Bacterial species and mycotoxin contamination associated with locust bean, melon and their fermented products in south-western Nigeria

International Journal of Food Microbiology 258 (2017) 73–80

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Bacterial species and mycotoxin contamination associated with locust bean, melon and their fermented products in south-western Nigeria

MARK

Bamidele S. Adedejia, Obinna T. Ezeokolib,c, Chibundu N. Ezekield,e,⁎, Adewale O. Obadinaa, Yinka M. Somorinf, Michael Sulyokd, Rasheed A. Adelekeb,c, Benedikt Warthg, Cyril C. Nwangburukah, Adebukola M. Omemui, Olusola B. Oyewolea, Rudolf Krskad a

Department of Food Science and Technology, Federal University of Agriculture, Abeokuta, P.M.B. 2240, Ogun State, Nigeria Microbiology and Environmental Biotechnology Research Group, Agricultural Research Council-Institute for Soil, Climate & Water, 600, Belvedere Street, Arcadia, 0001 Pretoria, South Africa c Unit for Environmental Science and Management, North-West University (Potchefstroom Campus), Private Bag x6001, Potchefstroom 2531, South Africa d Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU), Konrad Lorenzstr. 20, A-3430 Tulln, Austria e Department of Microbiology, School of Science and Technology, Babcock University, Ilishan Remo, Ogun State, Nigeria f Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland g University of Vienna, Faculty of Chemistry, Department of Food Chemistry and Toxicology, Waehringerstr. 38, 1090 Vienna, Austria h Department of Agriculture, School of Science and Technology, Babcock University, Ilishan Remo, Ogun State, Nigeria i Department of Hospitality and Tourism Management, Federal University of Agriculture, Abeokuta, P.M.B. 2240, Ogun State, Nigeria b

A R T I C L E I N F O

A B S T R A C T

Keywords: Bacterial diversity Food safety Locust beans Melon Natural toxins Public health

The microbiological safety of spontaneously fermented foods is not always guaranteed due to the undefined fermenting microbial consortium and processing materials. In this study, two commonly consumed traditional condiments (iru and ogiri) and their respective raw seeds (locust bean and melon) purchased from markets in south-western Nigeria were assessed for bacterial diversity and mycotoxin contamination using 16S rRNA gene sequencing and liquid chromatography tandem mass spectrometry (LC-MS/MS), respectively. Two hundred isolates obtained from the raw seeds and condiments clustered into 10 operational taxonomic units (OTUs) and spanned 3 phyla, 10 genera, 14 species and 2 sub-species. Bacillus (25%) and Staphylococcus (23.5%) dominated other genera. Potentially pathogenic species such as Alcaligenes faecalis, Bacillus anthracis, Proteus mirabilis and Staphylococcus sciuri subsp. sciuri occurred in the samples, suggesting poor hygienic practice during production and/or handling of the condiments. A total of 48 microbial metabolites including 7 mycotoxins [3-nitropropionic acid, aflatoxin B1 (AFB1), AFB2, beauvericin, citrinin, ochratoxin A and sterigmatocystin] were quantified in the food samples. Melon and ogiri had detectable aflatoxin levels whereas locust bean and iru did not; the overall mycotoxin levels in the food samples were low. There is a need to educate processors/vendors of these condiments on good hygienic and processing practices.

1. Introduction Traditional fermented foods from legumes and oilseeds form a major contribution to the protein requirements of poor households in rural populations across Africa and Asia. Many such fermented foods are used as condiments and widely consumed due to the aroma and flavour they impart on foods (Achi, 2005; Odunfa, 1988). The production of traditional fermented foods in many sub-Saharan African countries is still at small scale/household level (Adejumo et al., 2013; Odunfa, 1985a) and is mostly influenced by chance inoculants which

affects the quality characteristics of the finished product. African locust bean (Parkia biglobosa) seeds are very rich in proteins and are spontaneously fermented to produce iru (also known as dawadawa), a food condiment in Nigeria and parts of West Africa (Odunfa and Oyewole, 1986). Iru is important for its flavour and high protein content in soups and stews (Odunfa, 1988). Traditional processing of iru is mainly done by women and provides sustainable livelihood for their families (Adejumo et al., 2013). On the other hand, ogiri is produced by spontaneous fermentation of oil rich seeds such as those of melon (Colocynthis citrullus) (Odunfa, 1981), castor oil bean (Ricinus communis)

⁎ Corresponding author at: Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU), Konrad Lorenzstr. 20, A-3430 Tulln, Austria. E-mail address: [email protected] (C.N. Ezekiel).

http://dx.doi.org/10.1016/j.ijfoodmicro.2017.07.014 Received 4 March 2017; Received in revised form 6 July 2017; Accepted 24 July 2017 Available online 25 July 2017 0168-1605/ © 2017 Elsevier B.V. All rights reserved.

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2.2. Bacteriological analysis of food samples

(Odunfa, 1985b) and Telfairia occidentalis (Barber et al., 1989). Ogiri is rich in amino acids such as alanine, lysine and glutamic acid (Odunfa, 1981) and micronutrients (David and Aderibigbe, 2010). Bacillus species, especially Bacillus subtilis, have been implicated in the fermentation of both condiments (Barber et al., 1988; Odunfa, 1985b; Odunfa and Oyewole, 1986). Since iru and ogiri are widely consumed and often used in preparing meals by both households and food vendors, it is important to regularly monitor the safety of these food condiments. It has been previously established that the final microbiological quality and safety of spontaneously fermented food products are influenced by the quality of the raw materials (Steinkraus, 1983), the processing method (Sadiku, 2010) and hygiene of the personnel performing the art of fermentation (Iwuoha and Eke, 1996). Potentially pathogenic bacteria such as Bacillus cereus, Staphylococcus aureus, coliforms and enterococci (Aderibigbe et al., 2011; Falegan, 2011; Ijabadeniyi, 2007; Oguntoyinbo, 2012; Okanlawon et al., 2010) have been isolated from retailed ogiri. Most of the previous studies on microbial ecology of retailed iru and ogiri have been conducted based on classical biochemical identification methods for bacteria (Ajayi, 2014; Ajayi et al., 2015; Falegan, 2011; Ogunshe et al., 2008) which have several limitations, including the misidentification of species. Over a decade ago, toxigenic fungal species (e.g. Aspergillus flavus, A. ochraceus and Penicillium citrinum) and/or aflatoxins determined by thin-layer chromatography were reported in either melon or ogiri from Nigeria (Bankole et al., 2004, 2006, 2010) and ogiri from Sierra Leone (Jonsyn, 1990), but no such information is available on locust bean seeds or iru. More recently, diverse mycotoxins including aflatoxins, citrinin, cyclopiazonic acid and ochratoxin A have been reported in high levels in melon seeds analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (Ezekiel et al., 2016; Somorin et al., 2016); suggesting the need to analyse fermented condiments for mycotoxins. It is therefore imperative to study the bacterial community and spectrum of microbial metabolites occurring in these fermented condiments because the presence of bacterial pathogens and mycotoxins in iru and ogiri may constitute a public health concern for consumers. There was no need to study the fungal diversity as mycotoxin profile reflects the mycotoxicological risk posed by the contaminating moulds. Thus, this study aimed to assess the bacteriological and mycotoxicological safety of both food condiments regularly and widely consumed in Nigeria.

2.2.1. Isolation of bacteria Milled portions of the representative food samples were ten-fold serially diluted in sterile distilled water for the isolation of bacteria. Aliquots of serially diluted samples were pour-plated on a range of bacteriological media: plate count agar (PCA) (Oxoid, Basingstoke Hampshire, England) for aerobic bacteria; MacConkey agar (LAB M, Heywood Lancashire England) for coliforms; and mannitol salt agar (MSA) (Oxoid, Basingstoke, Hampshire, England) for staphylococci. All inoculated plates were incubated at 30 °C for 24–48 h. Distinct colonies were streaked on fresh PCA, MSA and MRS agar plates twice to obtain pure cultures, and then maintained on agar slants at 4 °C for further characterization studies. 2.2.2. Preliminary morphological assessment of bacterial isolates All purified bacterial isolates were microscopically assessed for cell morphology (Gram reaction, cell shape and arrangement). Colony growth pattern, colour, elevation and consistency were also examined. Isolates were then inoculated into nutrient broth E (LAB M, UK) supplemented with 40% glycerol (BDH, Poole, England) and stored at 4 °C for further characterization studies. 2.3. Molecular identification of isolates 2.3.1. DNA extraction Overnight cultures of pure isolates in Luria-Bertani broth (Acumedia, Michigan, USA) were centrifuged (10,000 ×g for 1 min) and genomic DNA was extracted using the ZR Fungal/Bacterial DNA MiniPrep extraction kit (Zymo Research, California, USA) according to the manufacturer's instructions. Briefly, cells were lysed by bead beating in a lysis buffer and the cell lysate was centrifuged. The supernatant was filtered prior to DNA binding to a column matrix. Bound DNA was further purified and eluted from the column matrix. The integrity of eluted DNA was verified by agarose gel electrophoresis, while quantification was performed using the Qubit 2.0 fluorometer (Thermo Fischer Scientific, Massachusetts, USA). 2.3.2. Partial 16S rRNA gene sequencing The partial 16S rRNA gene of isolates was amplified using universal primer sets 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′GGTTACCTTGTTACGACTT-3′). Each reaction tube contained 12.5 μL of 2× Master mix (Thermo Fisher Scientific, MA, USA), 0.2 μM of each of forward and reverse primers, 20 ng template DNA, and nuclease-free water to a total volume of 25 μL. The PCR protocol involved an initial denaturation at 94 °C for 5 min, 32 cycles of 94 °C for 30 s, 54 °C for 30 s and 72 °C for 2 min, and a final extension step of 94 °C for 5 min. All PCRs was performed in a thermal cycler (SimpliAmp, Applied Biosystems, California, USA). Amplicons (~ 1500 bp) were verified by agarose gel electrophoresis and purified using the NucleoFast 96 PCR clean-up kit (Macherey-Nagel, Duren, Germany) prior to sequencing the PCR products using forward primer 341F (5′-CCTACGGGAGGCAGCAG3′) and the Big Dye terminator sequencing v3.1 cycle sequencing kit (Applied Biosystems, UK). Sequencing amplicons were purified using Sephadex columns (Princeton Scientific, New Jersey, USA) and analyzed on a genetic analyzer (ABI3730xl, Applied Biosystems, CA, USA).

2. Material and methods 2.1. Food samples Locust bean seeds, shelled melon seeds, iru and ogiri samples were purchased from markets in Lagos (6.6084854 N 3.3915728 E), Ogun (7.1411394 N 3.3478305 E) and Oyo states (7.3905092 N 3.8687164 E), southwest of Nigeria in March 2016. Nine composite samples of each food material were collected to obtain a total of 36 composite samples. Each composite sample (300 g) consisted of three individual sub-samples aggregated from three food vendors. All samples were aseptically collected into sterile polyethylene bags and transported to the laboratory for analysis. Each composite sample was properly homogenized and quartered twice to yield 25 g representative sample for microbiological and mycotoxin analysis. All representative samples were comminuted, stored at 4 °C and processed within 24 h. Each representative sample was batched into two parts: batch A (15 g) for bacteriological analysis and batch B (10 g) for mycotoxin analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Batch B samples were kept at −20 °C until mycotoxin analysis.

2.3.3. Taxonomic assignment and phylogenetic reconstruction For taxonomic assignment, sequence electropherograms were inspected and manually edited using ChromasLite (v.2.1, Technelysium Pty Ltd). Edited sequences were aligned against the EzTaxon server (http://www.ezbiocloud.net; Kim et al., 2012) for identification of isolates on the basis of 16S rRNA gene sequence data. Sequences were further clustered into operational taxonomic units (OTUs) at a sequence similarity of 97% using Mothur software (Schloss et al., 2009) as previously described (Ezeokoli et al., 2016). For phylogenetic 74

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reconstruction, representative sequences along with closely related sequences in the Genbank were selected and aligned using ClustalX version 2.1 (Larkin et al., 2007) and edited for gaps in DAMBE software (Xia, 2013). Edited alignments were then used for the constructing of a neighbour-joining phylogenetic tree by using the Tamura–Nei substitution model and a thousand bootstrap replications in MEGA 7 (Kumar et al., 2016). All nucleotide sequences obtained in this study are available in the GenBank under accession numbers KX966428–KX966469. 2.4. Analysis of food samples for multiple microbial metabolites Multi-microbial analysis was performed by LC-MS/MS as previously described (Malachová et al., 2014). Each representative food sample (5 g) was weighed into a 50 mL polypropylene tube (Sarstedt, Germany) and extracted with 20 mL acetonitrile/water/acetic acid (79:20:1, v/v/v) on a GFL 3017 rotary shaker (GFL, Burgwedel, Germany) for 90 min. Extracts were diluted in an equal volume of extraction solvent and injected into the liquid chromatography (LC) instrument as described in detail by Malachová et al. (2014). The presence and levels of over 300 microbial metabolites (mainly mycotoxins) were determined using a QTrap 5500 LC-MS/MS System (Applied Biosystems, Foster City, CA, US) equipped with a TurboV electrospray ionization (ESI) source and a 1290 Series UHPLC System (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was performed at 25 °C on a Gemini® C18-column, 150 × 4.6 mm i.d., 5 μm particle size, equipped with a C18 security guard cartridge, 4 × 3 mm i.d. (all from Phenomenex, Torrance, CA, US). For spiking experiments, three samples (each 0.25 g) of locust beans and melon containing no or very minimal levels of the analytes were spiked with 100 μL a multianalyte standard at one concentration level and analyzed as described above. Microbial metabolites and mycotoxins were quantified in the samples by applying external calibration based on a serially diluted multicomponent stock solution. Apparent recoveries for data correction and limits of detection (LOD) of each analyte in the food matrices were estimated. The identification of positive analytes was confirmed by the acquisition of two MS/MS transitions which yielded 4.0 identification points according to the Commission decision 2002/657/EC. Furthermore, the LC retention time and the intensity ratio of the two MRM transitions agreed with the related values of an authentic standard within 0.1 min and 30% rel., respectively.

Fig. 1. Distribution of isolates into operational taxonomic Units (OTUs). For clustering of partial 16S rRNA gene sequences into OTUs, sequences of all isolates were aligned in ClustalX and edited in DAMBE software. Alignments were checked for Chimera (Bellerophon) and clustered de novo at a distance of 97% using Mothur.

the phyla Firmicutes (62%), Proteobacteria (36%) and Actinobacteria (2%). Taxonomic identity of the isolates is presented in Table 1; most of the isolates belonged to the genus Bacillus (25%) and Staphylococcus (23.5%). The occurrence of the isolates in the four food materials was so diverse that there was no particular isolate which was present in all sample categories. Proteus mirabilis and Staphylococcus sciuri subsp. sciuri were each found in three of the four food materials, while Alcaligenes faecalis, Bacillus pumilus, B. anthracis and Enterobacter cloacae subsp. cloacae were present in two food types. Achromobacter insuavis, B. subtilis subsp. inaquosorum, Brevundimonas olei, Bacillus licheniformis, Enterobacter cloacae subsp. dissolvens, Cellulosimicrobium cellulans, E. hormaechei, Klebsiella quasipneumoniae subsp. quasipneumoniae and Paenibacillus dendritiformis were unique to only one food type. 3.2. Bacterial association between pairs of raw seeds and fermented products The heat map in Fig. 2 depicts the association of bacterial isolates between food pairs (raw seeds and finished/fermented products). The map signals suggest that there was dissimilarity in bacterial species diversity and richness estimates obtained between melon seeds and ogiri as well as locust beans and iru pairs. The cluster dendrogram in Fig. 2 suggests that in terms of bacterial species richness, parent materials cluster together, while the fermented products cluster together.

2.5. Data analysis

3.3. Evolutionary relatedness of bacterial isolates

The abundance and associations of isolates within and between samples were depicted with heatmaps showing cluster dendrograms constructed using the average linkage hierarchical clustering of the Bray-Curtis dissimilarity matrix calculated for the full dataset. Heat map was constructed in R software (R Core Team, 2013) using the ‘gplots’ and ‘vegan’ packages. Data on multi-microbial metabolites were analyzed for descriptive statistics parameters using SPSS® 21.0 for Windows (IBM Corporation, New York, USA).

The phylogenetic dendrograms in Figs. 3 and 4 reveal the evolutionary relatedness of the isolates obtained in this study. The tree topology strongly suggests that some isolates are closely related to one or more known Genbank relatives. For example, isolate LMO1-B belongs to the same clade (monophyletic) as Bacillus subtilis, B. mojavensis and B. tequilensis (Fig. 3). Also, isolates AM03-3 and LL03 belong to a monophyletic clade as B. safensis and B. pumilus (Fig. S1). Furthermore, isolates LMO1 (Fig. 3) and AI02 (Fig. 4) are closely related to B. anthracis, which is a member of the B. anthracis taxonomic group consisting of B. cereus and B. thuringiensis among other Bacillus spp. Meanwhile, it is pertinent to mention that members of a taxonomic group are usually indistinguishable based on 16S rRNA gene (Wang et al., 2007).

3. Results 3.1. Taxonomic diversity of isolates in the food samples

3.4. Occurrence of mycotoxins and other microbial metabolites in the food samples

In total, 200 isolates were obtained from the raw seeds [locust bean (n = 15); melon (n = 69)] and fermented products [iru (n = 80); ogiri (n = 36)]. Based on partial 16S rRNA sequence of the bacterial isolates obtained from all categories of the food samples, the isolates clustered into 10 OTUs at ≥ 97% sequence similarity. The distribution of isolates into OTUs is shown in Fig. 1. OTU 2 had the highest number of isolates whereas OTU 8 and OTU 9 had the least number of isolates. Taxonomic assignment of OTUs revealed that isolates belonged to

Seven mycotoxins (3-nitropropionic acid (3-NPA), aflatoxin B1 (AFB1), AFB2, beauvericin (BEAU), citrinin (CIT), ochratoxin A (OTA) and sterigmatocystin) (Table 2) and 41 other metabolites from Aspergillus, Fusarium, Penicillium and other fungal genera (Table S1) were quantified in the four food matrices. Only the B-aflatoxins (AFB1 and 75

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Table 1 16S rRNA-based identification of bacterial isolates through EzTaxon server. Eztaxon closest match

Total no. of isolates

OTU no.a

Similarity (%)

Accession number of representative

Locust bean seeds Alcaligenes faecalis subsp. phenolicus Bacillus pumilus Staphylococcus sciuri subsp. sciuri

11 2 2

6 1 5

99.76 100 100

KX966447 KX966446 KX966469

Iru Bacillus anthracis Enterobacter cloacae subsp. cloacae Proteus mirabilis Staphylococcus sciuri subsp. sciuri

18 1 24 37

3 4 2 5

100 99.88 99.88 100

KX966448 KX966451 KX966450 KX966449

Melon seeds Achromobacter insuavis Bacillus pumilus B. anthracis B. subtilis subsp. inaquosorum Brevundimonas olei Cellulosimicrobium cellulans Enterobacter cloacae subsp. cloacae E. cloacae subsp. dissolvens E. hormaechei Klebsiella quasipneumoniae subsp. quasipneumoniae Proteus mirabilis Staphylococcus sciuri subsp. sciuri

1 7 10 7 1 4 11 13 1 5 1 8

8 1 3 1 9 10 4 4 4 4 2 5

99.76 100 100 100 99.88 100 100 99.52 99.65 99.76 99.22 100

KX966437 KX966467 KX966430 KX966436 KX966466 KX966445 KX966434 KX966431 KX966442 KX966441 KX966443 KX966440

Ogiri Alcaligenes faecalis subsp. phenolicus Bacillus licheniformis Paenibacillus dendritiformis Proteus mirabilis

1 6 3 26

6 1 7 2

99.76 100 99.65 100

KX966464 KX966460 KX966463 KX966458

a

OTU, operational taxonomic unit.

4. Discussion In the present study, we report the diversity of bacteria and spectrum of toxic microbial metabolites that could pose public health threats to consumers of two commonly consumed condiments used in preparation of local dishes across West Africa. This genotypic-based study of bacterial flora in the raw materials and their fermented products identified species which have been previously associated with the fermentation of legume seeds into condiments. Bacterial species of pathogenic relevance which pose potential risks to consumers of spontaneously fermented traditional food condiments were also identified. Several species of Bacillus including B. anthracis, B. subtilis, B. licheniformis and B. pumilus recovered from both raw seeds and fermented products in the present study substantiate previous reports on the association of Bacillus species notably, B. licheniformis and B. subtilis, with fermentation of iru (Adelekan and Esiobu, 2012; Aderibigbe et al., 2011; Adewumi et al., 2013; Antai and Ibrahim, 1986; Odunfa and Oyewole, 1986), ogiri (Barber et al., 1989; Odunfa, 1985b) and other fermented traditional foods (Chantawannakul et al., 2002; Ezeokoli et al., 2016; Omafuvbe et al., 2000; Sarkar et al., 1994). Bacillus licheniformis has also been previously isolated from soumbala (a fermented Parkia biglobosa condiment in Burkina Faso) (Ouoba et al., 2008) and Chungkook-Jang, a fermented traditional soybean paste indigenous to South Korea (Kim et al., 2004). In these food fermentations, B. licheniformis and B. subtilis contribute to the biochemical transformation of the seed substrate and subsequent generation of aromatic flavour compounds (Achi, 2005; Beaumont, 2002; Leejeerajumnean et al., 2001; Odunfa, 1985b). From a food safety perspective, B. subtilis may be considered safe in such fermented condiments, however, some strains of B. subtilis and B. licheniformis detected in foods may constitute food spoilage bacteria and opportunistic human pathogens (Fernández-No et al., 2011; Katina et al., 2002). Bacillus pumilus has been previously reported in iru (Odunfa and Oyewole, 1986). Although certain strains of B. pumilus are known human and plant pathogens (Kimouli et al., 2012;

Fig. 2. Heat map of isolates diversity between samples. Cluster dendrogram shown on the heat map was constructed using the average linkage hierarchical clustering of the BrayCurtis dissimilarity matrix calculated for the full dataset.

AFB2) were detected in the food samples. About 67% of the melon samples contained aflatoxins at mean concentrations of 5.6 μg/kg (range = 1.1–22.4 μg/kg) and 6.6 μg/kg (range = 1.1–27.7 μg/kg) for AFB1 and total aflatoxins (sum of AFB1 and AFB2 since the G-aflatoxins were not found in the samples), respectively. AFB1 was detected in only one sample of ogiri (7 μg/kg) while no detectable aflatoxin was recorded in locust beans and iru. Higher mean level of BEAU was observed in the raw food materials [locust beans (6 μg/kg) and melon (1.1 μg/kg)] than in their respective fermented products [iru (< LOD) and ogiri (0.5 μg/kg)]. CIT was found in all food matrices except melon while OTA was detected in only iru, both toxins occurred at very low levels (≤ 7 μg/kg). Only 11% of the 36 composite food samples contained AFB1 above the 2 μg/kg level stipulated for majority of the food matrices exported to the EU (Food and Agricultural Organization [FAO], 2004). 76

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Fig. 3. Unrooted neighbour-joining phylogenetic tree illustrating evolutionary relatedness of representative bacterial isolates obtained from melon seeds and ogiri with closely related Genbank sequences. The neighbour-joining phylogenetic tree was constructed by the Tamurai-Nei substitution model and a thousand bootstrap replications in MEGA 7 software. Isolates obtained in this study are indicated by shaded circles. Bootstrap values < 50 are not shown. Accession numbers of sequences are indicated in parenthesis.

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Fig. 4. Unrooted Neighbour-Joining phylogenetic tree illustrating evolutionary relatedness of representative bacterial isolates obtained from locust bean seeds and iru with closely related Genbank sequences. The neighbour-joining phylogenetic tree was constructed by the Tamurai-Nei substitution model and a thousand bootstrap replications in MEGA 7. Isolates obtained in this study are indicated by shaded triangles. Bootstrap values < 50 are not shown. Accession numbers of sequences are indicated in parenthesis.

Another bacterial species, Staphylococcus sciuri which is of importance in alkaline fermentation, was identified in our study. This species was the only staphylococcus found in the present study. Although S. sciuri has been previously reported to be involved in the fermentation of baobab seeds (Parkouda et al., 2010), it could also be pathogenic (Shittu et al., 2004). Staphylococcus sciuri is a coagulase negative skin commensal and its presence in fermented condiments can

Yuan and Gao, 2015), the proteases and metabolites of some strains are important in food and the food industry (Pan et al., 2004). On the other hand, B. anthracis is the etiological agent of anthrax (Turnbull, 1999). The detection of B. anthracis and/or isolates phylogenetically related to B. anthracis in iru and melon seed samples further corroborates previous concerns over the safety of spontaneously fermented indigenous foods (Adewumi et al., 2013; Iwuoha and Eke, 1996; Steinkraus, 1997).

Table 2 Mycotoxin concentrations in locust bean and melon seeds and their fermented products sold in southwestern Nigeria. Food category

Locust bean (na = 9)

Iru (na = 9) Melon (na = 9)

Ogiri (na = 9)

a b c d

Mycotoxins

b

LOD (μg/kg) Rc Nd Range (μg/kg) Mean (μg/kg) Nd Range (μg/kg) Mean (μg/kg) LODb (μg/kg) Rc Nd Range (μg/kg) Mean (μg/kg) Nd Range (μg/kg) Mean (μg/kg)

3-Nitropropionic acid

Aflatoxin B1

Aflatoxin B2

Beauvericin

Citrinin

Ochratoxin A

Sterigmatocystin

0.8 3.7 1 < LOD–5500.5 5500.5 0 < LOD – 0.8 18.7 2 177.9–1472.4 825.2 3 266.6–2761.1 1431.4

0.3 81.7 0 < LOD – 0 < LOD – 0.3 95.6 6 1.12–22.4 5.6 1 < LOD–6.99 6.99

0.4 89 0 < LOD – 0 < LOD – 0.4 93.3 2 0.75–5.30 3 0 < LOD –

0.008 85.8 6 0.12–29.3 6 0 < LOD – 0.008 99.1 7 0.19–2.1 1.09 2 0.24–0.67 0.46

0.16 83.2 2 0.9–6.6 3.7 1 < LOD–2.23 2.23 0.16 100 0 < LOD – 1 < LOD–4.49 4.49

0.4 78.9 0 < LOD – 1 < LOD–7.04 7.04 0.4 97.2 0 < LOD – 0 < LOD –

0.32 77.2 0 < LOD – 1 < LOD–1.71 1.71 0.32 105 4 0.64–12.8 3.98 5 1.5–8.2 3.67

Number of analyzed composite samples. Limit of detection [S/N = 3:1] expressed as μg/kg. Apparent recovery (%) determined by spiking experiment. Number of positive samples.

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pathogenic bacteria and mycotoxins into the condiments, these interventions are for household prevention of possible food-borne hazards that could result from consumption of these locally processed condiments. Suggested interventions include: the purchasing of quality raw materials/seeds, maintaining good personal hygiene among processors (handlers and vendors) of local condiments, soaking and repeated washing of condiments purchased from markets in brine prior to use in soup or food preparation, and the addition of condiments early enough during soup preparation so as to allow for proper boiling. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2017.07.014.

be attributable to lack of good hygiene in handling of fermented products and fermentation utensils/materials (Achi, 2005; Iwuoha and Eke, 1996). Furthermore, similar to this present study, Proteus mirabilis was found in raw materials and final product in pork balls and it was incriminated in a food poisoning outbreak (Wang et al., 2010). The similarity in the isolates from both fermented foods (iru and ogiri), which differ from the mix of isolates in the raw material, strongly suggests unhygienic handling of the marketed products and highlights the potential hazards associated with the consumption of fermented foods which are not further processed to eliminate these bacterial species. To the best of our knowledge, this is the first study reporting the association of A. insuavis and C. cellulans with melon seed or legumetype fermented condiments. Other isolates such as Al. faecalis subsp. phenolicus and Paenibacillus dendritiformis have been previously reported in some fermented foods including ogi, ikpiru and yanyanku (Agbobatinkpo et al., 2013; Antai and Ibrahim, 1986; Izah et al., 2016). These isolates along with other isolates of the phylum Proteobacteria including Proteus mirabilis, E. hormaechei, E. cloacae, K. quasipneumoniae and Br. olei are most likely contaminants of plant materials, soil or water used for processing and/or from human origin (Iwuoha and Eke, 1996). Importantly, majority of these microorganisms are of medical importance to humans (Abbott, 2011; Jacobsen et al., 2008; Podschun and Ullmann, 1998) and may further aggravate the public health concern of direct consumption of these fermented foods. Co-contamination of foodstuffs with mycotoxins consitutes an additional threat to public health besides pathogenic bacteria. In this study, the levels of the mycotoxins found in the raw materials and their fermented products sampled from the local markets were relatively low compared to data from other crops highly prone to aflatoxin studied in Nigeria (Atanda et al., 2013) except for the levels of 3-NPA which were quite high. Locust bean seeds did not contain aflatoxins; this needs to be further studied with a larger sample size to ascertain if this legume is less prone to aflatoxin contamination so as to be explored as an alternative protein source in regions with high aflatoxin risk. The levels of aflatoxins found in the melon samples and ogiri are similar to the concentrations reported in melon samples consumed in Ireland and the United Kingdom (Somorin et al., 2016) and ogiri samples collected in 2005 and 2006 in south-western Nigeria (Bankole et al., 2010). The levels of aflatoxins were however lower than levels reported by Ezekiel et al. (2016) in melon seeds from Nigeria. CIT and OTA levels in melon seed reported in the present study are similar to previous studies (Ezekiel et al., 2016; Somorin et al., 2016). Aflatoxins are potent liver carcinogens. AFB1 has been classified by the WHO International Agency for Research on Cancer as a group 1 carcinogen (International Agency for Research on Cancer [IARC], 2002). Thus, any level of this toxin in food is regarded a potential threat to consumer health considering the chronic effects that arise from ingestion of even low concentrations over time. Furthermore, considering that some mycotoxins may weaken the immune system through modulation/suppression (Bondy and Pestka, 2000; Council for Agricultural Science and Technology [CAST], 2003), their presence in foods may predispose the consumer to infections from pathogenic bacteria. Thus, stricter hygienic and sanitary measures need to be adopted by households or processors of local foods such as the fermented condiments under study in order to reduce the burden of food-borne diseases especially among poor and low-income families. It is noteworthy to mention that most investigations on hygiene focus on staple foods in economically-developed countries or on foods that are exported to developed countries. In this study, we have provided, for the first time, snapshot data on diversity of both bacteria and mycotoxins in two locally-processed and commonly consumed condiments that are rarely monitored by national regulatory agencies. In view of the findings of this study, we propose multiple food control interventions (listed below) for mitigating potential risks to consumers. Since we did not investigate the entry point (which could be at processing, transportation, storage or packaging) of the potentially

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