Introduction

Organophosphorus pesticides (OPPs) residues in food commodities become a world-wide concern of public health due to the increasing application of OPPs in agricultural production and management (Ecobichon 2001). Chemical pesticides can pollute the foods themselves directly, or pollute water and soil finally conferring indirect pollution to the foods (Zhang et al. 2012); so many commercial foods were found polluted by the pesticides (Wang et al. 1999; Melgar et al. 2010; Chowdhury et al. 2013). Pesticide residues are thus considered as one of the potential risks in the foods to the customer. Both animal- and plant-originated foods were detected to have OPPs of different levels. In the northwest region of Spain, OPPs residues were detected in 43 out of 312 milk samples (Melgar et al. 2010). However, OPPs residues were found more frequently in plant foods. In Bangladesh, chlorpyrifos, diazinon, malathion and dimethoate were among those OPPs detected more frequently in vegetables (Chowdhury et al. 2013). Phoxim and methamidophos of 0.0899–0.0365 mg kg−1 were detected in the vegetable samples collected from a city of China (Wang et al. 1999). In Kazakhstan, 18 out of 80 grain samples were reported to have pesticide residues, and chlorpyrifos methyl and pirimiphos methyl were the most frequently detected OPPs (Lozowicka et al. 2014). These reported results indicate that plant foods have potential safety risk of OPPs.

Food processing and handling show helpful effects on pesticide degradation (Bajwa and Sandhu 2014; Guha et al. 2015; Regueiro et al. 2015), and thereof have been widely investigated to obtain valuable information as well as to find out practical technical approaches to control pesticide residues in processed foods. It was found that dichlorvos and parathion methyl in coffee powder at 0.5–1 mg kg−1 were dissipated by 12–26 and 18–45 %, respectively, during infusion into coffee beverage (Oliviera et al. 2002). A study of Lalah and Wandiga (2002) showed that a significant percentage of malathion in the grains was eliminated during cooking treatment, and NaCl addition resulted in greater malathion elimination. Application of ozone micro-bubbles was found efficiently to remove fenitrothion from the treated leafy and fruity vegetables (Ikeura et al. 2011). It was reported that pH shifting to alkaline condition had effect on OPPs degradation in milk (Zhao and Wang 2012a).

Food fermentation is one of the oldest techniques, during which proteins, starches and lipids are degraded by the enzymes excreted from the applied microorganisms. Some edible microorganisms showed ability to degrade OPPs, indicating their potential application in fermented foods to eliminate OPPs residues. For example, lactic acid bacteria (LAB) and commercial yogurt starters enhanced OPPs degradation in the milk (Bo et al. 2011; Zhao and Wang 2012b; Zhou and Zhao 2015). Bread making process including yeast fermentation and final baking removed 47–89 % of the six pesticides from wheat flour (Sharma et al. 2005). Banna and Kawar (1982) reported that parathion in apple juice was dissipated by 80 % after 57 days of fermentation processing into vinegar. Ruediger et al. (2005) found that about 70 % chlorpyrifos in red wine was reduced during malolactic fermentation by Oenococcus oeni. In a recent study, Lu et al. (2013) observed OPPs degradation during cabbage pickling. However, OPPs degradation in some traditional fermented foods consumed very commonly in China and other areas of Asia, such as steamed breads, pickled Chinese cabbage and Mao-tofu, is still unclear so far, and needs a detailed investigation thereof.

In traditional preparation of steamed breads, yeast-mediated fermentation is an essential process for the preparation of wheat dough (Kim et al. 2009). In the preparation of pickled Chinese cabbage and Mao-tofu, two traditional fermented foods in Northeastern and Central China, LAB- and Mucor-mediated fermentation are two important biochemical processes (Zhao and Zheng 2009; Xiong et al. 2012). In the present study, degradation behaviours of four OPPs including chlorpyrifos, dichlorvos, phorate and trichlorphon in wheat dough, pickled Chinese cabbage and Mao-tofu were studied and compared. OPPs left in the prepared samples of different fermentation times were extracted, purified, and detected by a gas chromatography (GC). After then, degradation rate constants of the OPPs were calculated by a first-order reaction model and used for comparison. The objective of the present study was to clarify the potencies of the applied microorganisms to promote OPPs degradation in the three fermented food materials, and to provide more scientific evidence for safety control of traditional fermented foods.

Materials and methods

Materials and chemicals

Four OPPs standards, chlorpyrifos, dichlorvos, phorate and trichlorphon were purchased from Sigma-Aldrich (Schnelldorf, Germany) with declared purity of 94.5–99.8 %. The four OPPs were dissolved separately in acetone to prepare stock solutions (0.5 g L−1). The mixed working solutions required for the standard curve and food material spiking were prepared from the stock solutions by further dilution. Other chemicals used were of analytical grade while the solvents used were of chromatographic grade. Water used was prepared from Milli-Q PLUS (Millipore Corporation, New York, NY, USA).

Wheat flour, fresh Chinese cabbage and tofu were purchased from local market in Harbin, China. They were produced from local farms and found negative in the four investigated OPPs after GC detection.

Microorganisms

Lactobacillus plantarum used for pickling Chinese cabbage was supplied by the Centre of Lactic Acid Bacteria in Key Laboratory of Dairy Science (Northeast Agricultural University), Ministry of Education, located in Harbin, China. L. plantarum was cultured and sub-cultured on a normal lactobacilli (MRS) agar medium thrice to ensure its purity and viability before application. A strain of Mucor was isolated from commercial fresh Mao-tofu product from Anhui Province, China, and its spore suspension was used to prepare Mao-tofu. Commercial yeast powder used for wheat dough preparation was purchased from Angle Yeast Co. Ltd. (Beijing, China).

Sample preparation

Wheat dough was prepared as traditional procedure. Wheat flour of 1 kg containing yeast powder of 1.5 and 3 % (w/w), was mixed well with drinking water of 0.3 kg, to prepare two wheat dough samples (wheat dough I and II), respectively. The water was previously spiked by each OPP at 4 mg kg−1, to ensure dough samples with OPPs levels near 1 mg kg−1. Wheat dough I and II were fermented at 30 °C for 5 h. A portion of them were separated randomly at each hour as analysis samples. Water loss during dough fermentation was measured and used to correct analysis results. Control dough was prepared with the same conditions but without yeast powder addition, fermented and treated as the wheat dough I and II.

Chinese cabbage without rotten and broken parts was washed with drinking water, air-dried under room conditions for about one hour, and then cut into four parts with equal size. The cut Chinese cabbage of about 2 kg was put into a pickling jar; after that, pickling brine (NaCl solution of 1 %, w/w) of 3 kg was added. The pickling brine was previously spiked by each OPP at a level of 2 mg kg−1, to ensure OPPs levels in the samples close to 1 mg kg−1 . At the same time, the culture of L. plantarum was added at level of 2 % (w/w) on the basis of Chinese cabbage. The fermentation was carried out at 15 °C for 6 weeks. At every week, pickled Chinese cabbage of 100 g and pickling brine of 150 g were collected together as analysis sample. Water loss during fermentation was measured and used to correct analysis results. Control pickled Chinese cabbage was also prepared with the same conditions but without L. plantarum inoculation.

Fermentation of Mao-tofu was carried out as per the study (Zhao and Zheng 2009), but with minor modifications. After cooling, the cooked tofu cubes were immersed in the sterilized water spiked by each OPP at level of 2 mg kg−1 and left over night at 4 °C. Afterwards, the Mucor was inoculated into the surface of the cooked tofu by spreading Mucor inoculum (105 spores mL−1). All cubes were put in sterilized bamboo trays and fermented at 20 °C for 6 days. Some cubes were selected randomly at each day as analysis samples. Water loss during fermentation was measured and used to correct analysis results. Control tofu was prepared with the same conditions but without Mucor inoculation.

All analysis samples obtained were subjected to OPPs extraction, purification and GC detection immediately.

OPPs extraction, purification and detection

Extraction and purification of OPPs in wheat dough were performed as per the method (Uygun et al. 2007). Wheat dough of 5 g was mixed with ethyl acetate of 25 mL, and then extracted for 30 min. The extraction was repeated twice. The combined ethyl acetate phase was filtered through anhydrous Na2SO4 (5 g) and collected as purified OPPs extract.

Pickled Chinese cabbage of 100 g and pickling brine of 150 g were homogenized. The blend of 25 g was mixed with dichloromethane of 50 mL and extracted for 30 min. Liquid phase was transferred to a separatory funnel and set for 20 min. Dichloromethane phase was collected, while the left residues of liquid and solid were re-extracted by dichloromethane. The combined dichloromethane phase was filtered through anhydrous Na2SO4 (5 g) and collected as purified OPPs extract.

Mao-tofu sample was homogenized before OPPs extraction. The homogenate of 10 g was mixed with dichloromethane of 50 mL and extracted for 30 min. Forthcoming treatments were the same to those treatments used for the pickled Chinese cabbage.

The purified OPPs extract of 10 mL was evaporated to dryness under a stream of nitrogen at 30 °C. The residues were re-dissolved in acetone of 1 mL, and filtered through a 0.45 μm micro-porous membrane before GC analysis. OPPs analysis was performed using an Agilent 7890 Gas Chromatography (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a flame photometric detector in phosphorus mode, an Agilent Auto-sampler 7683, and a capillary column (DB-1701, 30 m × 0.250 mm × 0.25 μm). Nitrogen at a flow rate of 1 mL min−1 was used as carrying gas. GC temperature program and the temperature of injector and detector were the same to those used in a previous study (Zhou and Zhao 2015).

Calculation of kinetic parameters

Kinetic parameters of OPPs degradation were calculated by the kinetic equation of a first-order reaction model expressed as below. In the equation, C t is OPPs concentration (mg kg−1) at time t (hour or day), C 0 is initial OPPs concentration (mg kg−1), k is rate constant (hour−1 or day−1).

$$ {C}_t={C}_0{e}^{-kt} $$

Statistical analysis

All experiments and analyses were carried out three times. All data were expressed as means or means ± standard deviations. SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) was used in data analysis.

Results

GC detection of OPPs

GC analysis is a simple and rapid procedure widely used for OPPs quantification. Linear ranges of OPPs detection in the present analysis ranged from 0.1 to 8 mg kg−1 (r 2 > 0.995). Typical OPPs profiles for a standard solution and three assayed samples are depicted in Fig. 1. OPPs recoveries at three spiked levels (0.1, 0.5 and 1 mg kg−1) were 83.5–102.4, 90.8–99.0 and 91.6–101.0 % for wheat dough, pickled Chinese cabbage and Mao-tofu, respectively (Table 1). These results evidenced that the applied procedure and conditions were suitable for the present OPPs analysis, based on the recommended recoveries suggested by Putnam et al. (2003).

Fig. 1
figure 1

Typical GC profiles of the four organophosphorus pesticides from a standard solution (a), wheat dough (b), pickled Chinese cabbage (c) and Mao-tofu (d). Peaks 1–4 represent trichlorphon, dichlorvos, phorate and chlorpyrifos, respectively

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Table 1 Recoveries of four organophosphorus pesticides spiked in wheat dough, pickled Chinese cabbage and Mao-tofu
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OPPs degradation in wheat dough

OPPs degradation during wheat dough fermentation was observed, because residual OPPs in wheat dough I and II (yeast addition) as well as control dough (without yeast addition) showed decreasing trend as fermentation time progressed (Table 2). OPPs residual levels after a fermentation time of 5 h decreased by 7.3–11.1 % (control dough), 16.6–26.6 % (wheat dough I) and 23.4–31.8 % (wheat dough II).

Table 2 Measured concentrations and calculated degradation rate constants (k) of four organophosphorus pesticides in control dough and wheat dough samples at different fermentation times
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Degradation rate constants (k values) of the OPPs were calculated (Table 2). Yeast addition yielded greater OPPs degradation in wheat dough I and II, and yeast addition level also had clear effect on OPPs degradation. In control dough, the respective k values of chlorpyrifos, dichlorvos, phorate and trichlorphon were 1.56 × 10−2, 2.48 × 10−2, 1.48 × 10−2, and 1.94 × 10−2 hour−1. In wheat dough I (yeast added at 1.5 %), the respective k values were 3.80 × 10−2, 6.07 × 10−2, 4.17 × 10−2, and 3.53 × 10−2 hour−1. About 82–182 % increase in the k values was observed. If the yeast was added at higher level (e.g., 3 %), faster OPPs degradation occurred in the wheat dough II. The respective k values were 5.77 × 10−2, 7.83 × 10−2, 5.45 × 10−2 and 5.51 × 10−2 hour−1; that is, k values of the four OPPs were enhanced by 184–270 %. These results evidenced directly that yeast played an important role to promote OPPs dissipation in wheat dough. Based on these k values, it can be seen that the four OPPs had different stability during wheat dough fermentation. Dichlorvos had the highest k values but phorate mostly showed the lowest ones (Table 2), indicating dichlorvos and phorate were the most unstable and stable pesticides.

OPPs degradation in pickled Chinese cabbage

OPPs degradation in pickled Chinese cabbage was investigated for longer fermentation period (i.e., 42 days), as traditional preparation of pickled Chinese cabbage usually has a fermentation time near 2 months. The detected OPPs levels in pickled Chinese cabbage (Table 3) showed decreasing trend during the fermentation. After the fermentation, about 80.6–93.1 and 96.2–99.7 % of the four OPPs in the control pickled Chinese cabbage and pickled Chinese cabbage were dissipated, respectively.

Table 3 Measured concentrations and calculated degradation rate constants (k) of four organophosphorus pesticides in pickled Chinese cabbage (PCC) and control PCC at different fermentation times
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The calculated k values of the OPPs in control pickled Chinese cabbage and pickled Chinese cabbage are listed in Table 3, which showed that inoculation of L. plantarum enhanced OPPs degradation. Without L. plantarum inoculation, Chinese cabbage was fermented only by the wild LAB; k values of chlorpyrifos, dichlorvos, phorate and trichlorphon were 0.151 × 10−2, 0.276 × 10−2, 0.144 × 10−2 and 0.198 × 10−2 hour−1, respectively. If L. plantarum was inoculated, the respective k values were enhanced into 0.481 × 10−2, 0.617 × 10−2, 0.310 × 10−2 and 0.511 × 10−2 hour−1, yielding increasing levels about 115–219 %. Higher LAB inoculation density in the pickling brine (i.e., L. plantarum inoculation intendedly) brought about greater OPPs degradation for pickled Chinese cabbage. It is thus evidenced that L. plantarum promoted OPPs degradation in pickled Chinese cabbage, and LAB were capable of enhancing OPPs degradation.

Similarly to these results observed in dough fermentation, dichlorvos and phorate in pickled Chinese cabbage also showed the highest and lowest k values (Table 3), declaring again that they were the most sensitive and stable pesticides, respectively.

OPPs degradation in Mao-tofu

Traditional fermentation of Mao-tofu usually continues for 5–6 days; thereof, a fermentation time of 6 days was used in the present study. Residual levels of OPPs in Mao-tofu during fermentation are given in Table 4. OPPs degradation occurred in control tofu and Mao-tofu, as their OPPs levels behaved decreasing trend during fermentation. Finally, about 56.0–80.1 and 79.7–99.5 % of the OPPs were dissipated from control tofu and Mao-tofu, respectively. Clearly, Mucor inoculation resulted in greater OPPs dissipation in Mao-tofu.

Table 4 Measured concentrations and calculated degradation rate constants (k) of four organophosphorus pesticides in control tofu and Mao-tofu at different fermentation times
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The calculated k values of the OPPs are listed in Table 4. In control tofu, chlorpyrifos, dichlorvos, phorate and trichlorphon had k values of 0.682 × 10−2, 1.05 × 10−2, 0.549 × 10−2 and 0.776 × 10−2 hour−1, respectively. In Mao-tofu, the respective k values were 1.62 × 10−2, 3.40 × 10−2, 1.21 × 10−2 and 1.67 × 10−2 hour−1, yielding increasing levels of 115–224 %. It is elucidated that the Mucor played a positive role for OPPs degradation in Mao-tofu.

The data in Table 4 also revealed that dichlorvos and phorate had the highest and lowest k values, respectively. This suggests that they were the most labile and stable pesticides.

Discussion

Fermentation processing usually leads to declined pesticide levels in fermented foods, and microorganisms are primarily responsible for pesticide degradation (Sharma et al. 2005; Regueiro et al. 2015). It had been found that microorganisms of different taxonomic groups were potential to degrade OPPs (Briceño et al. 2012). The present study evidenced once again that the applied yeast, LAB and Mucor all could promote OPPs degradation during traditional fermentation of wheat dough, pickled Chinese cabbage and Mao-tofu.

The data (Tables 2, 3 and 4) revealed that if the investigated microorganisms were inoculated or added into the three food materials, they brought about enhanced OPPs dissipation but yeast and Mucor were more potent to enhance OPPs dissipation than LAB. Wheat dough and Mao-tofu samples were only fermented by yeast and Mucor for 5 h and 6 days, dissipation levels of the OPPs were 16.6–31.8 and 79.7–99.5 %, respectively. Chinese cabbage was fermented by LAB for much longer time (6 weeks), and dissipation levels of these OPPs were 96.2–99.7 %. Totally, residual OPPs in pickled Chinese cabbage were much lower than these in wheat dough and Mao-tofu. However, due to different fermentation times were applied to the three fermented food materials, degradation rate constants (i.e., k values) but not residual levels of OPPs were more useful and correct to show how fast the OPPs degraded during the fermentation. The k values of the OPPs in wheat dough and Mao-tofu were about (1.2–7.8) × 10−2 hour−1, nearly 4–12 folds greater than those of the OPPs in pickled Chinese cabbage [about (0.3–0.6) × 10−2 hour−1]. This fact indicates clearly that addition and inoculation of yeast and Mucor conferred greater OPPs degradation (i.e., higher k values), whereas LAB fermentation only brought about weaker OPPs degradation (i.e., lower k values). Yeast, LAB and Mucor thus showed different ability to enhance OPPs degradation; thereof, possible reason and mechanism should be investigated in future study to clarify why yeast and Mucor had better potency to degrade OPPs.

Some microorganisms degrade chemical pesticides by using them directly as a source of carbon, nitrogen and phosphorus, or by producing pesticide-degrading enzymes (Singh et al. 2004; Bhalerao and Puranik 2009). For example, Pseudomonas aeruginosa can utilize OPPs for growth (Ramu and Seetharaman 2014), while Aspergillus oryzae degrades monocrotophos by enzymatic metabolism (Bhalerao and Puranik 2009). As the one of widely used edible microorganisms in food processing, LAB had acceleration on the degradation of OPPs (Zhao and Wang 2012b) or organochlorine pesticides (Abou-Arab 1997) in some foods including pickled cabbage. Yeast is able to degrade chemical pesticides belonging to organochlorine and organophosphate groups (Abou-Arab 1997; Sharma et al. 2005). Enhanced OPPs dissipation was thus observed during wine (Cabras et al. 1995a) and bread making (Sharma et al. 2005). Fungi are also capable of degrading OPPs. It was reported that about 70 % of monocrotophos at 500 mg L−1 was dissipated by Aspergillus oryzae in the first 50 h (Bhalerao and Puranik 2009), while about 50–75 % of monocrotophos could be degraded by Aspergillus and Penicillium sp. after a culture time of 4 days (Zidan and Ramadan 1976). These mentioned studies provided important support for the present study. That is, yeast, LAB (or L. plantarum) and Mucor could bring about enhanced OPPs degradation in wheat dough, pickled Chinese cabbage and Mao-tofu.

Inoculum amount is a key factor to control pesticide biodegradation. Lower inoculum density could result in a small part of the microorganisms participating in pesticide degradation (Ramadan et al. 1990). These findings give important support to the present result: higher yeast addition (3 %, w/w) in wheat dough resulted in higher k values of the OPPs (Table 2). Similar result was also observed in the fermentation of pickled Chinese cabbage. In control pickled Chinese cabbage, the fermentation was carried out only by those wild LAB; in pickled Chinese cabbage, the fermentation was carried out by the inoculated L. plantarum and those wild LAB together. Consequentially, pickled Chinese cabbage other than control pickled Chinese cabbage showed greater OPPs degradation (Table 3).

In the present study, degradation kinetics of the four OPPs was fit by a first-order reaction model, as OPPs degradation was suggested as a first-order reaction (Zhou and Zhao 2015). Compared to the OPPs degradation in control samples, the four OPPs all showed higher k values (i.e., greater OPPs degradation) in wheat dough, pickled Chinese cabbage and Mao-tofu (Tables 2, 3 and 4). This declares an important role of these microorganisms in these food materials, to accelerate OPPs degradation. This conclusion was similar to those conclusions drawn for the LAB-, yeast- and Aspergillus oryzae-mediated OPPs degradation (Cabras et al. 1995a; Bhalerao and Puranik 2009; Zhao and Wang 2012b).

Chemical pesticides showed different stability towards food processing such as storage (Uygun et al. 2007), rinsing (Krol et al. 2000) and washing (Stepan et al. 2005). During yeast-mediated fermentation, dithiophosphates exhibited higher stability than others (Cabras et al. 1995b). During yoghurt fermentation, malathion was the most resistant (Bo et al. 2011). In the present study, the four OPPs showed different sensitivity to the applied fermentation. Dichlorvos and phorate were observed to be the most labile and stable pesticides. This finding provided important information about pesticide stability during food processing once more. Pesticides stability during food processing might be partly governed by their chemical nature. This also should be detailed clarified in future study.

Conclusion

Application of edible microorganisms in fermented food materials benefits flavor formation as well as texture and nutrition improvement. The present study demonstrated another benefit of the edible microorganisms; that is, to enhance OPPs degradation or to eliminate more OPPs residues. After fermentation, at least 16.6, 96.2 and 79.7 % of the investigated four OPPs were removed from wheat dough, pickled Chinese cabbage and Mao-tofu fermented by yeast, LAB and Mucor for 5 h, 42 and 6 days, respectively. Yeast and Mucor were more potent than LAB to degrade OPPs, resulting in faster OPPs degradation. These results evidenced the potentials of these microorganisms to control safety risk of these fermented food materials. Effect of the traditional fermentation of other food materials on pesticide degradation and the related mechanisms are suggested in future study.