Phylogenetic and toxinogenic characteristics of Bacillus cereus group members isolated from spices and herbs

The foodborne pathogen Bacillus (B.) cereus is a common contaminant in spices and herbs. To further characterise B. cereus and its closely related group members present in spices and herbs, we analysed presumptive B. cereus strains isolated from six different condiments with view to B. cereus group species, phylogenetic affiliation and toxinogenic potential.Of a total of 59 isolates 44 were identified as B. cereus sensu stricto (s.s.), four as B. toyonensis-like, five as B. thuringiensis, one as B. weihenstephanensis, two as B. pseudomycoides/B. mycoides and three as undefined B. cereus group species. A maximum of three different species occurred simultaneously in the same spice sample. The isolates comprised 33 multilocus (ML) sequence types (STs), which can be assigned to three different phylogenetic groups. Except two B. pseudomycoides/B. mycoides strains, all isolates were able to produce enterotoxins and one strain the emetic toxin cereulide as detected by an immunoassay and LC-MS, respectively. The prevalence of toxin genes was 96.6% for nheA, 94.9% for hblD, 50.8% for cytK-2 and 1.7% for ces. The emetic strain was characterised by ST 869, which for the first time was assigned to an emetic B. cereus (s.s.) strain and is not part of the previously known two emetic MLST clusters.Our results demonstrate that not only B. cereus (s.s.) but also toxin producing B. thuringiensis, B. weihenstephanensis and B. toyonensis-like strains could be detected in condiments. For some isolates MLST revealed disagreements between phylogenetic relationship and the classification as B. weihenstephanensis and B. mycoides based on previously described species markers.     

Culture-independent quantification of pathogenic bacteria in spices and herbs using real-time polymerase chain reaction

A culture-independent method was developed for quantification of pathogenic bacteria in spices and herbs. The method is based on DNA extraction using cetyltrimethylammonium bromide (CTAB) and on real-time polymerase chain reaction (PCR). When evaluated with spices (black pepper, paprika) and herbs (oregano, parsley) artificially contaminated with Staphylococcus aureus, Salmonella enterica or Escherichia coli, the method demonstrated quantitative response with linear calibration lines and quantification limits of 10²–10⁴ CFU/g. The developed method is suitable for rapid microbiological analysis of spices and herbs, taking 8–9 h.     

Tenacity of Bacillus cereus and Staphylococcus aureus in dried spices and herbs

Numerous studies examined the antimicrobial effects of spice and herb extracts, whereas little is known about the effects of dry condiments on the survival of microorganisms. This study investigated the impact of dried condiments on the survival of Bacillus cereus and B. thuringiensis spores as well as Staphylococcus aureus cells. In addition, the survival variability between different strains was evaluated. Condiments (allspice, basil, cinnamon, nutmeg, oregano, paprika, parsley and pepper) were artificially contaminated by a dry spiking method using sand as carrier matrix and as control. The results show that counts of B. cereus and B. thuringiensis spores (initial spore count 5.6 ± 0.2 log₁₀ cfu/g and 6.7 ± 0.1 log₁₀ cfu/g, respectively) did not decline significantly in all condiments over a period of 50 weeks. In contrast, in some of the spiked test materials, cell counts of S. aureus (initial cell count 8.1 ± 0.5 log₁₀ cfu/g) were reduced below the detection limit of 10 cfu/g within 10 weeks of storage. D values for S. aureus ranged between 5 and 31 days depending on the strain, condiment and initial contamination level. In conclusion, dried condiments may not affect the survival of spores but can significantly affect the survival of non-spore forming bacteria. As strain variability can occur, tenacity studies should be conducted including a variety of strains.     

Potential of high-throughput sequencing for broad-range detection of pathogenic bacteria in spices and herbs

A broad-range culture-independent method was developed and evaluated regarding its sensitivity of detection of pathogenic bacteria in spices and herbs, with focus on paprika powder. The method involved DNA extraction using cetyltrimethylammonium bromide (CTAB), 16S rDNA amplification using universal bacterial polymerase chain reaction, and high-throughput sequencing on Illumina MiSeq platform. The sensitivity of the method was evaluated with series of model samples contaminated at different levels with Salmonella enterica and Escherichia coli (as representatives of Gram-negative bacteria) and Staphylococcus aureus (as a representative of Gram-positive bacteria). For spices (paprika, black pepper), the method had a screening-level sensitivity with limits of detection in the range of 10⁴–10⁵ CFU/g, and a semi-quantitative response. Low sensitivity (LOD ≥10⁷ CFU/g) was observed with herbs (oregano, parsley). The developed method demonstrated a good potential for microbiological screening of spices, with a prospect of further improvement of sensitivity based on progress in high-throughput sequencing technology.     

Antimicrobial activities of spices and herbs against Salmonella Oranienburg

The detection of Salmonella spp. within dried spices and herbs is challenging due to their potent antimicrobial activity. In the present study nine condiments were investigated whereof oregano and cinnamon led to a complete inhibition of Salmonella Oranienburg at a 1:10 dilution in buffered peptone water, while towards allspice and thyme an adaptation was apparent. At a next step, a tenacity study was set up where the survival of S. Oranienburg in dry condiment samples was followed qualitatively and quantitatively during storage at 25 °C for 365 days. Generally, a higher susceptibility of S. Oranienburg to spices than herbs was determined. Furthermore, to the best of the author’s knowledge, this is the first study presenting a significant antimicrobial effect of paprika/chilli against Salmonella. Nevertheless, S. Oranienburg was able to persist during storage in several condiment samples with a low reduction of log₁₀ <1.5 colony-forming units g⁻¹. Thus, pointing to the outstanding ability of S. Oranienburg to survive dry storage conditions even spiked to condiments, known for their high antimicrobial activity.     

Detection of non-emetic and emetic Bacillus cereus by propidium monoazide multiplex PCR (PMA-mPCR) with internal amplification control

Bacillus cereus is an etiological agent of food-borne disease that can cause a type of emesis. To develop a sensitive and reliable diagnosis technique for detecting all the species of the B. cereus group, specific primers were designed to target a recently discovered part of the cereulide synthetase gene (cesB) for emetic B. cereus and 16S rRNA for non-emetic B. cereus. To detect PCR signals only from viable cells, propidium monoazide (PMA) was selected to eliminate the false-positive results. In addition, an internal amplification control (IAC) was applied to meet diagnostic multiplex PCR requirements that will prevent the occurrence of false-negative results. The inclusivity and exclusivity of the mPCR assay were estimated using a panel of 100 strains, including 2 emetic B. cereus, 77 non-emetic B. cereus and 21 non-Bacillus strains. The limit of detection (LOD) for dead B. cereus without PMA treatment in pure bacteria culture was 4.0 × 10² CFU/mL, as low as 7.5 × 10⁰ CFU/mL for viable B. cereus without PMA treatment, and 7.5 × 10¹ CFU/mL for viable B. cereus with PMA treatment. B. cereus in spiked food produce was detected specifically and sensitively at 1.0 × 10³ CFU/g which was the lowest concentration detected. This novel PMA-mPCR-IAC assay is rapid and reliable, providing an efficient diagnostic tool with promising application in monitoring food samples.     

Isolation, identification and monitoring of contaminant bacteria in Iranian Kefir type drink by 16S rDNA sequencing

Iranian Kefir type drink (IKTD) is a highly consumed, traditional Iranian, fermented milk product. To improve monitoring procedures for food safety 32 industrial Kefir type drinks from 4 brands and 8 different production dates as well as 32 samples from pasteurized milk of the same Kefir manufacturers and air of the production sites were analyzed for contaminations. 16S rDNA extraction from Kefir samples as well as 16S rDNA obtained from samples incubated on Columbia agar were analyzed using PCR/DGGE, cloning, sequencing and phylogenetic classification. Already DGGE analysis indicated contaminations including Bacillus strains. Subsequently analysis of cultured clones indicated contaminations with Bacillus sp. including Bacillus cereus, Bacillus thuringiensis and Paenibacillus sp. in 9 (28%) from all analyzed samples. Also 38% of pasteurized milk samples were contaminated with B. cereus. The average count of B. cereus was 74 ± 19 cfu/ml. B. cereus and B. thuringiensis were found as contaminant bacteria in the air of the all manufacturing sites. These results suggest that milk is one of the most important sources of contamination with Bacillus sp., especially B. cereus for Kefir products in Iran. But bacterial contamination in Kefir samples might also originate from the air of the production sites. 16S rDNA analysis accelerates monitoring strategies.     

Rapid detection of viable Bacillus cereus emetic and enterotoxic strains in food by coupling propidium monoazide and multiplex PCR (PMA-mPCR)

Bacillus cereus can cause emetic and diarrheal food poisoning. It is widespread in nature and therefore, considered a major foodborne pathogen. To develop a sensitive and reliable assay for detecting enterotoxin genes (nheA, entFM, hblD, cytK) and emetic toxin (ces), specific primers each targeting one individual gene were designed. Propidium monoazide (PMA) was coupled with the developed multiplex PCR (mPCR) for the detection of viable B. cereus. The inclusivity and exclusivity of the PMA-mPCR was confirmed using a panel of 44 strains including 17 emetic and 9 enterotoxic B. cereus reference strains and 18 non-target strains. The limit of detection (LOD) without PMA treatment in pure DNA was 2 pg/reaction tube. The LOD of mPCR assay in pure heat-killed dead bacteria was 4.0 × 10² CFU/mL. Also, the LOD on the viable bacteria with or without PMA treatment was similar (3.8 × 10² CFU/mL) showing that the PMA treatment did not significantly decrease sensitivity. Finally, the newly developed PMA-mPCR successfully detected 4.8 × 10³ and 3.6 × 10³ CFU/g of viable B. cereus F4810/72 (emetic) and B. cereus ATCC 12480 (enterotoxic) reference strains, respectively, in food samples. Hence, this study combines PMA and mPCR to detect viable B. cereus with a wide range of toxin detection (5 toxins). Thus, the novel PMA-mPCR assay developed in this study is a rapid and efficient diagnostic tool for the monitoring of viable B. cereus in food samples and potentially other samples via appropriate DNA extraction.     

Growth and inhibition by spices of growth from spores of enterotoxigenic Bacillus cereus in cooked rice

Bacillus cereus is a pathogenic spore-forming bacterium implicated in cases of diarrheal and emetic type of foodborne illness. We previously found that enterotoxigenic B. cereus is widely present in retail spices. Here we analyzed the spore heat resistance of nine diarrheal strains isolated from spices. Seven had D_95°C values ranging from 0.64 to 3.53 min while two emetic strains had D_95°C values of 7.04 min and 6.64 min. The ability of selected strains to grow in cooked rice at temperatures 20 °C, 17 °C and 12 °C was determined as well as their toxin expression capability. After 48 h, B. cereus levels of 1.26 × 10⁷ and 3.8 × 10⁷ CFU/g were obtained in cooked rice maintained at 17 °C and 20 °C respectively. At 12 °C, counts did not reach 10⁴ CFU/g even after 48 h of incubation. The increase in the aerobic, mesophilic bacterial population (APC) and B. cereus population naturally present in 0.1% crushed pepper added to cooked rice was determined over a period of 48 h at 20 °C and 17 °C. Levels of B. cereus in pepper/rice samples reached a maximum of 1600 MPN/g at 20 °C while the APC was 2.4 × 10⁸/g at this temperature. When thyme, containing the same initial natural level of B. cereus, was added to rice in place of pepper, more than a five-fold greater level of B. cereus was detected at 20 °C. Since thyme contained initial APC of 2.5 log₁₀ less than pepper we conclude that the high APC functions in a competitive-exclusion manner, minimizing the growth of B. cereus and potentially other agents of foodborne illness. This is particularly relevant in instances of temperature abuse of foods and may explain the low incidence of foodborne illness due to B. cereus despite its widespread presence shown in previous surveys of market spices.     

Extracellular enzyme production and enterotoxigenic gene profiles of Bacillus cereus and Bacillus thuringiensis strains isolated from cheese in Turkey

The aim of the present study was to investigate the biochemical characteristics, extracellular enzyme production and enterotoxigenic genes contents of 6 Bacillus cereus and 22 Bacillus thuringiensis strains, isolated from 100 cheese samples in Turkey. Crystal morphologies of B. thuringiensis strains were found either spherical (n = 12) or spherical and irregular-shaped (n = 10) by phase contrast microscopy. B. cereus and B. thuringiensis strains were found to produce extracellular enzymes, respectively: gelatinase (83% and 91%), DNase (83% and 77%), lecithinase (83% and 95%), protease on skim milk agar (100% and 100%), protease on milk agar (100% and 91%), protease on casein agar (83% and 77%), xylanase (100% and 45%), and cellulase (0% and 41%), and amylase (83% and 27%). All of the strains, except for Bt-D1, hydrolyzed Tween 20 (96%), but not Tween 80 or tributyrin. Pectinolytic activity was obtained to be the least frequent (4%). PCR analysis showed that all strains contained nheA, nheB, nheC and hblD genes. The hblA and hblC genes were present in 2 and 4 of B. thuringiensis strains, respectively. The bceT gene was detected in 1 B. cereus and 9 B. thuringiensis strains. The entFM gene was detected more frequently in B. thuringiensis (82%) than in B. cereus strains (50%). To our knowledge, this is the first report about the isolation and identification of enterotoxigenic B. cereus and B. thuringiensis strains from cheese samples in Turkey.     

Occurrence and diversity of Bacillus cereus and moulds in spices and herbs

Spices and herbs can contain toxin-producing bacteria and moulds, which can cause health problems for consumers and contribute to food spoilage and shelf-life reduction. The aims of the present work were (i) to determine the occurence and levels of B. cereus and moulds; (ii) to charactize the incidence and diversity of B. cereus emetic toxin (ces, CER), and diarrhoeal toxin-encoding genes (entFM, nheA, hblC, cytK) and toxigenic potential of Hbl toxin-producing B. cereus strains. Black ground pepper samples showed the most contamination with the highest concentration of B. cereus 2.49 log₁₀ CFU/g. Moreover, cumin contained the most prominent mould concentration level of 3.6 log₁₀ CFU/g. The most common moulds were Aspergillus and Penicillium spp. Compared to packaging type, all products acquired from the local market, except curry for B. cereus, exchibited high concentrations of B. cereus and moulds. Four genes were identified – 96% of B. cereus strains contained entFM, 94% nheA, 56% hblC, 42% cytK. None of the samples contained emetic toxin-encoding genes (ces, CER). Toxigenic potential of Hbl toxin was found in 72% of B. cereus strains. Different temperature, moisture levels and hygiene practices were observed at places of sale in local markets thus facilitating contamination and development of moulds. Moreover, the presence of B. cereus and its ability to produce toxins in spices and herbs, may suggest the need to establish microbiological criteria for mould and spore-forming bacteria in spices and herbs.     

The occurrence of polycyclic aromatic hydrocarbons in dried herbs and spices

A sensitive, selective, and robust method for the determination of four EU-regulated polycyclic aromatic hydrocarbons (benzo[a]pyrene, chrysene, benzo[b]fluoranthene, and benzo[a]anthracene) at trace levels in dried herbs and spices using gas chromatography coupled to tandem mass spectrometry was elaborated and validated in accordance with the performance criteria outlined in EU legislation. A total of 150 samples of oregano, basil, thyme, black pepper, paprika, and nutmeg were tested for the occurrence of four PAHs. These PAHs were detected in 86% of the samples. The benzo[a]pyrene content in seasonings ranged from non-detectable levels to 6.60 μg kg⁻¹. None of the samples exceeded the levels acceptable in EU for BaP and the sum of four regulated PAHs.     

Investigation of regional differences of the dominant microflora of spice paprika by molecular methods

As microbial contamination of spices comes primarily from the soil, and soil microbiota are dependent on climate, geography, agriculture, etc a qualitative focus on the dominant microflora of spices instead of the quantitative determination of commonly studied spoilage and pathogenic microflora may give a better insight into the geological origin of the sample. The aim of the present study was to identify using molecular microbiological methods the dominant bacteria of spice paprika produced at different countries and to look for species characteristic of spices from the given regions. According to our data, no differentiation could be made among spice paprika samples of different geographical origin using the total bacterial count or extent of mould contamination. However, when the dominant microflora is examined, bacterial species could be identified in the spice paprika samples characteristic of a particular climate. According to our study, the presence of Bacillus mycoides and Bacillus licheniformis was characteristic of the microflora of spice paprika grown in Central Europe; Bacillus safensis could be detected in all four paprika samples examined from the tropical monsoon climate; the species common to all three samples of the tropical climate group were Bacillus amyloliquefaciens subsp. plantarum and subsp. amyloliquefaciens, while Bacillus mojavensis was detected as being characteristic of Spanish origin for paprika. No common species were found in the paprika samples originating from China.     

Exposure assessment for Bacillus cereus in ready-to-eat Kimbab selling at stores

Kimbab, which is rolled cooked-rice and other foodstuffs in dried laver, is a representative ready-to-eat (RTE) food in Korea. Bacillus cereus is a significant hazard associated with Kimbab. However, the knowledge of the dose–response relationship for B. cereus is limited, now it is not possible to conduct a full microbial risk assessment. Therefore, our study may be focused on the exposure assessment from retail store to the point of consumption in a Kimbab contaminated with B. cereus. The survey data for preparing and selling of Kimbab, predictive growth model, and consumer eating patterns were combined with probabilistic modeling to simulate the level of B. cereus in store Kimbab at the time of consumption. The estimated contamination levels ranged from as minimum (5% percentile) −3.63 log cfu/g, median (50% percentile) 1.39 log cfu/g, mean 1.57 log cfu/g, and a maximum (95% percentile) 7.31 log cfu/g. However, there is always a paucity of data to conduct such full exposure assessments with associated variability and uncertainty, coupled with a validation step. Therefore, we suggest that additional studies to allow for a more realistic and accuracy exposure assessment in the future, based on data gaps we identify.     

Detection of Salmonella spp. in spices and herbs

The detection of microbial contaminations in spices and herbs is a challenging task due to their strong antimicrobial effects, which potentially increase the risk for false-negative results. Therefore, the present study mainly focuses on the detection of Salmonella spiked to cinnamon and oregano. Both condiments completely inhibited the proliferation of Salmonella at a 1:10 (w/w) dilution. Consequently, the supplementation of the buffered peptone water with K₂SO₃ as well as the application of higher initial dilutions was investigated. While no detrimental effect of K₂SO₃ was observed during the growth of 14 different Salmonella isolates, it even improved their detection in condiments. An effect, which was also determined with increased dilution ratios. For detection, a quantitative approach via enumeration of the colony-forming units (CFUs), and qualitative approaches via the culture-based detection according to ISO 6579 and via the nucleic acid-based detection with the 3M Molecular Detection System (MDS) were performed. Subsequently, the limit of detection (LOD) was determined, which was <5 CFU 25 g⁻¹ for both qualitative approaches. Furthermore, the persistence of Salmonella DNA spiked to parsley was determined with the MDS. Despite of the modifications, the LOD for Salmonella spiked to oregano was significantly lower prior than after enrichment, pointing to the requirement for further improvements. Last but not least, a ring trial was performed, which emphasized the importance for a reliable detection.     

Short communication: Fate of major foodborne pathogens and Bacillus cereus spores in sterilized and non-sterilized Korean turbid rice wine (Makgeolli)

The objective of this study was to examine the fate of foodborne pathogens (Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus) and B. cereus spores in Korean turbid rice wine (Makgeolli). Samples of sterilized and non-sterilized turbid rice wine were inoculated with each of the vegetative bacteria or B. cereus spores at 3–4 log CFU/ml. The samples were stored at 5 °C or 22 °C, and bacterial survival was monitored over 28 days. Despite the harsh environment (alcohol content: 6–7% and pH: 3.43–3.98), long-term survival of pathogens was observed. Survival time was different depending on the type of beverage (pathogens survived longer in sterilized wine than in non-sterilized wine), cellular state (spores survived longer than vegetative cells), species (B. cereus survived longer than other species), and storage temperature (pathogens survived longer at 5 °C then at 22 °C). The number of B. cereus spores remained constant at both temperatures. The vegetative B. cereus population declined rapidly within 1 day, but then remained steady for up to 28 days (1.20–1.55 log CFU/ml in sterile wine). These results indicate that B. cereus formed spores that survived for a long time; therefore, it is possible that B. cereus may exist as spores in turbid rice wine. E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus survived for up to 28, 14, 14, and 14 days, respectively, in sterilized wine at 5 °C. Thus, the health implications of the long-term survival of pathogens in alcoholic beverages should be carefully considered. The results provide new information that may be useful in predicting the potential microbiological hazards associated with turbid rice wine.     

Antimicrobial peptides derived from hen egg lysozyme with inhibitory effect against Bacillus species

In food industry, Bacillus species are encountered in deteriorating many food products thus shortening their shelf-life. Moreover, Bacillus cereus and the subtilis group (Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus) have been recognized as food poisoning agents. Lysozyme peptides preparation (LzP) is a commercially available as a natural food preservative. Although, LzP derived from lysozyme yet it showed only 11% of the lysozyme lytic activity. LzP at a concentration of 100 μg ml⁻¹ completely inhibited B. subtilis, B. licheniformis, B. megaterium, B. mycoides, B. pumilus, B. coagulans, B. amyloliquefaciens, B. polymexa and B. macerans. However, B. cereus and B. stearothermophilus showed a slightly higher resistance. Interestingly, LzP at concentration ⩾10 μg ml⁻¹ showed inhibitory effect on both vegetative and spore forms of B. subtilis. Moreover, LzP was stable at 95 °C for 30 min and at different pH values (4.5–7). In conclusion, LzP may be useful to control growth of Bacillus spoilage organisms.     

Radiation tolerance of Bacillus cereus pre-treated with carvacrol alone or in combination with nisin after exposure to single and multiple sub-lethal radiation treatment

Currently little information exists on the response of Bacillus cereus after repetitive exposure to combined mild treatments involving antimicrobials and gamma irradiation. Therefore, the aim of this present study was to evaluate the radiation stress on growth and physiology of B. cereus LSPQ 2872 vegetative cells at stationary phase, following exposure to single and repetitive sub-lethal γ-radiation treatment at 1 kGy simultaneously with carvacrol alone or combined with nisin at sub-inhibitory concentrations. Results indicated that the combination of carvacrol and nisin significantly enhance the radiation sensitivity of B. cereus since lower D₁₀ values were recorded after both single and repetitive irradiation treatments. Flow cytometric analysis of radiation-stressed B. cereus cells following repetitive treatment revealed the heterogenic behaviour of the bacterium leading to the induction of a radiation tolerance response. When compared to carvacrol alone, the combination of carvacrol and nisin developed also increased radioresistance if repetitively processed with γ-radiation at 1 kGy, since the decrease percentage of dead cells was accompanied by an increase in the number of injured cells. However, good agreement was not found between classical plate counting (log cfu reductions) and flow cytometry method. For both antimicrobials, the increase of radioresistance after repetitive mild treatment was not accompanied by augmentations of lag phase or growth rate. The structural changes of the outer membranes were assessed by TEM analysis and results revealed that radioresistance might be related to changes in the cell wall.     

A reliable screening of mycotoxins and pesticide residues in paprika using ultra-high performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry

In this study, an ultra-high performance liquid chromatography (UHPLC) coupled to a high resolution Orbitrap mass spectrometry (Orbitrap-HRMS) was demonstrated as a promising technique in high-throughput method development for the routine analysis and contamination control of mycotoxins and pesticide residues in spices. The method was applied for the analysis of fifty ground paprika samples containing blends of sweet and hot paprika harvested in Brazil and China. The efficiency and detection sensitivity of the used UHPLC-Orbitrap-HRMS technique were compared to the results obtained using a triple quadrupole tandem mass spectrometric detector (UHPLC-QqQ-MS/MS). The values of recovery (75–120%) and repeatability (8–15%) for both methods, calculated as the average (n = 5) from the results of spiked (10–500 μg kg⁻¹), paprika samples, were in good conformity to the relevant EU guidelines. The high resolution of the used Orbitrap-HRMS technique provided a better sensitivity for quantitative determination of several pesticide contaminants in paprika, compared to the results obtained by the QqQ-MS/MS method and were comparable in case of mycotoxins. The results of analysis demonstrated the ubiquitous presence of three mycotoxins (fumonisin B₁, ochratoxin A, and sterigmatocystin) and twelve pesticide residues in paprika. The concentrations of determined contaminants were below the MRLs set by the Regulations of the European Union with exception of iprovalicarb, which violated the EU MRL in two samples of hot paprika. In addition, a notable difference in the concentration of fumonisin B₁ was determined depending on the harvest period (2009–2013), reaching the maximum concentrations of 33 μg kg⁻¹ in sweet paprika and 140 μg kg⁻¹ in hot paprika. There was no significant correlation found between the determined mycotoxin contamination levels and the pesticide residues, with the sole exception of decreased fumonisin B₁ content in samples with an elevated concentration of metalaxyl fungicide.