Curr Microbiol DOI 10.1007/s00284-016-1000-5

Evaluation of Lactobacillus plantarum and Saccharomyces cerevisiae in the Presence of Bifenthrin Tijana M. Ðord¯evic´1



Rada D. Ðurovic´-Pejcˇev1

Received: 10 August 2015 / Accepted: 26 December 2015 Ó Springer Science+Business Media New York 2016

Abstract This work describes the effect of insecticide bifenthrin on Lactobacillus plantarum and Saccharomyces cerevisiae. Growths of used microorganisms in growth media supplemented with pesticide were studied. Determination of bacterial and yeast fermentation efficiency in wheat supplemented with bifenthrin was conducted. Additionally, investigation of bifenthrin dissipation during microbiological activity was performed. Experiments applying bifenthrin in different concentrations highlighted a negligible impact of the pesticide on the growth of L. plantarum and S. cerevisiae. This insecticide overall negatively affected the yeast fermentation of wheat, while its presence in wheat had a slight negative impact on lactic acid fermentation. The results of bifenthrin dissipation during lactic acid and yeast fermentations of wheat showed that activities of L. plantarum and S. cerevisiae caused lower pesticide reductions. Average bifenthrin residue reduction within samples fermented with L. plantarum was 5.4 % (maximum *16 %), while within samples fermented with S. cerevisiae, it was 11.6 % (maximum *17 %).

Introduction Bread baking using sourdough, dough made from cereals, liquid and microbes in an active state, is a common practice and has the advantage of improving the nutritional value and sensory qualities of products, increasing their shelf life & Tijana M. Ðord¯evic´ [email protected] 1

Institute of Pesticides and Environmental Protection, Banatska 31b, 11080 Belgrade, Serbia

by delaying the germination of bacterial and mould spores [18]. Nowadays, sourdough is already included in the manufacture of a variety products (breads, cakes, crackers, etc.), and its application is in continuous increase. A large variety of cereal flours are involved in sourdough production throughout the world, although wheat flours are the most commonly used. The microbiota of sourdoughs consists of specifically adapted lactic acid bacteria, mostly lactobacilli, as well as yeasts [19]. Since flour cannot be subjected to heat-sterilization during sourdough preparation, the occurrence and number of certain types of microorganisms strictly depend on a combination of available substrates and specific technological parameters [26]. Lactobacillus plantarum is one of the most frequently lactobacilli isolated from sourdoughs, and it contributes most to the process of dough acidification, while Saccharomyces cerevisiae, as sourdough yeasts, is primarily responsible for the leavening through CO2 production [19]. During wheat fermentation, the metabolic activity of the present microorganisms is in interaction with the grain constituents. The occurrence and growth of microorganisms in wheat are influenced by many factors [17] including the presence of pesticides. These agrochemicals had important role in the increase of agricultural productivity in developed world over the last few decades. Nowadays, a large proportion of stored cereal grain is protected against insect attack by residual pyrethroid insecticides, successful alternative to traditional organophosphorus insecticides. Limited environmental persistence, high insecticidal activity and low mammalian toxicity, gathered with cost effective simple application, enhance their popularity among chemicals for insect control [23]. Bifenthrin, registered in several European countries, is, among pyrethroids, one of the most commonly used active substances in plant protection in Serbia [20]. It

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has been investigated since the early 1990s as a potential grain protectant for stored grain in Europe, and proved to be very effective against a range of stored grain beetles [13]. However, in 2009, the European Parliament withdrawn pesticides containing bifenthrin from use in the European Union after the Swedish Chemicals Agency included bifenthrin in list of active substances in plant protection products which have been banned or withdrawn in Sweden during the period 1966–2000, because of its carcinogenic effect. In 2012, bifenthrin was reapproved for use in the European Union by the Standing Committee on the Food Chain and Animal Health [25]. Considering that this pesticide, as all chemicals for store cereal grain protection, is applied post-harvest to wheat, its application has been attracting much attention due to possible toxicity [30]. Besides endangering the health of consumer, non-adequate use of bifenthrin in agriculture may also cause some technological problems during sourdough production. Several studies indicated that pesticide residues can affect the growth of lactic acid bacteria, causing an inhibition of their fermentative capability at the same time [1, 2, 10]. There are numerous publications confirming the negative impact of pesticides on the yeast growth, as well. Most of the literature highlighted negative effect of fungicides on yeast [3, 12, 29], although there is a discrete number of studies reporting yeast growth inhibition in the presence of herbicides [4] and insecticides [28]. However, significant number of publications reported the lack of negative impact of pesticides on lactic acid and yeast fermentation efficiency, regardless of growth inhibitions obtained in media supplemented with agrochemicals [4, 6, 8, 9, 16, 21, 27, 28]. The metabolic activity of useful microorganisms is in direct interaction with the growth medium, and thus, it could be expected that in substrates supplemented with pesticides, microbial activity could cause a reduction of pesticide residues. Significant number of studies reported the ability of various microorganisms, including lactobacilli and yeast, to reduce pesticides residues in growth substrate by enzymatic activity [2, 11, 15, 16, 22, 24, 27]. Biodegradation of pesticides may be driven by nutrition and energy needs, or a need to detoxify the substrate. Considering all, the aim of this work was to study the effect of insecticide bifenthrin on L. plantarum and S. cerevisiae, as main constituents of sourdough microbiota. Bacterial and yeast growth in media supplemented with pesticide was evaluated. Determination of lactobacilli and yeast fermentation efficiency, in wheat supplemented with bifenthrin, was conducted. Additionally, investigation of the dissipation of bifenthrin during microbial activity was performed.

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Materials and Methods This article does not contain any studies with human participants or animals performed by any of the authors. Starter Culture, Analytical Standard and Working Solutions A lactobacilli strain—Lactobacillus plantarum (DSMZ 20174) and a yeast strain—Saccharomyces cerevisiae (WDCM 00058; CBS 2978) were maintained at 4–6 °C and activated for 24 h at 30 °C in MRS, i.e. YPD broth (Torlak Institute, Belgrade, Serbia). A stock solution (1.0 mg L-1) of an analytical standard of bifenthrin (purity 98.4 %) (Dr. Ehrenstorfer GmbH, Augsburg, Germany) prepared in acetone (J.T. Baker, Deventer, Netherlands) was stored at -18 °C, and working standard solutions were prepared daily by diluting stock with sterile distilled water. Effect of Bifenthrin on Lactobacillus plantarum and Saccharomyces cerevisiae Growth Cells of lactobacilli and yeast were incubated on 30 °C until exponential phases of growth were reached (24 h for L. plantarum and 5 h for S. cerevisiae) in MRS and YPD broth supplemented with bifenthrin at 0.25, 0.5, 1, 2.5, 5, 7.5 and 10.0 lg mL-1 rates, i.e. 1/2MRL, MRL, 2MRL, 5MRL, 10MRL, 15MRL and 20MRL for this pesticide in wheat, respectively. Cells growth was determined according procedure described previously [16]. Preparation and Fermentation of Wheat Substrate and Optimization of Fermentation Conditions Untreated grain of Triticum spelta (organic production by Jevtic´ farm, Bacˇko Gradisˇte, Serbia), used for preparation of wheat mash substrate for fermentation, and cells of lactobacilli and yeast, used for inoculations, were prepared according to the procedures described previously [15, 16]. The fermentation conditions were optimized using a Box-Behnken design containing three levels for each parameter: inoculums size (A)—6, 8 and 10 % (v/w), fermentation time (B)—24, 48 and 72 h and temperature (C)—23, 30 and 37 °C. The pH measurements (InoLab 730 pH meter WTW, Weilheim, Germany), determination of total titratable acidity (TTA) (for lactobacilli) and CO2 production (for yeast), and colony count—CFU (microorganism cells mg-1) were selected as responses for fermentation efficiency and performed according to the procedures described previously [15, 16].

T.M. Ðord¯evic´, R.D. Ðurovic´-Pejcˇev: Evaluation of Lactobacillus plantarum

Effect of Bifenthrin on Efficiency of Wheat Fermentation Untreated wheat grain was milled, fortified with 0, 0.5, 2.5 and 7.5 mg kg-1 rates of bifenthrin (i.e. MRL, 5MRL and 15MRL for wheat) and homogenized for 24 h and further prepared for fermentation as described previously [16]. Prepared cultures of microorganisms were inoculated individually at 8 % (v/w), and fermentation was carried out at 23, 30 and 37 °C, for 24, 48 and 72 h, and samples were analysed for fermentation efficiency. Effect of Fermentations on Bifenthrin Reduction in Wheat Wheat mash was prepared as described previously, with fortification at 7.5 mg kg-1 of bifenthrin (15MRL). For bifenthrin residue gas chromatograph–mass spectrometer (GC–MS) analyses, samples were prepared according analysis procedure described previously [14]. Two different controls were used: first consisted of sampling immediately after sterilization in autoclave to check the effect of sterilization on pesticide degradation and second consisted of wheat substrates held under the same incubation conditions, but without starter, to check the pesticide spontaneous chemical degradation. Acetone, methanol, ethyl acetate and anhydrous sodium sulphate (99.0 % purity), used for analysis, were purchased from J.T. Baker (Deventer, The Netherlands), while Florisil (60–100 mesh) was purchased from Serva Electrophoresis GmbH (Heidelberg, Germany). For the used method, average recoveries (at four fortification levels: 0.1, 0.5, 1 and 2 mg kg-1) ranged from 79.6 to 82.6 % with maximum relative standard deviation (RSD) of 7.4 %, while the limit of detection (LOD) and limit of quantification (LOQ) were 0.004 and 0.014 mg kg-1, respectively [14]. Instrumentation and Statistical Analysis A GC–MS (CP-3800/Saturn 2200; Varian, Melbourne, Australia), used as a detection device, operated under conditions previously described [16]. Ion (m/z) 181 was used for quantification, while ion (m/z) 166 was used for confirmation. All experiments were performed in triplicates. Statistical analysis was processed by Statistica 8.0 (StatSoft Inc., Tulsa, Oklahoma, USA) and ReliaSoft‘s DOE?? software (ReliaSoft Corporation, USA). A student’s t test (a = 0.05) was used to evaluate significant differences in bacterial and yeast growth in the media supplemented with different amounts of bifenthrin, comparing to control. This statistical test was also used to evaluate significance of lactic acid and yeast fermentations on bifenthrin reduction in wheat. Pareto charts of the

effects were used to determine the magnitude and the importance of effects of fermentation conditions on fermentation efficiency. One-way ANOVA (a = 0.05) was used for the evaluation of effect of bifenthrin on efficiency of wheat fermentation.

Results and Discussion Effect of Bifenthrin on Lactobacillus plantarum and Saccharomyces cerevisiae Growth Results presented in Fig. 1 indicated that neither lactobacilli nor yeast growth was highly inhibited by bifenthrin, regardless of the applied pesticide concentrations. Concerning L. plantarum, lower inhibition of colonyforming ability (up to 10.2 %) did occur in the presence of 10MRL concentration of bifenthrin, and at 15MRL, growth inhibition was slightly higher (18 %); however, comparing to control, no statistically relevant differences in number of viable cells were found. Significant decrease in CFU, up to 33.6 %, was recorded only in the presence of 20MRL concentration. Obtained results correspond with those recorded by Abou Ayana et al. [1] who stated that, although lactobacilli growth was reduced up to 50 % by fungicides mancozeb and metalaxyl, insecticides methomyl and chlorpyrifos-methyl and herbicides glyphosate and thiobencarb, still only extreme high concentrations of those pesticides in growth medium (60MRL and 300MRL for milk), caused overall significant reduction of CFU mL-1 higher than 10 %. In addition, the authors highlighted that growth inhibition is correlated directly to pesticide concentration, whereas lactobacilli overall were more resistant to pesticides in comparison to other bacteria. Also, AbouArab [2] indicated that, while the addition of DDT (5MRL) or lindane (100MRL) in medium had resulted in slightly decrease in viable counts of meat starter microorganisms (including L. plantarum) during the initial 24 h of incubation, microorganisms later recovered and began to grow logarithmically, although not as well as in normal situation. Despite that several studies confirmed correlation between glyphosate, chlorpyrifos and pirimiphos-methyl present in growth media and growth inhibition of various food microorganisms including Lactobacillus sp. [9, 10, 16], considering results obtained in this study, it could be concluded that L. plantarum response differently regarding the change of viable counts in the presence of different amounts of various pesticides. Thus, those interactions should always be tested prior further experiments dealing with fermentation efficiency. Growth of S. cerevisiae in media supplemented with pesticide at 1/2MRL, MRL, 2MRL and 5MRL concentrations was not at all inhibited; in fact, there was slight

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Fig. 1 Effect of bifenthrin on L. plantarum (a) and S. cerevisiae (b) growth in MRS and YPD broth presented as percentage of growth parameters inhibition. Data are expressed as the mean ± SEM (n = 3). ( log10 CFU ml-1, O.D.620nm)

growth stimulation at those dosages, most probably as result of cell stress response. Inhibition of colony-forming ability, of 8 %, began in the presence of 10MRL, reaching barely 12 % at the highest pesticide concentration, without statistically significant differences in the number of viable cells comparing with control sample. Obtained results are in correlation with those published by Cabras et al. [6], who indicated that residues of azoxystrobin, cyprodinil, fludioxonil, mepanipyrim, pyrimethanil and tetraconazole, at concentration 1.5 times higher than MRL for grapes and ten times higher than one found in field trials, did not influence the rate of yeast growth in YNBG medium. Jawich et al. [21] and Santos et al. [27] came to similar conclusion for penconazole in YP medium at concentrations up to 15MRL and mancozeb in growth medium at concentration 2 mg L-1, as in all trials, after increasing dose-dependent period of latency, growth resumptions were observed. Thus, the growth of S. cerevisiae in the presence of those fungicides was not affected by the final biomass production, but only by the growth rate. Braconi et al. [4] stated that herbicide treatments were able to affect yeast growth by affecting the enzymatic activity of catalase and superoxide dismutase, as well as by inducing oxidative modifications of proteins; however, only extreme high concentrations of fenoxaprop-P-ethyl (100MRL and 500MRL), tribenuron methyl (1000MRL) and glyphosate (500MRL) in growth medium caused overall significant reduction of CFU mL-1. Lower concentrations of those pesticides resulted only in a short-term reduction at the beginning of growth followed by growth resumption, and thus, biomass production was not significantly affected. Considering insecticides, it was confirmed that the presence of deltamethrin (at ‘MRL, MRL, 1.5MRL and 2MRL for wheat), chlorpyrifos (at 1/3MRL, 2/3MRL, MRL and 1.3MRL for wheat) and malathion (at max ‘MRL for wheat) in growth media did not show considerable effect on yeast growth, and inhibition of yeast growth observed in

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the presence of endosulfan was recorded only at significantly higher concentrations, i.e. 20MRL, 40MRL, 60MRL and 80MRL for wheat [24]. However, Shin et al. [29] found that some strains of S. cerevisiae did not grow in YPD medium supplemented with chlorothalonil at 1.5 mg ˇ usˇ and Raspor [12] indicated that pyrimethanil at L-1, C higher concentration (3MRL for grapes) diminished initial yeast growth (lag and exponential phase) in YM medium, and Bi Fai and Grant [3] established growth IC50 for S. cerevisiae in the presence of approximately 15 g L-1 of maneb, 40 g L-1 of captan, 120 g L-1 of picoxystrobin, 180 g L-1 of pyrazophos and 200 g L-1 of thiabendazole. Considering all, it is obvious that this yeast also responses differently regarding the change of viable counts in the presence of different amounts of various pesticides; thus, testing of those interactions is necessary prior to fermentations. Optimization of Fermentation Conditions The growth of a microorganism is strongly influenced by medium composition, and thus, the optimization of the growth is necessary in a fermentation experiments. To meet the optimal cell growth demands, it is necessary to set up the fermentation parameters and thus increase the fermentation efficiency. Limitations of one-at-a-time-parameter optimization can be eliminated by employing response surface method (RSM). Box-Behnken design, as RSM used to explain the combined factors in a fermentation process in this study, defines the effect of the independent process variables, alone or in combinations, and generates a mathematical model that describes the entire process. Results of optimization of wheat fermentation conditions showed that alterations of independent variables led to changes in responses selected for determination of fermentation efficiency. The magnitude and the importance of effects of fermentation conditions on fermentation

T.M. Ðord¯evic´, R.D. Ðurovic´-Pejcˇev: Evaluation of Lactobacillus plantarum

efficiency were presented through Pareto chart of the effects. This chart displays the absolute value of the standardized effects calculated by dividing each obtained coefficient of effect by its standard error. A reference line on the chart is value of two-tail t-distribution with degrees of freedom equal to the degrees of freedom for the model, and level of significance a = 0.05. Any effect that extends beyond this reference line is potentially important. Pareto charts presented in Fig. 2 indicated that alteration of fermentation time and temperature during fermentation with L. plantarum significantly affected pH alterations and TTA production. Prolonged fermentation at higher temperature led to an increase in TTA production, thus a decrease in pH. However, as can be seen from Fig. 2, inoculum size had no effect on pH and TTA. Obviously, there have been a sufficient number of cells required for a successful fermentation in all samples, regardless of initial inoculum size. As for colony-forming unit ability (CFU), none of tested fermentation parameters affected this response. This was not surprising considering prolonged stationary growth phase of L. plantarum [16] and effective temperature growth for this microorganism (15–40 °C). Considering S. cerevisiae, Pareto charts presented in Fig. 3 indicated that only alteration of temperature led to pH alterations (lower pH on higher temperatures), while CO2 production was influenced by fermentation time and temperature in correlation with fermentation time (both positive correlations). CFU was significantly affected by temperature and fermentation time. At 23 and 37 °C, CFU was lower, while the number of cells increased with fermentation time. This could be due to optimal temperature growth for this microorganism of 25–35 °C and sufficient amount of nutrients in substrate. However, initial inoculums size of yeast, same as for lactobacilli, had no effect on fermentation responses. Effect of Bifenthrin on Efficiency of Wheat Fermentation Considering that during optimization of fermentation conditions it was concluded that initial inoculums size had no effect on fermentation responses, for this experiment, prepared cultures of microorganisms were inoculated only at 8 % (v/w). Results presented in Fig. 4 showed that the presence of bifenthrin in the lowest applied concentration negatively affected CFU number, however, did not overall negatively affect the lactic acid fermentation in wheat (pH lowering and TTA production). Obtained P values (ANOVA, a = 0.01) of 4.07 9 10-7 for CFU, 2.4 9 10-3 for TTA production and 9.5 9 10-3 for pH, for fortification at different concentration levels (from 0 to 7.5 mg kg-1), indicated that this insecticide had lower, thus still significant, effect on all fermentation responses.

Reduction in CFU number, as well as less pH alterations and less TTA production, was in direct correlation with concentrations rate. However, considering that after 72 h TTA production and pH reached levels of those in the control samples, it could be cautiously stated that negative effect of bifenthrin on lactic acid fermentation of wheat was not concerning. Several studies previously reported that pesticide residues had some impact on the growth of lactic acid bacteria, causing an inhibition of their fermentative capability. Abou Ayana et al. [1] recorded that lactobacilli growth and their fermentation efficiency were affected by fungicides mancozeb and metalaxyl, insecticides methomyl and chlorpyrifos-methyl, and herbicides glyphosate and thiobencarb, whereby the growth inhibition and inhibition of fermentation efficiency correlated directly to pesticides’ concentration. In addition, the authors highlighted that insecticides were the most effective inhibitors of lactobacilli growth compared with herbicides or fungicides, and thus, it is obvious that different pesticides could impact on specific lactic acid bacteria biodiversity differently. Confirmation of negative impact of pesticides on lactobacilli growth can be found in several more publications [9, 16], whereby at the same time, there is lack of confirmation regarded negative impact of pesticide on lactic acid fermentation efficiency [7, 9, 16]. As can be seen from the results presented in Fig. 5, the presence of bifenthrin in all three applied concentrations overall negatively affected the yeast fermentation in wheat. Obtained P values (ANOVA, a = 0,01) of 1.67 9 10-7 for pH, 1.95 9 10-7 for CO2 production and 8.28 9 10-11 for CFU, for fortification with bifenthrin at different concentration levels (from 0 to 7.5 mg kg-1), indicated that this insecticide had significant effect on all fermentation responses. The presence of bifenthrin at all three concentration rates caused significant decrease in the number of cells per g of fermentation substrate and CO2 production. Reduced fermentation in those samples resulted with lower acidification; thus, pH of substrate was higher. However, this negative impact of bifenthrin was the least expressed in the presence of the smallest used amount of pesticide, i.e. at MRL level (0.5 mg kg-1), and thus, pH lowering in those samples, especially after 48 and 72 h of fermentation, and CO2 production, especially after 24 h of fermentation, were not significantly negatively affected. Further fortification with higher concentrations resulted in more expressive reduction of CFU number, as well as less pH alterations and less carbon dioxide production. Concerning S. cerevisiae, several studies indicated that various pesticides caused growth inhibition and fermentation efficiency of this yeast. Most of the literature highlighted negative effect of fungicides chlorothalonil [29], pyrimethanil [12], maneb, captan, picoxystrobin, pyrazophos and thiabendazole [3] on yeast growth or fermentation efficiency,

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T.M. Ðord¯evic´, R.D. Ðurovic´-Pejcˇev: Evaluation of Lactobacillus plantarum Fig. 2 Pareto chart of the effects—significance of variations of incubation parameters on L. plantarum fermentation efficiency. Bars display the absolute value of the standardized effects. A reference line on the chart represents t value of twotail t-distribution with df = 11 and a = 0.05. Any effect that extends beyond the reference line is potentially important

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T.M. Ðord¯evic´, R.D. Ðurovic´-Pejcˇev: Evaluation of Lactobacillus plantarum Fig. 3 Pareto chart of the effects—significance of variations of incubation parameters on S. cerevisiae fermentation efficiency. Bars display the absolute value of the standardized effects. A reference line on the chart represents t value of twotail t-distribution with df = 11 and a = 0.05. Any effect that extends beyond the reference line is potentially important

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T.M. Ðord¯evic´, R.D. Ðurovic´-Pejcˇev: Evaluation of Lactobacillus plantarum Fig. 4 Effect of bifenthrin on fermentation activity of L. plantarum in wheat substrate. Data are expressed as the mean ± SEM (n = 3). (d 0 mg kg-1 of bifenthrin, h 0.5 mg kg-1 of bifenthrin, u 2.5 mg kg-1 of bifenthrin, 4 7.5 mg kg-1 of bifenthrin)

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T.M. Ðord¯evic´, R.D. Ðurovic´-Pejcˇev: Evaluation of Lactobacillus plantarum Fig. 5 Effect of bifenthrin on fermentation activity of S. cerevisiae in wheat substrate. Data are expressed as the mean ± SEM (n = 3). (d 0 mg kg-1 of bifenthrin, h 0.5 mg kg-1 of bifenthrin, u 2.5 mg kg-1 of bifenthrin, 4 7.5 mg kg-1 of bifenthrin)

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although current literature also provides a discrete number of studies reporting the effects of herbicides fenoxaprop-Pethyl, tribenuron methyl and glyphosate [4], and insecticides endosulfan [28] on yeast biological parameters. However, concerning this microorganism, there are also opposite examples, and thus, Cabras et al. [6] indicated that residues of azoxystrobin, cyprodinil, fludioxonil, mepanipyrim, pyrimethanil and tetraconazole did not influence the rate of yeast fermentation activity. Chaves Lo´pez et al. [8] came to similar conclusion for quinoxyfen as the presence of this fungicide did not affect fermentation efficiency of yeast. Also, according to Braconi et al. [4], only extreme high concentrations of herbicides fenoxaprop-P-ethyl, tribenuron methyl and glyphosate caused overall significant reduction of CFU mL-1 and ethanol production. Lower concentrations resulted only in a shortterm reduction, at the beginning of growth, followed by growth resumption, and thus, rate constant was not significantly affected. Considering insecticides, it was confirmed that the presence of deltamethrin, chlorpyrifos and malathion did not show considerable effect on yeast fermentation efficiency [28]. Effect of Fermentations on Bifenthrin Reduction in Wheat Results obtained for determination of the effect of fermentations on bifenthrin reduction in wheat, together with two different controls (the effect of sterilization on pesticide degradation and the pesticide spontaneous chemical degradation), are shown in Fig. 6. Considering previously mentioned recoveries (of 79.6 to 82.6 %), it can be seen that the concentration of bifenthrin in the samples after

sterilization remained nearly intact. The loss of pesticide residues in substrate during heating obtain through some physico-chemical processes (evaporation, co-distillation and thermal degradation) which vary with the chemical nature of the pesticides [28]. The water contained in the sample could entrain pesticide molecules during the process (co-distillation), while heat can cause evaporation and degradation [5]. Obtained lack of bifenthrin degradation may be due to its lower vapour pressure and extremely low water solubility that cause remarkably low tendency for volatilization from wet material and a correspondingly strong tendency for binding to dry matrix. Further, during the incubation without microorganisms, bifenthrin was practically stable in wheat substrate, with no trend towards reduction of the analyte level with incubation temperature or time. Although the percentage of remaining bifenthrin was consistently lower than the value prior incubation (for approx. 1.5–2.4 %), the differences were not statistically significant (t test, a = 0.05). Those results were not unexpected considering that bifenthrin is generally persistent insecticide, with no recorded degradation during storage at ambient conditions for longer period of time. The results of bifenthrin reduction during lactic acid and yeast fermentations of wheat showed that degradation was negligible impacted by the inoculated L. plantarum and S. cerevisiae. Average bifenthrin residue reduction within samples fermented with L. plantarum was 24.5 % (maximum 34.9 %), and thus, concerning recovery, spontaneous degradation and one during sterilization, it can be concluded that lactobacilli were responsible for reduction of average 5,4 % (maximum *16 %). Average bifenthrin residue reduction within samples fermented with S. cerevisiae was overall somewhat higher -30.7 % (maximum

Fig. 6 Dissipation of bifenthrin residues during sterilization, incubation, lactic acid fermentation with L. plantarum (a) and yeast fermentation with S. cerevisiae (b) in wheat substrate samples fortified with 7.5 mg kg-1 of bifenthrin

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35.9 %), and thus, concerning recovery, spontaneous degradation and one during sterilization, it can be concluded that yeast was responsible for reduction of average 11.6 % (maximum *17 %). Naturally, various microorganisms, including lactobacilli and yeast, reduce the amount of pesticide in growth substrate by utilizing them as a source of nutritive constituents (i.e. carbon and phosphorus), or simply by degrading them due to detoxification of substrate. Considering that wheat is natural substrate for used microorganism with obviously enough nutrients, while higher amounts of bifenthrin did not show high toxicity for L. plantarum and S. cerevisiae, it could be cautiously concluded that those were the reasons for lack of degradation activity in this study.

Conclusion Experiments applying bifenthrin in different concentrations highlighted a negligible impact of the pesticide on the growth of L. plantarum. Lower inhibition of lactobacilli colony-forming ability, with statistically relevant differences in number of viable cells, comparing to control, occurred only in the presence of 20MRL concentration. As for L. plantarum fermentation efficiency, bifenthrin had significant, but lower, effect on all fermentation responses, with reduction in CFU number, less pH alterations and less TTA production being in direct correlation with pesticide concentrations rate. However, considering that after 72 h TTA production and pH reached levels of those in the control samples, it could be cautiously concluded that negative effect of bifenthrin on lactic acid fermentation of wheat was not concerning. In correlation, bifenthrin dissipation during lactic acid microbial activity was, as well, quite low. Concerning S. cerevisiae, growth inhibition was not at all inhibited by bifenthrin, as inhibition of colony-forming ability was, even at the highest applied pesticide concentration, low, and differences in number of viable cells were statistically insignificant comparing to control. However, the presence of this insecticide in wheat overall negatively affected the yeast fermentation. The presence of bifenthrin at all three concentration rates caused significant decrease in the number of cells per g of fermentation substrate and CO2 production. Reduced fermentation in samples supplemented with pesticide resulted with lower acidification, and thus, pH of those substrates was higher. Fortification with bifenthrin at higher concentrations rate resulted in more expressive reduction of fermentation efficiency. Accordingly, activities of S. cerevisiae caused somewhat higher pesticide reductions, which could be attributed to the urge for the detoxification of substrates.

Acknowledgments This study was carried out as a part of the project No TR31043, supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia. Compliance with Ethical Standards Conflict of interest of interest.

The authors declare that they have no conflict

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Evaluation of Lactobacillus plantarum and Saccharomyces cerevisiae in the Presence of Bifenthrin.

This work describes the effect of insecticide bifenthrin on Lactobacillus plantarum and Saccharomyces cerevisiae. Growths of used microorganisms in gr...
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