescine is one of the most common BAs in food. Its increased occurrence in food may lead to alimentary poisoning, due to enhancement of the toxic effects of other BAs, and also to lower quality of food, this amine is potentially carcinogenic. Increased occurrence of putrescine in food is mainly due to the bacterial metabolism of the Gram-negative as well as Gram-positive bacteria present. The bacterial metabolism of putrescine is very specific due to its complexity (in comparison with the metabolism of other BAs). There are 3 distinct known pathways leading toward the formation of putrescine, in some splices involving up to 6 different enzymes. The existence of more metabolic pathways and the possibility of their simultaneous use by different bacteria complicate the specification of the best conditions for food production and storage, which could lead to a lower content of putrescine. This review provides a summary of the existing knowledge about putrescine production and detection (mainly detection of specific genes for different enzymes using polymerase chain reaction) in both starter and contaminating microorganisms. Thus, this comprehensive review gives a useful overview for further research.

16S rRNA gene-based identification of cultured bacterial flora from host-seeking Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna ticks, vectors of vertebrate pathogens

A total of 151 bacterial isolates were recovered from different developmental stages (larvae, nymphs and adults) of field-collected ticks (67 strains from Ixodes ricinus, 38 from Dermacentor reticulatus, 46 from Haemaphysalis concinna). Microorganisms were identified by means of 16S rRNA gene sequencing. Almost 87 % of the strains belonged to G+ bacteria with predominantly occurring genera Bacillus and Paenibacillus. Other G+ strains included Arthrobacter, Corynebacterium, Frigoribacterium, Kocuria, Microbacterium, Micrococcus, Plantibacter, Rhodococcus, Rothia, and Staphylococcus. G− strains occurred less frequently, comprising genera Advenella, Pseudomonas, Rahnella, Stenotrophomonas, and Xanthomonas. Several strains of medical importance were found, namely Advenella incenata, Corynebacterium aurimucosum, Microbacterium oxydans, M. schleiferi, Staphylococcus spp., and Stenotrophomonas maltophilia. Data on cultivable microbial diversity in Eurasian tick species D. reticulatus and H. concinna are given, along with the extension of present knowledge concerning bacterial flora of I. ricinus.

The effect of lactose, NaCl and an aero/anaerobic environment on the tyrosine decarboxylase activity of Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis.

The aim of this work was to study, under model conditions, combined effects of the concentration of lactose (0-1% w/v), NaCl (0-2% w/v) and aero/anaerobiosis on the growth and tyramine production in 3 strains of Lactococcus lactis subsp. lactis and 2 strains of L. lactis subsp. cremoris. The levels of the factors tested were chosen with respect to the conditions which can occur during the real process of natural cheese production, including the culture temperature (10 ± 1°C). In all strains tested, tyrosine decarboxylation was most influenced by NaCl concentration; the highest production of tyramine was obtained within the culture with the highest (2% w/v) salt concentration applied. Two of the strains L. lactis subsp. lactis produced tyramine only in broth with the highest NaCl concentration tested. In the remaining 3 strains of L. lactis, tyramine was detected under all conditions applied. The tested concentration of lactose and aero/anaerobiosis had a less significant effect on tyramine decarboxylation. However, it was also found that at the same concentrations of NaCl and lactose, a higher amount of tyramine was detected under anaerobic conditions. In all strains tested, tyramine decarboxylation started during the active growth phase of the cells.

Monitoring of biogenic amines in cheeses manufactured at small-scale farms and in fermented dairy products in the Czech Republic

The aim of the study was the monitoring of six biogenic amines (histamine, tyramine, phenylethylamine, tryptamine, putrescine, and cadaverine) and two polyamines (spermidine and spermine) in 112 samples of dairy products purchased in the Czech Republic, namely in 55 cheeses made in small-scale farms and in 57 fermented dairy products. The products were tested at the end of their shelf-life period. Neither tryptamine nor phenylethylamine was detected in the monitored samples; histamine was found only in four cheese samples containing up to 25mg/kg. The contents of spermine and spermidine were low and did not exceed the values of 35 mg/kg. Significant amounts of tyramine, putrescine, and cadaverine occurred especially in cheeses produced from ewes milk or in long-term ripened cheeses. In about 10% of the tested cheeses, the total concentration of all the monitored biogenic amines and polyamines exceeded the level of 200mg/kg, which can be considered toxicologically significant. In fermented dairy products, the tested biogenic amines occurred in relatively low amounts (generally up to 30 mg/kg) that are regarded safe for the consumers health.

Xanthan and gellan degradation by bacteria of activated sludge

Owing to increasing amounts of xanthan and gellan in food, cosmetics and pharmaceuticals, as well as in some technical spheres, studies were carried out on the xanthan and gellan degrading bacteria present in activated sludge. The activated sludge used in the study was able to degrade both carbohydrates over 7 days, with levels of xanthan and gellan utilizing microbes estimated at 10(5) cells/g of dry sludge weight. Isolating key degrading bacteria revealed the important role of genus Paenibacillus in xanthan degradation and prosthecate bacterium Verrucomicrobium sp. GD, which was capable of gellan utilization. Further tests performed with both strains showed they were able to degrade other types of carbohydrate polymers, and that Verrucomicrobium sp. GD did not possess extracellular free gellan depolymerase.

Biogenic amine production by Lactococcus lactis subsp. cremoris strains in the model system of Dutch-type cheese

The aim of this study was to compare the biogenic amine production of two starter strains of Lactococcus lactis subsp. cremoris (strains from the Culture Collection of Dairy Microorganisms - CCDM 824 and CCDM 946) with decarboxylase positive activity in a model system of Dutch-type cheese during a 90-day ripening period at 10°C. During ripening, biogenic amine and free amino acid content, microbiological characteristics and proximate chemical properties were observed. By the end of the ripening period, the putrescine content in both samples with the addition of the biogenic amine producing strain almost evened out and the concentration of putrescine was >800mg/kg. The amount of tyramine in the cheeses with the addition of the strain of CCDM 824 approached the limit of 400mg/kg by the end of ripening. In the cheeses with the addition of the strain of CCDM 946 it even exceeded 500mg/kg. In the control samples, the amount of biogenic amines was insignificant.

Decarboxylation activity of enterococci isolated from rabbit meat and staphylococci isolated from trout intestines

The aim of the study was to explore production of seven biogenic amines (phenylethylamine, histamine, cadaverine, tyramine, putrescine, spermine and/or spermidine) by selected staphylococci and enterococci. Thirty three enterococcal strains isolated from rabbit meat (Oryctolagus cuniculus f. domesticus) and 21 staphylococcal strains isolated from intestinal content of trout (Salmo trutta morpha fario) were tested. Production of biogenic amines was evaluated after cultivation of the tested microorganisms in the de Man, Rogosa and Sharpe Broth (enterococci) or in the Brain Heart Infusion Broth (staphylococci). Both the above cultivation media were enriched with selected amino acids (histidine, tyrosine, arginine, ornithine and lysine; 2g/L each) serving as precursors of biogenic amines. After cultivation, levels of the monitored biogenic amines in broths were analysed by a high performance liquid chromatography equipped with a UV/VIS DAD detector. Among 21 staphylococci, 18 strains produced tyramine or cadaverine, 13 strains formed putrescine or phenylethylamine and only one strain generated histamine. Two staphylococcal strains produced cadaverine levels above 1000 mg/L. Among 33 enterococcal strains, 27 formed cadaverine, 18 strains produced tyramine, 10 strains generated phenylethylamine, and 2 strains gave putrescine. Most of the tyramine producing enterococci generated more than 1000 mg/L of this biogenic amine. Production of spermine or spermidine by the studied strains was not proved.

Novel touchdown-PCR method for the detection of putrescine producing gram-negative bacteria in food products.

Formation of biogenic amines may occur in food due to metabolic activities of contaminating Gram-negative bacteria. Putrescine is assumed to be the major biogenic amine associated with microbial food spoilage. Gram-negative bacteria can form putrescine by three metabolic pathways that can include eight different enzymes. The objective of this study was to design new sets of primers able to detect all important enzymes involved in the production of putrescine by Gram-negative bacteria. Seven new sets of consensual primers based on gene sequences of different bacteria were designed and used for detection of the speA, adiA, adi, speB, aguA, speC, and speF genes. A newly developed touchdown polymerase chain reaction (PCR) method using these primers was successfully applied on several putrescine-producers. Selected PCR products were sequenced and high similarity of their sequences (99-91%) with known sequences of the corresponding genes confirmed high specificity of the developed sets of primers. Furthermore, all the investigated bacteria produced both putrescine and agmatine, an intermediate of putrescine production, which was confirmed by chemical analysis. The developed new touchdown PCR method could easily be used to detect potential foodborne Gram-negative producers of putrescine. The newly developed sets of primers could also be useful in further research on putrescine metabolism in contaminating microbiota.

The influence of fat and monoacylglycerols on growth of spore-forming bacteria in processed cheese

Highly undesirable microbial contaminants of processed cheese are endospore-forming bacteria of the genera Bacillus and Clostridium. Survival of Bacillus subtilis, B. cereus, Clostridium butyricum and C. sporogenes was examined in model processed cheese samples supplemented with monoacylglycerols. In processed cheese samples, monoacylglycerols of undecanoic, undecenoic, lauric and adamantane-1-carboxylic acid at concentration of 0.15% w/w prevented the growth and multiplication of both Bacillus species throughout the storage period. The two species of Clostridium were less affected by monoacylglycerols in processed cheese samples and only partial inhibition was observed. The effect of milk fat content on microbial survival in processed cheese was also evaluated. The growth of Bacillus sp. was affected by the fat level of processed cheese while population levels of Clostridium sp. did not differ in processed cheese samples with 30, 40 and 50% fat in dry matter.