Pesticide Residues in Mandarins: Three-Year Monitoring Results.

The demand of plant production product use has increased because of the current system of citrus production, which prioritizes high agricultural yields. Therefore, the monitoring of pesticide residues in citrus fruits and other agricultural products and their impacts on human health and food security are of great concern. This study aims to determine multi-class pesticides including highly polar residues in satsuma mandarins. A total of 226 mandarin samples were collected over three consecutive harvesting years from 2019 to 2021 in the Izmir region of Turkey. Targeted compounds included pesticides and metabolites with European Union (EU) regulatory levels, plus other non-approved residues and highly polar compounds. The residues excluding highly polar substances were analyzed by applying the quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction and liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) determination for 434 analytes and gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) determination for 71 analytes. For six highly polar pesticides, sample preparation was based on Quick Polar Pesticides (QuPPe) extraction. The polar residues were determined by LC-MS/MS using internal standards. Forty different residues, including two highly polar substances, were recorded in mandarin samples through three harvesting years. In 8.4% of the samples, no quantifiable residues were detected, whereas 207 samples contained at least one residue. The maximum residue level (MRL) exceedances were recorded for 22.1% of the samples. The two most frequently found pesticides were phosphonic acid and spirotetramat, with an incidence rate of 48.7% and 46.5%, respectively. The concentration of phosphonic acid and spirotetramat in mandarin samples varied from 0.026 to 39.386 mg kg-1 and from 0.010 to 1.485 mg kg-1, respectively. The results will enable researchers and regulatory authorities to assess the extent of pesticide presence, identify potential risks, and take necessary measures to ensure the safety of satsuma mandarins for consumers.

Monitoring and Exposure Assessment of Fosetyl Aluminium and Other Highly Polar Pesticide Residues in Sweet Cherry.

Cherries are popular fruits due to their health benefits, organoleptic quality, and attractive appearance. Since highly polar pesticides are of low mass and amphoteric character, and are not amenable to traditional multi-residue extraction methods, they are more commonly not included in the pesticide monitoring program. This study aims to determine twelve highly polar pesticide residues in cherry samples intended for export from Turkey. A total of 16,022 cherry samples from 2018−2020 harvests in four production areas of Turkey were analyzed using a modification of the Quick Polar Pesticides method and liquid chromatography-tandem mass spectrometry. The method was validated at two fortification levels (0.01 and 0.05 mg kg−1), and good recoveries (87.4−111.4%) and relative standard deviations (<6%) were achieved for all analytes. The limits of quantification were in the range of 1.08−2.55 µg kg−1. Overall, 28.4% of the analyzed cherry samples were detected with phosphonic acid, calculated as fosetyl aluminium (fosetyl-Al) in amounts up to 77.7 mg kg−1. For 2304 samples (14.4%), the residues exceeded the European Union maximum residue level of 2 mg kg−1. There is no reason to be concerned about long-term exposure to phosphonic acid/fosetyl-Al, and the other highly polar pesticides through the consumption of sweet cherry.

Impact of different extraction processes on aflatoxin contamination in peanut oil.

Aflatoxins (AFs) are genotoxic carcinogens and are a growing concern in peanuts and peanut products. This study aims to impact of different extraction processes on the transition of AFs from peanuts to oils. Peanuts were collected from nine different factories in Osmaniye, Turkey, during the period of November 2017-May 2018. While no aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) were detected in peanuts, aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) were determined in all peanut samples at levels varying from 26.7 to 234.7 µg kg-1 and from 4.44 to 44.0 µg kg-1, respectively. No AFs were quantified in oils obtained by the industrial application method. The ratios of AFB1 transitions to oils obtained by solvent extraction, cold pressing of roasted peanuts and cold pressing methods were 9.0-79.8%, 11.3-75.3% and 9.3-77.6%, respectively. The concentrations of AFB2 in oils obtained by solvent extraction, cold pressing of roasted peanuts and cold pressing methods were 0.46-17.2 µg kg-1, 0.84-33.0 µg kg-1 and 1.02-36.3 µg kg-1, respectively. This is the first demonstration of the impact of different extraction processes on the transition of AFs from peanuts to oils.

Mycotoxins in spices and herbs-An update.

Spices and herbs have been used since ancient times as flavor and aroma enhancers, colorants, preservatives, and traditional medicines. There are more than 30 spices and herbs of global economic and culinary importance. Among the spices, black pepper, capsicums, cumin, cinnamon, nutmeg, ginger, turmeric, saffron, coriander, cloves, dill, mint, thyme, sesame seed, mustard seed, and curry powder are the most popular spices worldwide. In addition to their culinary uses, a number of functional properties of aromatic herbs and spices are also well described in the scientific literature. However, spices and herbs cultivated mainly in tropic and subtropic areas can be exposed to contamination with toxigenic fungi and subsequently mycotoxins. This review provides an overview on the mycotoxin risk in widely consumed spices and aromatic herbs.

Chloropropanols and Their Esters in Food: An Updated Review.

Chloropropanols, their fatty acid esters, and glycidol and its fatty acid esters (GEs) are process contaminants in foods that pose potential health risks. These contaminants typically arise during the deodorization process of vegetable oils, particularly in high concentrations within oils like palm oil and products derived from them, such as margarine, baked goods, pastries, and infant formula. Chloropropanol esters and GE can hydrolyze under the influence of lipases, forming chloropropanols. Elevated temperatures during food production can lead to the release of free 3-chloro-1,2-propanediol (3-MCPD) or free 2-chloro-1,3-propanediol (2-MCPD) in products containing both fat and salt. The exposure to these contaminants, especially for infants and young children, raises concerns about potential health hazards. While extensive research has focused on 3-MCPD, 2-MCPD, and GE, knowledge regarding other chloropropanols such as 1,3-dichloro-2-propanol (1,3-DCP), 2,3-dichloro-1-propanol (2,3-DCP), and their fatty acid esters remains limited. This review aims to provide a comprehensive overview encompassing formation mechanisms, analysis methods, toxicological implications, occurrence patterns, exposure levels, mitigation strategies, and legislative considerations concerning these contaminants.

Determination of Pesticide Residues in Vine Leaves Using the QuEChERS Method and Liquid Chromatography-Tandem Mass Spectrometry.

Commercial viticulture necessitates regular pesticide applications to manage diseases and pests, raising significant concerns regarding pesticide residues among stakeholders. Due to health risks associated with these residues in Turkish vine leaves, the European Commission has increased the frequency of official control from 20% to 50%. Thus, the aim of this study was to determine multi-class pesticide residues in brined vine leaves from Turkey. A total of 766 samples of vine leaves were collected between May 2022 and June 2023. More than 500 residues were analyzed using the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method, followed by liquid chromatography-tandem mass spectrometry. In-house validation data demonstrated that the analytical method exhibits fit-for-purpose performance in terms of linearity, accuracy, precision, and measurement uncertainty. Out of 766 samples analyzed, 180 samples (23.5%) contained one (131, 17.1%) or multiple (49, 6.4%) pesticides. Both the frequencies of occurrence and the rate of maximum residue level (MRL) exceedance increased in 2023 compared to 2022, with the MRL exceedance rate rising from 9.5% to 25.2%. Forty-three different residues were found in quantifiable concentrations and eight of them were non-approved. Among the residues, the non-systemic pyrethroid insecticides, lambda-cyhalothrin (8.0%) and cypermethrin (7.2%), were the two most frequently detected, with concentrations ranging from 0.010 to 0.248 mg kg-1 and from 0.011 to 0.533 mg kg-1, respectively. Turkey is a major exporter of vine leaves and these results provide crucial information regarding pesticide occurrence and quality assessment of vine leaves. The significant increase in both pesticide occurrence and MRL exceedance rates between 2022 and 2023 underscores the urgency for regulatory bodies to reassess current pesticide usage and monitoring practices. The findings emphasize the importance of implementing more stringent rules and improving enforcement methods in order to reduce the spread of unapproved pesticides and ensure adherence to global food safety standards.

Occurrence of Mycotoxins in Dried Fruits Worldwide, with a Focus on Aflatoxins and Ochratoxin A: A Review.

Dried fruits are popular and nutritious snacks consumed worldwide due to their long shelf life and concentrated nutrient content. However, fruits can be contaminated with various toxigenic fungal species during different stages, including cultivation, harvesting, processing, drying, and storage. Consequently, these products may contain high levels of mycotoxins. This risk is particularly pronounced in developed countries due to the impact of climate change. Several factors contribute to mycotoxin production, including the type of fruit, geographical location, climate conditions, harvest treatments, and storage management practices. The main mycotoxins in dried fruits are aflatoxins (AFs) and ochratoxin A (OTA), which can induce human health problems and economic losses. Mycotoxin contamination can vary significantly depending on the geographic origin of dried fruits (vine fruits, figs, dates, apricots, prunes, and mulberries). The aim of this review was to fill the knowledge gap by consolidating data from various regions to understand the global picture and identify regions with higher contamination risks. By consolidating research from various origins and stages of the supply chain, the review intends to shed light on potential contamination events during pre-harvest, drying, storage, and trading, while also highlighting the effects of storage conditions and climate change on mycotoxin contamination.

Quantification of 363 Pesticides in Leafy Vegetables (Dill, Rocket and Parsley) in the Turkey Market by Using QuEChERS with LC-MS/MS and GC-MS/MS.

Contamination of agricultural products with pesticide residues is a growing concern due to their adverse health effects and increasing worldwide usage of pesticides. In 2021 a total of 200 samples of green leafy vegetables, including 80 dill, 80 rocket and 40 parsley, purchased from greengrocer shops, markets and bazaars in Corum Province, Turkey, were monitored for pesticide residues. In green leafy vegetables, 363 pesticides were analyzed using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) sample preparation, followed by liquid chromatography coupled to mass spectrometry (LC-MS/MS) for 311 residues and gas chromatography coupled to mass spectrometry (GC-MS/MS) for 52 residues. The method was in-house validated at two fortification levels, and satisfactory recoveries and precisions were achieved for all residues. No quantifiable residues were found in 35% of the samples, whereas 43 residues belonging to 24 different chemical classes were detected in 130 green leafy vegetables. Among the green leafy vegetables, the highest occurrence frequency was recorded in the rocket, followed by dill and parsley. In 46% of the green leafy vegetables, the residue levels exceeded European Union Maximum Residue Levels (EU MRLs). The most frequently detected pesticides were pendimethalin (22.5%), diuron (38.7%) and pymetrozine (52.5%) in dill, rocket and parsley, respectively.

Effect of Three Different Preservatives on the Microbiota of Shalgam, a Traditional Lactic Acid Fermented Beverage.

Shalgam is a traditional Turkish beverage derived from the natural fermentation of purple carrots (Daucus carota) that boasts valuable antioxidant and prebiotic properties. These features of shalgam increase efforts to enhance its shelf life and ensure safe consumption. In this study, the effects of three different preservatives (sodium benzoate, potassium sorbate, or natamycin) on the physicochemical and microbiological properties of shalgam produced at laboratory scale and stored at room temperature for six months were investigated. Each preservative was used in four different concentrations (25, 100, 400, and 800 mg/L) to assess their impacts on the population of lactic acid bacteria (LAB) and yeast. After determining the total acidity and pH of the samples, colorimetric measurements were performed. The isolated LAB were defined using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) method. The addition of preservatives did not significantly affect the pH of the shalgam samples (3.44-3.52) compared to the control sample (3.43). However, a slight increase was observed in the total acidity of preservative-treated samples, with the highest level (5.61 g/L lactic acid) recorded in samples containing 100 mg/L sodium benzoate. Lacticaseibacillus paracasei subsp. paracasei, which has the potential to impart probiotic properties to shalgam, was the predominant LAB species in both non-treated and preservative-treated samples. The use of preservatives significantly reduced the total number of yeasts, which may cause spoilage in shalgam. The results indicate that using sodium benzoate at a concentration of 100 mg/L is the optimum method for shalgam production, resulting in the highest total acidity value obtained. Overall, the findings provide a significant contribution to prolonging the shelf life of shalgam, a beverage with immense production and consumption potential worldwide.

Determination of 355 Pesticides in Lemon and Lemon Juice by LC-MS/MS and GC-MS/MS.

While pesticides have become a primary tool in modern agriculture, these compounds remain a high priority on the list of consumer concerns regarding food safety. The use of pesticides in the production and post-harvesting of lemon fruits is widely used to ensure agricultural yield and fruit quality. Therefore, monitoring studies on citrus fruits to enforce regulatory compliance and ensure food safety is in great demand. The aim of this study was to monitor multi-class pesticide residues in lemon fruits commercialized in Turkey. The transmission of residues that existed on the outer surface of the fruit into its juice was also studied. Whole fruits and lemon juice samples were prepared using the quick, easy, cheap, effective, rugged and safe (QuEChERS) methodology prior to analysis. For the screening and quantification of 355 pesticide residues, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) were used. The analytical method has been shown to have a sufficiently low limit of quantification with respect to current maximum residue limits (MRLs) for all target analytes. The obtained recovery and precision parameters fulfilled the requirements in DG SANTE guidelines. The in-house validated analytical method was then applied for the determination of 355 pesticide substances in 100 whole fruit samples and their juices. Sixteen different residues were detected in 43% of lemon fruits, whereas 57 lemon samples were pesticide-free. The MRLs exceedances were recorded in 29 lemon samples. The most frequently detected (17%) pesticide in lemon fruits was chlorpyrifos-methyl, with a range of 0.013-0.098 mg kg-1. A lower frequency was detected for metamitron (10%, 0.027-0.118 mg kg-1), buprofezin (9%, 0.023-0.076 mg kg-1), pyriproxyfen (9%, 0.021-0.102 mg kg-1) and malathion (7%, 0.100-0.482 mg kg-1) in whole fruits. However, none of the pesticide residues were detected in lemon juice samples. These results showed that target analytes are unable to penetrate the lemon exocarp and/or endocarp.

A preliminary study on patulin contamination in spirit drinks.

A total of 120 spirit drinks and 40 apple juices were collected between 2018 and 2020 in three regions of Turkey and analysed by high-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA) for the incidence of patulin. The analytical method was successfully validated for both matrices. In 26.7% of apple juice samples, patulin was quantified at levels between 5.8 and 66.4 µg L-1. Only one regulatory level exceedance was identified for apple juice. The mean dietary exposure of the adult population to patulin ranged from 0.083 to 0.091 ng kg-1 bw day-1. With the HPLC-PDA method, patulin was measured in quantifiable concentrations in 11 out of 25 cognac, 5 out of 40 liqueur, and 1 out of 10 whisky samples, up to a level of 42.6 µg/l, whereas all vodka samples were free from patulin. In only one liqueur sample, the presence of patulin could be confirmed with LC-MS/MS, at a level of 8.9 µg L-1.

An introduction to the traditional fermented foods and beverages of Turkey.

Fermented foods and beverages, whether of plant or animal origin, play an important role in the diet of people in many parts of the world. Fermented foods not only provide important sources of nutrients but have also great potential in maintaining health and preventing diseases. Lactic acid bacteria and yeasts are the major group of microorganisms associated with traditional fermented foods. Many different types of traditional fermented foods and beverages are produced at household level in Anatolia. These include fermented milks (yoghurt, torba yoghurt, kurut, ayran, kefir, koumiss), cereal-based fermented food (tarhana), and non-alcoholic beverage (boza), fermented fruits, and vegetables (tursu, salgam, hardaliye), and fermented meat (sucuk). However, there are some differences in the preparation of traditional foods and beverages from region to region. The focus of this article is to describe the traditional fermented foods and beverages of Turkey.

Determination of more than 500 Pesticide Residues in Hen Eggs by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and Gas Chromatography-Tandem Mass Spectrometry (GC/MS/MS).

This study aims to validate a fast method of simultaneous analysis of 365 LC-amenable and 142 GC-amenable pesticides in hen eggs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS), respectively, operating in multiple reaction monitoring (MRM) acquisition modes. The sample preparation was based on quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction. Key method performance parameters investigated were specificity, linearity, limit of quantification (LOQ), accuracy, precision and measurement uncertainty. The method was validated at two spiking levels (10 and 50 µg/kg), and good recoveries (70%-120%) and relative standard deviations (RSDs) (≤20) were achieved for 92.9% of LC-amenable and 86.6% of GC-amenable pesticide residues. The LOQs were ≤10 µg/kg for 94.2% of LC-amenable and 92.3% of GC-amenable pesticides. The validated method was further applied to 100 egg samples from caged hens, and none of the pesticides was quantified.

Quantification of fosetyl-aluminium/phosphonic acid and other highly polar residues in pomegranates using Quick Polar Pesticides method involving liquid chromatography-tandem mass spectrometry measurement.

Interest in the quantification of highly polar substances in crops has noticeably increased in the last five years. This study was designed to assess quantification of six polar residues, chlorate, ethephon, fosetly-aluminium (fosetyl-Al), glyphosate, phosphonic acid and perchlorate. A total of 2513 pomegranate samples intended for export from Turkey were analysed using the Quick Polar Pesticides (QuPPe) method. The method was in-house validated with very good performance results. The limits of quantification (LOQs) for residues were much lower than the respective EU Maximum Residue Levels (MRLs). Phosphonic acid was detected in 38.5% of pomegranate samples at quantifiable concentrations, calculated as fosetyl-Al. The concentrations ranged between 0.005 and 12.9 mg kg-1. The 20% of pomegranate samples showed fosetyl-Al levels above the EU MRL of 2 mg kg-1. Other polar residues were not detected in any pomegranate samples. This is the first report about highly polar pesticides in pomegranates cultivated in Turkey.

A preliminary assessment of dietary exposure of ochratoxin A in Central Anatolia Region, Turkey.

The aim of this study was to determine dietary exposure to ochratoxin A (OTA) in Turkish adults. In this study, 500 food samples (50 rice, 50 wheat bread, 50 pasta, 50 raisins, 50 dried figs, 50 pistachios, 50 hazelnuts, 50 almonds, 50 chilli, 25 coffee, and 25 cocoa) collected from Turkey were analysed with a high-performance liquid chromatography (HPLC) method. Moreover, a total of 370 analytical results (110 cereal-based snacks, 95 wine, 35 beer, and 130 chocolate) collected from our previous observations were also used in the evaluation of exposure estimates. OTA was found in 52% of cocoa, 42% of raisins, 40% of coffee, 34% of chilli, 14% of dried figs, 10% of pasta, 8% of pistachios, 6% of wheat bread, 4% of rice, and 4% of hazelnuts. The chronic dietary exposure to OTA for Turkish adults, using lower bound (LB) and upper bound (UB) concentrations, varied from 0.683 to 4.487 ng/kg body weight (b.w.) per week for mean estimate and from 3.976 to 5.760 ng/kg b.w. per week for the 95th percentile (P95) estimate. Cereals and cereal-based products made the largest contribution (75.3-85.7%) to OTA exposure. Both mean and P95 chronic exposure to OTA were greatly below the tolerable weekly intake of 120 ng/kg b.w. per week and thus not a health concern for Turkish adults.

Assessment of dietary exposure to deoxynivalenol and fumonisin in the population of infants and toddlers in Turkey.

This study aims to estimate dietary exposure to deoxynivalenol and fumonisins (FBs) of infants and toddlers in Turkey. A total of 75 processed cereal-based foods intended for infants and toddlers collected between July and October 2018, were analysed by high performance liquid chromatography (HPLC). DON was determined in 21.3% of samples with mean middle bound (MB) level of 28.4 µg/kg. Of the 16 quantifiable samples, only one showed values above 200 µg/kg. Fumonisin B1 (FB1) was detected at quantifiable levels only in three samples, while FB2 was not found in any sample. Estimated mean MB chronic dietary exposures to DON in infants and toddlers were 0.161 and 0.118 µg/kg b.w. per day, while 95th percentile (P95) MB exposures to DON were estimated at 0.564 and 0.414 µg/kg b.w. per day, respectively. Mean MB dietary exposures to FBs for infants and toddlers, respectively, were 0.093 and 0.068 µg/kg b.w. per day; P95 exposure estimates were 0.079 and 0.058 µg/kg b.w. per day. Both for DON and FBs, mean and P95 exposures of infants and toddlers did not exceeded the threshold level of 1 µg/kg b.w. per day and are therefore not of health concern.

Biological strategies to counteract the effects of mycotoxins.

Mycotoxins are fungal secondary metabolites that if ingested can cause a variety of adverse effects on both humans and animals, ranging from allergic responses to death. Therefore, exposure to mycotoxins should be minimized. A variety of physical, chemical, and biological methods have been developed for decontamination and/or detoxification of mycotoxins from contaminated foods and feeds. This overview details the latest developments in the biological control of both fungal infection and mycotoxin formation and describes the detoxification of many of the most important mycotoxins by microorganisms. This review also addresses the potential for use of microorganisms as mycotoxin binders in the gastrointestinal tract of both humans and animals, thereby reducing the potential deleterious effects of exposure to these toxins.

Effects of probiotic bacteria on the bioaccessibility of aflatoxin B(1) and ochratoxin A using an in vitro digestion model under fed conditions.

In the present study, we aimed at determining the release of aflatoxin B(1) (AFB(1)) and ochratoxin A (OTA) from different food products in the gastro-intestinal tract in the absence and presence of probiotics, a possible adsorbent. The average bioaccessibility of AFB(1) and OTA without probiotics was about 90%, and 30%, respectively, depending on several factors, such as food product, contamination level, compound and type of contamination (spiked versus naturally contaminated). The six probiotic bacteria showed varying binding capacity to AFB(1) and OTA depending on the bacterial strain, toxin studied, type of food and contamination level. A reduction to a maximum of 37% and 73% as observed for the bioaccessibility of AFB(1) and OTA in the presence of probiotic bacteria, respectively. This is the first report on the effect of probiotic bacteria on reducing the fraction of mycotoxins available for absorption in the gastrointestinal tract from different food products.

Aflatoxins and ochratoxin A in chocolate products in Turkey.

This survey describes the occurrence and levels of AFs and OTA in chocolate products consumed in Turkey. A total of 130 samples, including bitter chocolate, milk chocolate and chocolate wafers were analysed for these mycotoxins by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD). The values of recovery (81-92%) and precision (RSD < 9%) fulfilled the requirements of EC Regulation No. 401/2006. OTA was the most prevalent mycotoxin, with an incidence of 46.7% in bitter chocolate, 22.8% in milk chocolate and 17.4% in chocolate wafers, ranging from 0.18 to 0.75 µg kg-1. AFs were detected in 13.3% of bitter chocolate, in 19.6% of milk chocolate and in 8.7% of chocolate wafers, in concentrations ranging from 0.15 to 2.04 µg kg-1.

Factors affecting the removal of aflatoxin M1 from food model by Lactobacillus and Bifidobacterium strains.

This paper describes the ability of six dairy strains of Lactobacillus and Bifidobacterium to remove aflatoxin M1 (AFM 1) from phosphate-buffered saline (PBS) and reconstituted milk. Bacteria were incubated in both PBS and reconstituted milk containing 5, 10 and 20 ng mL(-1) for 0, 4 and 24 h at 37 degrees C. After centrifugation the concentration of AFM 1 was determined in the supernatant fraction using high-performance liquid chromatography. The binding abilities of AFM 1 by viable (10(8) CFU mL(-1)) and heat-killed Lactobacillus and Bifidobacterium strains in PBS ranged from 10.22 to 26.65% and 14.04 to 28.97%, respectively. Similarly, AFM 1-binding capacity in reconstituted milk was found to range from 7.85 to 25.94% and from 12.85 to 27.31% for viable and heat-killed bacteria, respectively within 4 h. While B. bifidum Bb 13 was the best binder, the poorest removal was achieved by L. acidophilus NCC 68. Binding was reversible, and a small proportion of AFM 1 was released back into the solution. The toxin concentration and incubation period had no effect on the removal of AFM 1 by bacteria both in PBS and reconstituted milk.