Research Article Received: 9 February 2014
Revised: 19 April 2014
Accepted article published: 28 April 2014
Published online in Wiley Online Library: 21 May 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6710
Susceptibility of nine organophosphorus pesticides in skimmed milk towards inoculated lactic acid bacteria and yogurt starters Xin-Wei Zhoua and Xin-Huai Zhaoa,b,c* Abstract BACKGROUND: Previous research has shown that fresh milk might be polluted by some organophosphorus pesticides (OPPs). In this study the dissipation of nine OPPs, namely chlorpyrifos, chlorpyrifos-methyl, diazinon, dichlorvos, fenthion, malathion, phorate, pirimiphos-methyl and trichlorphon, in skimmed milk was investigated to clarify their susceptibility towards lactic acid bacteria (LAB) and yogurt starters. RESULTS: Skimmed milk was spiked with nine OPPs, inoculated with five strains of LAB and two commercial yogurt starters at 42 ∘ C for 24 and 5 h respectively and subjected to quantitative OPP analysis by gas chromatography. Degradation kinetic constants of these OPPs were calculated based on a first-order reaction model. OPP dissipation in the milk was enhanced by the inoculated strains and starters, resulting in OPP concentrations decreasing by 7.0–64.6 and 7.4–19.2% respectively. Totally, the nine OPPs were more susceptible to Lactobacillus bulgaricus, as it enhanced their degradation rate constants by 18.3–133.3%. Higher phosphatase production of the assayed stains was observed to bring about greater OPP degradation in the milk. CONCLUSION: Both LAB and yogurt starters could enhance OPP dissipation in skimmed milk, with the nine OPPs studied having different susceptibilities towards them. Phosphatase was a key factor governing OPP dissipation. The LAB of higher phosphatase production have more potential to decrease OPPs in fermented foods. © 2014 Society of Chemical Industry Keywords: organophosphorus pesticides; susceptibility; skimmed milk; lactic acid bacteria; yogurt starter; phosphatase
INTRODUCTION
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Organophosphorus pesticides (OPPs) are widely used in agricultural production.1,2 With respect to their chemical nature, OPPs are unstable compounds; however, excessive use of OPPs might eventually lead to widespread environmental and food contamination.3,4 Previous studies have indicated that milk products might be polluted by OPPs. Acephate and chlorpyrifos were detected in more than 100 milk samples from Italy, but at levels lower than the legal limits.5 Dichlorvos, phorate, chlorpyrifos and chlorfenvinphos at levels of 0.0051–0.0203 mg kg−1 were found in milk samples from Mexico.6 A recent study indicated that 43 out of 312 milk samples from northwestern Spain contained detectable OPPs.7 Another survey found that OPP levels in milk samples collected in Heilongjiang Province, China during 2008–2009 were less than 0.020 mg kg−1 .8 The residual chemical pesticides in milk products come from several possible routes, including external application for ectoparasite control directly on dairy cows,9 pest control in stables and factories6 and indirect contamination from polluted pasture, forages, feedstuffs or drinking water.10 – 12 OPPs in milk products might therefore pose a safety risk to consumers and should be kept under proper control before and during dairy processing. OPP levels in foods are affected by food processing such as fermentation, infusion, storage, cooking and drying,13 – 17 with OPPs showing different susceptibilities towards these treatments. Lu et al.18 found that washing had a positive effect on J Sci Food Agric 2015; 95: 260–266
pesticide reduction in green peppers. The fate of four OPPs in wheat during storage was investigated by Uygun et al.,19 who observed that chlorpyrifos-methyl, fenitrothion, malathion and pirimiphos-methyl were decreased by 76–88%. Banna and Kawar20 fortified apple juice with parathion at 25 mg kg−1 and found that only about 20% parathion was detectable after 57 days of fermentation process into vinegar. Some lactic acid bacteria (LAB) and commercial yogurt starters had accelerating effects on OPP degradation in milk, implying that OPPs were sensitive towards inoculated strains and starters.13,21,22 Also, pH shifting was observed to influence OPP degradation in fresh milk, with dimethoate, parathion-methyl and trichlorphon being highly susceptible to an alkaline pH value of 7.5.23
∗
Correspondence to: Xin-Huai Zhao, Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin 150030 China. E-mail: [email protected]
a Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin 150030, China b Department of Food Science, Northeast Agricultural University, Harbin 150030, China c Synergetic Innovation Center of Food Safety and Nutrition, Northeast Agricultural University, 150030, Harbin, China
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Susceptibility of nine organophosphorus pesticides in skimmed milk OPP degradation by soil or aquatic microorganisms has been well studied in the field of environmental science,24 – 26 whereas that by edible microorganisms has been less investigated in the field of food science. It is strongly suggested to characterize OPP degradation during food fermentation, especially to clarify the susceptibility of OPPs towards food microorganisms. This might provide a potential approach to control or decrease OPP residues in fermented foods. More importantly, biodegradation of OPPs during food processing could provide a safety guarantee to consumers. In this study, skimmed milk was spiked with nine OPPs, namely chlorpyrifos, chlorpyrifos-methyl, diazinon, dichlorvos, fenthion, malathion, phorate pirimiphos-methyl and trichlorphon, that had previously been detected in some foods. The spiked milk was inoculated by one of five LAB widely used in the preparation of yogurt, cheese and other fermented milk products or by one of two commercial direct vat-set yogurt starters. After detection of residual OPPs at different culture times by gas chromatography (GC) and calculation of degradation rate constants for the OPPs, OPP susceptibilities towards these strains and starters were clarified by comparing the increasing levels of degradation rate constants.
MATERIALS AND METHODS Materials and chemicals Nine OPP standards, i.e. chlorpyrifos, chlorpyrifos-methyl, diazinon, dichlorvos, fenthion, malathion, phorate, pirimiphos-methyl and trichlorphon, of declared purity 94.5–99.8% were purchased from Sigma-Aldrich (Schnelldorf, Germany). Stock solutions of the nine OPPs (0.5 g L−1 ) were prepared in acetone. The mixed working solutions required for the standard curve and milk spiking were prepared from the stock solutions by further dilutions. Skimmed milk power, prepared with fresh milk from dairy farms near Harbin city, was purchased from Harbin Longdan Dairy Co. Ltd (Harbin, China). Analysis showed that the powder was free of both antibiotics and the nine OPP residues. Other chemicals used were of analytical or chromatographic grade. Water was prepared using a Milli-Q Plus system (Millipore Corporation, New York, NY, USA). Strains and starters The five LAB, namely Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus), Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus and Streptococcus thermophilus, were supplied by the Centre of Lactic Acid Bacteria in Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University (Harbin, China). The two commercial yogurt starters, namely Lyofast Y250A and Y480F, were gifts from Beijing Duoaite Biotechnology Co. Ltd (Beijing, China). The lyophilized strains were rehydrated in 1 mL of sterilized rehydration medium (12% skimmed milk, w/w) with subsequent 5% (v/v) inoculation into normal DeMan/Rogosa/Sharpe (MRS) agar medium (Difco, Detroit, MI, USA) and then cultured at 37 ∘ C for 24 h. To ensure their purity and viability, the strains were cultured and subcultured in the MRS medium three times and then cultured in the skimmed milk (12%, w/w) for 24 h before further application.
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temperature for 30 min to ensure OPP distribution. The spiked milk (20 mL) was poured into glass bottles with a capacity of 50 mL, sterilized at 121 ∘ C for 20 min and cooled to ambient temperature. One of the five strains or a culture combination of S. thermophilus and L. bulgaricus (1:1, w/w) was inoculated into the milk at a level of 3% (w/w) and cultured at 42 ∘ C for 8, 12, 16, 20 and 24 h respectively. Some bottles were selected randomly as analysis samples, cooled in an ice water bath and subjected to OPP extraction and purification immediately. A control sample was also prepared with the same conditions and procedure but without strain inoculation. The spiked milk was also inoculated with each of the two yogurt starters as reported previously13 and fermented in glass cups at 42 ∘ C for 5 h. Some cups were selected randomly as analysis samples after each hour of fermentation and rapidly cooled in an ice water bath. The selected samples were subjected to OPP extraction and purification immediately. A control sample was also prepared with the same conditions and procedure but without starter addition. Extraction, purification and analysis of OPPs Extraction and purification of OPPs were carried out as described previously.21 The purified dichloromethane phase of 10 mL was evaporated to dryness under a stream of nitrogen at 30 ∘ C. The residues left were redissolved in 1 mL of acetone and filtered through a 0.45 μm microporous membrane before GC analysis. OPP detection was performed using an Agilent 7890 gas chromatograph (GC) (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a flame photometric detector, an Agilent 7683 autosampler and a capillary column (DB-1701, 30 m × 0.25 mm, 0.25 μm). Nitrogen was used as carrier gas at a flow rate of 1 mL min−1 . The GC temperature program consisted of an initial oven temperature of 50 ∘ C, held at 50 ∘ C for 1 min, elevated from 50 to 130 ∘ C at 20 ∘ C min−1 , from 130 to 200 ∘ C at 10 ∘ C min−1 and from 200 to 203 ∘ C at 0.5 ∘ C min−1 , then held at 203 ∘ C for 5 min. The injector and detector temperatures were set at 210 and 250 ∘ C respectively. Assay of phosphatase activity The spiked milk (10 g), cultured or fermented by the strains or the starters for 24 or 5 h respectively, was mixed well with 5 mL of carbonate/bicarbonate buffer (10 mmol L−1 , pH 10.5), stood at ambient temperature for 30 min and then centrifuged at 10 000 × g for 15 min at 4 ∘ C. The supernatant was subjected to sonication ten times (10 s duration at 15 s intervals) and measured for phosphatase activity as described previously,27 with some modifications. The reaction mixture comprised 1 mL of 5 mmol L−1 p-nitrophenyl phosphate (dissolved in buffer containing 1 mmol L−1 MgCl2 ), 1 mL of buffer and 1 mL of crude enzyme solution, diluted to a final volume of 5 mL with water. After a reaction time of 60 min at 37 ∘ C, the reaction was stopped by adding 3 mL of termination agent (0.1 mol L−1 NaOH and 5 mmol L−1 EDTA). p-Nitrophenol formed in the mixture was detected in a UV-2401 PC spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at 405 nm. Standard p-nitrophenol solutions (0.5–40.0 μmol L−1 ) were used to generate a standard curve. One unit (1 U) of phosphatase activity was expressed as the amount liberating 1 μmol p-nitrophenol min−1 at 37 ∘ C. Statistical analysis All experiments and analyses were carried out three times. All data were expressed as mean ± standard deviation (SD). Differences between mean values of multiple groups were analyzed
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Sample preparation Skimmed milk power was reconstituted into skimmed milk with a total solids content of 12% (w/w) and a pH value of 6.5. Each of the nine OPPs was added to the milk at a level of 1 mg kg−1 . The spiked milk was shaken vigorously and stood at ambient
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Table 1. Recoveries and limits of detection (LOD) of nine organophosphorus pesticides in skimmed milk
X-W Zhou, X-H Zhao
A
Recovery (%)a at three spiking levels (mg kg−1 ) Pesticide Chlorpyrifos Chlorpyrifos-methyl Diazinon Dichlorvos Fenthion Malathion Phorate Pirimiphos-methyl Trichlorphon
0.1 97.4 ± 2.4 94.0 ± 3.3 86.3 ± 2.8 85.7 ± 4.3 93.0 ± 2.3 87.3 ± 5.3 92.1 ± 5.7 86.7 ± 2.6 87.2 ± 6.0
0.5
LOD (mg kg−1 )b
1
96.5 ± 3.6 91.9 ± 0.9 97.5 ± 1.5 90.7 ± 1.6 87.8 ± 2.2 85.4 ± 2.7 83.9 ± 5.7 84.1 ± 2.0 93.0 ± 1.9 89.4 ± 5.2 84.9 ± 3.6 102.9 ± 1.3 98.6 ± 7.7 84.9 ± 1.1 86.8 ± 3.3 91.4 ± 3.5 89.5 ± 1.6 87.8 ± 1.0
0.006 0.005 0.002 0.001 0.007 0.008 0.002 0.005 0.003
B
a Values are expressed as mean ± SD. b Data were calculated based on a signal-to-noise ratio of 3.
by one-way analysis of variance (ANOVA) with Duncan’s multiple range tests. SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) was used in data analysis.
RESULTS AND DISCUSSION GC analysis of nine OPPs GC was used in the present study to detect residual OPPs in the milk samples. The linear range of detection of OPPs in the present analysis was 0.1–8.0 mg kg−1 (R2 > 0.995), while limits of detection ranged from 0.001 to 0.008 mg kg−1 based on a signal-to-noise ratio of 3 (Table 1). Typical OPP profiles for the standard solution, control and analysis samples are depicted in Fig. 1. OPP recoveries at three spiking levels (0.1, 0.5 and 1 mg kg−1 ) ranged from 83.9 to 102.9% (Table 1). These results indicated that the applied conditions and procedure were suitable for the present study, as a previous study suggested.28
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OPP susceptibility towards five LAB The residual OPPs detected in the milk inoculated with five LAB are given in Table 2. The data evidenced OPP dissipation in the milk, as the detected OPP concentrations showed a decreasing trend during a culture time of 24 h. In the control sample, chlorpyrifos and dichlorvos finally were lost by 6.2 and 32.6% respectively and appeared to be the most stable and unstable OPPs respectively. Chlorpyrifos inoculated with L. casei exhibited the least dissipation, as about 93% chlorpyrifos still remained in the milk, while dichlorvos inoculated with L. bulgaricus gave the greatest dissipation, as about 65% dichlorvos disappeared, i.e. chlorpyrifos and dichlorvos were the least and most susceptible to the inoculated strains respectively. Compared with OPP dissipation in the control sample, L. bulgaricus, L. rhamnosus and S. thermophilus totally had stronger acceleration on OPP dissipation and brought about increased dissipation by 22.9–160.0, 6.3–115.5 and 8.6–123.2% (except for malathion inoculated with L. rhamnosus or S. thermophilus) respectively. Lactobacillus acidophilus and L. casei showed weaker acceleration on OPP dissipation, as OPP dissipation levels were only increased by 11.7–86.6%. Among the five strains studied, both L. bulgaricus and S. thermophilus mostly showed greater acceleration on OPP dissipation. It is well known that S. thermophilus and
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C
Figure 1. Typical GC profiles of nine organophosphorus pesticides for (A) standard solution, (B) control milk and (C) skimmed milk. Peaks 1–9 represent trichlorphon, dichlorvos, phorate, diazinon, chlorpyrifos-methyl, pirimiphos-methyl, chlorpyrifos, fenthion and malathion respectively.
L. bulgaricus are two strains widely used in fermented dairy products, especially in yogurt. Streptococcus thermophilus and L. bulgaricus were thus combined and inoculated into the milk. Totally, this combination conferred slightly higher OPP dissipation than single S. thermophilus or L. bulgaricus did, as it resulted in an increased dissipation by 12.1–180.7%. Whether other strain combinations also have similar acceleration on OPP dissipation should be investigated in future studies.
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J Sci Food Agric 2015; 95: 260–266
Susceptibility of nine organophosphorus pesticides in skimmed milk
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Table 2. Residual concentrations and degradation rate constants (k) of nine organophosphorus pesticides in skimmed milk inoculated with lactic acid bacteria at 42 ∘ C Concentration (mg kg−1 )a detected at different culture times (h) Pesticide Chlorpyrifos
Chlorpyrifos-methyl
Diazinon
Dichlorvos
Fenthion
Malathion
Phorate
Pirimiphos-methyl
Strain L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus
0 1.010 ± 0.016 0.982 ± 0.009 0.981 ± 0.015 1.016 ± 0.011 1.020 ± 0.035 0.981 ± 0.018 0.946 ± 0.018 1.026 ± 0.023 0.995 ± 0.019 0.973 ± 0.003 1.031 ± 0.018 1.074 ± 0.023 0.972 ± 0.015 0.959 ± 0.030 1.027 ± 0.007 1.038 ± 0.011 1.079 ± 0.074 1.045 ± 0.041 0.959 ± 0.010 0.971 ± 0.026 0.953 ± 0.034 1.030 ± 0.025 1.005 ± 0.008 0.952 ± 0.012 1.042 ± 0.026 0.964 ± 0.012 0.979 ± 0.013 0.954 ± 0.036 0.991 ± 0.006 1.055 ± 0.034 1.024 ± 0.011 1.001 ± 0.016 0.968 ± 0.009 0.979 ± 0.016 0.948 ± 0.028 1.014 ± 0.046 1.023 ± 0.018 0.972 ± 0.018 1.048 ± 0.011 1.052 ± 0.035 0.967 ± 0.017 0.922 ± 0.026 1.055 ± 0.012 1.029 ± 0.061 1.037 ± 0.024 1.031 ± 0.038 0.994 ± 0.040 0.968 ± 0.005 0.956 ± 0.020 0.987 ± 0.021 1.068 ± 0.023 0.965 ± 0.012 0.997 ± 0.026 0.959 ± 0.004
8 0.984 ± 0.015 0.941 ± 0.014 0.959 ± 0.019 0.974 ± 0.025 0.971 ± 0.027 0.922 ± 0.012 0.928 ± 0.025 0.901 ± 0.054 0.861 ± 0.007 0.902 ± 0.007 0.907 ± 0.024 0.941 ± 0.036 0.881 ± 0.022 0.861 ± 0.028 1.005 ± 0.013 0.981 ± 0.006 1.027 ± 0.074 0.995 ± 0.039 0.909 ± 0.009 0.898 ± 0.028 0.927 ± 0.030 0.849 ± 0.064 0.838 ± 0.085 0.819 ± 0.050 0.772 ± 0.030 0.812 ± 0.011 0.798 ± 0.038 0.864 ± 0.075 0.949 ± 0.009 0.992 ± 0.039 0.986 ± 0.014 0.956 ± 0.023 0.936 ± 0.016 0.925 ± 0.021 0.930 ± 0.025 0.937 ± 0.065 0.921 ± 0.019 0.918 ± 0.034 0.962 ± 0.041 0.976 ± 0.035 0.879 ± 0.025 0.842 ± 0.035 1.002 ± 0.048 0.931 ± 0.054 0.931 ± 0.060 0.856 ± 0.034 0.930 ± 0.037 0.854 ± 0.014 0.897 ± 0.043 0.931 ± 0.033 1.018 ± 0.021 0.922 ± 0.021 0.929 ± 0.033 0.924 ± 0.009
12 0.971 ± 0.016 0.919 ± 0.020 0.951 ± 0.018 0.958 ± 0.022 0.935 ± 0.027 0.894 ± 0.014 0.917 ± 0.022 0.842 ± 0.039 0.803 ± 0.017 0.862 ± 0.011 0.861 ± 0.008 0.831 ± 0.030 0.814 ± 0.016 0.795 ± 0.034 0.979 ± 0.014 0.946 ± 0.016 0.999 ± 0.054 0.957 ± 0.046 0.879 ± 0.010 0.848 ± 0.015 0.897 ± 0.031 0.727 ± 0.043 0.701 ± 0.10 0.705 ± 0.037 0.647 ± 0.012 0.779 ± 0.010 0.700 ± 0.018 0.775 ± 0.013 0.934 ± 0.007 0.962 ± 0.040 0.973 ± 0.012 0.933 ± 0.018 0.914 ± 0.008 0.914 ± 0.023 0.921 ± 0.023 0.896 ± 0.026 0.860 ± 0.027 0.868 ± 0.048 0.912 ± 0.023 0.902 ± 0.019 0.830 ± 0.017 0.806 ± 0.02 0.924 ± 0.037 0.884 ± 0.046 0.907 ± 0.041 0.797 ± 0.036 0.858 ± 0.018 0.742 ± 0.055 0.835 ± 0.032 0.896 ± 0.030 0.952 ± 0.030 0.904 ± 0.021 0.916 ± 0.030 0.902 ± 0.010
16 0.959 ± 0.016 0.898 ± 0.020 0.941 ± 0.021 0.947 ± 0.017 0.917 ± 0.027 0.864 ± 0.019 0.898 ± 0.017 0.843 ± 0.043 0.754 ± 0.019 0.845 ± 0.014 0.827 ± 0.010 0.804 ± 0.036 0.761 ± 0.013 0.746 ± 0.032 0.933 ± 0.021 0.925 ± 0.011 0.981 ± 0.073 0.883 ± 0.046 0.864 ± 0.013 0.824 ± 0.015 0.880 ± 0.033 0.662 ± 0.057 0.565 ± 0.074 0.680 ± 0.024 0.555 ± 0.017 0.713 ± 0.009 0.611 ± 0.011 0.694 ± 0.036 0.919 ± 0.008 0.926 ± 0.028 0.964 ± 0.014 0.918 ± 0.009 0.903 ± 0.011 0.888 ± 0.027 0.902 ± 0.017 0.866 ± 0.029 0.816 ± 0.025 0.849 ± 0.048 0.899 ± 0.012 0.867 ± 0.017 0.797 ± 0.021 0.773 ± 0.010 0.900 ± 0.020 0.832 ± 0.055 0.890 ± 0.049 0.757 ± 0.039 0.819 ± 0.037 0.718 ± 0.043 0.805 ± 0.029 0.885 ± 0.026 0.927 ± 0.016 0.896 ± 0.005 0.905 ± 0.015 0.879 ± 0.014
20
24
0.949 ± 0.016 0.878 ± 0.016 0.922 ± 0.014 0.940 ± 0.019 0.903 ± 0.028 0.834 ± 0.020 0.894 ± 0.017 0.797 ± 0.033 0.702 ± 0.007 0.794 ± 0.013 0.788 ± 0.010 0.778 ± 0.026 0.710 ± 0.009 0.729 ± 0.023 0.884 ± 0.006 0.901 ± 0.004 0.951 ± 0.058 0.846 ± 0.035 0.821 ± 0.007 0.799 ± 0.017 0.850 ± 0.024 0.631 ± 0.050 0.441 ± 0.027 0.628 ± 0.048 0.500 ± 0.010 0.607 ± 0.008 0.539 ± 0.021 0.643 ± 0.021 0.905 ± 0.009 0.911 ± 0.021 0.950 ± 0.008 0.907 ± 0.010 0.891 ± 0.007 0.862 ± 0.020 0.886 ± 0.021 0.839 ± 0.032 0.779 ± 0.006 0.805 ± 0.036 0.874 ± 0.015 0.843 ± 0.025 0.743 ± 0.014 0.721 ± 0.014 0.867 ± 0.021 0.775 ± 0.061 0.835 ± 0.018 0.714 ± 0.033 0.759 ± 0.028 0.671 ± 0.034 0.780 ± 0.024 0.861 ± 0.033 0.911 ± 0.010 0.878 ± 0.015 0.886 ± 0.021 0.860 ± 0.006
0.939 ± 0.019 0.841 ± 0.008 0.913 ± 0.004 0.925 ± 0.006 0.889 ± 0.025 0.816 ± 0.016 0.887 ± 0.023 0.770 ± 0.023 0.651 ± 0.027 0.759 ± 0.008 0.734 ± 0.010 0.737 ± 0.017 0.658 ± 0.011 0.679 ± 0.035 0.843 ± 0.010 0.864 ± 0.021 0.932 ± 0.054 0.804 ± 0.036 0.806 ± 0.005 0.772 ± 0.018 0.841 ± 0.023 0.529 ± 0.017 0.356 ± 0.009 0.561 ± 0.014 0.457 ± 0.012 0.503 ± 0.009 0.464 ± 0.020 0.611 ± 0.028 0.886 ± 0.008 0.876 ± 0.043 0.934 ± 0.014 0.892 ± 0.016 0.872 ± 0.008 0.845 ± 0.020 0.879 ± 0.024 0.811 ± 0.042 0.725 ± 0.031 0.781 ± 0.016 0.856 ± 0.020 0.828 ± 0.026 0.709 ± 0.020 0.694 ± 0.023 0.834 ± 0.031 0.710 ± 0.041 0.800 ± 0.032 0.605 ± 0.031 0.733 ± 0.025 0.595 ± 0.011 0.759 ± 0.021 0.834 ± 0.029 0.882 ± 0.025 0.864 ± 0.018 0.868 ± 0.020 0.834 ± 0.006
k (h−1 )b 0.0031 0.0063 0.0030 0.0038 0.0059 0.0078 0.0028 0.0116 0.0175 0.0102 0.0136 0.0158 0.0165 0.0143 0.0085 0.0075 0.0062 0.0114 0.0074 0.0096 0.0056 0.0269 0.0446 0.0217 0.0352 0.0258 0.0312 0.0199 0.0045 0.0077 0.0037 0.0047 0.0043 0.0061 0.0033 0.0093 0.0142 0.0094 0.0084 0.0105 0.0130 0.0120 0.0102 0.0152 0.0103 0.0204 0.0134 0.0198 0.0101 0.0069 0.0082 0.0045 0.0054 0.0058
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Table 2. Continued Concentration (mg kg−1 )a detected at different culture times (h) Pesticide
Trichlorphon
Strain Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control
0 0.977 ± 0.011 0.956 ± 0.031 0.975 ± 0.008 0.984 ± 0.004 0.980 ± 0.020 1.055 ± 0.049 0.951 ± 0.010 0.967 ± 0.004 0.974 ± 0.024
8 0.899 ± 0.010 0.931 ± 0.022 0.815 ± 0.042 0.813 ± 0.012 0.776 ± 0.026 0.768 ± 0.010 0.795 ± 0.021 0.726 ± 0.016 0.945 ± 0.047
12 0.858 ± 0.017 0.920 ± 0.021 0.771 ± 0.029 0.737 ± 0.005 0.749 ± 0.039 0.669 ± 0.034 0.770 ± 0.010 0.630 ± 0.037 0.899 ± 0.039
16 0.834 ± 0.008 0.907 ± 0.016 0.743 ± 0.010 0.666 ± 0.028 0.721 ± 0.017 0.636 ± 0.012 0.765 ± 0.024 0.579 ± 0.042 0.849 ± 0.024
20
24
0.802 ± 0.013 0.888 ± 0.022 0.699 ± 0.013 0.586 ± 0.008 0.681 ± 0.032 0.625 ± 0.019 0.721 ± 0.017 0.524 ± 0.031 0.799 ± 0.017
0.773 ± 0.013 0.874 ± 0.026 0.656 ± 0.019 0.533 ± 0.010 0.616 ± 0.019 0.577 ± 0.027 0.704 ± 0.008 0.473 ± 0.020 0.747 ± 0.015
k (h−1 )b 0.0097 0.0038 0.0158 0.0258 0.0177 0.0240 0.0118 0.0294 0.0114
a Values are expressed as mean ± SD. b The coefficient (R2 ) for regression analysis of the rate constants ranged from 0.907 to 0.998.
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OPP degradation is reported as a first-order reaction.29 Degradation rate constants of the nine OPPs were therefore calculated (Table 2). Among the OPPs investigated, dichlorvos had the highest rate constants (0.0199–0.0446 h−1 ), while chlorpyrifos and fenthion gave lower ones (0.0028–0.0063 and 0.0033–0.0077 h−1 respectively). Thus dichlorvos was the most labile whereas chlorpyrifos and fenthion were more stable. Lactobacillus bulgaricus mostly showed the strongest acceleration on OPP dissipation, but two exceptions were phorate and diazinon with rate constants of 0.0075 and 0.0152 h−1 respectively. On the contrary, L. rhamnosus had the strongest acceleration on phorate and diazinon compared with the other strains. Based on the calculated degradation rate constants (Table 2), it was seen that chlorpyrifos, dichlorvos, fenthion, pirimiphos-methyl and trichlorphon were more susceptible to L. bulgaricus, as the increasing levels of degradation rate constants were up to 115.8–133.3%. At the same time, diazinon and phorate were more susceptible towards L. rhamnosus, as the degradation rate constants were increased by 103.6 and 102.0% respectively. However, chlorpyrifos-methyl and malathion were more resistant to all five strains compared with the other OPPs investigated. Totally, the nine OPPs showed greater susceptibility towards L. bulgaricus (as their degradation rate constants were enhanced by 18.3 − 133.3%), followed by L. rhamnosus and S. thermophilus, but less susceptibility towards L. acidophilus and L. casei. Abou-Arab30 applied streptococci, lactobacilli and yeasts isolated from Ras cheese to study dichlorodiphenyltrichloroethane (DDT) dissipation and found that DDT inoculated with lactobacilli had the highest decreasing level (i.e. DDT was more sensitive to lactobacilli). Our previous results showed that inoculation of some LAB into skimmed milk led to accelerated OPP degradation; among the seven OPPs investigated, phorate and trichlorphon were more unstable to S. thermophilus and Lactobacillus helveticus.22 More importantly, Cycon´ et al.1 studied diazinon biodegradation in sterilized soil and observed that Serratia liquefaciens, Serratia marcescens, Pseudomonas sp. and their combination resulted in rate constants of 0.032, 0.042, 0.048 and 0.082 day−1 respectively, i.e. diazinon showed different susceptibilities towards the applied strains and strain combination. The present study shared a similar conclusion with these mentioned studies and evidenced two facts: (1) LAB could enhance OPP degradation in the milk and (2) OPPs had different susceptibilities to the applied microorganisms. The present results also showed the
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potential of LAB to control or eliminate OPP residues in fermented foods.
OPP susceptibility towards two yogurt starters The impact of two yogurt starters on OPP dissipation in the milk was also assayed. Residual levels of the nine OPPs in the yogurt samples are recorded in Table 3, which showed OPP dissipation during yogurt fermentation. By a culture time of 5 h, trichlorphon inoculated with starter Y250A and fenthion inoculated with starter Y480F showed the greatest (19.2%) and least (7.4%) dissipation respectively (Table 3). Degradation rate constants of these OPPs during yogurt fermentation were also calculated (Table 3). Trichlorphon and dichlorvos had higher degradation rate constants (0.0212–0.0399 and 0.0233–0.0320 h−1 respectively) while malathion had lower ones (0.0164–0.0227 h−1 ), i.e. trichlorphon and dichlorvos were more susceptible while malathion was more resistant. Dichlorvos showed high instability to the applied strains (Table 2) and starters (Table 3) in the present study. Starters Y250A and Y480F had different strain compositions (the manufacturer did not give detailed information for commercial reasons), resulting in degradation rate constants of the OPPs of 0.0227–0.0399 and 0.0160–0.0233 h−1 respectively. It was thus concluded that starter Y250A was superior in OPP dissipation to its counterpart Y480F. Moreover, why the OPPs had different sensitivities to the strains and starters might be related to their chemical characteristics and should be clarified in detail in future studies. Bo et al.13 showed that two yogurt starters enhanced OPP degradation during yogurt processing and found that six out of seven OPPs were more sensitive to one starter applied. Zhang et al.31 investigated the degradation kinetic parameters of OPPs in apple juice subjected to ultrasonic treatment and found that chlorpyrifos was more labile than malathion. Another study reported that chlorpyrifos was more sensitive than methamidophos to pulsed electric fields.32 These mentioned results were consistent with the present result: OPPs had different susceptibilities to the applied treatment. The two starters used in the present study exhibited greater acceleration on OPP dissipation than the single strains did, implying that they might provide another benefit (food safety) to consumers besides yogurt quality, i.e. to decrease OPP residues in yogurt products.
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J Sci Food Agric 2015; 95: 260–266
Susceptibility of nine organophosphorus pesticides in skimmed milk
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Table 3. Residual concentrations and degradation rate constants (k) of nine organophosphorus pesticides in skimmed milk fermented by yogurt starters at 42 ∘ C Concentration (mg kg−1 )a detected at different fermentation times (h) Pesticide Chlorpyrifos Chlorpyrifos-methyl Diazinon Dichlorvos Fenthion Malathion Phorate Pirimiphos-methyl Trichlorphon
Starter Y250A Y480F Y250A Y480F Y250A Y480F Y250A Y480F Y250A Y480F Y250A Y480F Y250A Y480F Y250A Y480F Y250A Y480F
0
1
0.975 ± 0.023 0.942 ± 0.014 0.993 ± 0.012 0.947 ± 0.015 0.986 ± 0.020 0.966 ± 0.027 0.982 ± 0.030 0.958 ± 0.025 0.985 ± 0.013 0.980 ± 0.024 0.963 ± 0.024 0.963 ± 0.020 0.961 ± 0.015 0.993 ± 0.016 0.946 ± 0.008 0.955 ± 0.026 0.929 ± 0.032 0.969 ± 0.012
0.953 ± 0.030 0.936 ± 0.018 0.983 ± 0.015 0.940 ± 0.019 0.969 ± 0.017 0.961 ± 0.020 0.971 ± 0.021 0.944 ± 0.025 0.968 ± 0.022 0.977 ± 0.026 0.948 ± 0.023 0.956 ± 0.012 0.950 ± 0.020 0.963 ± 0.013 0.925 ± 0.012 0.945 ± 0.024 0.904 ± 0.023 0.956 ± 0.018
2 0.908 ± 0.028 0.932 ± 0.021 0.958 ± 0.019 0.920 ± 0.022 0.949 ± 0.012 0.940 ± 0.017 0.955 ± 0.027 0.929 ± 0.016 0.933 ± 0.027 0.960 ± 0.032 0.926 ± 0.027 0.938 ± 0.019 0.923 ± 0.032 0.947 ± 0.021 0.882 ± 0.016 0.942 ± 0.015 0.877 ± 0.016 0.939 ± 0.024
3 0.890 ± 0.018 0.908 ± 0.009 0.919 ± 0.024 0.894 ± 0.010 0.932 ± 0.025 0.926 ± 0.025 0.917 ± 0.019 0.913 ± 0.031 0.910 ± 0.017 0.950 ± 0.014 0.906 ± 0.019 0.925 ± 0.026 0.896 ± 0.009 0.939 ± 0.013 0.862 ± 0.011 0.918 ± 0.019 0.852 ± 0.026 0.916 ± 0.015
4
5
0.877 ± 0.036 0.895 ± 0.025 0.892 ± 0.011 0.882 ± 0.025 0.892 ± 0.023 0.908 ± 0.030 0.871 ± 0.031 0.875 ± 0.023 0.896 ± 0.024 0.925 ± 0.019 0.884 ± 0.031 0.903 ± 0.010 0.875 ± 0.018 0.917 ± 0.027 0.851 ± 0.019 0.899 ± 0.030 0.819 ± 0.030 0.897 ± 0.019
0.855 ± 0.031 0.858 ± 0.022 0.865 ± 0.014 0.862 ± 0.011 0.880 ± 0.014 0.893 ± 0.034 0.844 ± 0.013 0.856 ± 0.019 0.843 ± 0.015 0.908 ± 0.013 0.860 ± 0.021 0.891 ± 0.027 0.845 ± 0.023 0.915 ± 0.025 0.817 ± 0.024 0.880 ± 0.016 0.750 ± 0.015 0.872 ± 0.027
k (h−1 )b 0.0265 0.0179 0.0291 0.0196 0.0240 0.0166 0.0320 0.0231 0.0297 0.0159 0.0227 0.0164 0.0263 0.0160 0.0289 0.0167 0.0398 0.0212
a Values are expressed as mean ± SD. b The coefficient (R2 ) for regression analysis of the rate constants ranged from 0.895 to 0.995.
Table 4. Phosphatase activities detected in skimmed milk cultured with five lactic acid bacteria and two yogurt starters for 24 and 5 h respectively Strain/starter L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Y250A Y480F
Phosphatase activity (U mL−1 )a 0.081 ± 0.005g 0.150 ± 0.005b 0.097 ± 0.006f 0.116 ± 0.003e 0.127 ± 0.008d 0.175 ± 0.004a 0.137 ± 0.005c
a Mean values followed by different letters are significantly different (P < 0.05, one-way ANOVA).
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CONCLUSIONS The present results demonstrated that incubation of skimmed milk with five LAB and two yogurt starters could lead to enhanced dissipation of nine OPPs and higher degradation rate constants. It was
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Relationship between phosphatase production of strains and OPP susceptibility Phosphatase activities measured in the crude extracts of the cultured milk are given in Table 4, which shows that the five strains had different abilities to produce phosphatase (P < 0.05). Lactobacillus bulgaricus and L. acidophilus gave the highest and lowest phosphatase production (0.150 and 0.081 U mL−1 ) respectively. It was thus interesting to observe a potential relationship between phosphatase production of the strains and OPP degradation in the milk, i.e. inoculation of a strain of higher phosphatase production (e.g. L. bulgaricus) could bring about greater OPP dissipation. This might indicate that OPP susceptibility towards these strains partly or mostly depended on their phosphatase production. Other assay results in the present study further confirmed this finding. The extract from the yogurt with starter Y250A gave a higher phosphatase production of 0.175 U mL−1 , while that with starter Y480F
showed a lower value of 0.137 U mL−1 (Table 4). Starter Y250A thus might exhibit greater acceleration on OPP dissipation than starter Y480F did. The data in Table 3 show that starter Y250A did so, as expected. A brief conclusion is thus obtained that phosphatase production of LAB might be one of the key factors governing their ability to degrade OPPs. Further studies should provide additional support to this conclusion. It is well known that phosphatase is the most important enzyme during microbial cleavage of OPPs.33 One study found that Aspergillus oryzae with phosphatase activity was capable of degrading monocrotophos.27 Thengodkar and Sivakami34 reported that alkaline phosphatase obtained from the cell-free extract of Spirulina platensis could degrade chlorpyrifos by 78% during a time period of 1 h, while Kim et al.35 observed that the esterase from Candida cylindracea was important to malathion degradation. It was also reported that eight out of 12 commonly used OPPs could be hydrolyzed by the crude cell-free extract from a mixed bacterial culture, and seven OPPs showed higher hydrolysis rates than chemical hydrolysis by 0.1 mol L−1 NaOH.36 These results provide support to the present result. OPPs are ester compounds by chemical nature and are also target substrates for hydrolases (especially phosphatase). OPPs are thus susceptible to phosphatase-producing microorganisms, and LAB of different phosphatase production abilities might therefore give different accelerations on OPP hydrolysis (i.e. OPP degradation). This means that LAB of higher phosphatase production would have more potential to hydrolyze OPP residues in fermented foods.
www.soci.org clarified that these OPPs also had different susceptibilities towards the inoculated strains and starters, resulting in accelerated OPP degradation of different levels. Totally, the OPPs were more susceptive to L. bulgaricus but less sensitive to L. acidophilus and L. casei. The higher the phosphatase production of the strains, the greater was the OPP dissipation observed. Phosphatase should be a factor governing OPP degradation, and LAB of higher phosphatase production might be applied to decrease OPP residues in fermented foods efficiently.
ACKNOWLEDGEMENTS This study was funded by the National Key Technological Research and Development Program of China during the 11th Five-Year Plan Period (Project No. 2006BAD04A08). The authors thank the anonymous reviewers and editors for their valuable advice.
REFERENCES 1 Cycon´ M, Wójcik M and Piotrowska-Seget Z, Biodegradation of the organophosphorus insecticide diazinon by Serratia sp. and Pseudomonas sp. and their use in bioremediation of contaminated soil. Chemosphere 76:494–501 (2009). 2 Ecobichon DJ, Pesticide use in developing countries. Toxicology 160:27–33 (2001). 3 Kaushik G, Satya S and Naik SN, Food processing a tool to pesticide residue dissipation – a review. Food Res Int 42:26–40 (2009). 4 Li W, Qiu SP and Wu YJ, Triazophos residues and dissipation rates in wheat crops and soil. Ecotoxicol Environ Saf 69:312–316 (2008). 5 Bolles HG, Dixon-White HE, Peterson RK, Tomerlin JR, Day EW and Oliver GR, U.S. market basket study to determine residues of the insecticide chlorpyrifos. J Agric Food Chem 47:1817–1822 (1999). 6 Salas JH, Gon´zalez MM, Noa M, Pérez NA, Díaz G, Gutíerrez R, et al, Organophosphorus pesticide residues in Mexican commercial pasteurized milk. J Agric Food Chem 51:4468–4471 (2003). 7 Melgar MJ, Santaeufemia M and García MA, Organophosphorus pesticide residues in raw milk and infant formulas from Spanish northwest. J Environ Sci Health B 45:595–600 (2010). 8 Zhao XH, Bo LY, Wang J and Li TJ, Survey of seven organophosphorus pesticides in drinking water, feedstuffs and raw milk from dairy farms in the Province Heilongjiang during 2008–2009. Milk Sci Int 67:293–296 (2012). 9 Cardeal ZDL and Paes CMD, Analysis of organophosphorus pesticides in whole milk by solid phase microextraction gas chromatography method. J Environ Sci Health B 41:369–375 (2006). 10 Battu RS, Singh B and Kang BK, Contamination of liquid milk and butter with pesticide residues in the Ludhiana district of Punjab state, India. Ecotoxicol Environ Saf 59:324–331 (2004). 11 Tsiplakou E, Anagnostopoulos CJ, Liapis K, Haroutounian SA and Zervas G, Pesticides residues in milks and feedstuff of farm animals drawn from Greece. Chemosphere 80:504–512 (2010). 12 Toan PV, Sebesvari Z, Bläsing M, Rosendahl I and Renaud FG, Pesticide management and their residues in sediments and surface and drinking water in the Mekong Delta, Vietnam. Sci Total Environ 452:28–39 (2013). 13 Bo LY, Zhang YH and Zhao XH, Degradation kinetics of seven organophosphorus pesticides in milk during yoghurt processing. J Serb Chem Soc 76:353–362 (2011). 14 Ozbey A and Uygun U, Behaviour of some organophosphorus pesticide residues in peppermint tea during the infusion process. Food Chem 104:237–241 (2007).
X-W Zhou, X-H Zhao 15 Ticha J, Hajslova J, Jech M, Honzicek J, Lacina O, Kohoutkova J, et al, Changes of pesticide residues in apples during cold storage. Food Control 19:247–256 (2008). 16 Uygun U, Senoz B, Öztürk S and Koksel H, Degradation of organophosphorus pesticides in wheat during cookie processing. Food Chem 117:261–264 (2009). 17 Sood C, Jaggi S, Kumar V, Ravindranath SD and Shanker A, How manufacturing processes affect the level of pesticide residues in tea. J Sci Food Agric 84:2123–2127 (2004). 18 Lu HY, Shen Y, Sun X, Zhu H and Liu XJ, Washing effects of limonene on pesticide residues in green peppers. J Sci Food Agric 93:2917–2921 (2013). 19 Uygun U, Senoz B and Koksel H, Dissipation of organophosphorus pesticides in wheat during pasta processing. Food Chem 109:355–360 (2008). 20 Banna AA and Kawar NS, Behavior of parathion in apple juice processed into cider and vinegar. J Environ Sci Health B 17:505–514 (1982). 21 Zhao XH and Wang J, A brief study on the degradation kinetics of seven organophosphorus pesticides in skimmed milk cultured with Lactobacillus spp. at 42 ∘ C. Food Chem 131:300–304 (2012). 22 Zhao XH, Wang J, Li TJ and Zhang YH, Degradation behaviors of seven organophosphorus pesticides in skimmed milk inoculated with Streptococcus thermophilus or Lactobacillus helveticus. Milk Sci Int 67:399–401 (2012). 23 Zhao XH and Wang J, Degradation of seven organophosphorus pesticides in the fresh milk heated at 63 ∘ C and two pHs. Milk Sci Int 67:192–194 (2012). 24 Gundi VAKB and Reddy BR, Degradation of monocrotophos in soils. Chemosphere 62:396–403 (2006). 25 Briceño G, Fuentes MS, Palma G, Jorquera MA, Amoroso MJ and Diez MC, Chlorpyrifos biodegradation and 3,5,6-trichloro-2-pyridinol production by actinobacteria isolated from soil. Int Biodeter Biodegrad 73:1–7 (2012). 26 Sánchez ME, Estrada IB, Martínez O, Martín-Villacorta J, Aller A and Morán A, Influence of the application of sewage sludge on the degradation of pesticides in the soil. Chemosphere 57:673–679 (2004). 27 Bhalerao TS and Puranik PR, Microbial degradation of monocrotophos by Aspergillus oryzae. Int Biodeter Biodegrad 63:503–508 (2009). 28 Putnam RA, Nelson JO and Clark JM, The persistence and degradation of chlorothalonil and chlorpyrifos in a cranberry bog. J Agric Food Chem 51:170–176 (2003). 29 Vanclooster M, Ducheyne S, Dust M and Vereecken H, Evaluation of pesticide dynamics of the WAVE-model. Agric Water Manag 44:371–388 (2000). 30 Abou-Arab AAK, Effect of Ras cheese manufacturing on the stability of DDT and its metabolites. Food Chem 59:115–119 (1997). 31 Zhang YY, Xiao ZY, Chen F, Ge YQ, Wu JH and Hu XS, Degradation behavior and products of malathion and chlorpyrifos spiked in apple juice by ultrasonic treatment. Ultrason Sonochem 17:72–77 (2010). 32 Chen F, Zeng LQ, Zhang YY, Liao XJ, Ge YQ, Hu XS, et al, Degradation behaviour of methamidophos and chlorpyrifos in apple juice treated with pulsed electric fields. Food Chem 112:956–961 (2009). 33 Rosenberg A and Alexander M, Microbial cleavage of various organophosphorus insecticides. Appl Environ Microbiol 37:886–891 (1979). 34 Thengodkar RRM and Sivakami S, Degradation of chlorpyrifos by an alkaline phosphatase from the cyanobacterium Spirulina platensis. Biodegradation 21:637–644 (2010). 35 Kim YH, Ahn JY, Moon SH and Lee J, Biodegradation and detoxification of organophosphate insecticide malathion by Fusarium oxysporum f. sp. pisi cutinase. Chemosphere 60:1349–1355 (2005). 36 Munnecke DM, Enzymatic hydrolysis of organophosphate insecticides, a possible pesticide disposal method. Appl Environ Microbiol 32:7–13 (1976).
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J Sci Food Agric 2015; 95: 260–266
Revised: 19 April 2014
Accepted article published: 28 April 2014
Published online in Wiley Online Library: 21 May 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6710
Susceptibility of nine organophosphorus pesticides in skimmed milk towards inoculated lactic acid bacteria and yogurt starters Xin-Wei Zhoua and Xin-Huai Zhaoa,b,c* Abstract BACKGROUND: Previous research has shown that fresh milk might be polluted by some organophosphorus pesticides (OPPs). In this study the dissipation of nine OPPs, namely chlorpyrifos, chlorpyrifos-methyl, diazinon, dichlorvos, fenthion, malathion, phorate, pirimiphos-methyl and trichlorphon, in skimmed milk was investigated to clarify their susceptibility towards lactic acid bacteria (LAB) and yogurt starters. RESULTS: Skimmed milk was spiked with nine OPPs, inoculated with five strains of LAB and two commercial yogurt starters at 42 ∘ C for 24 and 5 h respectively and subjected to quantitative OPP analysis by gas chromatography. Degradation kinetic constants of these OPPs were calculated based on a first-order reaction model. OPP dissipation in the milk was enhanced by the inoculated strains and starters, resulting in OPP concentrations decreasing by 7.0–64.6 and 7.4–19.2% respectively. Totally, the nine OPPs were more susceptible to Lactobacillus bulgaricus, as it enhanced their degradation rate constants by 18.3–133.3%. Higher phosphatase production of the assayed stains was observed to bring about greater OPP degradation in the milk. CONCLUSION: Both LAB and yogurt starters could enhance OPP dissipation in skimmed milk, with the nine OPPs studied having different susceptibilities towards them. Phosphatase was a key factor governing OPP dissipation. The LAB of higher phosphatase production have more potential to decrease OPPs in fermented foods. © 2014 Society of Chemical Industry Keywords: organophosphorus pesticides; susceptibility; skimmed milk; lactic acid bacteria; yogurt starter; phosphatase
INTRODUCTION
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Organophosphorus pesticides (OPPs) are widely used in agricultural production.1,2 With respect to their chemical nature, OPPs are unstable compounds; however, excessive use of OPPs might eventually lead to widespread environmental and food contamination.3,4 Previous studies have indicated that milk products might be polluted by OPPs. Acephate and chlorpyrifos were detected in more than 100 milk samples from Italy, but at levels lower than the legal limits.5 Dichlorvos, phorate, chlorpyrifos and chlorfenvinphos at levels of 0.0051–0.0203 mg kg−1 were found in milk samples from Mexico.6 A recent study indicated that 43 out of 312 milk samples from northwestern Spain contained detectable OPPs.7 Another survey found that OPP levels in milk samples collected in Heilongjiang Province, China during 2008–2009 were less than 0.020 mg kg−1 .8 The residual chemical pesticides in milk products come from several possible routes, including external application for ectoparasite control directly on dairy cows,9 pest control in stables and factories6 and indirect contamination from polluted pasture, forages, feedstuffs or drinking water.10 – 12 OPPs in milk products might therefore pose a safety risk to consumers and should be kept under proper control before and during dairy processing. OPP levels in foods are affected by food processing such as fermentation, infusion, storage, cooking and drying,13 – 17 with OPPs showing different susceptibilities towards these treatments. Lu et al.18 found that washing had a positive effect on J Sci Food Agric 2015; 95: 260–266
pesticide reduction in green peppers. The fate of four OPPs in wheat during storage was investigated by Uygun et al.,19 who observed that chlorpyrifos-methyl, fenitrothion, malathion and pirimiphos-methyl were decreased by 76–88%. Banna and Kawar20 fortified apple juice with parathion at 25 mg kg−1 and found that only about 20% parathion was detectable after 57 days of fermentation process into vinegar. Some lactic acid bacteria (LAB) and commercial yogurt starters had accelerating effects on OPP degradation in milk, implying that OPPs were sensitive towards inoculated strains and starters.13,21,22 Also, pH shifting was observed to influence OPP degradation in fresh milk, with dimethoate, parathion-methyl and trichlorphon being highly susceptible to an alkaline pH value of 7.5.23
∗
Correspondence to: Xin-Huai Zhao, Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin 150030 China. E-mail: [email protected]
a Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin 150030, China b Department of Food Science, Northeast Agricultural University, Harbin 150030, China c Synergetic Innovation Center of Food Safety and Nutrition, Northeast Agricultural University, 150030, Harbin, China
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Susceptibility of nine organophosphorus pesticides in skimmed milk OPP degradation by soil or aquatic microorganisms has been well studied in the field of environmental science,24 – 26 whereas that by edible microorganisms has been less investigated in the field of food science. It is strongly suggested to characterize OPP degradation during food fermentation, especially to clarify the susceptibility of OPPs towards food microorganisms. This might provide a potential approach to control or decrease OPP residues in fermented foods. More importantly, biodegradation of OPPs during food processing could provide a safety guarantee to consumers. In this study, skimmed milk was spiked with nine OPPs, namely chlorpyrifos, chlorpyrifos-methyl, diazinon, dichlorvos, fenthion, malathion, phorate pirimiphos-methyl and trichlorphon, that had previously been detected in some foods. The spiked milk was inoculated by one of five LAB widely used in the preparation of yogurt, cheese and other fermented milk products or by one of two commercial direct vat-set yogurt starters. After detection of residual OPPs at different culture times by gas chromatography (GC) and calculation of degradation rate constants for the OPPs, OPP susceptibilities towards these strains and starters were clarified by comparing the increasing levels of degradation rate constants.
MATERIALS AND METHODS Materials and chemicals Nine OPP standards, i.e. chlorpyrifos, chlorpyrifos-methyl, diazinon, dichlorvos, fenthion, malathion, phorate, pirimiphos-methyl and trichlorphon, of declared purity 94.5–99.8% were purchased from Sigma-Aldrich (Schnelldorf, Germany). Stock solutions of the nine OPPs (0.5 g L−1 ) were prepared in acetone. The mixed working solutions required for the standard curve and milk spiking were prepared from the stock solutions by further dilutions. Skimmed milk power, prepared with fresh milk from dairy farms near Harbin city, was purchased from Harbin Longdan Dairy Co. Ltd (Harbin, China). Analysis showed that the powder was free of both antibiotics and the nine OPP residues. Other chemicals used were of analytical or chromatographic grade. Water was prepared using a Milli-Q Plus system (Millipore Corporation, New York, NY, USA). Strains and starters The five LAB, namely Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus), Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus and Streptococcus thermophilus, were supplied by the Centre of Lactic Acid Bacteria in Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University (Harbin, China). The two commercial yogurt starters, namely Lyofast Y250A and Y480F, were gifts from Beijing Duoaite Biotechnology Co. Ltd (Beijing, China). The lyophilized strains were rehydrated in 1 mL of sterilized rehydration medium (12% skimmed milk, w/w) with subsequent 5% (v/v) inoculation into normal DeMan/Rogosa/Sharpe (MRS) agar medium (Difco, Detroit, MI, USA) and then cultured at 37 ∘ C for 24 h. To ensure their purity and viability, the strains were cultured and subcultured in the MRS medium three times and then cultured in the skimmed milk (12%, w/w) for 24 h before further application.
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temperature for 30 min to ensure OPP distribution. The spiked milk (20 mL) was poured into glass bottles with a capacity of 50 mL, sterilized at 121 ∘ C for 20 min and cooled to ambient temperature. One of the five strains or a culture combination of S. thermophilus and L. bulgaricus (1:1, w/w) was inoculated into the milk at a level of 3% (w/w) and cultured at 42 ∘ C for 8, 12, 16, 20 and 24 h respectively. Some bottles were selected randomly as analysis samples, cooled in an ice water bath and subjected to OPP extraction and purification immediately. A control sample was also prepared with the same conditions and procedure but without strain inoculation. The spiked milk was also inoculated with each of the two yogurt starters as reported previously13 and fermented in glass cups at 42 ∘ C for 5 h. Some cups were selected randomly as analysis samples after each hour of fermentation and rapidly cooled in an ice water bath. The selected samples were subjected to OPP extraction and purification immediately. A control sample was also prepared with the same conditions and procedure but without starter addition. Extraction, purification and analysis of OPPs Extraction and purification of OPPs were carried out as described previously.21 The purified dichloromethane phase of 10 mL was evaporated to dryness under a stream of nitrogen at 30 ∘ C. The residues left were redissolved in 1 mL of acetone and filtered through a 0.45 μm microporous membrane before GC analysis. OPP detection was performed using an Agilent 7890 gas chromatograph (GC) (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a flame photometric detector, an Agilent 7683 autosampler and a capillary column (DB-1701, 30 m × 0.25 mm, 0.25 μm). Nitrogen was used as carrier gas at a flow rate of 1 mL min−1 . The GC temperature program consisted of an initial oven temperature of 50 ∘ C, held at 50 ∘ C for 1 min, elevated from 50 to 130 ∘ C at 20 ∘ C min−1 , from 130 to 200 ∘ C at 10 ∘ C min−1 and from 200 to 203 ∘ C at 0.5 ∘ C min−1 , then held at 203 ∘ C for 5 min. The injector and detector temperatures were set at 210 and 250 ∘ C respectively. Assay of phosphatase activity The spiked milk (10 g), cultured or fermented by the strains or the starters for 24 or 5 h respectively, was mixed well with 5 mL of carbonate/bicarbonate buffer (10 mmol L−1 , pH 10.5), stood at ambient temperature for 30 min and then centrifuged at 10 000 × g for 15 min at 4 ∘ C. The supernatant was subjected to sonication ten times (10 s duration at 15 s intervals) and measured for phosphatase activity as described previously,27 with some modifications. The reaction mixture comprised 1 mL of 5 mmol L−1 p-nitrophenyl phosphate (dissolved in buffer containing 1 mmol L−1 MgCl2 ), 1 mL of buffer and 1 mL of crude enzyme solution, diluted to a final volume of 5 mL with water. After a reaction time of 60 min at 37 ∘ C, the reaction was stopped by adding 3 mL of termination agent (0.1 mol L−1 NaOH and 5 mmol L−1 EDTA). p-Nitrophenol formed in the mixture was detected in a UV-2401 PC spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at 405 nm. Standard p-nitrophenol solutions (0.5–40.0 μmol L−1 ) were used to generate a standard curve. One unit (1 U) of phosphatase activity was expressed as the amount liberating 1 μmol p-nitrophenol min−1 at 37 ∘ C. Statistical analysis All experiments and analyses were carried out three times. All data were expressed as mean ± standard deviation (SD). Differences between mean values of multiple groups were analyzed
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Sample preparation Skimmed milk power was reconstituted into skimmed milk with a total solids content of 12% (w/w) and a pH value of 6.5. Each of the nine OPPs was added to the milk at a level of 1 mg kg−1 . The spiked milk was shaken vigorously and stood at ambient
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Table 1. Recoveries and limits of detection (LOD) of nine organophosphorus pesticides in skimmed milk
X-W Zhou, X-H Zhao
A
Recovery (%)a at three spiking levels (mg kg−1 ) Pesticide Chlorpyrifos Chlorpyrifos-methyl Diazinon Dichlorvos Fenthion Malathion Phorate Pirimiphos-methyl Trichlorphon
0.1 97.4 ± 2.4 94.0 ± 3.3 86.3 ± 2.8 85.7 ± 4.3 93.0 ± 2.3 87.3 ± 5.3 92.1 ± 5.7 86.7 ± 2.6 87.2 ± 6.0
0.5
LOD (mg kg−1 )b
1
96.5 ± 3.6 91.9 ± 0.9 97.5 ± 1.5 90.7 ± 1.6 87.8 ± 2.2 85.4 ± 2.7 83.9 ± 5.7 84.1 ± 2.0 93.0 ± 1.9 89.4 ± 5.2 84.9 ± 3.6 102.9 ± 1.3 98.6 ± 7.7 84.9 ± 1.1 86.8 ± 3.3 91.4 ± 3.5 89.5 ± 1.6 87.8 ± 1.0
0.006 0.005 0.002 0.001 0.007 0.008 0.002 0.005 0.003
B
a Values are expressed as mean ± SD. b Data were calculated based on a signal-to-noise ratio of 3.
by one-way analysis of variance (ANOVA) with Duncan’s multiple range tests. SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) was used in data analysis.
RESULTS AND DISCUSSION GC analysis of nine OPPs GC was used in the present study to detect residual OPPs in the milk samples. The linear range of detection of OPPs in the present analysis was 0.1–8.0 mg kg−1 (R2 > 0.995), while limits of detection ranged from 0.001 to 0.008 mg kg−1 based on a signal-to-noise ratio of 3 (Table 1). Typical OPP profiles for the standard solution, control and analysis samples are depicted in Fig. 1. OPP recoveries at three spiking levels (0.1, 0.5 and 1 mg kg−1 ) ranged from 83.9 to 102.9% (Table 1). These results indicated that the applied conditions and procedure were suitable for the present study, as a previous study suggested.28
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OPP susceptibility towards five LAB The residual OPPs detected in the milk inoculated with five LAB are given in Table 2. The data evidenced OPP dissipation in the milk, as the detected OPP concentrations showed a decreasing trend during a culture time of 24 h. In the control sample, chlorpyrifos and dichlorvos finally were lost by 6.2 and 32.6% respectively and appeared to be the most stable and unstable OPPs respectively. Chlorpyrifos inoculated with L. casei exhibited the least dissipation, as about 93% chlorpyrifos still remained in the milk, while dichlorvos inoculated with L. bulgaricus gave the greatest dissipation, as about 65% dichlorvos disappeared, i.e. chlorpyrifos and dichlorvos were the least and most susceptible to the inoculated strains respectively. Compared with OPP dissipation in the control sample, L. bulgaricus, L. rhamnosus and S. thermophilus totally had stronger acceleration on OPP dissipation and brought about increased dissipation by 22.9–160.0, 6.3–115.5 and 8.6–123.2% (except for malathion inoculated with L. rhamnosus or S. thermophilus) respectively. Lactobacillus acidophilus and L. casei showed weaker acceleration on OPP dissipation, as OPP dissipation levels were only increased by 11.7–86.6%. Among the five strains studied, both L. bulgaricus and S. thermophilus mostly showed greater acceleration on OPP dissipation. It is well known that S. thermophilus and
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C
Figure 1. Typical GC profiles of nine organophosphorus pesticides for (A) standard solution, (B) control milk and (C) skimmed milk. Peaks 1–9 represent trichlorphon, dichlorvos, phorate, diazinon, chlorpyrifos-methyl, pirimiphos-methyl, chlorpyrifos, fenthion and malathion respectively.
L. bulgaricus are two strains widely used in fermented dairy products, especially in yogurt. Streptococcus thermophilus and L. bulgaricus were thus combined and inoculated into the milk. Totally, this combination conferred slightly higher OPP dissipation than single S. thermophilus or L. bulgaricus did, as it resulted in an increased dissipation by 12.1–180.7%. Whether other strain combinations also have similar acceleration on OPP dissipation should be investigated in future studies.
© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 260–266
Susceptibility of nine organophosphorus pesticides in skimmed milk
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Table 2. Residual concentrations and degradation rate constants (k) of nine organophosphorus pesticides in skimmed milk inoculated with lactic acid bacteria at 42 ∘ C Concentration (mg kg−1 )a detected at different culture times (h) Pesticide Chlorpyrifos
Chlorpyrifos-methyl
Diazinon
Dichlorvos
Fenthion
Malathion
Phorate
Pirimiphos-methyl
Strain L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus
0 1.010 ± 0.016 0.982 ± 0.009 0.981 ± 0.015 1.016 ± 0.011 1.020 ± 0.035 0.981 ± 0.018 0.946 ± 0.018 1.026 ± 0.023 0.995 ± 0.019 0.973 ± 0.003 1.031 ± 0.018 1.074 ± 0.023 0.972 ± 0.015 0.959 ± 0.030 1.027 ± 0.007 1.038 ± 0.011 1.079 ± 0.074 1.045 ± 0.041 0.959 ± 0.010 0.971 ± 0.026 0.953 ± 0.034 1.030 ± 0.025 1.005 ± 0.008 0.952 ± 0.012 1.042 ± 0.026 0.964 ± 0.012 0.979 ± 0.013 0.954 ± 0.036 0.991 ± 0.006 1.055 ± 0.034 1.024 ± 0.011 1.001 ± 0.016 0.968 ± 0.009 0.979 ± 0.016 0.948 ± 0.028 1.014 ± 0.046 1.023 ± 0.018 0.972 ± 0.018 1.048 ± 0.011 1.052 ± 0.035 0.967 ± 0.017 0.922 ± 0.026 1.055 ± 0.012 1.029 ± 0.061 1.037 ± 0.024 1.031 ± 0.038 0.994 ± 0.040 0.968 ± 0.005 0.956 ± 0.020 0.987 ± 0.021 1.068 ± 0.023 0.965 ± 0.012 0.997 ± 0.026 0.959 ± 0.004
8 0.984 ± 0.015 0.941 ± 0.014 0.959 ± 0.019 0.974 ± 0.025 0.971 ± 0.027 0.922 ± 0.012 0.928 ± 0.025 0.901 ± 0.054 0.861 ± 0.007 0.902 ± 0.007 0.907 ± 0.024 0.941 ± 0.036 0.881 ± 0.022 0.861 ± 0.028 1.005 ± 0.013 0.981 ± 0.006 1.027 ± 0.074 0.995 ± 0.039 0.909 ± 0.009 0.898 ± 0.028 0.927 ± 0.030 0.849 ± 0.064 0.838 ± 0.085 0.819 ± 0.050 0.772 ± 0.030 0.812 ± 0.011 0.798 ± 0.038 0.864 ± 0.075 0.949 ± 0.009 0.992 ± 0.039 0.986 ± 0.014 0.956 ± 0.023 0.936 ± 0.016 0.925 ± 0.021 0.930 ± 0.025 0.937 ± 0.065 0.921 ± 0.019 0.918 ± 0.034 0.962 ± 0.041 0.976 ± 0.035 0.879 ± 0.025 0.842 ± 0.035 1.002 ± 0.048 0.931 ± 0.054 0.931 ± 0.060 0.856 ± 0.034 0.930 ± 0.037 0.854 ± 0.014 0.897 ± 0.043 0.931 ± 0.033 1.018 ± 0.021 0.922 ± 0.021 0.929 ± 0.033 0.924 ± 0.009
12 0.971 ± 0.016 0.919 ± 0.020 0.951 ± 0.018 0.958 ± 0.022 0.935 ± 0.027 0.894 ± 0.014 0.917 ± 0.022 0.842 ± 0.039 0.803 ± 0.017 0.862 ± 0.011 0.861 ± 0.008 0.831 ± 0.030 0.814 ± 0.016 0.795 ± 0.034 0.979 ± 0.014 0.946 ± 0.016 0.999 ± 0.054 0.957 ± 0.046 0.879 ± 0.010 0.848 ± 0.015 0.897 ± 0.031 0.727 ± 0.043 0.701 ± 0.10 0.705 ± 0.037 0.647 ± 0.012 0.779 ± 0.010 0.700 ± 0.018 0.775 ± 0.013 0.934 ± 0.007 0.962 ± 0.040 0.973 ± 0.012 0.933 ± 0.018 0.914 ± 0.008 0.914 ± 0.023 0.921 ± 0.023 0.896 ± 0.026 0.860 ± 0.027 0.868 ± 0.048 0.912 ± 0.023 0.902 ± 0.019 0.830 ± 0.017 0.806 ± 0.02 0.924 ± 0.037 0.884 ± 0.046 0.907 ± 0.041 0.797 ± 0.036 0.858 ± 0.018 0.742 ± 0.055 0.835 ± 0.032 0.896 ± 0.030 0.952 ± 0.030 0.904 ± 0.021 0.916 ± 0.030 0.902 ± 0.010
16 0.959 ± 0.016 0.898 ± 0.020 0.941 ± 0.021 0.947 ± 0.017 0.917 ± 0.027 0.864 ± 0.019 0.898 ± 0.017 0.843 ± 0.043 0.754 ± 0.019 0.845 ± 0.014 0.827 ± 0.010 0.804 ± 0.036 0.761 ± 0.013 0.746 ± 0.032 0.933 ± 0.021 0.925 ± 0.011 0.981 ± 0.073 0.883 ± 0.046 0.864 ± 0.013 0.824 ± 0.015 0.880 ± 0.033 0.662 ± 0.057 0.565 ± 0.074 0.680 ± 0.024 0.555 ± 0.017 0.713 ± 0.009 0.611 ± 0.011 0.694 ± 0.036 0.919 ± 0.008 0.926 ± 0.028 0.964 ± 0.014 0.918 ± 0.009 0.903 ± 0.011 0.888 ± 0.027 0.902 ± 0.017 0.866 ± 0.029 0.816 ± 0.025 0.849 ± 0.048 0.899 ± 0.012 0.867 ± 0.017 0.797 ± 0.021 0.773 ± 0.010 0.900 ± 0.020 0.832 ± 0.055 0.890 ± 0.049 0.757 ± 0.039 0.819 ± 0.037 0.718 ± 0.043 0.805 ± 0.029 0.885 ± 0.026 0.927 ± 0.016 0.896 ± 0.005 0.905 ± 0.015 0.879 ± 0.014
20
24
0.949 ± 0.016 0.878 ± 0.016 0.922 ± 0.014 0.940 ± 0.019 0.903 ± 0.028 0.834 ± 0.020 0.894 ± 0.017 0.797 ± 0.033 0.702 ± 0.007 0.794 ± 0.013 0.788 ± 0.010 0.778 ± 0.026 0.710 ± 0.009 0.729 ± 0.023 0.884 ± 0.006 0.901 ± 0.004 0.951 ± 0.058 0.846 ± 0.035 0.821 ± 0.007 0.799 ± 0.017 0.850 ± 0.024 0.631 ± 0.050 0.441 ± 0.027 0.628 ± 0.048 0.500 ± 0.010 0.607 ± 0.008 0.539 ± 0.021 0.643 ± 0.021 0.905 ± 0.009 0.911 ± 0.021 0.950 ± 0.008 0.907 ± 0.010 0.891 ± 0.007 0.862 ± 0.020 0.886 ± 0.021 0.839 ± 0.032 0.779 ± 0.006 0.805 ± 0.036 0.874 ± 0.015 0.843 ± 0.025 0.743 ± 0.014 0.721 ± 0.014 0.867 ± 0.021 0.775 ± 0.061 0.835 ± 0.018 0.714 ± 0.033 0.759 ± 0.028 0.671 ± 0.034 0.780 ± 0.024 0.861 ± 0.033 0.911 ± 0.010 0.878 ± 0.015 0.886 ± 0.021 0.860 ± 0.006
0.939 ± 0.019 0.841 ± 0.008 0.913 ± 0.004 0.925 ± 0.006 0.889 ± 0.025 0.816 ± 0.016 0.887 ± 0.023 0.770 ± 0.023 0.651 ± 0.027 0.759 ± 0.008 0.734 ± 0.010 0.737 ± 0.017 0.658 ± 0.011 0.679 ± 0.035 0.843 ± 0.010 0.864 ± 0.021 0.932 ± 0.054 0.804 ± 0.036 0.806 ± 0.005 0.772 ± 0.018 0.841 ± 0.023 0.529 ± 0.017 0.356 ± 0.009 0.561 ± 0.014 0.457 ± 0.012 0.503 ± 0.009 0.464 ± 0.020 0.611 ± 0.028 0.886 ± 0.008 0.876 ± 0.043 0.934 ± 0.014 0.892 ± 0.016 0.872 ± 0.008 0.845 ± 0.020 0.879 ± 0.024 0.811 ± 0.042 0.725 ± 0.031 0.781 ± 0.016 0.856 ± 0.020 0.828 ± 0.026 0.709 ± 0.020 0.694 ± 0.023 0.834 ± 0.031 0.710 ± 0.041 0.800 ± 0.032 0.605 ± 0.031 0.733 ± 0.025 0.595 ± 0.011 0.759 ± 0.021 0.834 ± 0.029 0.882 ± 0.025 0.864 ± 0.018 0.868 ± 0.020 0.834 ± 0.006
k (h−1 )b 0.0031 0.0063 0.0030 0.0038 0.0059 0.0078 0.0028 0.0116 0.0175 0.0102 0.0136 0.0158 0.0165 0.0143 0.0085 0.0075 0.0062 0.0114 0.0074 0.0096 0.0056 0.0269 0.0446 0.0217 0.0352 0.0258 0.0312 0.0199 0.0045 0.0077 0.0037 0.0047 0.0043 0.0061 0.0033 0.0093 0.0142 0.0094 0.0084 0.0105 0.0130 0.0120 0.0102 0.0152 0.0103 0.0204 0.0134 0.0198 0.0101 0.0069 0.0082 0.0045 0.0054 0.0058
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X-W Zhou, X-H Zhao
Table 2. Continued Concentration (mg kg−1 )a detected at different culture times (h) Pesticide
Trichlorphon
Strain Combination Control L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Combination Control
0 0.977 ± 0.011 0.956 ± 0.031 0.975 ± 0.008 0.984 ± 0.004 0.980 ± 0.020 1.055 ± 0.049 0.951 ± 0.010 0.967 ± 0.004 0.974 ± 0.024
8 0.899 ± 0.010 0.931 ± 0.022 0.815 ± 0.042 0.813 ± 0.012 0.776 ± 0.026 0.768 ± 0.010 0.795 ± 0.021 0.726 ± 0.016 0.945 ± 0.047
12 0.858 ± 0.017 0.920 ± 0.021 0.771 ± 0.029 0.737 ± 0.005 0.749 ± 0.039 0.669 ± 0.034 0.770 ± 0.010 0.630 ± 0.037 0.899 ± 0.039
16 0.834 ± 0.008 0.907 ± 0.016 0.743 ± 0.010 0.666 ± 0.028 0.721 ± 0.017 0.636 ± 0.012 0.765 ± 0.024 0.579 ± 0.042 0.849 ± 0.024
20
24
0.802 ± 0.013 0.888 ± 0.022 0.699 ± 0.013 0.586 ± 0.008 0.681 ± 0.032 0.625 ± 0.019 0.721 ± 0.017 0.524 ± 0.031 0.799 ± 0.017
0.773 ± 0.013 0.874 ± 0.026 0.656 ± 0.019 0.533 ± 0.010 0.616 ± 0.019 0.577 ± 0.027 0.704 ± 0.008 0.473 ± 0.020 0.747 ± 0.015
k (h−1 )b 0.0097 0.0038 0.0158 0.0258 0.0177 0.0240 0.0118 0.0294 0.0114
a Values are expressed as mean ± SD. b The coefficient (R2 ) for regression analysis of the rate constants ranged from 0.907 to 0.998.
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OPP degradation is reported as a first-order reaction.29 Degradation rate constants of the nine OPPs were therefore calculated (Table 2). Among the OPPs investigated, dichlorvos had the highest rate constants (0.0199–0.0446 h−1 ), while chlorpyrifos and fenthion gave lower ones (0.0028–0.0063 and 0.0033–0.0077 h−1 respectively). Thus dichlorvos was the most labile whereas chlorpyrifos and fenthion were more stable. Lactobacillus bulgaricus mostly showed the strongest acceleration on OPP dissipation, but two exceptions were phorate and diazinon with rate constants of 0.0075 and 0.0152 h−1 respectively. On the contrary, L. rhamnosus had the strongest acceleration on phorate and diazinon compared with the other strains. Based on the calculated degradation rate constants (Table 2), it was seen that chlorpyrifos, dichlorvos, fenthion, pirimiphos-methyl and trichlorphon were more susceptible to L. bulgaricus, as the increasing levels of degradation rate constants were up to 115.8–133.3%. At the same time, diazinon and phorate were more susceptible towards L. rhamnosus, as the degradation rate constants were increased by 103.6 and 102.0% respectively. However, chlorpyrifos-methyl and malathion were more resistant to all five strains compared with the other OPPs investigated. Totally, the nine OPPs showed greater susceptibility towards L. bulgaricus (as their degradation rate constants were enhanced by 18.3 − 133.3%), followed by L. rhamnosus and S. thermophilus, but less susceptibility towards L. acidophilus and L. casei. Abou-Arab30 applied streptococci, lactobacilli and yeasts isolated from Ras cheese to study dichlorodiphenyltrichloroethane (DDT) dissipation and found that DDT inoculated with lactobacilli had the highest decreasing level (i.e. DDT was more sensitive to lactobacilli). Our previous results showed that inoculation of some LAB into skimmed milk led to accelerated OPP degradation; among the seven OPPs investigated, phorate and trichlorphon were more unstable to S. thermophilus and Lactobacillus helveticus.22 More importantly, Cycon´ et al.1 studied diazinon biodegradation in sterilized soil and observed that Serratia liquefaciens, Serratia marcescens, Pseudomonas sp. and their combination resulted in rate constants of 0.032, 0.042, 0.048 and 0.082 day−1 respectively, i.e. diazinon showed different susceptibilities towards the applied strains and strain combination. The present study shared a similar conclusion with these mentioned studies and evidenced two facts: (1) LAB could enhance OPP degradation in the milk and (2) OPPs had different susceptibilities to the applied microorganisms. The present results also showed the
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potential of LAB to control or eliminate OPP residues in fermented foods.
OPP susceptibility towards two yogurt starters The impact of two yogurt starters on OPP dissipation in the milk was also assayed. Residual levels of the nine OPPs in the yogurt samples are recorded in Table 3, which showed OPP dissipation during yogurt fermentation. By a culture time of 5 h, trichlorphon inoculated with starter Y250A and fenthion inoculated with starter Y480F showed the greatest (19.2%) and least (7.4%) dissipation respectively (Table 3). Degradation rate constants of these OPPs during yogurt fermentation were also calculated (Table 3). Trichlorphon and dichlorvos had higher degradation rate constants (0.0212–0.0399 and 0.0233–0.0320 h−1 respectively) while malathion had lower ones (0.0164–0.0227 h−1 ), i.e. trichlorphon and dichlorvos were more susceptible while malathion was more resistant. Dichlorvos showed high instability to the applied strains (Table 2) and starters (Table 3) in the present study. Starters Y250A and Y480F had different strain compositions (the manufacturer did not give detailed information for commercial reasons), resulting in degradation rate constants of the OPPs of 0.0227–0.0399 and 0.0160–0.0233 h−1 respectively. It was thus concluded that starter Y250A was superior in OPP dissipation to its counterpart Y480F. Moreover, why the OPPs had different sensitivities to the strains and starters might be related to their chemical characteristics and should be clarified in detail in future studies. Bo et al.13 showed that two yogurt starters enhanced OPP degradation during yogurt processing and found that six out of seven OPPs were more sensitive to one starter applied. Zhang et al.31 investigated the degradation kinetic parameters of OPPs in apple juice subjected to ultrasonic treatment and found that chlorpyrifos was more labile than malathion. Another study reported that chlorpyrifos was more sensitive than methamidophos to pulsed electric fields.32 These mentioned results were consistent with the present result: OPPs had different susceptibilities to the applied treatment. The two starters used in the present study exhibited greater acceleration on OPP dissipation than the single strains did, implying that they might provide another benefit (food safety) to consumers besides yogurt quality, i.e. to decrease OPP residues in yogurt products.
© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 260–266
Susceptibility of nine organophosphorus pesticides in skimmed milk
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Table 3. Residual concentrations and degradation rate constants (k) of nine organophosphorus pesticides in skimmed milk fermented by yogurt starters at 42 ∘ C Concentration (mg kg−1 )a detected at different fermentation times (h) Pesticide Chlorpyrifos Chlorpyrifos-methyl Diazinon Dichlorvos Fenthion Malathion Phorate Pirimiphos-methyl Trichlorphon
Starter Y250A Y480F Y250A Y480F Y250A Y480F Y250A Y480F Y250A Y480F Y250A Y480F Y250A Y480F Y250A Y480F Y250A Y480F
0
1
0.975 ± 0.023 0.942 ± 0.014 0.993 ± 0.012 0.947 ± 0.015 0.986 ± 0.020 0.966 ± 0.027 0.982 ± 0.030 0.958 ± 0.025 0.985 ± 0.013 0.980 ± 0.024 0.963 ± 0.024 0.963 ± 0.020 0.961 ± 0.015 0.993 ± 0.016 0.946 ± 0.008 0.955 ± 0.026 0.929 ± 0.032 0.969 ± 0.012
0.953 ± 0.030 0.936 ± 0.018 0.983 ± 0.015 0.940 ± 0.019 0.969 ± 0.017 0.961 ± 0.020 0.971 ± 0.021 0.944 ± 0.025 0.968 ± 0.022 0.977 ± 0.026 0.948 ± 0.023 0.956 ± 0.012 0.950 ± 0.020 0.963 ± 0.013 0.925 ± 0.012 0.945 ± 0.024 0.904 ± 0.023 0.956 ± 0.018
2 0.908 ± 0.028 0.932 ± 0.021 0.958 ± 0.019 0.920 ± 0.022 0.949 ± 0.012 0.940 ± 0.017 0.955 ± 0.027 0.929 ± 0.016 0.933 ± 0.027 0.960 ± 0.032 0.926 ± 0.027 0.938 ± 0.019 0.923 ± 0.032 0.947 ± 0.021 0.882 ± 0.016 0.942 ± 0.015 0.877 ± 0.016 0.939 ± 0.024
3 0.890 ± 0.018 0.908 ± 0.009 0.919 ± 0.024 0.894 ± 0.010 0.932 ± 0.025 0.926 ± 0.025 0.917 ± 0.019 0.913 ± 0.031 0.910 ± 0.017 0.950 ± 0.014 0.906 ± 0.019 0.925 ± 0.026 0.896 ± 0.009 0.939 ± 0.013 0.862 ± 0.011 0.918 ± 0.019 0.852 ± 0.026 0.916 ± 0.015
4
5
0.877 ± 0.036 0.895 ± 0.025 0.892 ± 0.011 0.882 ± 0.025 0.892 ± 0.023 0.908 ± 0.030 0.871 ± 0.031 0.875 ± 0.023 0.896 ± 0.024 0.925 ± 0.019 0.884 ± 0.031 0.903 ± 0.010 0.875 ± 0.018 0.917 ± 0.027 0.851 ± 0.019 0.899 ± 0.030 0.819 ± 0.030 0.897 ± 0.019
0.855 ± 0.031 0.858 ± 0.022 0.865 ± 0.014 0.862 ± 0.011 0.880 ± 0.014 0.893 ± 0.034 0.844 ± 0.013 0.856 ± 0.019 0.843 ± 0.015 0.908 ± 0.013 0.860 ± 0.021 0.891 ± 0.027 0.845 ± 0.023 0.915 ± 0.025 0.817 ± 0.024 0.880 ± 0.016 0.750 ± 0.015 0.872 ± 0.027
k (h−1 )b 0.0265 0.0179 0.0291 0.0196 0.0240 0.0166 0.0320 0.0231 0.0297 0.0159 0.0227 0.0164 0.0263 0.0160 0.0289 0.0167 0.0398 0.0212
a Values are expressed as mean ± SD. b The coefficient (R2 ) for regression analysis of the rate constants ranged from 0.895 to 0.995.
Table 4. Phosphatase activities detected in skimmed milk cultured with five lactic acid bacteria and two yogurt starters for 24 and 5 h respectively Strain/starter L. acidophilus L. bulgaricus L. casei L. rhamnosus S. thermophilus Y250A Y480F
Phosphatase activity (U mL−1 )a 0.081 ± 0.005g 0.150 ± 0.005b 0.097 ± 0.006f 0.116 ± 0.003e 0.127 ± 0.008d 0.175 ± 0.004a 0.137 ± 0.005c
a Mean values followed by different letters are significantly different (P < 0.05, one-way ANOVA).
J Sci Food Agric 2015; 95: 260–266
CONCLUSIONS The present results demonstrated that incubation of skimmed milk with five LAB and two yogurt starters could lead to enhanced dissipation of nine OPPs and higher degradation rate constants. It was
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Relationship between phosphatase production of strains and OPP susceptibility Phosphatase activities measured in the crude extracts of the cultured milk are given in Table 4, which shows that the five strains had different abilities to produce phosphatase (P < 0.05). Lactobacillus bulgaricus and L. acidophilus gave the highest and lowest phosphatase production (0.150 and 0.081 U mL−1 ) respectively. It was thus interesting to observe a potential relationship between phosphatase production of the strains and OPP degradation in the milk, i.e. inoculation of a strain of higher phosphatase production (e.g. L. bulgaricus) could bring about greater OPP dissipation. This might indicate that OPP susceptibility towards these strains partly or mostly depended on their phosphatase production. Other assay results in the present study further confirmed this finding. The extract from the yogurt with starter Y250A gave a higher phosphatase production of 0.175 U mL−1 , while that with starter Y480F
showed a lower value of 0.137 U mL−1 (Table 4). Starter Y250A thus might exhibit greater acceleration on OPP dissipation than starter Y480F did. The data in Table 3 show that starter Y250A did so, as expected. A brief conclusion is thus obtained that phosphatase production of LAB might be one of the key factors governing their ability to degrade OPPs. Further studies should provide additional support to this conclusion. It is well known that phosphatase is the most important enzyme during microbial cleavage of OPPs.33 One study found that Aspergillus oryzae with phosphatase activity was capable of degrading monocrotophos.27 Thengodkar and Sivakami34 reported that alkaline phosphatase obtained from the cell-free extract of Spirulina platensis could degrade chlorpyrifos by 78% during a time period of 1 h, while Kim et al.35 observed that the esterase from Candida cylindracea was important to malathion degradation. It was also reported that eight out of 12 commonly used OPPs could be hydrolyzed by the crude cell-free extract from a mixed bacterial culture, and seven OPPs showed higher hydrolysis rates than chemical hydrolysis by 0.1 mol L−1 NaOH.36 These results provide support to the present result. OPPs are ester compounds by chemical nature and are also target substrates for hydrolases (especially phosphatase). OPPs are thus susceptible to phosphatase-producing microorganisms, and LAB of different phosphatase production abilities might therefore give different accelerations on OPP hydrolysis (i.e. OPP degradation). This means that LAB of higher phosphatase production would have more potential to hydrolyze OPP residues in fermented foods.
www.soci.org clarified that these OPPs also had different susceptibilities towards the inoculated strains and starters, resulting in accelerated OPP degradation of different levels. Totally, the OPPs were more susceptive to L. bulgaricus but less sensitive to L. acidophilus and L. casei. The higher the phosphatase production of the strains, the greater was the OPP dissipation observed. Phosphatase should be a factor governing OPP degradation, and LAB of higher phosphatase production might be applied to decrease OPP residues in fermented foods efficiently.
ACKNOWLEDGEMENTS This study was funded by the National Key Technological Research and Development Program of China during the 11th Five-Year Plan Period (Project No. 2006BAD04A08). The authors thank the anonymous reviewers and editors for their valuable advice.
REFERENCES 1 Cycon´ M, Wójcik M and Piotrowska-Seget Z, Biodegradation of the organophosphorus insecticide diazinon by Serratia sp. and Pseudomonas sp. and their use in bioremediation of contaminated soil. Chemosphere 76:494–501 (2009). 2 Ecobichon DJ, Pesticide use in developing countries. Toxicology 160:27–33 (2001). 3 Kaushik G, Satya S and Naik SN, Food processing a tool to pesticide residue dissipation – a review. Food Res Int 42:26–40 (2009). 4 Li W, Qiu SP and Wu YJ, Triazophos residues and dissipation rates in wheat crops and soil. Ecotoxicol Environ Saf 69:312–316 (2008). 5 Bolles HG, Dixon-White HE, Peterson RK, Tomerlin JR, Day EW and Oliver GR, U.S. market basket study to determine residues of the insecticide chlorpyrifos. J Agric Food Chem 47:1817–1822 (1999). 6 Salas JH, Gon´zalez MM, Noa M, Pérez NA, Díaz G, Gutíerrez R, et al, Organophosphorus pesticide residues in Mexican commercial pasteurized milk. J Agric Food Chem 51:4468–4471 (2003). 7 Melgar MJ, Santaeufemia M and García MA, Organophosphorus pesticide residues in raw milk and infant formulas from Spanish northwest. J Environ Sci Health B 45:595–600 (2010). 8 Zhao XH, Bo LY, Wang J and Li TJ, Survey of seven organophosphorus pesticides in drinking water, feedstuffs and raw milk from dairy farms in the Province Heilongjiang during 2008–2009. Milk Sci Int 67:293–296 (2012). 9 Cardeal ZDL and Paes CMD, Analysis of organophosphorus pesticides in whole milk by solid phase microextraction gas chromatography method. J Environ Sci Health B 41:369–375 (2006). 10 Battu RS, Singh B and Kang BK, Contamination of liquid milk and butter with pesticide residues in the Ludhiana district of Punjab state, India. Ecotoxicol Environ Saf 59:324–331 (2004). 11 Tsiplakou E, Anagnostopoulos CJ, Liapis K, Haroutounian SA and Zervas G, Pesticides residues in milks and feedstuff of farm animals drawn from Greece. Chemosphere 80:504–512 (2010). 12 Toan PV, Sebesvari Z, Bläsing M, Rosendahl I and Renaud FG, Pesticide management and their residues in sediments and surface and drinking water in the Mekong Delta, Vietnam. Sci Total Environ 452:28–39 (2013). 13 Bo LY, Zhang YH and Zhao XH, Degradation kinetics of seven organophosphorus pesticides in milk during yoghurt processing. J Serb Chem Soc 76:353–362 (2011). 14 Ozbey A and Uygun U, Behaviour of some organophosphorus pesticide residues in peppermint tea during the infusion process. Food Chem 104:237–241 (2007).
X-W Zhou, X-H Zhao 15 Ticha J, Hajslova J, Jech M, Honzicek J, Lacina O, Kohoutkova J, et al, Changes of pesticide residues in apples during cold storage. Food Control 19:247–256 (2008). 16 Uygun U, Senoz B, Öztürk S and Koksel H, Degradation of organophosphorus pesticides in wheat during cookie processing. Food Chem 117:261–264 (2009). 17 Sood C, Jaggi S, Kumar V, Ravindranath SD and Shanker A, How manufacturing processes affect the level of pesticide residues in tea. J Sci Food Agric 84:2123–2127 (2004). 18 Lu HY, Shen Y, Sun X, Zhu H and Liu XJ, Washing effects of limonene on pesticide residues in green peppers. J Sci Food Agric 93:2917–2921 (2013). 19 Uygun U, Senoz B and Koksel H, Dissipation of organophosphorus pesticides in wheat during pasta processing. Food Chem 109:355–360 (2008). 20 Banna AA and Kawar NS, Behavior of parathion in apple juice processed into cider and vinegar. J Environ Sci Health B 17:505–514 (1982). 21 Zhao XH and Wang J, A brief study on the degradation kinetics of seven organophosphorus pesticides in skimmed milk cultured with Lactobacillus spp. at 42 ∘ C. Food Chem 131:300–304 (2012). 22 Zhao XH, Wang J, Li TJ and Zhang YH, Degradation behaviors of seven organophosphorus pesticides in skimmed milk inoculated with Streptococcus thermophilus or Lactobacillus helveticus. Milk Sci Int 67:399–401 (2012). 23 Zhao XH and Wang J, Degradation of seven organophosphorus pesticides in the fresh milk heated at 63 ∘ C and two pHs. Milk Sci Int 67:192–194 (2012). 24 Gundi VAKB and Reddy BR, Degradation of monocrotophos in soils. Chemosphere 62:396–403 (2006). 25 Briceño G, Fuentes MS, Palma G, Jorquera MA, Amoroso MJ and Diez MC, Chlorpyrifos biodegradation and 3,5,6-trichloro-2-pyridinol production by actinobacteria isolated from soil. Int Biodeter Biodegrad 73:1–7 (2012). 26 Sánchez ME, Estrada IB, Martínez O, Martín-Villacorta J, Aller A and Morán A, Influence of the application of sewage sludge on the degradation of pesticides in the soil. Chemosphere 57:673–679 (2004). 27 Bhalerao TS and Puranik PR, Microbial degradation of monocrotophos by Aspergillus oryzae. Int Biodeter Biodegrad 63:503–508 (2009). 28 Putnam RA, Nelson JO and Clark JM, The persistence and degradation of chlorothalonil and chlorpyrifos in a cranberry bog. J Agric Food Chem 51:170–176 (2003). 29 Vanclooster M, Ducheyne S, Dust M and Vereecken H, Evaluation of pesticide dynamics of the WAVE-model. Agric Water Manag 44:371–388 (2000). 30 Abou-Arab AAK, Effect of Ras cheese manufacturing on the stability of DDT and its metabolites. Food Chem 59:115–119 (1997). 31 Zhang YY, Xiao ZY, Chen F, Ge YQ, Wu JH and Hu XS, Degradation behavior and products of malathion and chlorpyrifos spiked in apple juice by ultrasonic treatment. Ultrason Sonochem 17:72–77 (2010). 32 Chen F, Zeng LQ, Zhang YY, Liao XJ, Ge YQ, Hu XS, et al, Degradation behaviour of methamidophos and chlorpyrifos in apple juice treated with pulsed electric fields. Food Chem 112:956–961 (2009). 33 Rosenberg A and Alexander M, Microbial cleavage of various organophosphorus insecticides. Appl Environ Microbiol 37:886–891 (1979). 34 Thengodkar RRM and Sivakami S, Degradation of chlorpyrifos by an alkaline phosphatase from the cyanobacterium Spirulina platensis. Biodegradation 21:637–644 (2010). 35 Kim YH, Ahn JY, Moon SH and Lee J, Biodegradation and detoxification of organophosphate insecticide malathion by Fusarium oxysporum f. sp. pisi cutinase. Chemosphere 60:1349–1355 (2005). 36 Munnecke DM, Enzymatic hydrolysis of organophosphate insecticides, a possible pesticide disposal method. Appl Environ Microbiol 32:7–13 (1976).
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