Food Additives & Contaminants: Part B Surveillance

ISSN: 1939-3210 (Print) 1939-3229 (Online) Journal homepage: http://www.tandfonline.com/loi/tfab20

Fumonisins and fungi in dry soybeans (Glycine Max L.) for human consumption Laura P. Garcia, Geovana D. Savi, Karolina Santos & Vildes M. Scussel To cite this article: Laura P. Garcia, Geovana D. Savi, Karolina Santos & Vildes M. Scussel (2016): Fumonisins and fungi in dry soybeans (Glycine Max L.) for human consumption, Food Additives & Contaminants: Part B, DOI: 10.1080/19393210.2015.1135484 To link to this article: http://dx.doi.org/10.1080/19393210.2015.1135484

Accepted author version posted online: 04 Jan 2016. Published online: 28 Feb 2016. Submit your article to this journal

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Date: 26 March 2016, At: 00:03

FOOD ADDITIVES & CONTAMINANTS: PART B, 2016 http://dx.doi.org/10.1080/19393210.2015.1135484

Fumonisins and fungi in dry soybeans (Glycine Max L.) for human consumption Laura P. Garcia, Geovana D. Savi, Karolina Santos and Vildes M. Scussel

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Laboratory of Mycotoxicology and Food Contaminants, Food Science and Technology Department, Center of Agricultural Sciences, Federal University of Santa Catarina, Florianopolis, Brazil ABSTRACT

ARTICLE HISTORY

This survey reports the occurrence of fumonisins (FBs) and fungi in dry soybeans sold for human consumption. The variation levels were 138–1495 µg kg−1 and 178–552 µg kg−1 for FB1 and FB2, respectively. In addition, potentially toxigenic fungi as Fusarium, Aspergillus and Penicillium genera were isolated in the samples. These can be considered as indicator-toxin and can produce considerable amounts of mycotoxins. Despite FB presence in the soybeans for human consumption, there is no legal regulation. Therefore, it is important to emphasise the need for frequent monitoring of these contaminants in soybeans.

Received 19 October 2015 Accepted 19 December 2015

Introduction Fungi are responsible for raw and processed grain deterioration when exposed to optimal environmental conditions. In crops in the field it is possible to highlight the presence of Fusarium sp. and, during grain storage, the development of Aspergillus and Penicillium sp., which are often found in grains worldwide (Scaff & Scussel 2004; Ivić et al. 2009; Roigé et al. 2009; Riba et al. 2010; Savi et al. 2014; Scussel et al. 2014). The climatic conditions in soybean-growing regions, moderate mean temperature and relative humidity of 50–80%, provide optimal conditions for fungal growth. Soybean (Glyccine max L.) is often attacked by fungi during cultivation, which significantly decreases its productivity and quality in most production areas (Piotrowska et al. 2013). Fungi associated with cereal grains and oilseeds are important in assessing the potential risk of mycotoxin contamination. These secondary metabolites, produced by different fungi genera and species, can contaminate soybean either in the field or storage under high temperature/humidity conditions (Fessel et al. 2003). Fusarium species as F. verticillioides and F. proliferatum, under optimum growth conditions, can produce mycotoxins such as fumonisins (FBs) (Marin et al. 2004; Scaff & Scussel 2004; Savi et al. 2014). There have been concerns about the spread of FB contamination and its implications to humans and animal health (Boutigny et al. 2012). FBs have several toxic effects that trigger target organs specific in different

KEYWORDS

Soybean; Aspergillus; Penicillium; Fusarium; fumonisins

animal species, such as liver and kidney tumours in rats and neural tubes defects in mice (Voss et al. 2007; Gelineau-Van Waes et al. 2009). They also affect the reproductive system leading to delays on reaching sexual maturity and compromising the animal fertility potential (Gbore et al. 2012). The International Agency for Research on Cancer recognises FB1 in the Group 2B as “possible carcinogenic to humans” (IARC 1993). FB levels in maize and other cereals, apart from some soybeans and feed (swine/chicken), have been published (Castellá et al. 1999; Cançado 2004; Scaff & Scussel 2004; Kobashigawa 2010; Nones et al. 2014). However, when compared with the number of data on cereals, soybeans data are just a few. Most data are from other pulses, for example lentils, peas and black beans. As the main production aim of soybeans is oil extraction, known to be mycotoxin free due to the efficiency of the solvent extraction step, it is possible that the remaining soybean meal, known to keep the mycotoxins from the oil solvent extraction, thus less commercially utilised, which can lead to lack of interest on checking for mycotoxin contamination. Apart from the main use of soybean for oil production and its meal utilised as animal feed ingredient, it is an important protein source for vegans. Although they can get the protein from different vegetables, soybean protein is the most consumed, either itself as whole bean, or as its protein extract by-product from oil industries (Gomes et al. 2009). Soybeans have been rarely studied, when compared with cereals, in relation to mycotoxin contamination.

CONTACT Vildes M. Scussel [email protected] LABMICO, Food Science and Technology Department, Centre of Agricultural Sciences, Federal University of Santa Catarina, Rod. Admar Gonzaga, Itacorubi 1346, Florianopolis, Brazil © 2016 Taylor & Francis

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The risk of contamination with FBs seems to be higher for corn and wheat than for soybean (Ivić et al. 2009). However, due to the soybean’s chemical composition and climatic conditions in soybean-growing regions, it is particularly susceptible to microbial contamination, especially by filamentous fungi. Regarding regulation, there is no maximum limit (ML) set for this contaminant for soybeans (EC 2007; Brazil 2011). With the increase of soybean production worldwide, which occurred in the temperate zones, field mycotoxins tended to increase (Cast 2003). Considering the lack of information on food safety regarding fungi and FBs in dry whole soybeans intended for direct human consumption, this survey in commercialised beans was performed.

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Materials and methods Samples Dry soybean samples (N = 39), sold in packs (0.5–1 kg) and bulk, commercialised in the states of Rio Grande do Sul (RS) and Santa Catarina (SC) (Southern Brazil) were taken. These were produced in different Brazilian regions (Sao Paulo – SP, Parana – PR, RS and SC states). Table 1 shows sample characteristics.

Chemicals (a) Standards – FBs (FB1 and FB2) were purchased from Sigma Aldrich (St. Louis, MO, USA). (b) Solvents – Acetonitrile was purchased from Panreac (Castellar del Vallès, Barcelona, Spain); methanol from Vetec (Rio de Janeiro, Brazil), all HPLC grade; and ultrapure water from Millipore (Billerica, MA, USA). (c) Reagents – Phosphoric and acetic acids, 2-mercaptoethanol, ortho-phthalaldehyde (OPA) were purchased from Vetec, all HPLC grade. (d) Culture media – Potato dextrose agar (PDA), malt extract agar (MEA), czapeck yeast extract agar (CYA), glycerol nitrate 25% (G25 N), peptone and Dichloran Rose-Bengal Chloramphenicol Agar (DRBC) were purchased from Himedia (Curitiba, PR, Brazil).

Equipment High-performance liquid chromatography (HPLC) system was purchased from Gilson (Villiers-le-Bel, France) comprising of an isocratic pump (model 305), collector module (model 805) with 20 µL loop and fluorescence detector model 121. Aqua Lab 4TE (Decagon devices, São José dos Campos, SP, Brazil), vacuum solid phase extraction (SPE) filtration station (12 positions), AHO6023, Phenomenex (Torrance, CA, USA), Romer mill

Table 1. Collection, planting conditions and characteristics for fungi development in 39 soybean (Glycine max L.) samples. Planting condition

Sample collection

Characteristics

Collected Sold RS

Produced Brand P RS NB NA A PE A PE B PE B PE NB NA NB NA NB NA C PE C PE NB NA SC F PE SP NB NA NB NA NB NA D PE Total: 17 E PE SC RS A PE G PE C PE PR H PE H PE NB NA NB NA I PE NB NA NA NB2 SC NB NA NB NA NB NA NB NA J PE K PE F PE L PE SP NB P NB P5 M PE Total: 22 I PE

B L NA NA NA NA L L L NA NA L NA L L L NA NA NA NA NA NA NA L L NA L L L L L L NA NA NA NA NA NA NA NA

Humidity O NA NA NA NA NA NA NA NA O O NA NA NA NA NA NA NA NA NA O O1 NA NA NA NA NA NA NA NA NA NA NA NA NA O4 NA NA NA NA

C C C C C C C C C NA NA C C C C C C C C C NA NA C C C C C C C C C C C3 C C NA C C C C

pH 6.53 6.53 6.46 6.38 6.47 6.46 6.34 6.46 6.49 6.50 6.38 6.50 6.40 6.48 6.44 6.53 6.56 6.53 6.41 6.50 6.57 6.53 6.56 6.45 6.40 6.44 6.34 6.48 6.52 6.49 6.46 6.49 6.39 6.39 6.44 6.40 6.40 6.40 6.39

aw mc (%) 0.69 12.40 0.66 12.10 0.66 12.02 0.57 9.44 0.64 10.84 0.61 10.33 0.63 10.88 0.63 11.35 0.69 10.58 0.68 12.02 0.68 12.71 0.56 9.42 0.62 10.73 0.61 10.52 0.62 10.38 0.63 10.79 0.52 7.82 0.65 11.22 0.62 10.21 0.71 12.89 0.56 9.61 0.58 9.53 0.70 12.86 0.69 12.00 0.66 11.67 0.66 11.64 0.43 3.02 0.59 9.99 0.58 9.63 0.58 9.71 0.63 11.07 0.60 9.38 0.65 11.01 0.63 10.86 0.64 11.52 0.64 11.65 0.56 8.49 0.65 11.47 0.60 10.16

Collected: P, packed; PE, polyethylene; L, bulk (loose); O, organic (no pesticide application); C, commercial; NA, not applicable; NB, no brand specified; RS, Rio Grande do Sul; SP, Sao Paulo; SC, Santa Catarina; PR, Parana; aw, water activity; mc, moisture content (n = 3). 1Organic [IDB seal]; 2barbecue flavoured; 3non-transgenic; 4claims to be organic, not government certified though; 5match and peeled.

model 1301 (Romer Labs, Union, MO, USA), blender model Lar. 2 (Metvisa, Brusque, SC, Brazil) were also purchased. Light microscopes (LM), CH-Bl45-2, Olympus (Shinjuku, Tokyo, Japan); autoclave, Phoenix (Araraquara, SP, Brazil); microwave oven, Philco (Sao Paulo, SP, Brazil); laminar flow cabinet, Veco (Campinas, SP, Brazil); fume cabinet, Quimis (Diadema, SP, Brazil); stomacher, Marconi (Piracicaba, SP, Brazil); and microbiological incubator, Quimis were obtained.

Other materials Solid phase extraction (SPE) column quaternary amino (500 mg, 6 mL; Applied Separations, Allentown, PA,

FOOD ADDITIVES & CONTAMINANTS: PART B

USA); C18 chromatographic column of 250 × 4.6 mm (length × diameter), 5 µm (particle size), model 201 TP54 (Vydac, Hesperia, CA, USA); nylon membrane filter (0.45 µm pore size, 13 mm diameter); and syringe filter, 0.45 mm pore size, 4 mm diameter (Waters, Mildford, MS, USA) were obtained.

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Sample collection Soybeans were collected randomly from different shops and food stores of RS and SC states from March to October 2013. Beans were of different brands and types (whole, peeled and peeled with herbs), from organic (no pesticides applied) and conventional (regular crop treatments) planting systems. They were purchased in polyethylene packs heat sealed (0.5–1 kg) or in bulk (1 kg) after filth removal and drying. The samples from bulk batches were collected using a grains auger from different points of the bulk batches, with a minimum final weight of ca. 1 kg. Samples were identified and stored under refrigeration (4 ± 1°C) for analysis. Table 1 shows sample details regarding origin, where they were purchased and also produced, brands, crop conditions and number of samples.

Sample characteristics (a) pH: Performed according to the method of IAL (2008); (b) Humidity – Moisture content (mc): By the gravimetric method of AOAC (2005a), (n = 3); and water activity (aw): By the Aqua Lab 4TE (n = 3; Table 1).

FBs (FB1 and FB2) analysis Each soybean sample was ground in a Romer mill with automatic quartering and portions of 50 g were taken for FB analysis, performed according to AOAC method 995.15 (2005b). Briefly, mycotoxins were extracted with methanol:water (80:20) in a blender, filtered and cleaned up with a SPE column previously conditioned with methanol and methanol:water 80:20 (v/v). The extract was loaded and the mycotoxins eluted with methanol:acetic acid 99:1 and dried under nitrogen stream. Prior to HPLC FBs were derivatised with OPA solution and 20 µL injected into the HPLC with fluorescence detection at emission interval 430–470 nm and excitation interval 305–395 nm. Separation was accomplished in isocratic mode with methanol:dihydrogen phosphate buffer of sodium (0.1 M) 77:23 mobile phase, pH adjusted to 3.3 with phosphoric acid, at a flow rate of 1 mL/min. FB quantification was performed by measurement of peak area at FB retention time compared with the standard solutions used for the

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calibration curve. For quality control of the routine analytical process, samples were analysed on five different days. The analytical work was performed in LABMICO, which is accredited by MAPA (Ministry of Agriculture and Food Supplies), following ISO/IEC 17025 (2005). Proficiency testing was performed in an interlaboratory study with matrix reference materials of Romer Labs, following ISO/IEC 17043 (2010) with satisfactory z-scores (–1 to 1) for FB1 and FB2. Measurement uncertainty (data shown in the database) was calculated according to European Commission Regulation No. 401/2006 (EC 2006).

Mycology tests (a) Total fungi count – Carried out according to the method reported by ICMSF (1986)/Da Silva et al. (2012). Briefly, to each sample (25 g) sterile peptone water was added, stirred on a stomacher (2 min) and dilutions (10–1, 10–2, 10–3, 10–4) were prepared. Aliquots of each dilution (0.1 mL) were spread (n = 2) on a PDA medium surface containing chloramphenicol and incubated for 7 days, at 28°C, in the dark. Data were reported as colony forming units per gram (CFU g−1) in dilution 10–1. (b) Fungi genera and species identification – The strains isolated were inoculated individually in PDA, MEA, G25 N and CYA media. Species identification was performed by a microculture method in agar carnation leaf for Fusarium and Czapek-Dox for Aspergillus and Penicillium (Weber & Pitt 2000). The isolates were examined through a light microscope (100 and 400× magnification) and the species identification was carried out according to the taxonomic keys of available guides (Raper & Fennel 1965; Pitt 1979; Nelson et al. 1983; Pitt & Hocking 2009).

Results and discussion From the data obtained it was possible to observe that FBs were detected in some of the dry soybean samples for human consumption, and some different fungi species were also detected (Table 2).

Characteristics of the soybean samples and humidity levels Most of the soybean samples were from conventional crops, being only 11% and 9% from organic crop systems sold in the RS and SC states, respectively. Those samples were grown in different states (SP, PR, RS, SC) though. It was observed that the soybean samples purchased in the shops presented variations on pH and humidity levels, conditions that may allow

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Table 2. Soybean (Glycine max L.) fumonisin levels and isolated fungi. Fumonisins (µg kg−1)

Sample N 1–10 11 12 13–16 17 18 19

Sold RS

SC SP SC

20 21 22

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Produced RS

23 24–27 28 29 30 31 32 33–35 36 37 38,39 Positive(%)/total samples

RS

PR

SC

SP

Fungi

FB1