- Email: [email protected]
Use of Lactobacillus spp to Degrade Pesticides in Milk
C H A P T E R
25
Use of Lactobacillus spp to Degrade Pesticides in Milk Mónica Calderón-Santiago, María Dolores Luque de Castro Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, University of Córdoba, Córdoba, Spain
CHAPTER POINTS • M any foods can be contaminated by pesticides, with milk as the main source of pesticide residues in human diets. • Microorganisms present in cheese can degrade pesticides during storage. • Pesticide residues and some nutrients present in milk can be degraded by heating for a long time. • A strategy to degrade pesticides in milk by use of microorganisms was recently proposed. • An organophosphorus hydrolase exhibiting high activity on pesticides was obtained by cloning a Lactobacillus brevis gen.
INTRODUCTION The growing demand for agricultural products has raised the need to use pesticides to prevent pest and insect growth. Pesticides are generally deemed safe for crops and animals by virtue of their relatively fast degradation. However, the local environmental conditions can alter their degradation rate and extend their persistence over long periods (Ragnarsdottir, 2000). Although a number of foods can be contaminated by pesticides, those of animal origin are the most difficult to free from these substances; this is especially the case with milk, which is the main source of pesticide residues in human diet owing to the high complexity of its matrix (Kampire et al., 2011). Pesticides can reach milk-producing animals through water, forage, and the environment. Animals can metabolize and detoxify most pesticides except
Processing and Impact on Active Components in Food http://dx.doi.org/10.1016/B978-0-12-404699-3.00025-1
chlorinated hydrocarbons, which are stored in body fat and contaminate meat and milk for human consumption as a result (Li and Bradley, 1969). In fact, pesticides have been found in milk from cows, buffalo, and sheep (Bulut et al., 2011), and even from women, where placenta and milk are the preferential excretory routes of lipophilic pesticides (Siddiqui and Saxena, 1985). Organophosphorus pesticides are the most frequently encountered in milk as a result of their widespread use as herbicides and for cow ectoparasite control; however, they are more rapidly degraded than other pesticides. Figure 25.1 shows the organochlorine and organophosphorus pesticides most frequently found in milk. Although pesticides concentrate mainly in the fatty fraction of milk, they rarely disappear when the fat is removed by cooking or other processing steps. All steps by which milk is transformed into dairy products or made suitable for marketing have been studied in depth in order to find ways to efficiently remove pesticides. Also, various strategies for complete pesticide removal from milk have been proposed. The presence of pesticides in food must be avoided or at least controlled because most of them are mutagenic and teratogenic, and their long-term, low-dose exposure is linked to effects on human health such as immunosuppression, hormone disruption, diminished intelligence, reproductive abnormalities, and cancer (Aktar et al., 2009). For these reasons, national and transnational governments have set maximum residue levels of pesticides in or on food and feed of plant and animal origin intended for human or animal consumption. Since pesticide use can not be suppressed altogether without placing food production at risk, a number of strategies have been developed to remove pesticides from contaminated food.
207
Copyright © 2015 Elsevier Inc. All rights reserved.
208
25. LACTOBACILLUS FOR PESTICIDE DEGRADATION
FIGURE 25.1 Pesticides most commonly encountered in milk.
One key aspect in dealing with pesticides in food is the way in which they are analyzed. A number of sensitive, selective methods for milk analysis exist to ensure compliance with legal limits and facilitate the detection of pesticides and their removal. However, existing methods fail to identify the nature of the products formed by pesticides in response to milk treatments.
Butter Butter production is not a useful step for pesticide removal. In fact, some pesticides such as DDT and lindane have been found to undergo preconcentration during butter production due to their high affinity for the fatty fraction of milk (Li et al., 1970). Diminishing butter contamination by pesticides therefore requires an additional step to remove them.
HOW COMPOSITION IS ALTERED
Cheese Pesticide degradation during cheese production occurs mainly at the heating and salting stages. For example, heating and salting were found to remove 98% of leptophos (an organophosphorus pesticide) present in milk used for cheese production (Abu-Elamayem et al., 1979). Pesticide degradation is also influenced by the presence of microorganisms, which act mainly d uring storage. Thus, lindane and DDT levels were found to fall by 37 and 70–60%, respectively, after six months of storage of cheese from cow milk (Abou-Arab, 1999). Conversely, lindane
Influence of Milk Processing and Storage on Pesticide Removal Efficiency Safety concerns have raised the need to examine the behavior of pesticides in milk during its processing and storage. Milk processing typically involves pasteurization, boiling, sterilization and/or skimming. Butter, cheese, and yogurt production are also included here as they are these dairy products are among the most popular ingredients of human diet.
3. DAIRY AND EGGS
Other Ways in which Composition is Altered
levels in feta cheese from ovine milk underwent no change during cheese ripening and storage for up to 240 days, possibly because the pesticide was bound to other cheese components (Mallatou et al., 2002). Yogurt Bo et al. (2011) studied the degradation of the organophosphorus pesticides dimethoate, fenthion, malathion, methyl parathion, monocrotophos, phorate, and t richlorphon in bovine milk during yogurt processing and identified time and temperature as the most influential factors on the pesticide concentrations. The nature of the fermentation starter proved highly influential as well. Pesticide degradation was ascribed to the action of microorganisms used to obtain yogurt. Milk Kampire et al. (2011) showed heat treatments such as pasteurization to efficiently degrade residual organochlorine pesticides. Bo and Zhao (2010) studied pesticide degradation by heating at a variable temperature and concluded that increased processing temperatures led to faster degradation. At 100°C, the half-lives of denthion, dimethoate, malathion, methyl parathion, monocrotophos, phorate, and trichlorfon ranged from 4 to 6 h.
OTHER WAYS IN WHICH COMPOSITION IS ALTERED Available Methods for the Removal or Degradation of Pesticides in Milk Available methods for pesticide removal are mainly based on their physical and chemical properties (especially volatility and polarity). Bills and Sloan (1967) found molecular distillation to remove 95–99% of chlorinated pesticides such as DDT, DDD, or DDE. Their method uses a temperature of 200°C and a pressure of 5 × 10−4 torr, and is similar to one previously proposed by Kroger (1968) called ‘steam deodorization’. Kroger’s method, which uses a temperature of 180–195°C and a pressure of 0.01–0.5 mmHg, completely removes heptachlor epoxide and dieldrin from milk. A number of polarity-based methods use a sorbent to remove pesticides from food; most, however, have only been applied to water. The main problem with milk is that its large fatty fraction can be co-extracted with the pesticides. Thus, pinus bark is able to adsorb 97% of heptachlor, aldrin, endrin, dieldrin, DDD, DDT, and DDE—but only 37% of lindane—and activated carbon efficiently removes 97% of all these pesticides (Brás et al., 1999), but both are useless for the removal of pesticides from milk. This is also the case with the acid-treated date stones used by Bakouri et al. (2009), who conducted a
209
comprehensive physico-chemical study of the adsorption of aldrin, dieldrin, and endrin on this sorbent, albeit in aqueous solutions of the pesticides rather than milk. As regards pesticide degradation, Basfar and Mohamed (2012) have patented a method using ionizing radiation that is applicable to aqueous solutions, fruits, and vegetables, and also to milk. Pesticide degradation by exposure to non-ionizing radiation (ultraviolet light) is also possible. Thus, Li and Bradley (1969) used UV energy to destroy organophosphate and organochlorine pesticides, which are photosensitive, in milk and butter oil. UV light was used to degrade six chlorinated hydrocarbon pesticides of which only methoxychlor was significantly destroyed. The main disadvantage of using UV energy for this purpose is that it can also degrade some nutrients such as vitamins and diminish food quality as a result. A more effective strategy here may be provided by some microorganisms (especially fungi and bacteria), which have for some time been used to degrade pesticides in food, water, and soil. As stated above, microorganisms are responsible for pesticide degradation during cheese ripening and storage, and also during yogurt fermentation. Therefore, they may be useful to degrade pesticides in milk as well. Langlois et al. (1970) studied the degradation of DDT in skimmed milk in the presence of three different species (Bacillus, Escherichia coli, and Enterobacter aerogenes), none of which, however, was capable of degrading DDT in this matrix. Abou-Arab (2002) found Lactobacillus plantarum to degrade lindane and DDT in fermented sausage and tryptone soya broth. More recently, Zhao and Wang (2012) assessed the degradation potential of Lactobacillus delbruekii ssp. bulgarius, Lactobacillus paracasei, and Lactobacillus plantarum on seven pesticides spiked to skimmed milk (at a 0.5-mg/ kg level for monocrotophos and phorate, and 1.2 mg/ kg for the other five pesticides, all in a 12% w/w solution) incubated at 42°C. As can be seen from Table 25.1, dimethoate and methyl parathion were the most stable pesticides, whereas malathion was the most labile. Lactobacillus bulgarius proved especially active on dimethoate, fenthion, and monocrotophos, whereas L. plantarum was more effective on malathion, methyl parathion, and trichlorphon. As can also be seen from Table 25.1, the pesticide half-lives ranged from 16 to 45 h with Lactobacillus; the effect was not significant, however, since heating at 42°C without microorganism inoculation led to half-lives of 32–50 h. In any case, the results showed that each Lactobacillus species was effective at removing some pesticides and not others, so a combination of microorganisms may be needed to efficiently degrade a mixture of pesticides. In vitro studies can be useful to assess the pesticide degradation efficiency of microorganisms, identify the resulting degradation products, and evaluate their
3. DAIRY AND EGGS
Pesticide (Amount Spiked) Dimethoate (1.2 mg/kg)
Fenthion (1.2 mg/kg)
3. DAIRY AND EGGS
Methyl parathion (1.2 mg/kg)
Monocrotophos (0.5 mg/kg)
Phorate (0.5 mg/kg)
Trichlorphon (1.2 mg/kg)
Degradation Kinetic Parameters
Stain Inoculated
8
16
24
k (per h)
R2
t1/2 (h)
L. bulgaricus
1.100 ± 0.025
0.909 ± 0.046
0.808 ± 0.039
0.0184
0.975
37.7
L. paracasei
1.178 ± 0.042
0.986 ± 0.031
0.897 ± 0.024
0.0169
0.974
41.0
L. plantarum
0.990 ± 0.017
0.841 ± 0.048
0.735 ± 0.032
0.0168
0.933
41.2
Control
1.050 ± 0.021
0.908 ± 0.035
0.808 ± 0.037
0.0165
0.996
42.0
L. bulgaricus
0.996 ± 0.036
0.872 ± 0.046
0.679 ± 0.044
0.0247
0.972
28.1
L. paracasei
0.992 ± 0.035
0.843 ± 0.048
0.711 ± 0.013
0.0226
0.974
30.7
L. plantarum
1.019 ± 0.033
0.804 ± 0.043
0.698 ± 0.017
0.0236
0.959
29.4
Control
0.934 ± 0.051
0.800 ± 0.024
0.667 ± 0.037
0.0210
0.990
33.0
L. bulgaricus
0.987 ± 0.026
0.853 ± 0.042
0.677 ± 0.046
0.0233
0.986
29.7
L. paracasei
0.991 ± 0.034
0.728 ± 0.032
0.614 ± 0.041
0.0279
0.964
24.8
L. plantarum
0.912 ± 0.044
0.663 ± 0.036
0.484 ± 0.033
0.0420
0.979
16.5
Control
0.986 ± 0.046
0.821 ± 0.034
0.699 ± 0.056
0.0218
0.998
31.8
L. bulgaricus
0.919 ± 0.056
0.838 ± 0.035
0.683 ± 0.051
0.0204
0.844
34.0
L. paracasei
1.096 ± 0.026
0.977 ± 0.034
0.867 ± 0.038
0.0153
0.992
45.3
L. plantarum
1.163 ± 0.027
0.942 ± 0.045
0.807 ± 0.043
0.0213
0.955
32.5
Control
1.051 ± 0.033
0.919 ± 0.028
0.822 ± 0.049
0.0153
0.998
45.3
L. bulgaricus
0.423 ± 0.036
0.388 ± 0.012
0.303 ± 0.034
0.0220
0.937
31.5
L. paracasei
0.438 ± 0.013
0.348 ± 0.031
0.326 ± 0.345
0.0188
0.994
36.9
L. plantarum
0.432 ± 0.044
0.377 ± 0.031
0.311 ± 0.029
0.0199
0.986
34.8
Control
0.439 ± 0.047
0.353 ± 0.030
0.320 ± 0.027
0.0196
0.962
35.4
L. bulgaricus
0.422 ± 0.032
0.369 ± 0.029
0.295 ± 0.047
0.0200
0.916
34.6
L. paracasei
0.411 ± 0.029
0.346 ± 0.042
0.291 ± 0.037
0.0230
0.984
30.1
L. plantarum
0.412 ± 0.039
0.342 ± 0.016
0.297 ± 0.029
0.0204
0.988
34.0
Control
0.392 ± 0.035
0.341 ± 0.034
0.288 ± 0.032
0.0185
0.964
37.4
L. bulgaricus
1.210 ± 0.041
0.988 ± 0.033
0.877 ± 0.053
0.0229
0.926
30.3
L. paracasei
1.136 ± 0.025
0.900 ± 0.011
0.778 ± 0.019
0.0233
0.977
30.1
L. plantarum
1.230 ± 0.033
0.990 ± 0.014
0.847 ± 0.012
0.0242
0.979
28.6
Control
1.036 ± 0.030
0.892 ± 0.034
Not detected
0.0140
0.874
49.5
k, degradation rate constant, R2, regression coefficient, t1/2 half-life. Adapted with permission from Zhao and Wang (2012).
25. LACTOBACILLUS FOR PESTICIDE DEGRADATION
Malathion (1.2 mg/kg)
Residual Concentration at Different Times (h)
210
TABLE 25.1 Residual Concentration (Mean ± SD, mg/kg) and Degradation Kinetic Parameters of Seven Organophosphorus Pesticides in Skimmed Milk Cultured with Lactobacillus spp. at 42°C for Variable Lengths of Time
211
Analytical Techniques
toxicity in relation to their precursors. Harishankar et al. (2012) studied the in vitro degradation of the organophosphorus pesticide chlorpyrifos by five intestinal bacteria (viz. Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus fermentum, Escherichia coli, and Enterococcus faecalis). As can be seen from Figure 25.2, L. fermentum degraded chlorpyrifos to 3,5,6-trichloro-2-pyridol by 70% and L. lactis degraded chlorpyrifos to chlorpyrifos oxon by 61%. The study, which used liquid chromatography–mass spectrometry (LC–MS) with ion trapping and atomic pressure chemical ionization, revealed differences in degradation (metabolite) profiles between bacterial strains, which clearly shows the need to accurately identify metabolites in order assess toxicity and the m etabolic pathways behind each microorganism. Cho et al. (2009) studied the microorganisms potentially responsible for chlorpyrifos degradation during fermentation of ‘kimchi’, a vegetable-based fermented food. They identified four organisms as responsible for chlorpyrifos degradation, namely: Leuconostoc mesenteroides WCP907, Lactobacillus brevis WCP902, Lactobacillus plantarum WCP931, and Lactobacillus sakei WCP904. These microorganisms degraded chlorpyrifos by over 80% in 3 days and 100% in 9 days. Islam et al. (2010) cloned the genes of Lactobacillus brevis and obtained an organophosphorus hydrolase potentially useful for decontaminating raw food materials after testing its degrading effect on ‘kimchi’ production. The enzyme exhibited high hydrolase activity against methyl parathion, diazinon, chlorpyrifos, coumaphos, and parathion. One other important subject to be studied in using bacteria to degrade pesticides is their nutritional requirements. Barragán-Huerta and RodríguezVázquez (2010) used green bean coffee as nutrient source for Pseudomonas aeruginosa isolated from this
natural product and found the microorganism to remove 51% of endosulfan and 67% of DDT after 7 days of incubation. As can be seen, pesticide-degrading bacteria are useful to decontaminate not only milk and dairy products, but also other fermented foods or even soil and water, where pesticide accumulation can lead to animal and vegetable contamination. Contamination in this context can be assessed by metabolomics and fate analysis. The former can help identify the pathways involved in pesticide degradation and determine the physico-chemical properties of the resulting metabolites, and the latter to predict the fate of pesticides under specific environmental conditions.
ANALYTICAL TECHNIQUES Methods for Determining Pesticides in Milk As stated above, the most common pesticides in milk belong to the organochlorine or organophosphorus families; therefore, most pesticide determination methods focus on these two groups. MS in combination with capillary electrophoresis, gas chromatography, or LC for separation is the preferred choice for pesticide determination; some methods, however, use an electron capture detector instead. In any case, the sample treatment is complex and similar, whichever the choice. Figure 25.3 shows the most common methods for determining pesticides in milk and depicts their sample treatment process. For example, Armendáriz et al. (2004) developed a method to determine up to 15 organochlorine pesticides in milk by gas chromatography coupled to electron capture detection and involving fat separation, solid-phase extraction, and purification as sample FIGURE 25.2 Bar graph showing the chlorpyrifos concentration tolerated by various bacterial strains. With permission from Harishankar et al. (2012).
3. DAIRY AND EGGS
212
25. LACTOBACILLUS FOR PESTICIDE DEGRADATION
FIGURE 25.3 Most common strategies for determining pesticides in milk.
treatment. More recently, Zheng et al. (2009) developed a method for the determination of 128 pesticides in milk by LC coupled to MS. The former method requires removing fat by centrifugation prior to extraction of the pesticides on a glass column; by contrast, the latter requires no fat separation but liquid–liquid extraction with acetonitrile prior to clean-up by passage through solid-phase extraction cartridges. Both use a concentration step before analysis.
Acknowledgments Funding of this research by Spain’s Ministry of Economy and Competitiveness and the FEDER Program through project CTQ2012-37428 is gratefully acknowledged. M. Calderón Santiago is also grateful to Spain’s Ministry of Science and Innovation for award of an FPU scholarship.
References Abou-Arab, A.A.K., 1999. Effects of processing and storage of dairy products on lindane residues and metabolites. Food Chem. 64, 467–473. Abou-Arab, A.A.K., 2002. Degradation of organochlorine pesticides by meat starter in liquid media and fermented sausage. Food Chem. Toxicol. 40 (1), 33–41. Abu-Elamayem, M.M., Abdel-All, A., Tantawy, G.A., Marei, A.S.M., 1979. Fate of leptophos in milk and wheat during the processing steps. Alexandria J. Agric. Res. 27 (3), 659–663. Aktar, M.W., Sengupta, D., Chowdhury, A., 2009. Impact of pesticides use in agriculture: their benefits and hazards. Interdiscip. Toxicol. 2 (1), 1–12. Armendáriz, C., Pérez de Ciriza, J.A., Farré, R., 2004. Gas chromatographic determination of organochlorine pesticides in cow milk. Int. J. Food Sci. Nutr. 55 (3), 215–221.
Bakouri, H.E., Usero, J., Morillo, J., Rojas, R., Ouassini, A., 2009. Drin pesticides removal from aqueous solutions using acid-treated date stones. Bioresour. Technol. 100, 2676–2684. Barragán-Huerta, B.E., Rodríguez-Vázquez, R., 2010. Green bean coffee as nutrient source for pesticide-degrading bacteria. In: MéndezVilas, A. (Ed.), Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, vol. 2. Formatex, Badajoz, Spain, pp. 1322–1327. Basfar, A.A., Mohamed, K.A., 2012. Removal of pesticide residues in food by ionizing radiation. U. S. Patent Appl. 20120021103. Bills, D.D., Sloan, J.L., 1967. Removal of chlorinated insecticide residues from milk fat by molecular distillation. J. Agric. Food Chem. 15 (4), 676–678. Bo, L.Y., Zhang, Y.H., Zhao, X.H., 2011. Degradation kinetics of seven organophosphorus pesticides in milk during yoghurt processing. J. Serb. Chem. Soc. 76 (3), 353–362. Bo, L.Y., Zhao, X.H., 2010. Preliminary study on the degradation of seven organophosphorus pesticides in bovine milk during lactic acid fermentation or heat treatment. Afr. J. Microbiol. Res. 4 (11), 1171–1179. Brás, I.P., Santos, L., Alves, A., 1999. Organochlorine pesticides removal by pinus bark sorption. Environ. Sci. Technol. 33 (4), 631–634. Bulut, S., Akkaya, L., Gök, V., Konuk, M., 2011. Organochlorine pesticide (OCP) residues in cow’s, buffalo’s, and sheep’s milk from Afyonkarahisar region. Turkey. Environ. Monit. Assess. 181, 555–562. Cho, K.M., Math, R.K., Islam, S.M.A., Lim, W.J., Hong, S.Y., Kim, J.M., Yun, M.G., Cho, J.J., Yun, H.D., 2009. Biodegradation of chlorpyrifos by lactic acid bacteria during kimchi fermentation. J. Agric. Food Chem. 57 (5), 1882–1889. Harishankar, M.K., Sasikala, C., Ramya, M., 2012. Efficiency of the intestinal bacteria in the degradation of the toxic pesticide chlorpyrifos. 3 Biotech. http://dx.doi.org/10.1007/s13205-012-0078-0. Islam, S.M., Math, R.K., Cho, K.M., Lim, W.J., Hong, S.Y., Kim, J.M., Yun, M.G., Cho, J.J., Yun, H.D., 2010. Organophosphorus hydrolase (OpdB) of Lactobacillus brevis WCP902 from Kimchi is able to degrade organophosphorus pesticides. J. Agric. Food Chem. 58, 5380–5386. Kampire, E., Kiremire, B.T., Nyanzi, S.A., Kishimba, M., 2011. Organochlorine pesticide in fresh and pasteurized cow’s milk from Kampala markets. Chemosphere 84, 923–927.
3. DAIRY AND EGGS
References
Kroger, M., 1968. Effect of various physical treatments on certain organochlorine hydrocarbon insecticides found in milk fat. J. Dairy Sci. 51 (2), 196–198. Langlois, B.E., Collins, J.A., Sides, K.G., 1970. Some factors affecting degradation of organochlorine pesticides by bacteria. J. Dairy Sci. 53 (12), 1671–1675. Li, C., Bradley Jr., R.L., Schultz, L.H., 1970. Fate of organochlorine pesticides during processing of milk into dairy products. J. Assoc. Off. Agric. Food Chem. 53 (1), 127–139. Li, C.F., Bradley, R.L., 1969. Degradation of chlorinated hydrocarbon pesticides in milk and butteroil by ultraviolet energy. J. Dairy Sci. 52 (1), 27–30. Mallatou, H., Pappas, C.P., Albanis, T.A., 2002. Behaviour of pesticides lindane and methyl parathion during manufacture, ripening and storage of feta cheese. J. Dairy Technol. 55 (4), 211–216.
213
Ragnarsdottir, K.V., 2000. Environmental fate and toxicology of organophosphate pesticides. J. Geol. Soc. 157 (4), 859–876. Siddiqui, M.K., Saxena, M.C., 1985. Placenta and milk as excretory routes of lipophilic pesticides in women. Hum. Toxicol. 4 (3), 249–254. Zhao, X.H., Wang, J., 2012. A brief study on the degradation kinetics of seven organophosphorus pesticides in skimmed milk cultured with Lactobacillus spp. at 42 °C. Food Chem. 131 (1), 300–304. Zheng, J., Pang, G., Fan, C., Wang, M., 2009. Simultaneous determination of 128 pesticide residues in milk by liquid chromatographytandem electrospray mass spectrometry. Chin. J. Chromatogr. 27 (3), 254–263.
3. DAIRY AND EGGS
25
Use of Lactobacillus spp to Degrade Pesticides in Milk Mónica Calderón-Santiago, María Dolores Luque de Castro Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, University of Córdoba, Córdoba, Spain
CHAPTER POINTS • M any foods can be contaminated by pesticides, with milk as the main source of pesticide residues in human diets. • Microorganisms present in cheese can degrade pesticides during storage. • Pesticide residues and some nutrients present in milk can be degraded by heating for a long time. • A strategy to degrade pesticides in milk by use of microorganisms was recently proposed. • An organophosphorus hydrolase exhibiting high activity on pesticides was obtained by cloning a Lactobacillus brevis gen.
INTRODUCTION The growing demand for agricultural products has raised the need to use pesticides to prevent pest and insect growth. Pesticides are generally deemed safe for crops and animals by virtue of their relatively fast degradation. However, the local environmental conditions can alter their degradation rate and extend their persistence over long periods (Ragnarsdottir, 2000). Although a number of foods can be contaminated by pesticides, those of animal origin are the most difficult to free from these substances; this is especially the case with milk, which is the main source of pesticide residues in human diet owing to the high complexity of its matrix (Kampire et al., 2011). Pesticides can reach milk-producing animals through water, forage, and the environment. Animals can metabolize and detoxify most pesticides except
Processing and Impact on Active Components in Food http://dx.doi.org/10.1016/B978-0-12-404699-3.00025-1
chlorinated hydrocarbons, which are stored in body fat and contaminate meat and milk for human consumption as a result (Li and Bradley, 1969). In fact, pesticides have been found in milk from cows, buffalo, and sheep (Bulut et al., 2011), and even from women, where placenta and milk are the preferential excretory routes of lipophilic pesticides (Siddiqui and Saxena, 1985). Organophosphorus pesticides are the most frequently encountered in milk as a result of their widespread use as herbicides and for cow ectoparasite control; however, they are more rapidly degraded than other pesticides. Figure 25.1 shows the organochlorine and organophosphorus pesticides most frequently found in milk. Although pesticides concentrate mainly in the fatty fraction of milk, they rarely disappear when the fat is removed by cooking or other processing steps. All steps by which milk is transformed into dairy products or made suitable for marketing have been studied in depth in order to find ways to efficiently remove pesticides. Also, various strategies for complete pesticide removal from milk have been proposed. The presence of pesticides in food must be avoided or at least controlled because most of them are mutagenic and teratogenic, and their long-term, low-dose exposure is linked to effects on human health such as immunosuppression, hormone disruption, diminished intelligence, reproductive abnormalities, and cancer (Aktar et al., 2009). For these reasons, national and transnational governments have set maximum residue levels of pesticides in or on food and feed of plant and animal origin intended for human or animal consumption. Since pesticide use can not be suppressed altogether without placing food production at risk, a number of strategies have been developed to remove pesticides from contaminated food.
207
Copyright © 2015 Elsevier Inc. All rights reserved.
208
25. LACTOBACILLUS FOR PESTICIDE DEGRADATION
FIGURE 25.1 Pesticides most commonly encountered in milk.
One key aspect in dealing with pesticides in food is the way in which they are analyzed. A number of sensitive, selective methods for milk analysis exist to ensure compliance with legal limits and facilitate the detection of pesticides and their removal. However, existing methods fail to identify the nature of the products formed by pesticides in response to milk treatments.
Butter Butter production is not a useful step for pesticide removal. In fact, some pesticides such as DDT and lindane have been found to undergo preconcentration during butter production due to their high affinity for the fatty fraction of milk (Li et al., 1970). Diminishing butter contamination by pesticides therefore requires an additional step to remove them.
HOW COMPOSITION IS ALTERED
Cheese Pesticide degradation during cheese production occurs mainly at the heating and salting stages. For example, heating and salting were found to remove 98% of leptophos (an organophosphorus pesticide) present in milk used for cheese production (Abu-Elamayem et al., 1979). Pesticide degradation is also influenced by the presence of microorganisms, which act mainly d uring storage. Thus, lindane and DDT levels were found to fall by 37 and 70–60%, respectively, after six months of storage of cheese from cow milk (Abou-Arab, 1999). Conversely, lindane
Influence of Milk Processing and Storage on Pesticide Removal Efficiency Safety concerns have raised the need to examine the behavior of pesticides in milk during its processing and storage. Milk processing typically involves pasteurization, boiling, sterilization and/or skimming. Butter, cheese, and yogurt production are also included here as they are these dairy products are among the most popular ingredients of human diet.
3. DAIRY AND EGGS
Other Ways in which Composition is Altered
levels in feta cheese from ovine milk underwent no change during cheese ripening and storage for up to 240 days, possibly because the pesticide was bound to other cheese components (Mallatou et al., 2002). Yogurt Bo et al. (2011) studied the degradation of the organophosphorus pesticides dimethoate, fenthion, malathion, methyl parathion, monocrotophos, phorate, and t richlorphon in bovine milk during yogurt processing and identified time and temperature as the most influential factors on the pesticide concentrations. The nature of the fermentation starter proved highly influential as well. Pesticide degradation was ascribed to the action of microorganisms used to obtain yogurt. Milk Kampire et al. (2011) showed heat treatments such as pasteurization to efficiently degrade residual organochlorine pesticides. Bo and Zhao (2010) studied pesticide degradation by heating at a variable temperature and concluded that increased processing temperatures led to faster degradation. At 100°C, the half-lives of denthion, dimethoate, malathion, methyl parathion, monocrotophos, phorate, and trichlorfon ranged from 4 to 6 h.
OTHER WAYS IN WHICH COMPOSITION IS ALTERED Available Methods for the Removal or Degradation of Pesticides in Milk Available methods for pesticide removal are mainly based on their physical and chemical properties (especially volatility and polarity). Bills and Sloan (1967) found molecular distillation to remove 95–99% of chlorinated pesticides such as DDT, DDD, or DDE. Their method uses a temperature of 200°C and a pressure of 5 × 10−4 torr, and is similar to one previously proposed by Kroger (1968) called ‘steam deodorization’. Kroger’s method, which uses a temperature of 180–195°C and a pressure of 0.01–0.5 mmHg, completely removes heptachlor epoxide and dieldrin from milk. A number of polarity-based methods use a sorbent to remove pesticides from food; most, however, have only been applied to water. The main problem with milk is that its large fatty fraction can be co-extracted with the pesticides. Thus, pinus bark is able to adsorb 97% of heptachlor, aldrin, endrin, dieldrin, DDD, DDT, and DDE—but only 37% of lindane—and activated carbon efficiently removes 97% of all these pesticides (Brás et al., 1999), but both are useless for the removal of pesticides from milk. This is also the case with the acid-treated date stones used by Bakouri et al. (2009), who conducted a
209
comprehensive physico-chemical study of the adsorption of aldrin, dieldrin, and endrin on this sorbent, albeit in aqueous solutions of the pesticides rather than milk. As regards pesticide degradation, Basfar and Mohamed (2012) have patented a method using ionizing radiation that is applicable to aqueous solutions, fruits, and vegetables, and also to milk. Pesticide degradation by exposure to non-ionizing radiation (ultraviolet light) is also possible. Thus, Li and Bradley (1969) used UV energy to destroy organophosphate and organochlorine pesticides, which are photosensitive, in milk and butter oil. UV light was used to degrade six chlorinated hydrocarbon pesticides of which only methoxychlor was significantly destroyed. The main disadvantage of using UV energy for this purpose is that it can also degrade some nutrients such as vitamins and diminish food quality as a result. A more effective strategy here may be provided by some microorganisms (especially fungi and bacteria), which have for some time been used to degrade pesticides in food, water, and soil. As stated above, microorganisms are responsible for pesticide degradation during cheese ripening and storage, and also during yogurt fermentation. Therefore, they may be useful to degrade pesticides in milk as well. Langlois et al. (1970) studied the degradation of DDT in skimmed milk in the presence of three different species (Bacillus, Escherichia coli, and Enterobacter aerogenes), none of which, however, was capable of degrading DDT in this matrix. Abou-Arab (2002) found Lactobacillus plantarum to degrade lindane and DDT in fermented sausage and tryptone soya broth. More recently, Zhao and Wang (2012) assessed the degradation potential of Lactobacillus delbruekii ssp. bulgarius, Lactobacillus paracasei, and Lactobacillus plantarum on seven pesticides spiked to skimmed milk (at a 0.5-mg/ kg level for monocrotophos and phorate, and 1.2 mg/ kg for the other five pesticides, all in a 12% w/w solution) incubated at 42°C. As can be seen from Table 25.1, dimethoate and methyl parathion were the most stable pesticides, whereas malathion was the most labile. Lactobacillus bulgarius proved especially active on dimethoate, fenthion, and monocrotophos, whereas L. plantarum was more effective on malathion, methyl parathion, and trichlorphon. As can also be seen from Table 25.1, the pesticide half-lives ranged from 16 to 45 h with Lactobacillus; the effect was not significant, however, since heating at 42°C without microorganism inoculation led to half-lives of 32–50 h. In any case, the results showed that each Lactobacillus species was effective at removing some pesticides and not others, so a combination of microorganisms may be needed to efficiently degrade a mixture of pesticides. In vitro studies can be useful to assess the pesticide degradation efficiency of microorganisms, identify the resulting degradation products, and evaluate their
3. DAIRY AND EGGS
Pesticide (Amount Spiked) Dimethoate (1.2 mg/kg)
Fenthion (1.2 mg/kg)
3. DAIRY AND EGGS
Methyl parathion (1.2 mg/kg)
Monocrotophos (0.5 mg/kg)
Phorate (0.5 mg/kg)
Trichlorphon (1.2 mg/kg)
Degradation Kinetic Parameters
Stain Inoculated
8
16
24
k (per h)
R2
t1/2 (h)
L. bulgaricus
1.100 ± 0.025
0.909 ± 0.046
0.808 ± 0.039
0.0184
0.975
37.7
L. paracasei
1.178 ± 0.042
0.986 ± 0.031
0.897 ± 0.024
0.0169
0.974
41.0
L. plantarum
0.990 ± 0.017
0.841 ± 0.048
0.735 ± 0.032
0.0168
0.933
41.2
Control
1.050 ± 0.021
0.908 ± 0.035
0.808 ± 0.037
0.0165
0.996
42.0
L. bulgaricus
0.996 ± 0.036
0.872 ± 0.046
0.679 ± 0.044
0.0247
0.972
28.1
L. paracasei
0.992 ± 0.035
0.843 ± 0.048
0.711 ± 0.013
0.0226
0.974
30.7
L. plantarum
1.019 ± 0.033
0.804 ± 0.043
0.698 ± 0.017
0.0236
0.959
29.4
Control
0.934 ± 0.051
0.800 ± 0.024
0.667 ± 0.037
0.0210
0.990
33.0
L. bulgaricus
0.987 ± 0.026
0.853 ± 0.042
0.677 ± 0.046
0.0233
0.986
29.7
L. paracasei
0.991 ± 0.034
0.728 ± 0.032
0.614 ± 0.041
0.0279
0.964
24.8
L. plantarum
0.912 ± 0.044
0.663 ± 0.036
0.484 ± 0.033
0.0420
0.979
16.5
Control
0.986 ± 0.046
0.821 ± 0.034
0.699 ± 0.056
0.0218
0.998
31.8
L. bulgaricus
0.919 ± 0.056
0.838 ± 0.035
0.683 ± 0.051
0.0204
0.844
34.0
L. paracasei
1.096 ± 0.026
0.977 ± 0.034
0.867 ± 0.038
0.0153
0.992
45.3
L. plantarum
1.163 ± 0.027
0.942 ± 0.045
0.807 ± 0.043
0.0213
0.955
32.5
Control
1.051 ± 0.033
0.919 ± 0.028
0.822 ± 0.049
0.0153
0.998
45.3
L. bulgaricus
0.423 ± 0.036
0.388 ± 0.012
0.303 ± 0.034
0.0220
0.937
31.5
L. paracasei
0.438 ± 0.013
0.348 ± 0.031
0.326 ± 0.345
0.0188
0.994
36.9
L. plantarum
0.432 ± 0.044
0.377 ± 0.031
0.311 ± 0.029
0.0199
0.986
34.8
Control
0.439 ± 0.047
0.353 ± 0.030
0.320 ± 0.027
0.0196
0.962
35.4
L. bulgaricus
0.422 ± 0.032
0.369 ± 0.029
0.295 ± 0.047
0.0200
0.916
34.6
L. paracasei
0.411 ± 0.029
0.346 ± 0.042
0.291 ± 0.037
0.0230
0.984
30.1
L. plantarum
0.412 ± 0.039
0.342 ± 0.016
0.297 ± 0.029
0.0204
0.988
34.0
Control
0.392 ± 0.035
0.341 ± 0.034
0.288 ± 0.032
0.0185
0.964
37.4
L. bulgaricus
1.210 ± 0.041
0.988 ± 0.033
0.877 ± 0.053
0.0229
0.926
30.3
L. paracasei
1.136 ± 0.025
0.900 ± 0.011
0.778 ± 0.019
0.0233
0.977
30.1
L. plantarum
1.230 ± 0.033
0.990 ± 0.014
0.847 ± 0.012
0.0242
0.979
28.6
Control
1.036 ± 0.030
0.892 ± 0.034
Not detected
0.0140
0.874
49.5
k, degradation rate constant, R2, regression coefficient, t1/2 half-life. Adapted with permission from Zhao and Wang (2012).
25. LACTOBACILLUS FOR PESTICIDE DEGRADATION
Malathion (1.2 mg/kg)
Residual Concentration at Different Times (h)
210
TABLE 25.1 Residual Concentration (Mean ± SD, mg/kg) and Degradation Kinetic Parameters of Seven Organophosphorus Pesticides in Skimmed Milk Cultured with Lactobacillus spp. at 42°C for Variable Lengths of Time
211
Analytical Techniques
toxicity in relation to their precursors. Harishankar et al. (2012) studied the in vitro degradation of the organophosphorus pesticide chlorpyrifos by five intestinal bacteria (viz. Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus fermentum, Escherichia coli, and Enterococcus faecalis). As can be seen from Figure 25.2, L. fermentum degraded chlorpyrifos to 3,5,6-trichloro-2-pyridol by 70% and L. lactis degraded chlorpyrifos to chlorpyrifos oxon by 61%. The study, which used liquid chromatography–mass spectrometry (LC–MS) with ion trapping and atomic pressure chemical ionization, revealed differences in degradation (metabolite) profiles between bacterial strains, which clearly shows the need to accurately identify metabolites in order assess toxicity and the m etabolic pathways behind each microorganism. Cho et al. (2009) studied the microorganisms potentially responsible for chlorpyrifos degradation during fermentation of ‘kimchi’, a vegetable-based fermented food. They identified four organisms as responsible for chlorpyrifos degradation, namely: Leuconostoc mesenteroides WCP907, Lactobacillus brevis WCP902, Lactobacillus plantarum WCP931, and Lactobacillus sakei WCP904. These microorganisms degraded chlorpyrifos by over 80% in 3 days and 100% in 9 days. Islam et al. (2010) cloned the genes of Lactobacillus brevis and obtained an organophosphorus hydrolase potentially useful for decontaminating raw food materials after testing its degrading effect on ‘kimchi’ production. The enzyme exhibited high hydrolase activity against methyl parathion, diazinon, chlorpyrifos, coumaphos, and parathion. One other important subject to be studied in using bacteria to degrade pesticides is their nutritional requirements. Barragán-Huerta and RodríguezVázquez (2010) used green bean coffee as nutrient source for Pseudomonas aeruginosa isolated from this
natural product and found the microorganism to remove 51% of endosulfan and 67% of DDT after 7 days of incubation. As can be seen, pesticide-degrading bacteria are useful to decontaminate not only milk and dairy products, but also other fermented foods or even soil and water, where pesticide accumulation can lead to animal and vegetable contamination. Contamination in this context can be assessed by metabolomics and fate analysis. The former can help identify the pathways involved in pesticide degradation and determine the physico-chemical properties of the resulting metabolites, and the latter to predict the fate of pesticides under specific environmental conditions.
ANALYTICAL TECHNIQUES Methods for Determining Pesticides in Milk As stated above, the most common pesticides in milk belong to the organochlorine or organophosphorus families; therefore, most pesticide determination methods focus on these two groups. MS in combination with capillary electrophoresis, gas chromatography, or LC for separation is the preferred choice for pesticide determination; some methods, however, use an electron capture detector instead. In any case, the sample treatment is complex and similar, whichever the choice. Figure 25.3 shows the most common methods for determining pesticides in milk and depicts their sample treatment process. For example, Armendáriz et al. (2004) developed a method to determine up to 15 organochlorine pesticides in milk by gas chromatography coupled to electron capture detection and involving fat separation, solid-phase extraction, and purification as sample FIGURE 25.2 Bar graph showing the chlorpyrifos concentration tolerated by various bacterial strains. With permission from Harishankar et al. (2012).
3. DAIRY AND EGGS
212
25. LACTOBACILLUS FOR PESTICIDE DEGRADATION
FIGURE 25.3 Most common strategies for determining pesticides in milk.
treatment. More recently, Zheng et al. (2009) developed a method for the determination of 128 pesticides in milk by LC coupled to MS. The former method requires removing fat by centrifugation prior to extraction of the pesticides on a glass column; by contrast, the latter requires no fat separation but liquid–liquid extraction with acetonitrile prior to clean-up by passage through solid-phase extraction cartridges. Both use a concentration step before analysis.
Acknowledgments Funding of this research by Spain’s Ministry of Economy and Competitiveness and the FEDER Program through project CTQ2012-37428 is gratefully acknowledged. M. Calderón Santiago is also grateful to Spain’s Ministry of Science and Innovation for award of an FPU scholarship.
References Abou-Arab, A.A.K., 1999. Effects of processing and storage of dairy products on lindane residues and metabolites. Food Chem. 64, 467–473. Abou-Arab, A.A.K., 2002. Degradation of organochlorine pesticides by meat starter in liquid media and fermented sausage. Food Chem. Toxicol. 40 (1), 33–41. Abu-Elamayem, M.M., Abdel-All, A., Tantawy, G.A., Marei, A.S.M., 1979. Fate of leptophos in milk and wheat during the processing steps. Alexandria J. Agric. Res. 27 (3), 659–663. Aktar, M.W., Sengupta, D., Chowdhury, A., 2009. Impact of pesticides use in agriculture: their benefits and hazards. Interdiscip. Toxicol. 2 (1), 1–12. Armendáriz, C., Pérez de Ciriza, J.A., Farré, R., 2004. Gas chromatographic determination of organochlorine pesticides in cow milk. Int. J. Food Sci. Nutr. 55 (3), 215–221.
Bakouri, H.E., Usero, J., Morillo, J., Rojas, R., Ouassini, A., 2009. Drin pesticides removal from aqueous solutions using acid-treated date stones. Bioresour. Technol. 100, 2676–2684. Barragán-Huerta, B.E., Rodríguez-Vázquez, R., 2010. Green bean coffee as nutrient source for pesticide-degrading bacteria. In: MéndezVilas, A. (Ed.), Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, vol. 2. Formatex, Badajoz, Spain, pp. 1322–1327. Basfar, A.A., Mohamed, K.A., 2012. Removal of pesticide residues in food by ionizing radiation. U. S. Patent Appl. 20120021103. Bills, D.D., Sloan, J.L., 1967. Removal of chlorinated insecticide residues from milk fat by molecular distillation. J. Agric. Food Chem. 15 (4), 676–678. Bo, L.Y., Zhang, Y.H., Zhao, X.H., 2011. Degradation kinetics of seven organophosphorus pesticides in milk during yoghurt processing. J. Serb. Chem. Soc. 76 (3), 353–362. Bo, L.Y., Zhao, X.H., 2010. Preliminary study on the degradation of seven organophosphorus pesticides in bovine milk during lactic acid fermentation or heat treatment. Afr. J. Microbiol. Res. 4 (11), 1171–1179. Brás, I.P., Santos, L., Alves, A., 1999. Organochlorine pesticides removal by pinus bark sorption. Environ. Sci. Technol. 33 (4), 631–634. Bulut, S., Akkaya, L., Gök, V., Konuk, M., 2011. Organochlorine pesticide (OCP) residues in cow’s, buffalo’s, and sheep’s milk from Afyonkarahisar region. Turkey. Environ. Monit. Assess. 181, 555–562. Cho, K.M., Math, R.K., Islam, S.M.A., Lim, W.J., Hong, S.Y., Kim, J.M., Yun, M.G., Cho, J.J., Yun, H.D., 2009. Biodegradation of chlorpyrifos by lactic acid bacteria during kimchi fermentation. J. Agric. Food Chem. 57 (5), 1882–1889. Harishankar, M.K., Sasikala, C., Ramya, M., 2012. Efficiency of the intestinal bacteria in the degradation of the toxic pesticide chlorpyrifos. 3 Biotech. http://dx.doi.org/10.1007/s13205-012-0078-0. Islam, S.M., Math, R.K., Cho, K.M., Lim, W.J., Hong, S.Y., Kim, J.M., Yun, M.G., Cho, J.J., Yun, H.D., 2010. Organophosphorus hydrolase (OpdB) of Lactobacillus brevis WCP902 from Kimchi is able to degrade organophosphorus pesticides. J. Agric. Food Chem. 58, 5380–5386. Kampire, E., Kiremire, B.T., Nyanzi, S.A., Kishimba, M., 2011. Organochlorine pesticide in fresh and pasteurized cow’s milk from Kampala markets. Chemosphere 84, 923–927.
3. DAIRY AND EGGS
References
Kroger, M., 1968. Effect of various physical treatments on certain organochlorine hydrocarbon insecticides found in milk fat. J. Dairy Sci. 51 (2), 196–198. Langlois, B.E., Collins, J.A., Sides, K.G., 1970. Some factors affecting degradation of organochlorine pesticides by bacteria. J. Dairy Sci. 53 (12), 1671–1675. Li, C., Bradley Jr., R.L., Schultz, L.H., 1970. Fate of organochlorine pesticides during processing of milk into dairy products. J. Assoc. Off. Agric. Food Chem. 53 (1), 127–139. Li, C.F., Bradley, R.L., 1969. Degradation of chlorinated hydrocarbon pesticides in milk and butteroil by ultraviolet energy. J. Dairy Sci. 52 (1), 27–30. Mallatou, H., Pappas, C.P., Albanis, T.A., 2002. Behaviour of pesticides lindane and methyl parathion during manufacture, ripening and storage of feta cheese. J. Dairy Technol. 55 (4), 211–216.
213
Ragnarsdottir, K.V., 2000. Environmental fate and toxicology of organophosphate pesticides. J. Geol. Soc. 157 (4), 859–876. Siddiqui, M.K., Saxena, M.C., 1985. Placenta and milk as excretory routes of lipophilic pesticides in women. Hum. Toxicol. 4 (3), 249–254. Zhao, X.H., Wang, J., 2012. A brief study on the degradation kinetics of seven organophosphorus pesticides in skimmed milk cultured with Lactobacillus spp. at 42 °C. Food Chem. 131 (1), 300–304. Zheng, J., Pang, G., Fan, C., Wang, M., 2009. Simultaneous determination of 128 pesticide residues in milk by liquid chromatographytandem electrospray mass spectrometry. Chin. J. Chromatogr. 27 (3), 254–263.
3. DAIRY AND EGGS