Development and validation of an improved method for the detection of Salmonella in cinnamon bark and oregano leaves using the adsorbent beta zeolite in the pre-enrichment media.

The detection of Salmonella in spices is challenging due to the presence of antibacterial components. In this study, we evaluated the use of an adsorbent beta zeolite in pre-enrichment media to improve the recovery of Salmonella from cinnamon bark and oregano leaves. Samples (25 g) were spiked with varying levels of S. Montevideo or S. Senftenberg. After 2 weeks of stabilization at RT, betazeolite was added to cinnamon and oregano samples prior to the addition of 225 mL or 475 mL of pre-enrichment media, respectively. Detection sensitivity and rate of the test method were compared to the FDA Bacteriological Analytical Manual (BAM) method which requires the use of 2.5 L pre-enrichment broth. While Salmonella could not be detected in the test method using the reduced volume of pre-enrichment media alone, the addition of beta zeolite resulted in a positivity rate of 62% and 72.6% for cinnamon bark and oregano leaves respectively (all spike levels and both serovars combined). Furthermore, while there were differences in the LOD50 compared to the BAM method, there was no significant difference in the minimum level of detection between the betazeolite and the BAM methods. Our results demonstrate that the use of betazeolite in the pre-enrichment media offers a method with reduced media volumes without compromising on the sensitivity or efficiency of Salmonella detection in cinnamon bark and oregano leaves.

How can packaging, source and food safety management system affect the microbiological quality of spices and dried herbs? The case of a developing country.

Spices and herbs are widely used in almost all types of food preparation and their microbial contamination may cause spoilage and pose public health risk. Thus, the aim of this study was to assess the effect of packaging, source and a food safety management system (FSMS) on the microbiological quality of spices and dried herbs in a developing country, like Lebanon. For this, a total of 96 composite samples of thirteen most commonly consumed types of spices and dried herbs were collected twice at three-month interval. Each type was purchased in 5 common brands from 4 categories: packaged in companies with FSMS, packaged in companies without FSMS, packaged imported, and unpackaged. Total aerobic mesophilic bacteria (TAMB), sulfite reducing anaerobic bacteria, C. perfringens, coliforms, E. coli, yeasts and molds were found in 89%, 43%, 18%, 15%, 1% and 54% of the samples, respectively. All samples were negative for Salmonella. One per cent, 4%, 6%, 1% and 7% of the samples had unacceptable levels of TAMB, coliforms, sulfite reducing anaerobic bacteria, E. coli, yeasts and molds, respectively. Among the four categories, imported samples had the lowest microbiological load, followed by locally packaged in companies with FSMS, then locally packaged in companies without FSMS and the highest microbiological load was for the unpackaged spices and dried herbs. This study highlighted the importance of storage conditions, good hygienic practices, process controls and FSMSs in the spices and dried herbs sector.

A single serving of mixed spices alters gut microflora composition: a dose-response randomised trial.

Short-term changes in dietary intake can induce changes in gut microbiome. While various dietary polyphenols have been shown to modulate gut microflora, the acute influence of polyphenol-rich mixed spices has not been explored in a controlled setting. We investigated the effects of a single serving of mixed spices Indian curry consumption, in two separate doses, on the gut microbiome in 15 healthy, Singaporean Chinese males, with age and BMI of 23.5 ± 2.4 years and 22.9 ± 2.2 kg/m2 respectively. We found that a low-polyphenol, no spices Dose 0 Control (D0C) meal led to an increase in Bacteroides and a decrease in Bifidobacterium. In comparison to D0C, there was significant suppression of Bacteroides (p < 0.05) and an increase in Bifidobacterium (p < 0.05) with increasing doses of curry meal Dose 1 Curry (D1C) and Dose 2 Curry (D2C) containing 6 g and 12 g mixed spices respectively. Significant correlations were also found between bacterial changes and plasma phenolic acids. No differences between treatments were observed in the alpha-diversity of the gut microflora. This study has shown that a single serving of mixed spices can significantly modify/restore certain commensal microbes, particularly in people who do not regularly consume these spices.

Inactivation of Salmonella enterica and Enterococcus faecium NRRL B2354 on cumin seeds using gaseous ethylene oxide.

The objectives of this study were to investigate the effects of processing parameters (relative humidity (RH), temperature, and exposure time) on the ethylene oxide (EtO) microbial inactivation of Salmonella spp. and to evaluate Enterococcus faecium NRRL B2354 as a suitable surrogate for Salmonella inactivation on cumin seeds. Five grams of cumin seeds inoculated with either Salmonella or E. faecium were treated with EtO at different temperatures (46, 53, and 60 °C) and RH (30, 40, and 50%) levels for different exposure time to investigate the effects of process parameters on the microbial inactivation. The Weibull model fit the survival data of both bacteria with a shape parameter p < 1, which showed a tailing effect with concave shape indicating that the sensitive cells were inactivated first, and the sturdy ones survived at low RH treatment conditions. In general, the log reductions of both bacteria on cumin seeds increased with the increasing RH and temperature for EtO treatment. RH is a critical factor for successful EtO inactivation treatment. RH must be higher than 40% to implement a successful and efficient EtO decontamination of cumin seeds. E. faecium consistently showed lower log reductions than those of Salmonella under all EtO treatment conditions investigated in this study, demonstrating that E. faecium is a suitable surrogate for Salmonella. Twenty minutes of EtO treatment at 50% RH achieved ~5 log reductions of both bacteria at all three temperatures. A response surface model was developed to predict the log reductions of both bacteria under different treatment conditions and the contour plots representing log reductions were created. Inactivation is positively correlated to temperature and RH. Therefore, a higher temperature is required to achieve the desired log reduction at lower RH and vice versa. The developed response surface model is a valuable tool for the spice industry in identifying the possible combinations of EtO process parameters (temperature, RH, and exposure time) required to achieve a desired microbial reduction of Salmonella for ensuring microbial food safety of spices.

Influence of water activity on the heat resistance of Salmonella enterica in selected low-moisture foods.

Low-moisture foods (LMF with water activity, aw < 0.85) including pet foods and black pepper powder have consistently been associated with foodborne disease caused by Salmonella enterica. Increased heat resistance and prolonged survival at low-moisture conditions, however, remain major challenges to achieve effective inactivation of Salmonella in low-moisture foods. At low water activity (aw) conditions, heat resistance of Salmonella is greatly enhanced when compared to high aw conditions. This study aimed to quantify the effect of aw on the heat resistance of Salmonella enterica in pet food pellets and black pepper powder. Pet food pellets were inoculated with two strains of heat resistant S. enterica and black pepper powder was inoculated with a 5-strain cocktail of Salmonella. Both inoculated food samples were equilibrated at 0.33, 0.54, and 0.75 aw in controlled humidity chambers. Inoculated pet food pellets and black pepper powder in closed aluminum cells were heat treated at specific temperatures for selected times. The results showed that the Weibull model fitted well the inactivation data. At a specific temperature, the rate of inactivation increased with the increase in the aw from 0.33 to 0.75, and the 3-log reduction times decreased for Salmonella in both food samples with the increase in aw. Water adsorption isotherms of pet food pellets and black pepper powder at initial and treatment temperatures were developed to understand the change in aw during heat treatments. The change in aw during heat treatment was dependent on the type of food matrix, which possibly influenced the thermal inactivation of Salmonella in pet food pellets and black pepper powder. The quantitative analysis of heat reduction of Salmonella with respect to aw aids in selection of the appropriate initial aw to develop effective heat treatment protocols for adequate reduction of Salmonella in pet foods and black pepper powder.

Prevalence and genetic characteristics of Cronobacter spp. from food and human clinical stool samples in Wenzhou, China 2008-2018.

Pathogenic Cronobacter species are responsible for life-threatening illness in neonates. A ten-year comprehensive survey was conducted to examine the population structure and antimicrobial resistant patterns of Cronobacter isolates from food (n = 78) and clinical (n = 12) sources in Wenzhou, China. A total of 90 (4.4%) isolates were recovered from 2051 collected samples. The occurrence of Cronobacter spp. was highest in spices with a rate of 22% (26/119), whereas the lowest contamination rate of 1% was found in powered infant and toddler formula (7/494), special medical infant formula (1/95) and human stool samples (12/1024). Cronobacter strains revealed a high degree of genetic diversity among the isolates tested. Pulsed-field gel electrophoresis (PFGE) distinguished 75 clonal groups, and the biggest cluster consisted of four strains. Multilocus sequence typing (MLST) method displayed 43 sequence types (STs), of which ST1, ST4, ST8, ST64, ST148 and ST201 were most frequently identified. Meanwhile, two new sequence types were discovered and added to the PubMLST international database. Resistance to ceftriaxone, cefotaxiv, amoxicillin, ampicillin, cefoxitin, tetracycline, streptomycin, azithromycin, chloramphenicol, as well as multidrug resistance, was noted. Taken together, this large-scale surveillance study highlights the wide dissemination and diverse molecular features of Cronobacter spp. in Wenzhou China.

Identification of antimicrobial resistant bacteria from plant-based food products imported into Canada.

The role of plant-based foods in the epidemiology of antimicrobial resistance has been inadequately studied. In this investigation, resistant organisms from vegetables, fruits and spices imported into Canada were identified and characterized. A total of 143 products imported from primarily Asian and African countries were purchased from international markets in Saskatoon, Saskatchewan. Samples were selectively cultured for bacterial species where resistance is known to be emerging. The proportions of samples positive for each organism were as follows: E. coli (n = 13, 9.1%), Salmonella spp. (n = 2, 1.4%), ESBL producing Enterobacter spp. (n = 2, 1.4%) and K. pneumoniae (n = 2, 1.4%), S. aureus (n = 7, 4.9%) and Enterococcus spp. (n = 66, 46.2%). Antimicrobial minimum inhibitory concentrations were determined by broth micro-dilution and agar-dilution. Based on the susceptibility of each organism, isolates were screened for resistance genes (ß-lactamases and plasmid mediated quinolones resistance determinants) by PCR. Extended-spectrum ß-lactamase producing Enterobacteriaceae and methicillin resistant S. aureus (MRSA) were identified from 6/143 (4.2%) and 2/143 (1.4%) of samples respectively. The qnrB, qnrS and aac(6')-Ib-cr plasmid mediated quinolone resistance determinants were identified in 2/143 (1.4%) of samples tested. None of the Enterobacteriaceae isolates were resistant to meropenem or colistin. Similarly, all Enterococcus isolates remained susceptible to ampicillin, penicillin and vancomycin. Finding multi-drug resistant bacteria which are frequently isolated from human infections is concerning, although the contribution of the global food trade to the dissemination of resistance remains cryptic. These results suggest that imported plant-based foods may be an underappreciated source of clinically relevant resistant organisms. Further study is required to address these gaps in our understanding of the epidemiology of resistance, and the magnitude of the risk posed to human health by these organisms.

Optimization of Salmonella detection in garlic, onion, cinnamon, red chili pepper powders and green tea.

Salmonella is the causative agent of many outbreaks related to spice consumption. However, because of the antimicrobial properties of various spices which hinders recovery and detection, Salmonella detection in spices remains a challenge. The objective of this study was to optimize an enrichment broth for Salmonella growth in different spices and tea, in order to maintain an adequate pH and decrease the antimicrobial effects of spices during Salmonella enrichment and subsequent detection. Salmonella contaminated spice and tea dried samples were prepared and the detection of Salmonella was assessed using the developed broth and automated DNA extraction and RT-PCR. Double strength Buffered Peptone Water (BPW) was used to maintain pH, and L-cysteine and DL-serine were added to the broth to reduce the effects of antimicrobial compounds in spices. The modified enrichment broth allowed the growth of Salmonella from each spice sample. Sample to broth ratios varied from 1:9 (garlic powder, chili peppers and tea), to 1:20 (cinnamon). The pH value of each enrichment varied but remained above 4.8. The addition of L-cysteine (30 mmol/L) allowed Salmonella recovery and growth in garlic and onion samples and the addition of DL-serine (11.23 mmol/L) allowed the recovery and growth in cinnamon. The results indicated that Salmonella detection was achieved in <24 h in the modified (BPW + L-cysteine and DL-serine) enrichment broth followed by detection by RT-PCR. This protocol could allow for a more rapid, robust, and sensitive enrichment method for Salmonella in spices.

Management of Mycotoxins in Spices.

Spices are very important for cuisines around the world as well as for health enhancement. The Egyptians, Chinese, and Indians have used spices in medicinal remedies and procedures starting in around 2000 BC. Through the centuries, spices have found use as food ingredients to modify the aroma and taste of the final products; however, some spices are suitable substrates for mold growth and mycotoxin development, which could be detrimental to human and animal health. This report covers regulatory control of mycotoxins in food and spices by means of monitoring and regulatory limits, sampling and analysis, management, and prevention of mycotoxins from plant growth (preharvest) through harvest and postharvest as well as decontamination for mycotoxins when necessary. There is no one single-best strategy that can solve mycotoxin contamination problems, but a well-designed and integrated plan of all these strategies could result in a substantial reduction of mycotoxins in spices to regulation safety levels.

Genetic diversity and delineation of Salmonella Agona outbreak strains by next generation sequencing, Bavaria, Germany, 1993 to 2018.

BackgroundIn 2017, a food-borne Salmonella Agona outbreak caused by infant milk products from a French supplier occurred in Europe. Simultaneously, S. Agona was detected in animal feed samples in Bavaria.AimUsing next generation sequencing (NGS) and three data analysis methods, this study's objectives were to verify clonality of the Bavarian feed strains, rule out their connection to the outbreak, explore the genetic diversity of Bavarian S. Agona isolates from 1993 to 2018 and compare the analysis approaches employed, for practicality and ability to delineate outbreaks caused by the genetically monomorphic Agona serovar.MethodsIn this observational retrospective study, three 2017 Bavarian feed isolates were compared to a French outbreak isolate and 48 S. Agona isolates from our strain collections. The later included human, food, feed, veterinary and environmental isolates, of which 28 were epidemiologically outbreak related. All isolates were subjected to NGS and analysed by: (i) a publicly available species-specific core genome multilocus sequence typing (cgMLST) scheme, (ii) single nucleotide polymorphism phylogeny and (iii) an in-house serovar-specific cgMLST scheme. Using additional international S. Agona outbreak NGS data, the cluster resolution capacity of the two cgMLST schemes was assessed.ResultsWe could prove clonality of the feed isolates and exclude their relation to the French outbreak. All approaches confirmed former Bavarian epidemiological clusters.ConclusionEven for S. Agona, species-level cgMLST can produce reasonable resolution, being standardisable by public health laboratories. For single samples or homogeneous sample sets, higher resolution by serovar-specific cgMLST or SNP genotyping can facilitate outbreak investigations.

Atividade antimicrobiana de óleos essenciais de diferentes especiarias / Antimicrobial activity of essential oils of different spices

As especiarias são utilizadas na alimentação, conferindo sabor e conservação prolongada aos alimentos, pois apresentam propriedades antimicrobianas provenientes dos óleos essenciais de sua constituição. O objetivo deste estudo foi avaliar a atividade antimicrobiana dos óleos essenciais de cravo, louro, manjericão, noz moscada e orégano frente a seis bactérias patogênicas e deteriorantes por meio da técnica de difusão em poços e determinação das Concentrações Inibitória Mínima (CIM) e Bactericida Mínima (CBM). O óleo essencial de orégano evidenciou forte atividade antibacteriana (CIM 50 - 800 μg.mL-1), seguido do cravo (CIM 800 - 3200 μg.mL-1), com atividade moderada para todos os microrganismos. Os outros óleos apresentaram baixa ação (CIM 400 - 3200 μg.mL-1), não apresentando atividade sobre todos as bactérias. Desta forma os óleos essenciais de cravo e orégano apresentaram melhor atividade antibacteriana e se apresentam como promissores para a aplicação e uso em alimentos.

Radiofrequency pasteurization process for inactivation of Salmonella spp. and Enterococcus faecium NRRL B-2354 on ground black pepper.

Salmonella persistence in ground black pepper has caused several foodborne outbreaks and created public concern about the safety of low water activity (aw) foods. In this study, radiofrequency (RF) processing was evaluated for pasteurization of ground black pepper. Stability and homogeneity tests were done for both Salmonella spp. and E. faecium during moisture equilibration before RF heating to evaluate the inoculation method. Moisture content of samples were conditioned such that the final moisture content after RF heating reached the optimal storage moisture. RF heating was shown to provide more than 5.98 log CFU/g reduction for Salmonella spp. and the reduction of 3.89 log CFU/g for E. faecium with a 130 s of treatment time. The higher thermal resistance of E. faecium indicated its suitability as surrogate for Salmonella spp. during RF heating of ground black pepper. Piperine, total phenolics, volatile compounds, and antioxidant activity were assessed as quality parameters for ground black pepper. The results demonstrated that the RF processing provided effective inactivation of Salmonella spp. with insignificant (p > 0.05) quality deterioration.

Morphologic, molecular and metabolic characterization of Aspergillus section Flavi in spices marketed in Lebanon.

Spices are used extensively in Lebanon not only to flavour foods but also for their medicinal properties. To date, no data are available regarding the nature of the toxigenic fungal species that may contaminate these products at the marketing stage in this country. Eighty samples corresponding to 14 different types of spices were collected throughout Lebanon to characterize the Aspergillus section Flavi contaminating spices marketed in Lebanon and the toxigenic potential of these fungal species. Most fungal genera and species were identified as belonging to Aspergillus section Flavi. Aspergillus flavus was the most frequent species, representing almost 80% of the isolates. Although identified as A. flavus by molecular analysis, some strains displayed atypical morphological features. Seven strains of A. tamarii and one A. minisclerotigenes were also isolated. Analyses of toxigenic potential demonstrated that almost 80% of strains were able to produce mycotoxins, 47% produced aflatoxins, and 72% produced cyclopiazonic acid, alone or in combination with aflatoxins.

Microbiological Safety of Dried Spices.

Spices in the desiccated state provide an environment that allows the survival of many foodborne pathogens. Currently, the incidence of pathogen-positive spices imported into the United States is 1.9 times higher than for any other imported food. Correspondingly, imported spices have been associated with numerous foodborne outbreaks and multiple product recalls. Despite the association with recalls and outbreaks, the actual pathogen populations in spices, when found, are frequently extremely small. In addition to pathogenic bacterial species, toxigenic molds have been frequently recovered from spices, and aflatoxins have been found in as many as 58% of the spices sampled. The presence of toxigenic molds is especially problematic to the immunocompromised or those on immunosuppressive therapy and has been linked to gut aspergillosis. Numerous detection methods, including both traditional and advanced DNA regimes, are being tested to optimize recovery of pathogens from spices. Further, a number of new inactivation intervention methods to decontaminate spices are examined and discussed.

Occurrence and species diversity of human-pathogenic Mucorales in commercial food-stuffs purchased in Paris area.

Mucormycoses are life-threatening fungal diseases that affect a variety of patients including those with diabetes mellitus or hematological malignancies. The responsible agents, the Mucorales, are opportunistic pathogens originating from the environment such as soil or decaying organic matter. The aim of the present study was to assess the prevalence and diversity of human-pathogenic species of Mucorales in commercially available foodstuffs in France. All food samples were purchased from January 2014 to May 2015 in France. A total of 159 dried food samples including spices and herbs (n = 68), herbal tea (n = 19), cereals (n = 19), vegetables (n = 14), and other foodstuffs (n = 39) were analyzed. Each strain of Mucorales was identified phenotypically, and molecular identification was performed by ITS sequencing. From the 28 (17.6%) samples that were culture-positive for Mucorales, 30 isolates were recovered. Among the isolates, 13 were identified as Rhizopus arrhizus var. arrhizus, 10 R. arrhizus var. delemar, two Rhizopus microsporus, one Lichtheimia corymbifera, three Lichtheimia ramosa, and one Syncephalastrum racemosum. Culture-positive samples originated from different countries (Europe, Asia) and brands. The samples most frequently contaminated by Mucorales were spices and herbs (19/68, 27.9%), followed by herbal tea (2/19, 10.5%), cereals (2/19, 10.5%), other food products (5/39, 12.8%). The present study showed that human-pathogenic Mucorales were frequently recovered from commercially available foodstuffs in France with a large diversity of species. The potential danger represented by Mucorales present in food for immunocompromised patients should be further analyzed.

Contamination of Cronobacter spp. in Chinese Retail Spices.

Crononbacter spp. is an opportunistic foodborne pathogen that causes infections in neonates, infants, and immunocompromised adults. Although the contamination of spices with Cronobacter has been previously reported in some countries, there have been no studies on Cronobacter contamination in China. Therefore, this study aimed to investigate the prevalence of Cronobacter spp. in Chinese retail spices. Fifty-six packaged Chinese spices were collected from different markets, and 32 of these were found to be contaminated with Cronobacter. Five species were identified from the 54 isolates of the 32 positive samples: Cronobacter sakazakii (n = 35), Cronobacter muytjensii (n = 8), Cronobacter malonaticus (n = 6), Cronobacter turicensis (n = 3), and Cronobacter dublinensis (n = 2). Pulsed-field gel electrophoresis demonstrated high genetic diversity, as 53 PFGE profiles were revealed for the 54 isolates. Multilocus sequence typing analysis revealed 46 sequence types, and of these, 26 were newly identified. Most of the isolates were sensitive to antibiotics (n = 15), with the exception of cefazolin. This study revealed that the contamination of Chinese retail spices by Cronobacter spp. poses a potential risk to the consumer.

Imported edible leaves collected at retail sale in England during 2017 with an emphasis on betel and curry leaves: microbiological quality with respect to Salmonella, Shiga-toxin-producing E. coli (STEC) and levels of Escherichia coli.

AIMS: To investigate the microbiological quality of imported fresh leaves on retail sale during 2017 with respect to Salmonella, Shiga-toxin-producing Escherichia coli (STEC) and levels of E. coli. METHODS AND RESULTS: Two hundred and seventy-nine samples of imported edible leaves (69 banana, 77 betel, 118 curry and 15 other types) were tested. Salmonella spp. were confirmed by whole-genome sequencing and isolated from 44 samples, 26% from curry leaves, 14% from betel and 2·4% from all other leaf types: 80% of all samples contained ≥102 , 44% ≥103 and 22% ≥104 CFU of E. coli CFU per g. All samples where Salmonella were detected also yielded ≥20 CFU of E. coli/g. 54 samples were tested for STEC which was detected in six samples and isolated from three: one was identified as STEC O157:H7. CONCLUSIONS: This report further highlights an ongoing problem of Salmonella contamination of imported fresh edible leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: Among all food tested by Public Health England (approximately 11 000 per annum), curry leaves were the herb most commonly contaminated with Salmonella, and betel leaves were the most commonly contaminated ready-to-eat food. The high proportion with unsatisfactory E. coli levels and the detection of STEC suggests risks of contamination by multiple enteric pathogens.

Aflatoxigenic and ochratoxigenic fungi and their mycotoxins in spices marketed in Brazil.

During their processing, spices usually remain close to the ground for drying, a fact that disposes to fungal contamination, as well as moisture transferred from the tropical environment can allow their multiplication and synthesis of mycotoxins. The objective of this study was to evaluate the presence of potentially toxigenic fungi and mycotoxins in spices marketed in Brazil. The fungal contamination was evaluated by direct plating for samples of clove, black and white peppers. Spread plate was used for the samples of rosemary, cinnamon, fennel, pepperoni pepper and oregano. Analyses were performed in triplicate in DG18 media with incubation at 25°C for 7days. The isolation and identification of fungi followed specific recommendations of culture media and incubation period for each genus. The presence of mycotoxins in spices was verified by high-performance liquid chromatography (HPLC) coupled to fluorescence. The frequency of species potentially toxigenic was high in white and black peppers with presence of both aflatoxigenic and ochratoxigenic fungi. Only rosemary and fennel showed contamination with aflatoxin B1 and there was a positive correlation (ρ<0.01) between the rosemary contamination with the presence of AFB1 and A. flavus. Even in the presence of ochratoxigenic fungi, ochratoxin A was not detected in the samples. The presence of natural components with antimicrobial activity could justify the low presence of mycotoxins, even in the presence of known toxigenic fungi in the samples. Mycotoxins were not detected in spices covered by Brazilian regulation of mycotoxins. On the other hand, these contaminants were present in other spices consumed by population and not mentioned in the regulation, which could be considered a cause to concern.

Fungi in spices and mycotoxigenic potential of some Aspergilli isolated.

The aim of this study was to identify fungal species present in 200 samples of rosemary, fennel, cinnamon, clove, pepperoni, black and white pepper and oregano and evaluate the mycotoxigenic potential of the some Aspergilli isolated. Clove, black and white peppers were analyzed by direct plating. For rosemary, cinnamon, fennel, pepperoni pepper and oregano samples were used spread plate. Mycotoxigenic capacity was verified by the agar plug method. With the exception of clove, all the spices showed high fungal contamination, especially by Aspergillus sp., Penicillium sp. and Cladosporium sp. Frequency of toxigenic Aspergillus spp. was intense in white and black peppers, with presence of Aspergillus flavus (up to 32%), Aspergillus nomius (up to 12%), Aspergillus parasiticus (up to 4%), Aspergillus niger complex (up to 52%), Aspergillus ochraceus (up 12%) and Aspergillus carbonarius (up to 4%). 14,2% of A. flavus isolated from black pepper were aflatoxins producers. In the white pepper, 66.7% of A. flavus isolates and 100% of A. nomius were aflatoxigenic. Oregano showed the highest number of A. niger complex isolates (49), however, only 2.04% produced ochratoxin A. This study showed a huge fungal presence in spices, which could compromise the sensorial quality of these products and represent a hazard for consumers.

LAMP-based group specific detection of aflatoxin producers within Aspergillus section Flavi in food raw materials, spices, and dried fruit using neutral red for visible-light signal detection.

Aflatoxins can be produced by 21 species within sections Flavi (16 species), Ochraceorosei (2), and Nidulantes (3) of the fungal genus Aspergillus. They pose risks to human and animal health due to high toxicity and carcinogenicity. Detecting aflatoxin producers can help to assess toxicological risks associated with contaminated commodities. Species specific molecular assays (PCR and LAMP) are available for detection of major producers, but fail to detect species of minor importance. To enable rapid and sensitive detection of several aflatoxin producing species in a single analysis, a nor1 gene-specific LAMP assay was developed. Specificity testing showed that among 128 fungal species from 28 genera, 15 aflatoxigenic species in section Flavi were detected, including synonyms of A. flavus and A. parasiticus. No cross reactions were found with other tested species. The detection limit of the assay was 9.03pg of A. parasiticus genomic DNA per reaction. Visual detection of positive LAMP reactions under daylight conditions was facilitated using neutral red to allow unambiguous distinction between positive and negative assay results. Application of the assay to the detection of A. parasiticus conidia revealed a detection limit of 211 conidia per reaction after minimal sample preparation. The usefulness of the assay was demonstrated in the analysis of aflatoxinogenic species in samples of rice, nuts, raisins, dried figs, as well as powdered spices. Comparison of LAMP results with presence/absence of aflatoxins and aflatoxin producing fungi in 50 rice samples showed good correlation between these parameters. Our study suggests that the developed LAMP assay is a rapid, sensitive and user-friendly tool for surveillance and quality control in our food industry.