Article pubs.acs.org/JAFC

Rapid Turbidimetric Detection of Milk Powder Adulteration with Plant Proteins Peter F. Scholl,* Samantha M. Farris, and Magdi M. Mossoba Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 5100 Paint Branch Parkway, HFS-707, Room BE-006, College Park, Maryland 20740, United States S Supporting Information *

ABSTRACT: Development of assays to screen milk for economically motivated adulteration with foreign proteins has been stalled since 2008 due to strong international reactions to the melamine poisoning incident in China and the surveillance emphasis placed on low molecular weight nitrogen-rich adulterants. New screening assays are still needed to detect high molecular weight foreign protein adulterants and characterize this understudied potential risk. A rapid turbidimetric method was developed to screen milk powder for adulteration with insoluble plant proteins. Milk powder samples spiked with 0.03−3% by weight of soy, pea, rice, and wheat protein isolates were extracted in 96-well plates, and resuspended pellet solution absorbance was measured. Limits of detection ranged from 100 to 200 μg, or 0.1−0.2% of the sample weight, and adulterant pellets were visually apparent even at ∼0.1%. Extraction recoveries ranged from 25 to 100%. Assay sensitivity and simplicity indicate that it would be ideally suitable to rapidly screen milk samples in resource poor environments where adulteration with plant protein is suspected. KEYWORDS: allergen, deterrent, economically motivated adulteration, infrared, legume, mass spectrometry, milk, pea, plant storage protein, protein, protein concentrate, protein isolate, rapid screening, rice, simple, soy, tetraborate, turbidimetry, wheat

■

INTRODUCTION Milk product adulteration dramatically came to the world’s attention in 2008 when children in the People’s Republic of China were hospitalized after consuming formula containing melamine.1,2 This nitrogen-rich molecule fraudulently boosted the assayed protein concentration, a metric that can be used to determine the price of milk, as measured by Kjeldahl or combustion (Dumas) total nitrogen analyzers or infrared instrumentation calibrated to them. This public health tragedy revealed weaknesses of protein nitrogen assays to manipulation and the need to apply more effective surveillance methods.3 To decrease reliance on total nitrogen assays, the United States Pharmacopeial Convention (USP) has advocated development of a holistic suite of tools to detect the economically motivated adulteration (EMA) of skim milk powder (SMP).4 This toolbox concept strives to rely on the analysis of intrinsic SMP properties and multivariate statistical methods to facilitate nontargeted adulterant detection strategies.5,6 Methods ranging from early rapid screening to more extensive compendial authentication procedures are required. EMA detection methods have been reviewed, and a food fraud reporting database is available.7,8 Milk powder is the second most likely commodity to be adulterated, following olive oil.8 In 2002 and 2007, reports from the European Union indicated plant proteins were used to adulterate SMP, and these fostered interest in developing methods for their detection.9−13 However, development of assays to screen milk products for EMA with foreign protein since then has been overshadowed by international reactions to the 2008 melamine incident in China, the recall of melamine contaminated pet food in North America in 2007, and the subsequent emphasis placed on assays for low molecular weight nitrogen-rich adulterants.14−19 New This article not subject to U.S. Copyright. Published 2014 by the American Chemical Society

screening assays are still needed to detect high molecular weight foreign protein adulterants and characterize this understudied potential risk. EMA tactics are anticipated to change under this surveillance pressure with a shift toward cheap and easy to disguise plant protein sources. Plant protein isolates are used as food additives to control product characteristics (e.g., viscosity, flavor, nutrition).20,21 Soy, rice, and almond protein are processed into milk-like products and sold as coffee creamer and dairy milk replacers for vegans and individuals with allergies or lactose intolerance.20,22 Soy, wheat, and almond proteins are statutorily defined as allergens by the Food Allergen Labeling and Consumer Protection Act (FALCPA) of 2004, but proteins from other plants (e.g., pea, rice, lupin, and maize) are clinically recognized allergens, even though not designated by statute.23−25 Preruminant calf fatalities have resulted from the consumption of unlabeled weaning formula containing plant protein.26 The development of rapid, inexpensive, and robust new surveillance tools to broadly detect plant proteins in dairy products is needed because it is a process that could be proactively used to help manage this uncharacterized EMA risk involving nonstatutory and statutorily defined allergens. Methods for detecting plant proteins in dairy products have been reported in fewer than approximately 50 publications, and these are biased toward soy and pea isolates. Chromatographic, ELISA, near-infrared (NIR) spectroscopic, and mass spectrometric (MS) methods have been reviewed.27−29 Hewedy et al. Received: Revised: Accepted: Published: 1498

December 13, 2013 January 30, 2014 January 31, 2014 January 31, 2014 dx.doi.org/10.1021/jf405617f | J. Agric. Food Chem. 2014, 62, 1498−1505

Journal of Agricultural and Food Chemistry

Article

Extraction and Turbidimetric Analysis in 96-Well Plates. Immediately after vortex mixing, SMP solution and PI/SMP suspensions in buffer (300 μL, 300 mg/mL) were loaded in 96-well plate column 1 wells, rows B−G, using a multichannel pipettor to deliver 90 mg of SMP test sample and progressively diluted 2-fold with buffer (150 μL) in columns 2−3, to respectively deliver 45 and 22.5 mg of each SMP test sample. Rows B through G were respectively loaded with test samples containing 0−3% PI/SMP. A schematic of the 96-well plate sample layout is provided as Supplemental Figure S1 in the Supporting Information. Wells in columns 4−6, 7−9, and 10− 12 were similarly used to prepare replicates of samples prepared in columns 1−3 (rows B−G). Row A was used to prepare duplicate standard curves using only the plant PI without any SMP. Immediately after vortexing, PI solution (300 μL, 15 mg/mL) was deposited in wells A1 and A7. These test portions were progressively diluted 3-fold with buffer in columns A2−6 and A8−12. Buffer was added to produce a final volume of 300 μL in all wells. Solvent blank wells in row H were filled with 300 μL of buffer. Two 96-well plates were prepared for each type of PI/SMP mixture to provide 8 data points at each spike level and 4 data points at each PI (alone) concentration. Plates were centrifuged (Sorvall Evolution RC, Thermo Electron Corporation) at 1,500 rpm (466g) for 5 min at 20 °C in a swinging microplate carrier. Supernatants were removed taking care not to disturb sample pellets. Pellets were resuspended in fresh buffer (150 μL) by pipetting them up and down three times using aerosol barrier pipet tips. Samples were centrifuged again, and the supernatants were removed. This washing process was repeated three times. Pellets were finally resuspended in fresh buffer (150 μL), and the absorbance (360 ± 2 nm) was promptly measured, without shaking, for 1 s per well using a VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA) at 20 °C. Plant Protein Isolate Turbidimetric Standard Curves. Individual plant PI powders without SMP were suspended in buffer (5 mg/mL) and serially diluted 2-fold in 96-well plates so that 300 μL aliquots delivered 0−1.5 mg (dry powder weight). Four technical replicate wells were prepared at 12 concentrations. Test portions were not extracted before measuring the absorbance. Standard curves were prepared by linear regression of the baseline corrected absorbance data, limited to values approximately less than 1 OD unit. The LOD was estimated as three times the ratio of the standard error of the absorbance divided by the slope. The limit of quantitation (LOQ) was estimated as ten times this ratio. Standard curves were used to estimate the recovery of adulterants from samples extracted with tetraborateEDTA buffer. Fourier Transform Infrared Spectroscopy. Transmission Fourier transform infrared (FT-IR) measurements were carried out on an Agilent Technologies Inc. (Wilmington, DE; formerly Varian, Randolph, MA) FTS 7000e IR spectrometer operating with Resolution Pro 4.0 software. The optical bench included a Michelson interferometer with an air bearing moving mirror, a KBr substrate beam splitter, and a linearized mercury cadmium telluride liquid N2 cooled detector. In the absence of a test sample, a 3 min data collection at 4 cm−1 resolution from 2200 to 2000 cm−1 yielded a peak-to-peak noise level pyro-Glu (N-terminal Q), Glu > pyro-Glu (N-terminal E); peptide charge = +2, +3; peptide and MS/ MS tol. = ±0.8 Da; instrument type = ESI-trap. Individual proteins were inferred to be present if at least two different peptide sequences exceeding the critical peptide MASCOT score were detected.

linearly increased with the addition of soy adulterant to SMP from ∼0.1−1.0 OD units. Liquid and soy beverage powder added to SMP at 5% by weight yielded strong positive results with absorbance values ranging from 0.4 to 1.2 OD (data not shown). By contrast, negative control samples extracted from 5% sorbitol, chitosan, gelatin, whey, egg protein, and infant formula containing partially hydrolyzed whey in SMP were turbidimetrically and visually indistinguishable from the SMP blank. The observed lack of a pellet in these samples demonstrates that the extraction method is to some degree selective for plant proteins over other sources of added protein, polysaccharides, or monosaccharides potentially encountered in EMA schemes. Instead of longer visible wavelengths often used in turbidimetric assays, 360 nm was used because it was empirically found to yield the strongest signal. It is more direct to specify the weight of spiked adulterant, instead of a weight percentage, because different SMP sample weights were used to explore the assay’s dynamic range. The spiked adulterant weight and extracted sample absorbance data for soy in Figure 1 are replotted in Figure 2, along with pea,

■

Figure 2. Turbidimetric detection of soy, pea, rice, and wheat protein isolates extracted from 22.5 to 90 mg of skim milk powder samples spiked with 0−3% plant protein isolate by weight. Data points represent the mean (n = 8) ± one sample standard deviation. Protein isolate symbol legend: rice (triangles), pea (squares), soy (closed circles), wheat (diamonds).

RESULTS AND DISCUSSION Soy, pea, rice, and wheat protein isolates extracted from SMP/ PI suspensions in 96-well plates were turbidimetrically detected using a semitargeted technique for the rapid screening of milk powder for EMA with insoluble foreign proteins. Because previous reports described adulterant concentration as a percentage of the total sample weight, soy data are initially presented using this metric to facilitate comparison with published work.5,13,33 Extracted sample absorbance in Figure 1

rice, and wheat protein isolate data. In Figure 2, absorbance begins to exhibit nonlinearity with increases in the theoretical amount of plant protein deposited above ∼900 μg (assuming 100% extraction efficiency). This parallels the onset of nonlinearity in Figure 1 above 1% soy in 90 mg SMP samples. Extraction Efficiency. Plant protein extraction efficiency and gel formation can be affected by intra- and intermolecular protein interactions.40−42 Changes in the resuspended particle size distribution could affect turbidimetric responses because light scattering is a function of particle size.43 To survey for the influence of these effects and measure extraction efficiencies, absorbance differences between nonextracted and extracted plant adulterant standards alone (without SMP) and extracted adulterant-spiked SMP (Figure 2) were compared. Linear regression of adulterant weight versus absorbance data was performed from 0.1 to 1.0 OD. The extracted to nonextracted sample linear regression slope ratio was multiplied by 100 to calculate adulterant extraction efficiency in the presence and absence of SMP. Linear regression, LOD, LOQ, and extraction efficiency results are presented in Table 1. Recovery estimates were constrained to absorbance values nominally equal to the LOD (∼200 μg of plant protein isolate). LODs were similar for

Figure 1. Turbidimetric detection of soy protein isolate extracted from skim milk powder in 96-well plates. Powder sample weight loaded: 22.5 mg (open circles), 45 mg (shaded circles), 90 mg (filled circles). Data points represent the mean (n = 8) ± one sample standard deviation. 1500

dx.doi.org/10.1021/jf405617f | J. Agric. Food Chem. 2014, 62, 1498−1505

Journal of Agricultural and Food Chemistry

Article

Table 1. Turbidimetric Assay Performance Metrics for Detection of Protein Isolates recovery (%)

a

protein isolate

slope (OD 360 nm/mg)

Y-intercept (OD)

R2

LODa (% wt)

LOD (μg)

LOQ (μg)

with SMP

without SMP

soy rice wheat pea

0.7 1.6 0.6 0.7

0.0020 −0.0472 0.0015 0.014

0.9829 0.9728 0.9296 0.9525

0.2 0.1 0.2 0.2

190 115 191 204

575 350 580 618

50 100 25 90

65 96 47 46

90 mg powder sample basis.

in extraction efficiency, baseline absorbance of SMP, and the optimal number of pellet washes need to be investigated to extend analyses to lower adulterant concentrations. Effects Extraction Buffer Composition on Pellet Washing. The sequential extraction of storage proteins from plant sources by adjusting salt concentrations, pH, and organic solvent composition, known as Osborne fractionation, has been extensively studied, but mechanism(s) are not known through which EDTA-tetraborate buffer selectively precipitates plant proteins from dairy samples.13,32,42 The formation of insoluble protein−boronate complexes via carbohydrate modified plant proteins and dissociation of casein micelles by EDTA may be involved.45,46 On the basis of HPLC−fluorescence analysis of soy and pea protein isolate extracted from SMP, storage protein hydrophobicity may also be responsible.13 Studies of protein extraction from soybean meal do not support isoelectric precipitation as being involved at pH 8.3.47 To determine the optimal number of pellet washes and the effect of extraction buffer composition during assay development, SMP samples (n = 4) spiked with 0 and 2.5% soy protein isolate were extracted with water, EDTA (40 mM, pH 8.3), tetraborate (30 mM, pH 8.3), or EDTA (40 mM)/tetraborate (30 mM)/pH 8.3. The average resuspended pellet absorbance (n = 4) after each of five successive washes is presented in Supplemental Figure S4 in the Supporting Information. Water alone failed to yield differences between SMP and spiked sample absorbance after five washes. Tetraborate alone failed to yield significant (p < 0.05) differences between SMP and spiked test samples until the third wash. The absorbance of both samples continued to decrease in the fifth wash. EDTA alone and EDTA-tetraborate buffer were both very effective for extracting plant proteins and worked equally well. Large stable differences between SMP and spiked samples were apparent after the first wash. No changes occurred after the third wash, where absorbance of SMP-only samples decreased to 0.10 ± 0.00 OD (average ± SD). Three washes also produced optimal results in SMP samples spiked with pea protein isolate (data not shown). The insolubility of soy protein isolate at pH 8.3 in 30−40 mM salt solutions due to hydrophobicity, instead of boronate−plant protein complex insolubility, appears to more significantly contribute to the effectiveness of this extraction technique.41 SMP solutions prepared in water exhibit a characteristic milky white appearance, but addition of EDTA under conditions of the assay produces transparent colorless solutions. Collapse of milk casein micelles due to EDTA chelation of Ca2+ minimizes light scattering, produces transparent SMP solutions, and enables the visual and turbidimetric detection of insoluble material that is otherwise obscured.48 Detailed EDTA−SMP titration studies were not performed, but this process can be saturated by SMP to produce samples of varying turbidity in the absence of plant proteins, unless EDTA is maintained at some undetermined stoichiometric excess of Ca2+. This is important if the amount of SMP loaded in the assay is increased.

all isolates, ranging from 0.1 to 0.2% by weight (for 90 mg SMP load), and corresponded to 115−205 μg of spiked protein (independently of SMP load). This analytical sensitivity is comparable to or better than that offered by other reported ELISA, SDS−PAGE, and CE assays for detecting dairy product EMA with plant proteins and is achieved more rapidly using less equipment.30−33,44 The extraction efficiencies of soy and rice proteins were not substantially affected by presence of SMP matrix in Table 1. However, wheat and pea protein extraction efficiencies were respectively decreased and increased by 50% in the presence of SMP. Soy extraction efficiency independence from SMP is consistent with HPLC−fluorescence data reported by Luykx, but, for unknown reasons, the dependency of pea extraction is not.13 The 96-well plate assay format detected adulteration of 90 mg SMP samples spiked with ∼200 μg of plant protein isolate, a level (0.2% by weight) generally considered satisfactory for EMA detection. If adulterated samples are blended in complex supply chains, detection at even lower concentrations may be required. A Larger Capacity Microcentrifuge Assay Format. Linear relationships between the protein spike concentration and absorbance in Figure 1 indicate that even lower amounts of adulterant, on a percentage of sample weight basis, could be detected by extracting larger amounts of sample. However, the maximum amount of SMP that could be precisely loaded in a single 96-well plate well was limited by three factors: solution viscosity, bubble formation, and well volume. The viscosity of nonspiked SMP solutions exceeding ∼600 mg/mL interfered with pipetting, and these samples formed bubbles. Aerosolization occurred when bubbles burst during pipetting of even lower concentration samples and gradually led to deposit accumulation inside the pipettor. The use of aerosol barrier pipet tips eliminated this problem. Because sensitivity is limited by the amount of SMP loaded in a well, a microcentrifuge tube assay format was briefly examined to increase the maximum sample load from 90 mg (300 μL of 300 mg of solids/mL) in the 96-plate format to 540 mg (1.8 mL × 300 mg of solids/ mL). Photographs of pellets extracted using the 96-well plate and microcentrifuge tube assay formats are provided as Supplemental Figures S2 and S3 in the Supporting Information. Resuspended microcentrifuge pellet aliquots were transferred to 96-well plates to measure absorbance. The same linear dependence of absorbance on the amount of protein isolate spiked was observed in the microcentrifuge tube assay format without change in the SMP baseline signal. LODs in the microcentrifuge tube format ranged from 100 to 200 μg as was observed in the 96-well plate assay format in Figure 1. The microcentrifuge tube format is useful for detecting adulteration at low percent weight concentrations due to the more readily visible pellet obtained from larger samples. This enables spot tests to be performed if a microplate reader is not available. Although not explored here, larger samples could be used to achieve LODs at even lower % spike concentrations. Changes 1501

dx.doi.org/10.1021/jf405617f | J. Agric. Food Chem. 2014, 62, 1498−1505

Journal of Agricultural and Food Chemistry

Article

Other Analytical Tools. To determine the composition of pellets detected in the turbidimetric assay and demonstrate a general work-flow for characterization of EMA with insoluble foreign proteins, additional SDS−PAGE, FT-IR, and LC−MS/ MS analyses were performed. SDS−PAGE gel images of SMP and spiked (3% by weight) SMP test samples before and after extraction are presented in Figure 3. Nonextracted samples

related factors (e.g., heat treatment, storage time) yield sufficiently large SMP matrix variability and compromise chemometric detection models, sample processing may be required to improve adulterant detection. Removal of milk components and concentration of plant proteins via tetraborate-EDTA extraction is expected to simplify IR spectra and facilitate the detection of insoluble foreign proteins but at the cost of lowering assay throughput. The FT-IR spectra of SMP, soy protein isolate, and extracted soy protein isolate are presented in Figure 4. Extracted protein isolate pellets exhibit

Figure 3. Reducing SDS−PAGE analysis of skim milk powder samples spiked with 3% plant protein isolate by weight before and after extraction with tetraborate-EDTA buffer. SMP = skim milk powder. Plant protein isolates: soy (S), pea (P), rice (R), wheat (W), molecular weight ladder (L).

Figure 4. FT-IR KBr pellet transmission spectra of nonextracted skim milk powder and soy protein isolate reference samples, and a pellet extracted from skim milk powder spiked with soy protein isolate at 0.1% by weight. The atmospheric CO2 stretching band (2360 cm−1) appears as an artifact of the analyte to background single-beam normalization process. Nonextracted reference samples: skim milk powder (top dotted line), soy protein isolate (middle dashed line). Extracted pellet: skim milk powder spiked with soy protein isolate (bottom solid line).

(lanes 1−5) are dominated by intense bands (25−37 kDa) that correspond to milk proteins (α-S1, α-S2, and β-caseins) known to comprise ∼72% of normal milk protein. All nonextracted samples also exhibit bands (10−20 kDa) corresponding to βlactoglobulin and α-lactalbumin that normally contribute a total of ∼15% to all milk protein. Nonextracted soy and pea spiked SMP samples (lanes 2 and 3) exhibit additional faint bands (50−75 kDa) that are also observed in extracted spiked samples (lanes 8 and 9) and the corresponding individual protein isolate standards analyzed without SMP (Supplemental Figure S5 in the Supporting Information). These bands are interpreted as being plant protein-specific proteins and are indicative of the protein isolate spike. This contrasts with nonextracted rice and wheat (lanes 4 and 5) where there are no clear differences from nonspiked SMP (lane 1). Comparison of nonextracted (lanes 1−5) and extracted (lanes 7−10) samples in Figure 3 shows that extraction produces distinct banding pattern changes and enables discrimination of spiked from nonspiked samples. The appearance of Figure 3 is consistent with previously reported SDS−PAGE data for soy and pea samples prepared using tetraborate-EDTA precipitation.13 Transmission FT-IR of Extracted SMP. Rapid screening of SMP using mid- and near-IR spectroscopy and chemometrics to detect EMA with low and high molecular weight compounds (e.g., melamine and soy protein isolate) is an active research area.5,6 This is an appealing strategy because the NIR analysis of dry powder does not require samples to be dissolved or extracted. This lends itself to high sample throughput and automation. However, if seasonal or other manufacturing

strong amide I (1600−1700 cm−1) and amide II (1500−1600 cm−1) bands and the loss of the intense C−O stretching bands (1000−1200 cm−1) that are uniquely characteristic of milk lactose and detected only in nonextracted SMP. The corresponding pea and rice data are provided as Supplemental Figures S6 and S7 in the Supporting Information. Subtle differences in protein bands between extracted soy, pea, and rice pellets were observed. These data principally indicate that extraction removes soluble lactose and milk proteins and yields pellets from spiked samples that exhibit IR spectra confirming the presence of insoluble proteins. The analysis of pellets using SDS−PAGE and LC−MS/MS, discussed below and in related work by Luykx et al., further indicates that these characteristic amide absorbance bands do not substantially arise from coprecipitated milk proteins.13 The spectrum of nonextracted SMP in Figure 4 is similar to a published spectrum.49 LC−MS/MS Identification of Peptides and Detection of Proteins. To identify peptides and detect proteins in extracted pellets, trypsin digested samples were analyzed by nLC−MS/MS and protein database searching. Table 2 presents a list of proteins detected in the pellet obtained from SMP spiked with soy protein isolate. These soy storage proteins account for ∼70% of all seed protein.27 Virtually identical lists 1502

dx.doi.org/10.1021/jf405617f | J. Agric. Food Chem. 2014, 62, 1498−1505

Journal of Agricultural and Food Chemistry

Article

Table 2. Proteins Detected in Pellets Isolated from SMP Spiked with Soy Protein Isolate Uniprot protein accession name GLYG2_SOYBN GLYG1_SOYBN GLCA_SOYBN GLYG4_SOYBN GLCB_SOYBN GLYG3_SOYBN SBP_SOYBN GLYG5_SOYBN LOX3_SOYBN GLCAP_SOYBN LOX2_SOYBN ITRA_SOYBN LOX1_SOYBN 7SB1_SOYBN 2SS_SOYBN

protein glycinin G2 glycinin G1 β-conglycinin, αchain glycinin G4 β-conglycinin, βchain glycinin G3 sucrose-binding protein glycinin G5 seed lipoxygenase-3 β-conglycinin, αchain seed lipoxygenase-2 trypsin inhibitor A seed lipoxygenase-1 basic 7S globulin 2S albumin

MASCOT protein score

protein mass

protein sequence coverage (%)

no. of peptide sequences exceeding MASCOT identity score

1112 973 669

54357 55672 70250

49.3 40.2 40.8

10 11 9

504 463

63548 50521

29.7 41.2

7 6

454 426

54208 60485

37 31.5

6 7

375 340 295

57921 96698 74280

21.5 16.9 20.2

5 4 4

240 198 159 118 95

97085 23990 94310 46363 18448

11.4 28.2 9.9 15.7 13.9

5 5 5 2 2

of soy proteins were previously reported in tetraborate-EDTA extracted pellets from different soy isolate samples, except that α-S1-casein and β-lactoglobulin were not detected in the present study.13,34 This is presumably due to more extensive washing of the pellet in the 96-well plate assay format presented here. The corresponding protein detection data for pea and rice isolate-spiked SMP samples are presented in Supplemental Tables SI and SII in the Supporting Information. A corresponding abundance of pea and rice storage proteins were detected in these pellets, and the pea list matched those reported in earlier studies.13,34 The detection of extracted soy, pea, and rice storage proteins amid little to no milk protein confirms the selectivity of tetraborate-EDTA buffer reported in this and previous studies. MASCOT search results for extracted soy, pea, and rice spiked SMP pellets are provided as Supplemental Data Files in the Supporting Information. Analysis of pellets from SMP spiked with wheat protein isolate failed to identify wheat peptides. The addition of glutamine and asparagine deamidation as variable modifications to protein database searches did not improve search results. Wheat storage proteins are generally less soluble in low salt aqueous conditions than soy, pea, and rice and contain many fewer trypsin cleavage sites. The use of more effective organic solvent (alcohol) and endoproteases (e.g., chymotrypsin) that yield a larger number of wheat peptide sequences for MS detection may be required to address this problem in future studies. Turbidimetric detection of plant proteins extracted from SMP samples offers an inexpensive, fast, and simple procedure to detect a class of foreign proteins suspected of being used in EMA schemes. This is a semitargeted screening approach in that it is not plant-specific and relies on an intrinsic property of SMP, namely, the failure to form a precipitate under assay conditions. It does not require chemometric treatment of data, special equipment, or expertise. It exploits the use of EDTA to render adulterated opaque milk solutions transparent and the insolubility of plant protein isolates to rapidly produce samples for measurement by optical absorbance. The assay is scalable in that different amounts of SMP can be used to achieve different LODs on a percent weight basis. Other plant protein (e.g., zein, lupin, sorghum, millet) sources are expected to yield

comparable turbidimetric results due to chemical similarities among storage proteins. Although SMP was studied, other imported high dairy protein concentration products such as milk protein concentrate, whey protein concentrate, and caseinates along with high value finished products such as quark, skyr, and greek yogurt are of special interest. Even without the use of a 96-well plate reader, the addition of as little as 0.1−0.2% by weight of plant protein isolate to SMP was visually detected. These features potentially make the assay useful for performing spot tests in resource poor environments. Very small, portable and inexpensive centrifuges that accommodate 2 mL sample tubes can be purchased for less than $300 (US). Hand-held spectrophotometers suitable for field deployment can be purchased for less than ∼$2000 (US). Although data reported here were acquired in a laboratory, the simplicity of the assay should make it possible to obtain comparable results using these less expensive tools in field applications. Given the ubiquitous presence of smart phones, their adaptation to enable even lower cost absorbance measurements in the field is expected.50 It has been pointed out that it is not possible to “test your way to absolute food safety.”3 However the selective application of this simple assay to the riskiest high protein dairy products may act as a deterrent and mitigate EMA risk imposed by insoluble and potentially allergenic proteins.

■

ASSOCIATED CONTENT

S Supporting Information *

The 96-well plate sample template, photographs of pellets extracted using the 96-well plate and microcentrifuge tube turbidimetric assay formats, a figure presenting the effect of extraction buffer composition and wash number on turbidimetric response, two tables listing peptides identified in pellets extracted from SMP spiked with pea and rice protein isolates, and MASCOT .dat files (converted to .csv format). This material is available free of charge via the Internet at http:// pubs.acs.org. 1503

dx.doi.org/10.1021/jf405617f | J. Agric. Food Chem. 2014, 62, 1498−1505

Journal of Agricultural and Food Chemistry

■

Article

(11) Sanchez, L.; Perez, M. D.; Puyol, P.; Calvo, M.; Brett, G. Determination of vegetal proteins in milk powder by enzyme-linked immunosorbent assay: Interlaboratory study. J. AOAC Int. 2002, 85, 1390−1397. (12) Haasnoot, W.; Marchesini, G. R.; Koopal, K. Spreeta-based biosensor immunoassays to detect fraudulent adulteration in milk and milk powder. J. AOAC Int. 2006, 89, 849−855. (13) Luykx, D. M. A. M.; Cordewener, J. H. G.; Ferranti, P.; Frankhuizen, R.; Bremer, M. G. E. G.; Hooijerink, H.; America, A. H. P. Identification of plant proteins in adulterated skimmed milk powder by high-performance liquid chromatography-mass spectrometry. J. Chromatogr., A 2007, 1164, 189−197. (14) Xu, Q.; Wei, H. P.; Du, S.; Li, H. B.; Ji, Z. P.; Hu, X. Y. Detection of Subnanomolar Melamine Based on Electrochemical Accumulation Coupled with Enzyme Colorimetric Assay. J. Agric. Food Chem. 2013, 61, 1810−1817. (15) Gao, F.; Ye, Q. Q.; Cui, P.; Zhang, L. Efficient Fluorescence Energy Transfer System between CdTe-Doped Silica Nanoparticles and Gold Nanoparticles for Turn-On Fluorescence Detection of Melamine. J. Agric. Food Chem. 2012, 60, 4550−4558. (16) Turnipseed, S.; Casey, C.; Nochetto, C.; Heller, D. N. Determination of melamine and cyanuric acid residues in infant formula using LC-MS/MS. US FDA Laboratory Information Bulletin; Laboratory Information Bulletin, LIB No. 4421; 2008; Vol. 24. (17) Abernethy, G.; Higgs, K. Rapid detection of economic adulterants in fresh milk by liquid chromatography−tandem mass spectrometry. J. Chromatogr., A 2013, 1288, 10−20. (18) MacMahon, S.; Begley, T. H.; Diachenko, G. W.; Stromgren, S. A. A liquid chromatography-tandem mass spectrometry method for the detection of economically motivated adulteration in proteincontaining foods. J. Chromatogr., A 2012, 1220, 101−107. (19) ISOIDF, Technical specification 15495: Milk, milk products and infant formulae--Guidelines for the Quantitative determination of melamine and cyanuric acid by LC-MS/MS. In ISO: 2010. (20) Soybeans: Chemistry, Production, Processing, and Utilization; AOCS Press: Urbana, IL, USA, 2008. (21) Morr, C. V. Technical Problems and Opportunities in Using Vegetable Proteins in Dairy-Products. J. Am. Oil Chem. Soc. 1979, 56, 383−385. (22) Kolar, C. W.; Cho, I. C.; Watrous, W. L. Vegetable Protein Application in Yogurt, Coffee Creamers and Whip Toppings. J. Am. Oil Chem. Soc. 1979, 56, 389−391. (23) Sanchez-Monge, R.; Lopez-Torrejon, G.; Pascual, C. Y.; Varela, J.; Martin-Esteban, M.; Salcedo, G. Vicilin and convicilin are potential major allergens from pea. Clin. Exp. Allergy 2004, 34, 1747−1753. (24) Satoh, R.; Nakamura, R.; Komatsu, A.; Oshima, M.; Teshima, R. Proteomic analysis of known and candidate rice allergens between non-transgenic and transgenic plants. Regul. Toxicol. Pharmacol. 2011, 59, 437−444. (25) Goggin, D. E.; Mir, G.; Smith, W. B.; Stuckey, M.; Smith, P. M. Proteomic analysis of lupin seed proteins to identify conglutin β as an allergen, Lup an 1. J. Agric. Food Chem. 2008, 56, 6370−6377. (26) Schoonderwoerd, M.; Misra, V. Detection and Quantitation of Pea and Soy-Derived Proteins in Calf Milk Replacers. J. Dairy Sci. 1989, 72, 157−161. (27) Saz, J. M.; Marina, M. L. High performance liquid chromatography and capillary electrophoresis in the analysis of soybean proteins and peptides in foodstuffs. J. Sep. Sci. 2007, 30, 431−451. (28) Reid, L. M.; O’Donnell, C. P.; Downey, G. Recent technological advances for the determination of food authenticity. Trends Food Sci. Technol. 2006, 17, 344−353. (29) De la Fuente, M. A.; Juarez, M. Authenticity assessment of dairy products. Crit. Rev. Food Sci. Nutr. 2005, 45, 563−585. (30) Hewedy, M. M.; Smith, C. J. Detection of soy milk in pasteurized bovine milk. Food Hydrocolloids 1989, 3, 399−405. (31) Hewedy, M.; Smith, C. Modified immunoassay for the detection of soy milk in pasteurized skimmed bovine milk. Food Hydrocolloids 1990, 3, 485−490.

AUTHOR INFORMATION

Corresponding Author

*Phone: (240) 402-2167. Fax: (301) 436-2624. E-mail: peter. [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors are grateful to Jeffery Moore at the USP for providing materials from the USP library of standards and helpful discussions. Further thanks are extended to Jonathan DeVries of the Medallion Laboratories at General Mills and John Sheehan of the US FDA for many helpful discussions.

■

ABBREVIATIONS USED CE, capillary electrophoresis; CID, collision-induced dissociation; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; EMA, economically motivated adulteration; FALCPA, Food Allergen Labeling and Consumer Protection Act; FT-IR, Fourier transform infrared; IAA, iodoacetamide; LC, liquid chromatography; LOD, limit of detection; LOQ, limit of quantitation; NIR, near-infrared; NFDM, nonfat dry milk powder; PI, plant protein isolate; PPI, pea protein isolate; RPI, rice protein isolate; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; SMP, skim milk powder; SPI, soy protein isolate; SPR, surface plasmon resonance; USP, United States Pharmacopia; WPI, wheat protein isolate

■

REFERENCES

(1) Xin, H.; Stone, R. Tainted milk scandal. Chinese probe unmasks high-tech adulteration with melamine. Science 2008, 322, 1310−1311. (2) Everstine, K.; Spink, J.; Kennedy, S. Economically Motivated Adulteration (EMA) of Food: Common Characteristics of EMA Incidents. J. Food Protect. 2013, 76, 723−735. (3) Kennedy, S. Epidemiology. Why Can’t We Test Our Way to Absolute Food Safety? Science 2008, 322, 1641−1643. (4) Moore, J.; DeVries, J.; Lipp, M.; Griffiths, J.; Abernethy, D. Total Protein Methods and Their Potential Utility to Reduce the Risk of Food Protein Adulteration. Compr. Rev. Food Sci. Food Saf. 2010, 9, 330−357. (5) Moore, J.; Ganguly, A.; Smeller, J.; Botros, L.; Mossoba, M.; Bergana, M. Standardisation of non-targeted screening tools to detect adulterations in skim milk powder using NIR spectroscopy and chemometrics. NIR news 2012, 23, 9−11. (6) Botros, L. L.; Jablonski, J.; Chang, C.; Bergana, M. M.; Wehling, P.; Harnly, J. M.; Downey, G.; Harrington, P.; Potts, A. R.; Moore, J. C. Exploring authentic skim and nonfat dry milk powder variance for the development of nontargeted adulterant detection methods using near-infrared spectroscopy and chemometrics. J. Agric. Food. Chem. 2013, 61, 9810−9818. (7) Ellis, D. I.; Brewster, V. L.; Dunn, W. B.; Allwood, J. W.; Golovanov, A. P.; Goodacre, R. Fingerprinting food: current technologies for the detection of food adulteration and contamination. Chem. Soc. Rev. 2012, 41, 5706−5727. (8) Moore, J.; Spink, J.; Lipp, M. Development and Application of a Database of Food Ingredient Fraud and Economically Motivated Adulteration from 1980 to 2010. J. Food Sci. 2012, 77, R108−R116. (9) Maraboli, A.; Cattaneo, T. M. P.; Giangiacomo, R. Detection of vegetable proteins from soy, pea and wheat isolates in milk powder by near infrared spectroscopy. J. Near Infrared Spectrosc. 2002, 10, 63−69. (10) Manso, M. A.; Cattaneo, T. M.; Barzaghi, S.; Olieman, C.; Lopez-Fandino, R. Determination of vegetal proteins in milk powder by sodium dodecyl sulfate-capillary gel electrophoresis: Interlaboratory study. J. AOAC Int. 2002, 85, 1090−1095. 1504

dx.doi.org/10.1021/jf405617f | J. Agric. Food Chem. 2014, 62, 1498−1505

Journal of Agricultural and Food Chemistry

Article

(32) Cattaneo, T. M. P.; Feroldi, A.; Toppino, P. M.; Olieman, C. Sample Preparation for Selective and Sensitive Detection of Soya Proteins in Dairy-Products with Chromatographic and Electrophoretic Techniques. Neth. Milk Dairy J. 1994, 48, 225−234. (33) Lopez-Tapia, J.; Garcia-Risco, M. R.; Manso, M. A.; LopezFandino, R. Detection of the presence of soya protein in milk powder by sodium dodecyl sulfate capillary electrophoresis. J. Chromatogr., A 1999, 836, 153−160. (34) Cordewener, J. H. G.; Luykx, D. M. A. M.; Frankhuizen, R.; Bremer, M. G. E. G.; Hooijerink, H.; America, A. H. P. Untargeted LC-Q-TOF mass spectrometry method for the detection of adulterations in skimmed-milk powder. J. Sep. Sci. 2009, 32, 1216− 1223. (35) Krusa, M.; Torre, M.; Marina, M. L. A reversed-phase highperformance liquid chromatographic method for the determination of soya bean proteins in bovine milks. Anal. Chem. 2000, 72, 1814−1818. (36) Haasnoot, W.; Olieman, K.; Cazemier, G.; Verheijen, R. Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder. J. Agric. Food Chem. 2001, 49, 5201−5206. (37) García, M.; Marina, D. M. Rapid detection of the addition of soybean proteins to cheese and other dairy products by reversed-phase perfusion chromatography. Food Addit. Contam. 2006, 23, 339−347. (38) Haasnoot, W.; du Pre, J. G. Luminex-based triplex immunoassay for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powder. J. Agric. Food Chem. 2007, 55, 3771−3777. (39) Chambers, M. C.; Maclean, B.; Burke, R.; Amodei, D.; Ruderman, D. L.; Neumann, S.; Gatto, L.; Fischer, B.; Pratt, B.; Egertson, J.; Hoff, K.; Kessner, D.; Tasman, N.; Shulman, N.; Frewen, B.; Baker, T. A.; Brusniak, M. Y.; Paulse, C.; Creasy, D.; Flashner, L.; Kani, K.; Moulding, C.; Seymour, S. L.; Nuwaysir, L. M.; Lefebvre, B.; Kuhlmann, F.; Roark, J.; Rainer, P.; Detlev, S.; Hemenway, T.; Huhmer, A.; Langridge, J.; Connolly, B.; Chadick, T.; Holly, K.; Eckels, J.; Deutsch, E. W.; Moritz, R. L.; Katz, J. E.; Agus, D. B.; MacCoss, M.; Tabb, D. L.; Mallick, P. A cross-platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol. 2012, 30, 918− 920. (40) Utsumi, S.; Kinsella, J. E. Structure-Function-Relationships in Food Proteins - Subunit Interactions in Heat-Induced Gelation of 7s, 11s, and Soy Isolate Proteins. J. Agric. Food Chem. 1985, 33, 297−303. (41) Hayakawa, S.; Nakai, S. Relationships of Hydrophobicity and Net Charge to the Solubility of Milk and Soy Proteins. J. Food Sci. 1985, 50, 486−491. (42) Mills, E. N. C.; Jenkins, J. A.; Bannon, G. A. Plant Seed Globulin Allergens. In Plant Food Allergens; Mills, E. N. C., Ed.; Blackwell Publishing: Bodmin, Cornwall, U.K., 2004; pp 141−157. (43) Kolthoff, I. M.; Sandell, E. B. Textbook of Quantitative Inorganic Analysis; Macmillan Co.: New York, NY, USA, 1967. (44) Sanchez, L.; Perez, M. D.; Puyol, P.; Calvo, M.; Brett, G. Determination of vegetal proteins in milk powder by enzyme-linked immunosorbent assay: interlaboratory study. J. AOAC Int. 2002, 85, 1390−1397. (45) Sengupta, C.; Deluca, V.; Bailey, D. S.; Verma, D. P. S. Posttranslational processing of 7S and 11S components of soybean storage proteins. Plant Mol. Biol. 1981, 1, 19−34. (46) Vaia, B.; Smiddy, M. A.; Kelly, A. L.; Huppertz, T. Solventmediated disruption of bovine casein micelles at alkaline pH. J. Agric. Food Chem. 2006, 54, 8288−8293. (47) Wolf, W. J. Soybean Proteins - Their Functional, Chemical, and Physical Properties. J. Agric. Food Chem. 1970, 18, 969−976. (48) Griffin, M. C. A.; Lyster, R. L. J.; Price, J. C. The Disaggregation of Calcium-Depleted Casein Micelles. Eur. J. Biochem. 1988, 174, 339− 343. (49) Kher, A.; Udabage, P.; McKinnon, I.; McNaughton, D.; Augustin, M. A. FTIR investigation of spray-dried milk protein concentrate powders. Vib. Spectrosc. 2007, 44, 375−381. (50) Choodum, A.; Kanatharana, P.; Wongniramaikul, W.; Daeid, N. N. Using the iPhone as a device for a rapid quantitative analysis of trinitrotoluene in soil. Talanta 2013, 115, 143−149.

1505

dx.doi.org/10.1021/jf405617f | J. Agric. Food Chem. 2014, 62, 1498−1505