A Biotin–Streptavidin Amplified Enzyme-Linked Immunosorbent Assay with Improved Sensitivity for Rapid Detection of Ractopamine in muscular tissue Development and Nonspecific Adsorption Reduction

An effective biotin–streptavidin amplified enzyme-linked immunosorbent assay (BA-ELISA) was optimized and characterized for the rapid detection of Ractopamine (RAC) residue in muscular tissue. Purification of the RAC antiserum by protein A-Sepharose 4B followed with bovine serum albumin (BSA)-Sepharose 4B affinity chromatography enhanced the sensitivity and reduce the background adsorption. Blocking with 0.5% skimmed milk power and diluting streptavidin–HRP conjugates with 0.5% BSA/phosphate-buffered saline (PBS) effectively remove the nonspecific adsorption in biotin–streptavidin amplified ELISA system. The established method allowed RAC determination with an IC₅₀ value of 0.3 ± 0.02 ng ml⁻¹ and a limit of detection of 0.02 ± 0.003 ng ml⁻¹, more sensitive than the other reported methods. The variation coefficients of intra-assay and inter-assay were all below 7%. RAC residue in pig muscular tissue could be quantified without matrix effects after a 5-fold extraction and 2-fold dilution with PBS. Recoveries of RAC in pig muscular tissue ranged from 75% to 82.75%. The results were also compared with those from HPLC and a good correlation was obtained (r ² = 0.9822). The characters show that the established biotin–streptavidin amplified ELISA could be potentially useful in rapid detection of RAC in animal-derived foods.     

Occurrence of aflatoxin M1 in raw, pasteurized and UHT milk commercialized in Esfahan and Shahr-e Kord, Iran

Between September 2006 and September 2007, 236 samples of raw (n = 140), pasteurized (n = 48) and UHT (n = 48) milk were collected from supermarkets and from bulk milk tanks of eight dairy plants in the cities of Esfahan and Shahr-e Kord, Iran. All samples were analyzed for aflatoxin M₁ (AFM₁) contamination by ELISA and 213 (90.3%) were positive with mean concentrations 65 ng.l⁻¹. These concentrations are lower than the standards of Codex Alimentarius and FDA (500 ng.l⁻¹), but 119 samples (55.9%) had higher concentrations than the maximum tolerance accepted by some European countries (50 ng.l⁻¹). Mean concentrations of AFM₁ in raw, pasteurized and UHT milk were 68, 56, and 65 ng.l⁻¹, respectively. Mean concentrations of AFM₁ in autumn and winter samples were significantly higher (P < 0.05) than those of spring and summer but differences between AFM₁ concentrations of spring and summer samples were not significantly different. Concentrations of AFM₁ in milk from Shahr-e Kord were significantly lower (P ≤ 0.05) than those from Esfahan.     

Comparative Examination of the Analysis of β-Lactam Antibiotic Residues in Milk by Enzyme, Receptor–Enzyme, and Inhibition Procedures

Antibiotic residues in milk are of great concern to dairy farmers, milk processors, regulatory agencies, and consumers. The aim of this study was to compare the screening tests for residue detection in milk with the purpose of choosing the most sensitive test that could prove residue quantities at maximum residue limits (MRL). The Penzyme S, Delvo-X-press β-lactam II, Delvo SP test, and diffusion test were examined. Milk samples (218) were collected from different segments of milk production chain: farms, milk collection points, dairy, and market. The limit of detection (LOD) of all methods for penicillin G, ampicillin, and amoxicillin were within the MRL, except the LOD for cloxacillin (diffusion test and Delvo-X-press), which were above the MRL value of 30 ng/g. Agreement between test results evaluated by kappa statistic for all tests was substantial (κ = 0.61–0.80). We suggest the samples to be examined by Delvo SP test in the dairy. Positive milk samples should be retested by Penzyme S or Delvo-X-press test because of the high probability that residues are β-lactams, and these tests can prove it. By recurrent examining procedure, starting with the milk samples from pickup trucks, bulk milk tanks in collection points, or bulk milk tanks at farm and from individual producers, the specific milk that was contaminated by antibiotics can be traced.     

Magnetic nanoparticle‐based fluorescent immunoassay for determination of progesterone in milk

A sensitive and rapid magnetic nanoparticle‐based fluorescent immunoassay (MNPs‐FIA) for determination of progesterone in raw milk was developed. The PG‐3‐CMO‐BSA and PG‐3‐CMO‐BSA‐FITC conjugates were obtained. The optimal amount of immobilised antibody on MNPs (0.375 mg) and optimal concentration of PG‐3‐CMO‐BSA‐FITC conjugate (7.5 μg/mL) for MNPs‐FIA were determined. The optimal time and temperature for progesterone MNPs‐FIA were found to be – 12 min and 37 °C. The analytical parameters of immunoassay in cow milk were in the concentration range 0.25‐25 ng/mL and detection limit of 0.065 ng/mL for progesterone. The MNPs‐FIA was applied to evaluate progesterone in three types of milk (UHT, pasteurised and raw cow's milk).     

A real-time immuno-PCR assay for the detection of transgenic Cry1Ab protein

The adaptation and production of transgenic crops harboring Cry1Ab protein is increasing every year globally due to their potent insecticidal activity. The Cry1Ab protein produced by Bacillus thuringiensis confers resistance against several lepidopteran insect pests. The release of transgenic crops/produce in the market worldwide has increased the need of regulatory affairs to monitor and verify the presence of transgenic protein in crops/produce. In this regard, the real-time immuno-PCR (IPCR) assay was developed for the detection and quantification of Cry1Ab protein. The IPCR assay showed high sensitivity with minimum detection limit of 100 pg/mL (0.1 ppb) and found to be 10 times more sensitive than sandwich ELISA. Under the optimized assay conditions, Cry1Ab protein can be determined in the concentration ranged from 100 pg/mL to 100 ng/mL. As results suggest, this assay could be a powerful tool for the detection of even trace amounts of Cry1Ab protein in transgenic crops.     

Quantification of Nitrite/Nitrate in Food Stuff Samples Using 2-Aminobenzoic Acid as a New Amine in Diazocoupling Reaction

2-Aminobenzoic acid has been used as an amine in diazocoupling reaction to form an azo dye in the quantification of nitrite/nitrate at trace level. The formed azo dye has an absorption maximum at 550 nm in aqueous phase, and the resulted dye can be extracted into organic solvent to lower the detection limit. The method obeys Beer's law in the concentration range 0–10 μg of nitrite in 25 ml of aqueous solution with a molar absorptivity of 3.6 × 10³ L mol⁻¹ cm⁻¹ and 0–2 μg of nitrite in 5 ml of organic phase. The detection limit of the dye has been found to be 0.056 μg ml⁻¹. Nitrate is determined by reducing it to nitrite after passing through a copperized cadmium reductor column. The effect of interfering ions on the determination of nitrite/nitrate has been described. The developed method has been applied to determine the nitrite/nitrate trace level in vegetable, fruit juice, and milk powder samples.     

Pulsed Electric Field Induced Aggregation of Food Proteins: Ovalbumin and Bovine Serum Albumin

Ovalbumin (OVA), bovine serum albumin (BSA), and a mixture of the two proteins (OVA + BSA) in solution were exposed to pulsed electric field (PEF) to investigate the protein interaction and aggregation. The results demonstrated the self-aggregation of OVA through disulfide bond due to the exposure of sulfhydryl groups and intermolecular disulfide interactions when PEF intensity exceeded 25 kV cm⁻¹. However, no protein self-aggregation of BSA was observed under PEF treatments of 20 to 35 kV cm⁻¹, which might be ascribed to the less sulfhydryl groups in BSA molecule. The mixture of the two proteins (OVA + BSA) were also subjected to the same PEF treatments. The results showed both OVA and BSA molecules were involved in the protein interaction and aggregation when PEF intensity exceeded 25 kV cm⁻¹. The BSA which was not sensitive to PEF was incorporated covalently into protein aggregates through disulfide cross-links with the other proteins under PEF.     

Development of a rapid and sensitive immunomagnetic-bead based assay for detecting <Emphasis Type="Italic">Bacillus cereus</Emphasis> in milk

Bacillus cereus is a major food-born pathogen in Taiwan and its major syndromes include vomiting, fever and diarrhea. To minimize the possibility of exposing consumers to pathogenic B. cereus, this study develops a rapid and sensitive assay that utilizes immunoliposomal nanovesicles (IMLNs) and immunomagnetic beads (IMBs). In this work, fluorescent dyes (sulforhodamine B)-loaded IMLNs were employed to increase the detection signal; anti-B. cereus antibody-conjugated IMBs were applied to capture B. cereus in samples. Hence in this assay, a sandwich complex was formed as “IMBs-B. cereus-IMLNs”. The optimal IMLNs had a diameter of 300 nm with a conjugated antibody molar percentage (mol%) of 0.25 mol%. The limit of detection (LOD) of this developed assay reaches 10 CFU/mL of B. cereus with the false negative value as zero in 20 parallel assays in milk samples. To evaluate the specificity of this assay, nine Gram positive and negative bacteria were tested and found to cause no significant interference problems. In conclusion, this study elucidates the feasibility of using a novel IMB/IMLN assay for detecting B. cereus and its LOD without pre-enrichment could amount to 10 CFU/mL within 4 h.     

Effect of Calcium on Acrylamide Level and Sensory Properties of Cookies

Thermal process contaminants including acrylamide and hydroxymethylfurfural have been an intensive area of research in recent years. The main pathway of acrylamide formation is linked to the Maillard reaction. The first step is the formation of Schiff base between the carbonyl and α-amino group of asparagine. Presence of cations partially or completely eliminates the formation of Schiff base. This study aimed to investigate the effects of calcium chloride and calcium lactate on acrylamide and hydroxymethylfurfural levels in cookies. The effects of calcium derivatives on the sensory properties of cookies were also investigated. A direct relationship was determined between the amount of calcium in recipe and acrylamide formed in cookies. Addition of 1.0% of Puracal Act 100 decreased acrylamide concentration of cookies from 128 ± 10 ng/g to 24 ± 4 ng/g. In the same time, hydroxymethylfurfural concentration increased from 2.0 ± 0.19 mg/kg to 3.3 ± 0.24 mg/kg by the addition of 1.0% of Puracal Act 100. The calcium derivatives had no effect on cookie diameter and thickness, but the surface colors were different. The use of calcium significantly increased the lightness (L*) parameter, but decreased the redness (a*; p < 0.05). The sensory properties of cookies in terms of sweetness, saltiness and bitterness were not significantly affected by the addition of calcium derivatives at dosages up to 0.5% (p > 0.05).     

PTR-TOF-MS Analysis for Influence of Milk Base Supplementation on Texture and Headspace Concentration of Endogenous Volatile Compounds in Yogurt

In the present study, the effects of milk fat (0.3% and 3.5% w/w), solids non-fat (8.4% and 13% w/w), and modified tapioca starch (0%, 0.5%, 1.0%, 1.5%, and 2.0% w/w) concentrations on the textural and physicochemical properties as well as the concentration of several endogenous flavor compounds in the headspace of set and stirred yogurts were investigated. The novel proton transfer reaction time-of-flight mass spectrometry technique was implemented for the non-invasive determination of the amounts of volatile organic compounds in the samples headspace. Milk fat and skim milk powder supplementation of the milk samples increased significantly the firmness and adhesiveness of yogurts (p < 0.001) and improved the stability of the formed gels by increasing their water holding capacity and reducing the amounts of expulsed whey (3.94 and 5.1 g for the milk fat and SNF-fortified samples). Acetaldehyde was significantly (p < 0.001) higher in the low fat-unfortified systems (6.15 ± 0.48 and 5.6 ± 0.60 ppmv, respectively). A similar trend was also reported in the case of 2-propanone (0.91 ± 0.11 and 1.13 ± 0.07 ppmv), diacetyl (334 ± 37 and 350 ± 34 ppbv), 2,3-pentanedione (54 ± 6 and 55 ± 6 ppbv), and 2-butanone (56 ± 7 and 68 ± 5 ppbv) for the same systems. In contrast, the concentration of flavor compounds in the headspace with hydroxyl groups (ethanol and acetoin) increased (p < 0.001) by solid non-fat fortification of milk base (350 ± 32 and 206 ± 7 ppbv, respectively, for the systems fortified with skim milk powder). Modified tapioca starch addition improved the textural properties and gel stability of yogurts whereas affected only the ethanol concentration (222 ± 16 and 322 ± 55 for the control and 2.0% w/w containing systems, respectively). Our data suggested that the reinforcement of textural and structural properties combined with the protein binding affinity of the flavor compounds seemed to be responsible for the aforementioned observations. In the case of stirred yogurts, the gel breakdown did not provoke significant changes in the headspace concentration of the most compounds, with the exception of ethanol, acetoin, and 2,3-pentanedione being significantly (p < 0.05) higher in the stirred yogurts (267 ± 29, 153 ± 11, and 38 ± 1 ppbv, respectively) than set style ones (232 ± 19, 134 ± 9, and 45 ± 3 ppbv, respectively).     

Application of magnetic immuno-polymerase chain reaction assay for detection of <Emphasis Type="Italic">Salmonella</Emphasis> spp. in chicken meats

Since contaminated chicken meats have been the principal foodborne source of the contamination of Salmonella to human beings and cultural detection methods are labor-intensive and time-consuming, a study evaluating the performance of the combination of two techniques that are immunomagnetic separation (IMS) and polymerase chain reaction (PCR) for the detection of Salmonella in chicken meats was conducted. The IMS and PCR assay combines selective extraction of Salmonella by specific antibodies with primer-specific (primer pair based on the sequence of invA gene) PCR amplification. Initially chicken meat samples, in which no Salmonella contamination had been determined by using ISO 6579 reference method, were inoculated with Salmonella Enteritidis culture and subsequently the shortest non-selective pre-enrichment time, that had been needed for the detection of approximately 1 or 10 CFU/mL chicken meat levels of target bacteria by magnetic immuno-PCR assay, was found by using 14, 12, 10 and 8-h periods. In conclusion, it was found that magnetic immuno-PCR assay was able to detect 1–10 CFU Salmonella/25 g chicken meat, after only incorporating a non-selective pre-enrichment period of 12 h. Therefore, an overall 16-h (magnetic immuno-PCR assay in conjunction with 12-h non-selective pre-enrichment) magnetic immuno-PCR assay statistically evaluated as sufficient (p = 0.182 > 0.05) for rapid and sensitive detection of approximately 1–10 CFU Salmonella from 25 g chicken meat samples. Accordingly, 16-h magnetic immuno-PCR assay can be promising for routine use in the detection of Salmonella in chicken meat samples, and it consequently may prevent the risk of Salmonella infections in regard to chicken meats.     

Use of different thermal indices to assess the quality of pasteurized milks

 Lactoperoxidase activity and lactulose, furosine and undenatured whey protein contents were determined in Spanish commercial milks labelled as pasteurized (group A) and high-temperature pasteurized (group B), in order to assess their quality. Three samples of group A and all of the samples of group B were lactoperoxidase negative. Most samples of group A had measurable amounts of lactulose, even though their concentrations of undenatured β-lactoglobulin were higher than 2600 mg/l. In general, samples of group B showed higher lactulose and furosine and lower undenatured whey protein contents. High levels of furosine and lactulose accompanied by high levels of undenatured β-lactoglobulin could indicate the addition of milk heated at high temperatures, whereas high levels of furosine and relatively low levels of lactulose may have been due to the presence of reconstituted milk powder.     

Screening of Fluoroquinolone Residues in Caprine Milk Using a 5-kg Luminescence Photometer

Fluoroquinolone (FQ) residues in caprine milk were screened by terbium-sensitized luminescence (TSL). After extraction and cleanup using Oasis HLB columns, TSL was measured at λ _ex = 300 nm and λ _em = 546 nm using a 5-kg luminescence photometer. A common threshold was established at x _F50–3σ _F50, where x _F50 and σ _F50 were the mean and standard deviation, respectively, of TSL intensities of milk samples (n = 18) spiked with flumequine at 50 ng/g, its maximum residue limits (MRL) set by the European Union. Enrofloxacin, ciprofloxacin, and danofloxacin at their respective MRLs had higher TSL responses, so could be screened below their MRLs. Among 48 blind samples, each randomly spiked with one FQ at up to 200% of its MRL, 36 were screened correctly without false negative. This rapid protocol can reduce a sample pool to a small fraction for confirmation, hence improve throughput and save assay costs.     

Use of different thermal indices to assess the quality of pasteurized milks

 Lactoperoxidase activity and lactulose, furosine and undenatured whey protein contents were determined in Spanish commercial milks labelled as pasteurized (group A) and high-temperature pasteurized (group B), in order to assess their quality. Three samples of group A and all of the samples of group B were lactoperoxidase negative. Most samples of group A had measurable amounts of lactulose, even though their concentrations of undenatured β-lactoglobulin were higher than 2600 mg/l. In general, samples of group B showed higher lactulose and furosine and lower undenatured whey protein contents. High levels of furosine and lactulose accompanied by high levels of undenatured β-lactoglobulin could indicate the addition of milk heated at high temperatures, whereas high levels of furosine and relatively low levels of lactulose may have been due to the presence of reconstituted milk powder. Received: 29 June 1998     

Gold nanoparticle-based lateral flow assay for detection of staphylococcal enterotoxin B

The lateral flow assay (LFA), a rapid, sensitive, and reproducible technique, was successfully applied to detect staphylococcal enterotoxin B (SEB). The assay was based on a double-antibody sandwich format on a porous nitrocellulose membrane. When SEB-containing samples were applied to the LFA-device, the toxin initially reacted with polyclonal antibody (Pab)-coated colloidal gold particles and then reacted with the fixed Pab on the membrane. These reactions resulted in a red line at the detection zone, with intensity proportional to the SEB concentration (under 100 ng/ml). With this method, 1 ng/ml of SEB can be detected in less than 5 min and was highly reproducible. Signal can be amplified to 10 pg/ml by silver enhancement. This assay also showed no cross-reaction with other SEs, such as SEA, SEC, SED and SEE. The assay was significantly faster than the ELISA or real-time PCR assay and should facilitate early and rapid SEB detection in clinical and food samples.     

Effect of Processing Methods on the In Vitro Protein Digestibility and Vitamin Content of Edible Winged Termite (<Emphasis Type="Italic">Macrotermes subhylanus</Emphasis>) and Grasshopper (<Emphasis Type="Italic">Ruspolia differens</Emphasis>)

The influence of processing methods of toasting and solar drying on the in vitro protein digestibility and vitamins content of edible winged termites, green grasshoppers, and brown grasshoppers consumed in Siaya, district of Kenya, was determined using standard methods. Analysis was done on fresh, toasted, toasted dried, and fresh dried insect samples. There was no significant change (p > 0.05) in protein digestibility in the termite samples, while a significant decrease (p ≤ 0.05) in the grasshopper samples was observed on toasting and drying. There was a significant reduction (p ≤ 0.05) in riboflavin content with 4.18 mg/100 g in fresh termites, 2.76 mg/100 g in toasted termites, 2.26 mg/100 g in fresh dried termites, and 1.50 mg/100 g in toasted dried termites on processing. There was also a significant reduction (p ≤ 0.05) in niacin content in the grasshoppers with 3.61 mg/100 g in fresh green grasshopper, 3.28 mg/100 g in toasted green grasshopper, 3.22 mg/100 g in fresh dried green grasshoppers, and 3.06 mg/100 g in toasted dried green grasshoppers. A significant reduction (p ≤ 0.05) in retinol content with 2.24 μg/g in fresh termites, 1.56 μg/g in toasted termites, 1.02 μg/g in toasted dried termites, and 0.98 μg/g in fresh dried termites was also reported. The processing methods of the insects affected their nutrient potential as evidenced by the changes in protein digestibility and vitamins content. Therefore, optimal processing methods need to be investigated even as we promote commercialization of these insects.     

Simultaneous Determination of 69 Pesticide Residues in Coffee by Gas Chromatography?CMass Spectrometry

A method using gel permeation chromatography (GPC) combined with solid-phase extraction (SPE) cleanup followed by gas chromatography–mass spectrometry (GC-MS) has been established for quantitative determination of 69 pesticide residues in coffee. Based on an appraisal of the characteristics of GC-MS, validation experiments were conducted for 69 pesticides. In the method, 2.0 g samples were mixed with 5 ml water and 1 g sodium chloride and extracted with 5 ml of ethyl acetate by blender homogenization, centrifugation, and filtration. Evaporation was conducted and the sample was injected into a 250 mm × 10 mm S-X3 GPC column, with ethyl acetate–n-hexane (1:2 v/v) as the mobile phase at a flow rate of 3 ml/min. The 4–15 min fraction was collected for the SPE cleanup, which was Envi-Carb SPE cartridge coupled with NH₂-LC SPE cartridge with acetone–ethyl acetate (2:5 v/v) as the eluted solvent. The eluents were collected and then evaporated to dryness, which was redissolved in 0.5 ml ethyl acetate for GC-MS analysis. For the 69 pesticides determined by GC-MS, the portions collected from GPC were concentrated to 0.5 ml and exchanged with 5 ml n-hexane. In the linear range of each pesticide, the correlation coefficient was R ² ≥ 0.99. At the low, medium, and high fortification levels of 0.05–1.0 mg/kg, recoveries fell within 60–120%. The relative standard deviation was between 1.3% and 22.3% for all 69 pesticides. The limits of detection for the method were 10 μg/kg to 150 μg/kg, depending on each pesticide.     

Development of a Green Chromatographic Method for Simultaneous Determination of Food Colorants

A green chromatographic method for the successful separation and determination of eight synthetic food colorants (Tartrazine E 102, Quinoline Yellow E 104, Sunset Yellow E 110, Carmoisine E 122, Ponceau 4R E 124, Allura Red E 129, Indigo Carmine E 132 and Brilliant Blue E 133) was developed. A C8 stationary phase was used and the mobile phase was a mixture of 50 mM phosphate buffer at pH 7 containing triton X-100 (0.25% v/v). The method was validated as regards its selectivity, linearity, precision, accuracy, limit of detection (LOD) and quantification (LOQ). LOD of colorants varied between 0.17 μg mL⁻¹ in Allura Red and 1.91 μg mL⁻¹ in Quinoline Yellow. In the case of LOQ, it was ranged from 0.52 in the Allura Red to 5.79 in the Quinoline Yellow. The method applicability was verified by the determination of colorants present in 22 samples. The 15 samples were only unicolor and the color concentration in these samples varied from 18.426 ± 0.100 to 610.390 ± 4.711 ppm. The method can be used successfully to the determination of binary and ternary color food and drug samples too. This method provides substantial green benefits without using organic solvents in extraction procedure and in both liquid and paper chromatographic methods.     

Concentrations of cadmium and lead in different types of milk

 Concentrations of Pb and Cd were determined in samples of human, raw and pasteurized cow's and goat's milk and powdered infant formula. The following mean Cd concentrations (and ranges) were recorded: in human milk, 2.70 μg/l (0.6–11.3, n=55); in raw cow's milk, 4.88 μg/l (0.7–23.1, n=47); in pasteurized cow's milk, 4.30 μg/l (3.4–5.9, n=6); in goat's milk, 7.81 μg/l (1.0–18.4, n=38); and in powdered, infant formula, 3.81 μg/l (3.4–4.1, n=5). The concentrations (and ranges) of Pb were: in human milk, 8.34 μg/l (0.1–32.3, n=55); in raw cow's milk, 14.82 μg/l (1.3–39.1, n=28); in pasteurized cow's milk, 10.25 μg/l (6.9–19.6, n=6); in goat's milk, 11.86 μg/l (0.4–38.5, n=36); and in powdered, infant formula, 8.30 μg/l (5.1–10.6, n=5). Our data were within the normal ranges for each kind of milk. The Cd and Pb concentrations in goat's milk were significantly higher than the concentrations observed in the other milks, whereas human milk and powdered infant formula presented the lowest Cd and Pb concentrations. A considerable decrease in the concentration of Cd with the stage of lactation was observed. The concentrations of Cd and Pb in human, cow's and goat's milk also varied according to the time of year. The concentrations of Pb and Cd in the different milks did not present any risk to human health (infants or adults). Received: 26 May 1998