The effect of Saccharomyces cerevisiae on the ... - Wiley Online Library

Letters in Applied Microbiology 2005, 40, 133–137

doi:10.1111/j.1472-765X.2004.01633.x

The effect of Saccharomyces cerevisiae on the stability of the herbicide glyphosate during bread leavening F.L. Low1,2, I.C. Shaw1,2 and J.A. Gerrard1 1

School of Biological Sciences, University of Canterbury, Christchurch, New Zealand, and 2Institute of Environmental Science and Research, Christchurch Science Centre, Christchurch, New Zealand

2004/0282: received 11 March 2004, revised 8 October 2004 and accepted 11 October 2004

ABSTRACT F . L . L O W , I . C . S H A W A N D J . A . G E R R A R D . 2004.

Aims: To investigate the ability of baker’s yeast (Saccharomyces cerevisiae) to degrade the herbicide glyphosate during the fermentation cycle of the breadmaking process. Methods and Results: Aqueous glyphosate was added to bread ingredients and kneaded by commercially available breadmaking equipment into dough cultures. Cultures were incubated in the breadmaker throughout the fermentation cycle. The recovery of glyphosate levels following fermentation was determined, thus allowing an estimation of glyphosate degradation by yeast. Conclusions: It was shown, for the first time, that S. cerevisiae plays a role in metabolizing glyphosate during the fermentation stages of breadmaking. Approximately 21% was degraded within 1 h. Significance and Impact of the Study: As a result of projected increases in the glyphosate use on wheat and the role of bread as a dietary staple, this may contribute to more informed decisions being made relating to the use of glyphosate on glyphosate-resistant wheat, from a public health/regulatory perspective. Keywords: bakers’ yeast, breadmaking, consumers, glyphosate, risk.

INTRODUCTION Most food commodities undergo some form of processing prior to consumption. The baking of wheat flour into bread is of particular interest as it includes both microbiological processes and food chemistry. Such food processing steps will usually reduce the amount of pesticide residues on the raw commodity. This has been found with pesticides of varying chemical characteristics, on a diverse range of crops (Çelik et al. 1995; Schattenberg et al. 1996; Knio et al. 2000). However, certain pesticides may actually produce a more toxic product upon breakdown. For example, the ethylenebisdithiocarbamate fungicides break down into ethylenethiourea, a highly toxic compound, upon being heated or boiled (Marshall 1977). To make accurate risk assessments for consumption of pesticides in food, the quantity and toxicity of both the parent pesticide and Correspondence to: Dr Juliet A. Gerrard, School of Biological Sciences, University of Canterbury, PO Box 4800, Christchurch, New Zealand (e-mail: [email protected]).

ª 2004 The Society for Applied Microbiology

breakdown products must be determined. This premise is not taken into account by pesticide monitoring programmes that analyse raw, unprocessed food, as their aim is assessing farmer compliance relating to pesticide usage, rather than determining the consumer’s actual dietary intake of pesticide residues. Thus, it is imperative to elucidate the behaviour of pesticides during food processing. Glyphosate [N-(phosphonomethyl)glycine] is a nonselective, systemic herbicide that is used on a large variety of plants (Kamrin 1997). Glyphosate is a major pesticide within the agrochemical market and its usage is predicted to increase in the future. This is mainly because of the introduction of glyphosate-resistant crops, several of which are already in the market (Shaner 2000). Conventional usage of glyphosate requires that contact with crop plants be avoided because of its broad spectrum property. However, during the cultivation of genetically modified crops that are resistant to glyphosate, glyphosate may be liberally applied over both weeds and crop plants. This new form of usage is likely to result in increased residues being retained on the crop.

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Wheat (Triticum spp.) is a staple commodity and it is used as a basic ingredient in making dietary staples like bread (using Triticum aestivum), pasta and breakfast cereal. The release of glyphosate-resistant wheat is expected shortly (ACDI/VOCA 2002; see also Monsanto website: ‘Products and solution: product pipeline’. Available at http://www. monsanto.com/monsanto/layout/sci_tech/prod_pipeline/ productpipeline.asp. Accessed 9 November 2004) which may result in increased residues present on wheat grains destined for bread production. However, the behaviour of glyphosate residues that remain on the crop during food processing is, at present, remarkably understudied. Bacterial metabolism pathways of glyphosate are well understood and their respective end-products have been identified (Rueppel et al. 1977; Liu et al. 1991; Dick and Quinn 1995). However, yeast metabolism of glyphosate had not been studied to date and it became interesting to investigate if this in vivo element could influence glyphosate’s degradative behaviour. We therefore investigated the stability of the herbicide glyphosate during the fermentation cycle of the breadmaking process. In doing so, we aimed to assess the level of glyphosate exposure from consuming bread made from wheat containing glyphosate residues.

Victor Ltd equipment (Sydney, Australia). Freeze-drying was achieved using the Centrifugal Freeze Dryer (Model 30 P.2./822) from Edwards High Vacuum Ltd (SanyoGallenkamp, Sussex, UK). High-performance liquid chromatography (HPLC) analyses were performed using a Shimadzu (Kyoto, Japan) system controller (SCL-10ADvp), solvent delivery module (LC-10ATvp), auto injector (SIL-10ADvp) and degasser (DGU-14A). The system controller was connected to a Hitachi F1000 Fluorescence Spectrophotometer (Hitachi, Tokyo, Japan). This setup was interfaced with a personal computer running Shimadzu CLASS-VP version 5.032 operating software. The column used was a 220 mm · 4Æ6 mm amino column (Applied Biosystems, Foster City, CA, USA), which was coupled with a guard column (Brownlee New Guard Cartridge column RP-18, Aquapore ODS, 7 lM; Perkin Elmer, Boston, MA, USA). The mobile phase consisted of 35 : 65 (v/v) acetonitrile–0Æ05 mol l)1 phosphate, pH 5Æ5. Analyses were performed at a flow rate of 1Æ5 ml min)1, using injection volumes of 100 ll. The fluorescence detector was set at 263 nm (excitation) and 317 nm (emission). All glassware used, including HPLC vials, was silanized with Sigmacote silanizing agent (Sigma, Steinheim, Germany), according to the manufacturer’s instructions.

M A T E R I A LS A N D M E T H O D S All materials were obtained from Sigma Chemical Company (Steinheim, Germany), Aldrich Chemical Company (Milwaukee, WI, USA) or BDH Laboratory Supplies (Poole, UK). The exception was acetonitrile, which was acquired from Mallinckrodt Baker Incorporated (Paris, USA). All solvents and chemicals were of analytical grade. Commercial breadmaking flour (organic white flour, stone ground) was purchased from New Zealand Bio Grains Ltd. Yeast (Edmonds active yeast; Saccharomyces cerevisiae) was purchased from Bluebird Foods Ltd (Auckland, New Zealand), sugar (Chelsea white sugar) from New Zealand Sugar Company Ltd (Auckland, New Zealand) and salt (Cerebos iodized table salt) from Cerebos-Skellerup Ltd (Auckland, New Zealand). All breadmaking ingredients were stored at ambient temperature, away from sunlight. Glyphosate in its solid form (95–96% purity; Sigma or 96% purity; Aldrich) was used for all experiments. Dough cultures were produced with the commercially available Breville Baker’s Oven (Model no. BB290; Hwy Pty, Botany, Australia). Sample homogenization was carried out using the Chiltern Flask Shaker (Chiltern Scientific, Auckland, New Zealand), set at a speed of 3, for 10 min. Samples were centrifuged (Centaur 2 MSE; Henderson Biomedical, Kent, UK) at 2440 g for 10 min. Sonication was accomplished with equipment from Julabo Labortechnik GMBH (Seelbach, Germany) and autoclaving with Watson

Bread dough Ingredients used to produce the dough were 300 ml (50 mg kg)1) aqueous glyphosate, 25Æ9 g sugar, 8Æ3 g salt, 450Æ0 g flour and 5Æ7 g yeast, added in the order listed. Yeast was excluded from the set of control doughs. Immediately after addition of yeast, the breadmaker was set to option 1A (normal loaf, light crust). Proofing temperatures in the breadmaking chamber ranged from 31 to 34C during the fermentation cycle. Samples (ca 5 g each) were removed at 10-min intervals, the first commencing 2 min after the start of the breadmaking cycle (this was to ensure adequate mixing of the ingredients prior to sample removal). Immediately after removal, samples were kept at )20C for at least 2 h. They were then stored at )80C for a minimum of 2 h, and freeze-dried for at least 11/2 days. Samples were ground into a fine, consistent powder with a mortar and pestle before analysis. Glyphosate extraction The extraction procedures utilized were based on a protocol modified from Hogendoorn et al. (1999). One gram (±0Æ005) of ground, freeze-dried dough was added to a 100-ml round-bottomed flask. Twenty millilitres of distilled water was added and the contents shaken vigorously for 10 min on a flask shaker. The mixture was left

ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 40, 133–137, doi:10.1111/j.1472-765X.2004.01633.x

GLYPHOSATE DEGRADATION BY SACCHAROMYCES CEREVISIAE

RESULTS In order to establish the proof of the principle that glyphosate could be degraded by yeast within the dough matrix, a relatively high dose of the herbicide was employed in these experiments. This level is unlikely to be met in actual food systems. However, if the yeast in the dough was able to metabolize a significant fraction of this high glyphosate concentration, further experiments to assess the impact of this process at likely exposure levels is justified. The dose was just chosen such that glyphosate could be simply detected using unlabelled glyphosate and established HPLC protocols. The changes in glyphosate levels extracted from dough over time are shown in Figs 1 and 2. Interestingly, yeasted dough consistently gave a significant decrease in glyphosate levels, showing a 21% mean decline 120

Percentage original (glyphosate)

100 80 60

y = –0·3775x + 101·52

40 20 0 0

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Incubation time (min)

Fig. 1 Change in glyphosate levels extracted from yeasted dough spiked at 50 mg kg)1 (wet weight) and incubated at 31–34C. Error bars represent the standard deviation about the mean of triplicates

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Percentage original (glyphosate)

to stand overnight at ambient temperature (away from sunlight), after which it was centrifuged at 3600 rev min)1 for 10 min. Two millilitres of supernatant was passed through a 0Æ45 lM syringe filter using over pressure. A 1Æ5ml of filtered supernatant was used for the derivatization procedure as follows. Two hundred microlitres of 0Æ125 mol l)1 borate buffer (pH 9Æ0) and 1Æ0 ml of 9-fluorenylmethyl chloroformate (FMOC-Cl) solution (2 mg ml)1 in acetonitrile) were added to 1Æ5 ml of sample solution. The mixture was swirled and left at room temperature for 60 min. A 1Æ0-ml of the reaction mixture was then added to 3 ml of 0Æ025 mol l)1 borate buffer (pH 9Æ0). The resultant mixture was swirled thoroughly and 1Æ0 ml was pipetted into a vial for HPLC analysis within an hour. Statistical analyses were performed with the Student’s t-test, using a ¼ 0Æ05.

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80 60

y = –0·0822x + 98·431

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0

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Fig. 2 Change in glyphosate levels extracted from nonyeasted dough spiked at 50 mg kg)1 (wet weight) and incubated at 31–34C. A 4Æ3% decline was observed after 50 min of incubation. Error bars represent the standard deviation about the mean of triplicates

in recovery after 60 min of incubation. In contrast, nonyeasted dough showed no significant decrease in glyphosate levels. This strongly suggests that yeast is involved in the reduction of glyphosate during the breadmaking process. It is also very unlikely that binding is a cause of the decline in extracted glyphosate from yeasted dough. This is because the decline in glyphosate levels was gradual over the incubation period and as the number of yeast cells does not significantly increase under dough fermentation conditions (Hui 1992), there would be a sharper decline at the onset of incubation if binding was involved. DISCUSSION Saccharomyces cerevisiae has been used as a eucaryotic model for examining the toxicity of various pesticides (Iwahashi et al. 2000; Kitagawa et al. 2002; Cabral et al. 2003). For example, introduction of the fungicide thiuram induces genes responsible for detoxification (Kitagawa et al. 2002). Saccharomyces cerevisiae has also been found to be able to metabolize the herbicide atrazine (Hack et al. 1997), albeit at a rate of t1/2 ¼ 98 days. Glyphosate does not appear to affect aromatic acid biosynthesis in baker’s yeast at concentrations of 2 mmol l)1 (Roisch and Lingens 1980), suggesting that it has no toxic effects at that concentration. It is therefore possible that yeast could metabolize glyphosate via detoxification mechanisms, perhaps resulting in the production of novel degradation compounds. Glyphosate is resistant to degradation when exposed to temperatures up to 121C, and pH environments from 2 to 10 (International Program on Chemical Safety 1994). However, this research has shown for the first time that glyphosate is susceptible to degradation by S. cerevisiae


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during the fermentation stages of breadmaking. Thus, if glyphosate residues are present in wheat flour, proofing of yeasted dough for 1 h will reduce the levels by approximately one-fifth. It is interesting to note that yeasted dough reduced glyphosate by approximately the same amount as boiling aqueous glyphosate for 7 h (21 and 20% respectively; data not shown). This is a good illustration of biotic factors catalysing the degradation of glyphosate. The ability of yeast to degrade glyphosate is indeed in line with knowledge that glyphosate is liable to bacterial degradation. It is expected that, in parallel with bacterial degradation, the major breakdown product generated by glyphosate degradation was aminomethylphosphonic acid (AMPA). Further work is underway to investigate this possibility, using radiolabelled glyphosate. The results of this study have several implications with respect to food safety and risk assessment. First, glyphosate has a relatively low toxicity (acceptable daily intake, ADI ¼ 0Æ3 mg kg bw)1), although the reduction in glyphosate level during fermentation is small, the reduced level will further lower the risk of consuming bread made from glyphosatecontaminated flour. In addition, certain types of bread, such as those produced by the straight dough system, require longer proofing times of ca 3 h (Kent 1975). This is likely to reduce glyphosate levels even further. Secondly, existing regulatory standards for the appraisal of glyphosate residues in wheat and wheat end-products may be affected. As glyphosate-resistant wheat has not yet been commercially released, knowledge of glyphosate’s behaviour in the breadmaking system could provide information necessary to make informed regulatory decisions when considering the possibility of commercial release. The low toxicity of glyphosate means that residue levels on wheat grain is of low toxicological significance. This could allay concerns about glyphosate usage on wheat, and give confidence to both the bread industry and consumers that the bread being produced for consumption or eaten is safe. The maximum permitted levels in wheat or bread can be reevaluated in relation to consumer safety. In this way, the regulatory authorities are better equipped to disseminate more accurate information to the public. The results from this research have opened new avenues for investigation. Current investigations are focusing on the use of radiolabelled glyphosate to enable simple detection of the herbicide and its breakdown products at lower concentrations. In tandem with this work, in vitro assays to elucidate the chemistry of glyphosate metabolism by yeast will be undertaken. There is relatively little literature on the ability of yeast to metabolize other pesticides and it will be interesting to see if this finding is replicated in other pesticide models. In addition, it may be prudent to analyse wholemeal and multigrain breads, as the flour from which

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