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ARTICLE Spore immobilization and its analytical performance for monitoring of aflatoxin M1 in milk Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Auckland on 12/06/14 For personal use only.
V.K. Singh, N.A. Singh, N. Kumar, H.V. Raghu, Pradeep Kumar Sharma, K.P. Singh, and Avinash Yadav
Abstract: Immobilization of Bacillus megaterium spores on Eppendorf tubes through physical adsorption has been used in the detection of aflatoxin M1 (AFM1) in milk within real time of 45 ± 5 min using visual observation of changes in a chromogenic substrate. The appearance of a sky-blue colour indicates the absence of AFM1 in milk, whereas no colour change indicates the presence of AFM1 in milk at a 0.5 ppb Codex maximum residue limit. The working performance of the immobilized spores was shown to persist for up to 6 months. Further, spores immobilized on 96-well black microtitre plates by physical adsorption and by entrapment on sensor disk showed a reduction in detection sensitivity to 0.25 ppb within a time period of 20 ± 5 min by measuring fluorescence using a microbiological plate reader through the addition of milk and fluorogenic substrate. A high fluorescence ratio indicated more substrate hydrolysis due to spore-germination-mediated release of marker enzymes of spores in the absence of AFM1 in milk; however, low fluorescence ratios indicated the presence of AFM1 at 0.25 ppb. Immobilized spores on 96-well microtitre plates and sensor disks have shown better reproducibility after storage at 4 °C for 6 months. Chromogenic assay showed 1.38% false-negative and 2.77% false-positive results while fluorogenic assay showed 4.16% false-positive and 2.77% false-negative results when analysed for AFM1 using 72 milk samples containing raw, pasteurized, and dried milk. Immobilization of spores makes these chromogenic and fluorogenic assays portable, selective, cost-effective for real-time detection of AFM1 in milk at the dairy farm, reception dock, and manufacturing units of the dairy industry. Key words: spore, immobilization, chromogenic, fluorogenic, aflatoxin M1. Résumé : On a eu recours a` l’immobilisation de spores de Bacillus megaterium sur des tubes Eppendorf par adsorption physique aux fins de détection de l’aflatoxine M1 dans le lait, réalisée par observation visuelle en temps réel de la conversion d’un substrat chromogène sur une période de 45 ± 5 minutes. L’apparition d’une couleur bleu ciel signale l’absence d’AFM1 dans le lait, tandis que l’absence de changement de couleur signale la présence d’AFM1 dans le lait au-dela` de la LMR (limite maximale de résidu) de 0,5 ppb du Codex. La performance fonctionnelle des spores immobilisées a perduré jusqu’a` 6 mois. De plus, on a également immobilisé des spores sur une plaque de microtitration noire de 96 puits par adsorption physique et par piégeage sur disque capteur. Cette approche a entraîné une baisse de la sensibilité de détection qui est descendue a` 0,25 ppb sur une période de 20 ± 5 min telle que mesurée par analyse de la fluorescence a` l’aide d’un lecteur de plaques microbiologiques après l’ajout de lait et d’un substrat fluorogène. Le rapport de fluorescence élevé témoigne d’une hydrolyse de substrat plus prononcée en raison de la libération d’enzymes révélatrices issues de la germination des spores en l’absence d’AFM1 dans le lait. En contrepartie, un rapport de fluorescence plus bas signale la présence d’AFM1 a` une concentration de 0,25 ppb. Les spores immobilisées sur plaques de microtitration de 96 puits et sur disques capteurs affichaient une meilleure reproductibilité après 6 mois de remisage a` 4 °C. L’épreuve chromogène a présenté un taux de résultats faussement positifs de 1,38 % et un taux de résultats faussement négatifs de 2,77 % tandis que l’épreuve fluorogène s’est soldée par 4,16 % de faux positifs et 2,77 % de faux négatifs sur la foi d’une analyse de la présence d’AFM1 dans 72 échantillons de lait cru, pasteurisé ou déshydraté. L’immobilisation de spores permet a` ces épreuves chromogènes et fluorogènes de devenir portatives, sélectives et économiquement efficientes eu égard a` la détection en temps réel d’AFM1 dans le lait, qu’on se trouve a` la ferme laitière, au quai de réception ou dans les usines de transformation de l’industrie laitière. [Traduit par la Rédaction] Mots-clés : spore, immobilisation, chromogène, fluorogène, aflatoxine M1.
Introduction The practical utility of whole-cell biosensors in field applications requires that bacteria be formulated at a lower cost in a way that maintains their sustainability and recognition competency while enabling their long-term storage and transport (Belkin 2003). Several techniques have been reported in recent years for preservation of cells, including immobilization in different types of matrices, freeze-drying, and formation of spores. Among these, the use of Bacillus spores has emerged as a promising approach for
long-term storage of whole-cell biosensing systems for arsenic, zinc (Date et al. 2007), and bacitracin antibiotic (Fantino et al. 2009) at ambient temperature as well as in extreme environmental conditions. Immobilization is a general term used for a wide variety cell attachment or entrapment methods (Lopez et al. 1997). Cell immobilization has been defined as the physical localization of viable microbial cells to a defined region in such a way as to limit their free movement while retaining their catalytic activities (Covizzi et al. 2007). Various techniques used for immobilization
Received 17 July 2014. Revision received 16 September 2014. Accepted 17 September 2014. V.K. Singh, N.A. Singh, N. Kumar, H.V. Raghu, P.K. Sharma, and A. Yadav. Microbial Biosensors and Food Safety Laboratory, Dairy Microbiology Division, National Dairy Research Institute, Indian Council of Agricultural Research, Karnal 132001, Haryana, India. K.P. Singh. Department of Biotechnology, Shri Ram College, Muzaffarnagar, Uttar Pradesh, India. Corresponding author: H.V. Raghu (e-mail: [email protected]). Can. J. Microbiol. 60: 793–798 (2014) dx.doi.org/10.1139/cjm-2014-0465
Published at www.nrcresearchpress.com/cjm on 22 September 2014.
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of microorganisms include gel bead entrapment, carrier binding, adsorption to solid surface, encapsulation, and biofilm development (Chien et al. 2012). Adsorption is the simplest method of reversible immobilization based on van der Waals forces, ionic binding, hydrophobic interactions, and hydrogen bonds. However, it is based on weak forces but still facilitates an efficient binding process (Flickinger and Drew 1999). The immobilization of enzymes could also be done by reversible physical adsorption of natural and synthetic organic polymers. Natural polymers include polysaccharides (cellulose, agar, agarose, and alginate), proteins (collagen, albumin), and carbon, whereas synthetic polymers contain polystyrenes, polyacrylamides, polyamides, and vinyl and allyl polymers (Brena and Batista-Viera 2006). Covalent bonding is one of the most widely used methods for enzyme immobilization and comes under the irreversible immobilization category. Despite all advantages that this method presents when applied to enzymes, it is rarely applied for immobilization of cells because agents used for covalent bond formation are usually cytotoxic and it is difficult immobilize cells without any damage (Flickinger and Drew 1999). Encapsulation is another irreversible immobilization method, in which biocatalysts are restricted by the membrane walls (usually in a form of a capsule) but are free-floating within the core space (Bickerstaff 1997; Cao 2005). Entrapment is an irreversible method, where immobilized particles or cells are entrapped in a support matrix or inside fibres (Górecka and Jastrzêbska 2011). An advantage of using immobilized spores as biocatalysts is that there is an increase in the provision of higher cell density to increase overall substrate conversion rates and shelf life (Chien et al. 2012). Aflatoxin M1 (AFM1) is the 4-hydroxy derivative of aflatoxin B1 and is secreted in the milk of mammals that consume feed containing aflatoxin B1 (Tajik et al. 2007). AFM1 exhibits a high level of genotoxic activity with serious health risk in the dairy food chain; the accumulation of this mycotoxin damages DNA (Shundo and Sabino 2006; Viegas et al. 2012). AFM1 induces DNA adducts, leading to genetic changes in the target cells, which then cause DNA strand breakage, DNA base damage, and oxidative damage that may ultimately lead to cancer (Hamid et al. 2013). According to the International Agency for Research on Cancer (IARC 2002), aflatoxin is classified as a Group 1 human carcinogens, and its actionable level 15–20 ppb in animal feed and 0.5 ppb (AFM1) in dairy products has been established by Codex Alimentarius Commissions (2001). The European Union specified an AFM1 limit of 0.05 ppb in milk for all European Union member states, and 25 ppt for baby food (Cucci et al. 2007). Various biosensor methods used for detection and quantification of AFM1 in milk have been developed (Micheli et al. 2005; Wanga et al. 2009; Dinckaya et al. 2011), and few commercial systems like enzyme-linked immunosorbent assay (Mohammadian et al. 2010; Omar 2012), radio-immune-based Charm assay (Offiah and Adesiyun 2007), high-performance liquid chromatography (HPLC) with fluorescence detection are available. But all of these methods have inherent limitations for being costly and require huge infrastructure and experienced personnel while processing samples. These innovations have limited scope in their application at field level where milk is being produced, collected, chilled, and transported further to dairy plants for their processing (Singh et al. 2013). Crude aflatoxin at 30 ppm was found to inhibit 12 species of the genera Bacillus, Clostridium, and Streptomyces (Burmeister and Hesseltine 1965). Bacillus spp. were reported to be highly sensitive to aflatoxin (Madhyastha et al. 1994). Escherichia coli, Bacillus subtilis, and Enterobacter aerogenes were found to have moderate sensitivity towards a new aflatoxin, namely aflatoxin B2b (Wang et al. 2012). Whole-cell-based sensing systems have been applied in environmental monitoring (not for AFM1), but on-site monitoring of analytes is restricted because of their limited shelf life and transportability (Date et al. 2010). From our previous studies, we have
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developed a spore-inhibition-based enzyme substrate assay for monitoring of AFM1 in milk using Bacillus megaterium 2949 spores (Kumar et al. 2010; Singh et al. 2013). These spores possess a unique ability to sense environmental changes in response to specific a “germinant”, and their transformation into growing vegetative cells in real time has an enormous scope for their application in biosensing of contaminants in food products. The spore germination concept involves the release of marker enzymes and their action on specific chromogenic or fluorogenic substrates as a means for the detection of AFM1 in milk systems (Singh et al. 2013). In our present study, attempts were made to immobilize the same B. megaterium 2949 spores in different formats to increase its shelf life for the detection of AFM1 in milk using chromogenic and fluorogenic assay. These assays owing to their portability and costeffectiveness can be used in dairy plants as well as in research and development.
Materials and methods Chemicals and instrumentation Eppendorf tubes (1.5 mL) were procured from Axygen, USA, and 96-well microtitre plates were procured from Greiner, Germany. The sensor disks were prepared by using glass fibre paper purchased from Millipore (India), which was cut into 6.5-mmdiameter size. AFM1 was procured from Sigma Aldrich (India) and acetone from Rankem (India). Diacetyl fluorescein (DAF) obtained from SigmaAldrich (India). Distilled water used during the experiments was produced in Bioage-Labpure ultra plus (BIO-AGE, Punjab, India). Incubation was done in an Eppendorf Innova 42R shaker (Germany). For spore immobilization, lyophilizer procured from Hanil Science Co., Ltd., Korea, and a vacuum drier from Thermo Scientific, USA, were used. For absorbance of spores and fluorescence measurement, a Victor X3 2030 microbiological plate reader from PerkinElmer (USA) was used. Validation work was done by radioimmunoassay, i.e., Charm system 6602 (Charm Science Inc., USA). The data processing was done by using Microsoft excel (Window 7) data analysis. Spore preparation Fifteen strains of B. megaterium (MTCC 2444, 2412, 428, 453, 1684, 3165, 4911, 6129, 6130, 6131, 7163, 7349, 2949, and ATCC 9885, 14581) were screened for minimal inhibitory concentrations against AFM1 using a disc assay (Singh et al. 2013). Bacillus megaterium MTCC 2949 was streaked on nutrient agar plates for activation overnight at 37 °C followed by transfer into 5.0 mL propagation medium, i.e., tryptone glucose yeast extract (TGYE) broth. Further, indicator B. megaterium 2949 strain was propagated in the TGYE broth medium and spore production was done in the newly developed sporulation medium, as per the protocol explained by Singh et al. (2013). The final pellet obtained was analysed for total viable count and spore count (Downes and Ito 2001). The spore suspension was refrigerated at 2–8 °C and immobilized for its application. Immobilization of spores by different techniques By physical adsorption in Eppendorf tubes Bacillus megaterium spores were immobilized in Eppendorf tubes at a concentration of 107 spores/mL (Allam et al. 2011) and were then covered with parafilm and punctured with a needle. These Eppendorf tubes containing spores were lyophilized at 900g 20 °C, under 670 mm pressure for 1 ± 0.15 h. By physical adsorption in 96-well microtitre plates Bacillus megaterium spores were immobilized in 96-well microtitre plates at a concentration of 108 spores/mL (Allam et al. 2011) by vacuum drying for 1.30 h. Since 96-well microtitre plates are Published by NRC Research Press
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made up of polystyrene, the spores were physically adsorbed by this synthetic polymer.
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By entrapment in sensor disks Sensor disks (size 6.5 mm diameter) were placed in sterile glass Petri plates. Spores at a concentration of 108 spores/mL (Allam et al. 2011) were immobilized by entrapment in sensor disks by air-drying in an incubator at 37 °C for 4 h. Disks without spores and having the same quantity of buffer were labelled as control disks and were dried under similar conditions (Rotman and Cote 2003). Monitoring of analytical performance of immobilized spores Out of different sets of immobilized spores, refrigerated at 4 °C, one set was taken at different intervals from each of the experimental settings, and spores were germinated to vegetative cells in the presence of milk by the reaction of released enzyme, i.e., acetyl esterase, on chromogenic or fluorogenic substrate without any exogenous germinating agents, and the signal was measured visually or by a microbiological plate reader (Victor X3). Chromogenic assay Three batches (A, B, C) of immobilized spores were analysed for their analytical performance after storage at different time intervals. Four Eppendorf tubes were taken for checking immobilized spore performance by chromogenic assay. Raw milk without AFM1 was taken as negative sample; the same milk spiked with AFM1 at 0.5 ppb (Codex limit) was the positive sample and its recovery (94%–100%) was checked by a reference method, i.e., radioimmunoassay, as depicted in Fig. 2 (Offiah and Adesiyun 2007). In radioimmunoassay, 300 L of AF solution (provided by Charm Sciences, USA) was added in a test tube. Then 3/4 of the tube was filled with the milk sample (cooled at 4 ± 2 °C), mixing followed by centrifugation at 3150g for 5 min. The fat layer of the milk sample was removed and a White (receptor) tablet was added to the test tube, followed by 300 ± 100 L of distilled water. Mixing was done for 10 s, and 5.0 mL of the centrifuged milk sample was added. A purple tablet was added immediately and mixed by swirling milk up and down for 15 s. The sample was incubated at 35 ± 2 °C for 3 min followed by centrifugation for 5 min at 3150g. The sample was removed from the centrifuge, and the milk poured off. The fat ring was removed and wiped dry with swabs. A 300 ± 100 L volume of water was added and mixed thoroughly to break up the tablet. Scintillation fluid (300 ± 0.5 mL) was added and mixed for uniform appearance. Counts per minute was taken in the Charm system for 60 s on a [3H] channel. Negative and positive milk samples were pretreated at 80 °C for 15 min, followed by cooling (room temperature) under tap water in a test tube. Negative and positive milk samples (1 mL) and chromogenic substrate (25 mL) were added to Eppendorf tubes containing immobilized spores, followed by incubation at 37 °C for 45 min, and were observed for colour change along with the control without spores, i.e., milk and substrate (Singh et al. 2013). Intertesting of 6 different batches of immobilized spores was performed by chromogenic assay after 1 month of storage. Fluorogenic assay using 96-well microtitre plates One milligram of fluorogenic substrate, i.e., DAF, was dissolved in 2.5 mL of acetone; a second stock was prepared by the addition of 50 L of first stock to 475 L of 10 mmol/L potassium phosphate buffer. Further stock of DAF (11.2 nmol/L) was prepared in milk. A fluorogenic assay was performed in microwell plates containing spores by the addition of 100 L of pretreated milk with DAF, (C of equation 1), along with the controls, i.e., spores with DAF (A of equation 1) and milk with DAF (B of equation 1). After incubation, fluorescence was measured at specific excitation/emission spectra of 485 nm / 535 nm by using a Victor X3 2030 microbiological plate reader.
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A control point for the fluorogenic assay for detection of AFM1 was established by negative milk (without AFM1) and positive milk (AFM1 spiked at 0.25 ppb, as fluorogenic assay was found sensitive up to ≥0.25 ppb). Fluorogenic enzyme assays are more sensitive than chromogenic assays (Zhou et al. 1997) owing to the higher interaction of spores with substrate in a small quantity of milk. The results obtained were analysed for enzyme activity in terms of fluorescence using the following equation: (1)
Enzyme activity (ratio) ⫽ C/A ⫹ B
where A is enzyme activity (photon count per 0.1 s) of spores, B is enzyme activity of milk (photon count per 0.1 s), and C is enzyme activity of spores with milk (photon count per 0.1 s), Control Point of fluorogenic assay at 0.25–0.35 ppb. An enzyme activity (ratio) of ≥0.35 indicates the sample is negative for the presence of AFM1, while ≤0.35 indicates the presence of AFM1 in milk. Fluorogenic assay using sensor disk The spore-sensor disks were placed in 96-well microtitre plates. A mixture of milk and DAF at the rate of 100 L per well was added to microwell plates, followed by incubation at 37 ± 2 °C for 20 ± 5 min. After incubation, fluorescence was measured at specific excitation/emission spectra of 485 nm / 535 nm by using a Victor X3 2030 microbiological plate reader. The results obtained were analysed for enzyme activity in terms of fluorescence using equation 1 above. Experimental design and statistical analysis Experiments were designed using CRD (Critical Random Design) with 3 replicates. The data were analysed statistically and expressed as the mean (±SD) of 3 replicates, and correlation coefficient was calculated according to Snedecor and Cochran (1980).
Results and discussion Spore preparation Out of 15 strains of B. megaterium, strain MTCC 2949 was found to be the most sensitive against AFM1 at 0.25–0.5 ppb, so this strain was used in our assays. In TGYE broth medium, 75% sporulation has been achieved (Singh et al. 2013). Chromogenic assay Three different batches of immobilized spores were analysed monthly. Negative milk samples (i.e., without AFM1) turned skyblue colour while positive milk samples (i.e., with AFM1) showed no colour change. These 3 batches gave a distinguishable colour difference between positive (≥0.5 ppb) and negative samples for up to 5.5–6 months (Fig. 1a), but after 6 months the intensity of the sky-blue colour decreased, resulting in less differentiation between positive and negative samples. After 1 month of storage, 6 batches of immobilized spores showed similar results by chromogenic assay (Fig. 1b), indicating no significant variation in colour intensity was found in the different batches. Negative milk (without AFM1) and positive milk (with AFM1) samples used for analytical performance of chromogenic and fluorogenic assays were selected based on Charm assay results. A control point of AFM1 at 0.5 ppb was set by running a 0.5 ppb positive sample and a 0 ppb control standard (aflatoxin-free) provided by Charm Sciences (3 replicates). Readings at the maximum residue limit (MRL) dose and 0 ppb control were 317 ± 7 and 1100 ± 20, (mean ± SE, n = 3), respectively. So any reading ≤317 was considered positive for AFM1, and a reading between 318 and 1100 or above was considered negative for AFM1. The Charm reading was found inversely proportional to aflatoxin concentration, meaning that the greater the Charm reading the lower the aflatoxin conPublished by NRC Research Press
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Fig. 1. Analytical performance of immobilized spores by chromogenic assay. (a) Working performance of 3 batches. (b) Intertesting of different batches for chromogenic assay (n = 3).
centration in the sample, as depicted in the calibration graph (Fig. 2). Fluorogenic assay using 96-well micro-titre plates Milk (positive or negative) and DAF were added to 96-well microtitre plates containing spores, followed by fluorescence measurement at 485 nm / 535 nm excitation/emission spectra by using a microbiological plate reader (Victor X3 2030). Fluorescence, based on the acetyl esterase enzymatic activity of B. megaterium, was increased after spore germination in the presence of negative milk, but a decreasing trend was observed with positive milk, as the spores were being inhibited by AFM1 (Singh et al. 2013). Analytical performance of immobilized spores in 96-well microtitre plates was analysed for up to 6 months. These spores worked well with a negative sample, with a high fluorescence ratio, ratio decrease with an increase of AFM1 level in milk with 0.25 ppb detection limit up to 6 months, as depicted in Fig. 3. Although spores were immobilized in many wells of the microtitre plates, during the monthly analysis, only 3 microwells were used to perform the fluorogenic assay. Although the whole microtitre plate was incubated, only those spores treated with milk and DAF germinated, while other spores were unaffected. Fluorogenic assay using sensor disks A fluorogenic assay was performed by the addition of 100 L of pretreated milk plus DAF (11.2 nmol/L) on sensor disk immobilized spores (C), along with the controls, i.e., disks containing spores with DAF only (A) and disks containing spores with milk and DAF (B). After incubation for 20 ± 5 min at 37 ± 2 °C, fluorescence was measured at 485 nm / 535 nm excitation/emission spectra. The results obtained were analysed for enzyme activity in terms of fluorescence (ratio) = C/A+B. No significant differences (positive milk critical difference = 0.00234, and negative milk critical difference = 0.00154, ␣ ≤ 0.05, n = 3) were found in the viability and sensing ability of the vegetative cells upon storage of the spores at 4 °C in refrigerated conditions. During their fluorogenic performances, they retain the difference of high and low ratio between negative and positive samples respectively (Fig. 4). These results support that of Date et al. (2007), who demonstrated that a sporebased biosensing mechanism was stable for its analytical performance for a period of 6–8 months when stored at room temperature.
Fig. 2. Charm reading against aflatoxin M1 concentration.
Fig. 3. Fluorescence ratio after storage of immobilized spores, as determined by fluorogenic assay using microwell plates (n = 3).
Fig. 4. Fluorescence ratio after storage of immobilized spores, as determined by fluorogenic assay using sensor disks (n = 3).
Comparative evaluation with real food samples The above 3 immobilization methods are compared in Table 1 in terms of sensitivity, shelf life, limitations, etc. During analytical performance of chromogenic and fluorogenic assays using 72 milk samples containing raw, pasteurized, and dried milk it was observed that the chromogenic assay showed an incidence of AFM1 in 20% of the milk samples at the 0.5 ppb Codex MRL limit, while the fluorogenic assay showed an incidence of AFM1 in 23.6% Published by NRC Research Press
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Table 1. Comparison of spore immobilization methods. Property
In Eppendorf tubes
96-well microtitre plates
Sensor disks
Mechanism
Physical adsorption on polypropylene synthetic polymer 6 months 0.5 ppb Qualitative —
Physical adsorption on polystyrene synthetic polymer 6 months 0.25 ppb Semiquantitative For 1 trial, 3 wells containing spores are needed, but the whole plate is incubated because we cannot separate wells
Entrapment in sensor disk
Shelf life Sensitivity* Detection Limitations
6 months 0.25 ppb Semiquantitative —
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*ppb, parts per billion.
Fig. 5. Comparative results of fluorogenic and chromogenic assays with radioimmunoassay.
Table 2. Comparison of reference methods available for aflatoxin M1 (AFM1) analysis with our developed assays. Detection method
Mechanism
HPLC
Immuno-affinity columns containing antibodies against AFM1 ELISA Antigen–antibody reaction Radioimmunoassay Binding of radio-labeled aflatoxin with microbial receptor sites Charm ROSA Lateral flow assay Chromogenic assay Spore-inhibition-based enzyme (present work) substrate reaction Fluorogenic assay Spore-inhibition-based enzyme (present work) substrate reaction
Assay time (min) Sensitivity* 60
Limitations
6 ppt
75 14
Problem in extraction, costly, laboratory-bound, need experienced person 50 ppt Costly, laboratory-bound, need experienced person 0.5 and 0.25 ppb Costly, laboratory-bound, need experienced person
11 45
0.5 ppb 0.5 ppb
Costly, laboratory-bound, need experienced person —
20
0.25 ppb
—
*ppt, parts per trillion; ppb, parts per billion.
of the samples at ≤0.25 ppb (≤Codex MRL limit). The same samples were also analysed by the Charm-based method, and 22.2% of the samples of raw milk, pasteurized milk, and dried milk were found positive at 0.5 ppb AFM1. By comparing the results of the chromogenic-based assay with the radioimmunoassay (Charm assay), the chromogenic assay was found to yield 1.38% falsenegative and 2.77% false-positive results. When the fluorogenic assay was compared with the radioimmunoassay (Charm assay), the fluorogenic assay generated false-positive results in 4.16% of the samples and false-negative results in 2.77% of the samples (Fig. 5). However, the chromogenic assay and fluorogenic assay were found to correlate 0.997 and 0.999, respectively, with the radioimmunoassay (Charm assay). Duarte et al. (2013) investigated 40 samples of pasteurized and ultra-high temperature processed milk for AFM1 and found that 27.5% of the samples were above the cutoff limit of 0.05 ppb of European Union MRL, using ELISA. The developed concept of chromogenic assay for detection of AFM1 detection was validated and a correlation of 0.97 was established with AOAC approved Charm 6602 and ELISA at Codex MRL, with minimal false-positive and -negative results (Singh et al. 2013). After a comparative analysis of all the reference methods avail-
able on the market for AFM1 analysis with our chromogenic and fluorogenic assays, it can be concluded that our developed methods can be used in dairy, research and development, and milk chilling centres because of their portability, cost-effectiveness, sensitivity, etc. (Table 2).
Conclusion The present study reports spore immobilization through different formats, i.e., by physical adsorption in Eppendorf tubes and in 96-well black microtitre plates and by entrapment in sensor disk. These immobilized spores were germinated in the presence of milk, followed by enzyme release and its action on the substrate, i.e., chromogenic or fluorogenic. Analytical performance of these spores through analysis by chromogenic and fluorogenic assays on a monthly basis showed that in addition to being sensitive, reproducible, robust, easy to use, and cost-effective, the spores also maintain their viability and sensing capability during storage in refrigerated conditions. Spore immobilization provides portability to these assays, making it suitable for the detection of AFM1 in milk at the dairy farm, reception dock, manufacturing unit, Published by NRC Research Press
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and research and development centres. Overall, the analytical performance of chromogenic and fluorogenic assays in terms of the detection limit and reproducibility did not change significantly during the 6 months of storage in refrigerated conditions.
Acknowledgements The National Agricultural Innovative Program (NAIP) is greatly acknowledged for supporting this research work. The Director of the NDRI is thankfully acknowledged for providing facilities at NDRI, Karnal.
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References Allam, R.F., Shafei, M.S., Refai, A.H. El., Ali, M.I., and Mohamed, S.S. 2011. 11-Hydroxylation of cortexolone (Reichstein’s compound S) to hydrocortisone using immobilized Cunninghamella elegans spores. Malays. J. Microbiol. 7(1): 7–13. Belkin, S. 2003. Microbial whole-cell sensing systems of environmental pollutants. Curr. Opin. Microbiol. 6: 206–212. doi:10.1016/S1369-5274(03)00059-6. PMID:12831895. Bickerstaff, GF. 1997. Immobilization of enzymes and cells. In Methods in biotechnology. Edited by G.F. Bickerstaff. Humana Press, Totowa, N.J., USA. pp. 7–19. Brena, B.M., and Batista-Viera, F. 2006. Immobilization of enzymes — a literature survey. In Methods in biotechnology: immobilization of enzymes and cells. Edited by J. M. Guisan, Humana Press Inc., Totowa, N.J., USA., p. 15–30. Burmeister, H.R., and Hesseltine, C.W. 1965. Survey of the sensitivity of microorganisms to aflatoxin. Appl. Microbiol. 14(3): 403–404. PMID:5970826. Cao, L. 2005. Carrier-bound immobilized enzymes: principles, applications and design. John Wiley & Sons, Weinheim, Germany. doi:10.1002/3527607668. Chien, M., Nakahata, R., Ono, T., Miyauchi, K., and Endo, G. 2012. Mercury removal and recovery by immobilized Bacillus megaterium MB. Front. Chem. Sci. Eng. 6(2): 192–197. doi:10.1007/s11705-012-1284-3. Codex Alimentarius Commissions (CAC). 2001. Comments submitted on the draft maximum level for AFM1 in milk. Codex Committee on Food Additives and Contaminant 33rd sessions, Hague, The Netherlands. Available from ftp://ftp.fao.org/codex/Meetings/CCFAC/ccfac33/fa01_20e.pdf. Covizzi, L.G., Giese, E.C., Gomes, E., Dekker, R.F.H., and Silva, R. 2007. Immobilization of microbial cells and their biotechnological applications. Semina: Ciências Exatas Tecnológicas. 28: 143–160. Cucci, C., Mignani, A.G., Dall’Asta, C., Pela, R., and Dossena, A. 2007. A portable fluorometer for the rapid screening of M1 aflatoxin. Sens. Actuators, B, 126: 467–472. doi:10.1016/j.snb.2007.03.036. Date, A., Pasini, P., and Daunert, S. 2007. Construction of spores for portable bacterial whole-cell biosensing systems. Anal. Chem. 79: 9391–9397. doi:10. 1021/ac701606g. PMID:18020369. Date, A., Pasini, P., and Daunert, S. 2010. Fluorescent and bioluminescent cellbased sensors: strategies for their preservation. Adv. Biochem. Eng. Biotechnol. 117: 57–75. doi:10.1007/10_2009_22. PMID:20091290. Dinckaya, E., Kimik, O., Sezginturk, M.K., Altug, C., and Akkoca., A. 2011. Development of an impedimetric aflatoxin M1 biosensor based on a DNA probe and gold nanoparticles. Biosens. Bioelectron. 26: 3806–3811. doi:10.1016/ j.bios.2011.02.038. PMID:21420290. Downes, F.P., and Ito, K. (Editors). 2001. Compendium of methods for the microbiological examination of foods. American Public Health Association, Washington, D.C., USA. Duarte, S.C., Almeida, A.M., Teixeira, A.S., Pereira, A.L., Falcao, A.C., Pena, A., and Lino, C.M. 2013. Aflatoxin M1 in marketed milk in Portugal: assessment of human and animal exposure. Food Cont. 30(2): 411–417. doi:10.1016/j. foodcont.2012.08.002. Fantino, J.R., Barras, F., and Denizot, F. 2009. Sposensor: a whole-bacterial biosensor that uses immobilized Bacillus subtilis spores and a one-step incubation/detection process. J. Mol. Microbiol. Biotechnol. 17: 90–95. doi:10.1159/ 000206634. PMID:19258707.
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Flickinger, M.C., and Drew, S.W. (Editors). 1999. Enzyme, immobilization methods. In Encyclopedia of bioprocess technology: fermentation, biocatalysis and bioseparation, John Wiley & Sons, New York. pp. 1062–1063. Górecka, E., and Jastrzêbska, M. 2011. Immobilization techniques and biopolymer carriers. Biotechnol. Food Sci. 75(1): 65–86. Hamid, A.S., Tesfamariam, I.G., Zhang, Y., and Zhang, Z.G. 2013. Aflatoxin B1induced hepatocellular carcinoma in developing countries: geographical distribution, mechanism of action and prevention. Oncol. Lett. 5: 1087–1092. PMID:23599745. IARC. 2002. Some mycotoxins, naphthalene and styrene. In IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol 82. International Agency for Research on Cancer, Lyon, France. pp. 171–300. Kumar, N., Singh, N.A., Singh, V.K., Bhand, S., and Malik, R.K. 2010. Development of spore inhibition based enzyme substrate assay (SIB-ESA) for monitoring AFM1 in milk. Indian Patent 3064/DEL/2010. Office of the Controller General of Patents, Designs and Trade Marks, Mumbai, India. Lopez, A., Lazaro, N., and Marques, A.M. 1997. The interphase technique: a simple method of cell immobilization in gel-beads. J. Microbiol. Methods, 30: 231–234. doi:10.1016/S0167-7012(97)00071-7. Madhyastha, M.S., Marquardt, R.R., Masi, A., Borsa, J., and Frohlich, A.A. 1994. Comparison of toxicity of different mycotoxins to several species of bacteria and yeasts: use of Bacillus brevis in a disc diffusion assay. J. Food Prot. 57(1): 48–53. Micheli, L., Grecco, R., Badea, M., Moscone, D., and Palleschi, G. 2005. An electrochemical immunosensor for aflatoxin M1 determination in milk using screen- printed electrodes. Biosens. Bioelectron. 21(4): 588–596. doi:10.1016/ j.bios.2004.12.017. PMID:16202872. Mohammadian, B., Khezri, M., Ghasemipour, N., Mafakheri, S., and Langroudi, P. 2010. Aflatoxin M1 contamination of raw and pasteurized milk produced in Sanandaj, Iran. Arch. Razi Inst. 65(2): 99–104. Offiah, N., and Adesiyun, A. 2007. Occurrence of aflatoxins in peanuts, milk, and animal feed in Trinidad. J. Food Prot. 70(3): 771–775. PMID:17388075. Omar, S.S. 2012. Incidence of aflatoxin M1 in human and animal milk in Jordan. J. Toxicol. Environ. Health, Part A, 75: 1404–1409. doi:10.1080/15287394.2012. 721174. PMID:23095158. Rotman, B., and Cote, M.A. 2003. Application of a real-time biosensor to detect bacteria in platelet concentrates. Biochem. Biophys. Res. Commun. 300: 197– 200. doi:10.1016/S0006-291X(02)02828-0. PMID:12480543. Shundo, L., and Sabino, M. 2006. AFM1 in milk by immune affinity column cleanup with TLC/HPLC determination. Braz. J. Microbiol. 37: 164–167. doi:10. 1590/S1517-83822006000200013. Singh, N.A., Kumar, N., Raghu, H.V., Sharma, P.K., Singh, V.K., Khan, A., and Raghav, N. 2013. Spore inhibition-based enzyme substrate assay for monitoring of aflatoxin M1 in milk. Toxicol. Environ. Chem. 95(5): 765–777. doi:10. 1080/02772248.2013.807540. Snedecor, G.W, and Cochran, W.G. 1980. Statistical methods. 7th ed. The Iowa State University Press, Iowa. Tajik, H., Rohani, S.M.R., and Moradi, M. 2007. Detection of aflatoixn M1 in raw and commercial pasteurized milk in Urmia, Iran. Pak. J. Biol. Sci. 10: 4103– 4107. doi:10.3923/pjbs.2007.4103.4107. PMID:19090287. Viegas, S., Veiga, L., Malta-Vacas, J., Sabino, R., Figueredo, P., Almeida, A., et al. 2012. Occupational exposure to aflatoxin (AFB1) in poultry production. J. Toxicol. Environ. Health, Part A, 75: 1330–1340. doi:10.1080/15287394.2012. 721164. PMID:23095151. Wang, H., Lu, Z., Qu, H.J., Liu, P., Miao, C., Zhu, T., et al. 2012. Antimicrobial aflatoxins from the marine-derived fungus Aspergillus flavus 092008. Arch. Pharm. Res. 35: 1387–1392. doi:10.1007/s12272-012-0808-1. PMID:22941481. Wanga, Y., Dostalek, J., and Knalla, W. 2009. Long range surface plasmonenhanced fluorescence spectroscopy for the detection of aflatoxin M1 in milk. Biosens. Bioectron. 24(7): 2264–2267. doi:10.1016/j.bios.2008.10.029. PMID:19095432. Zhou, M., Diwu, Z., Panchuk-Voloshina, N., and Haugland, R.P. 1997. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem. 253: 162–168. doi:10. 1006/abio.1997.2391. PMID:9367498.
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ARTICLE Spore immobilization and its analytical performance for monitoring of aflatoxin M1 in milk Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Auckland on 12/06/14 For personal use only.
V.K. Singh, N.A. Singh, N. Kumar, H.V. Raghu, Pradeep Kumar Sharma, K.P. Singh, and Avinash Yadav
Abstract: Immobilization of Bacillus megaterium spores on Eppendorf tubes through physical adsorption has been used in the detection of aflatoxin M1 (AFM1) in milk within real time of 45 ± 5 min using visual observation of changes in a chromogenic substrate. The appearance of a sky-blue colour indicates the absence of AFM1 in milk, whereas no colour change indicates the presence of AFM1 in milk at a 0.5 ppb Codex maximum residue limit. The working performance of the immobilized spores was shown to persist for up to 6 months. Further, spores immobilized on 96-well black microtitre plates by physical adsorption and by entrapment on sensor disk showed a reduction in detection sensitivity to 0.25 ppb within a time period of 20 ± 5 min by measuring fluorescence using a microbiological plate reader through the addition of milk and fluorogenic substrate. A high fluorescence ratio indicated more substrate hydrolysis due to spore-germination-mediated release of marker enzymes of spores in the absence of AFM1 in milk; however, low fluorescence ratios indicated the presence of AFM1 at 0.25 ppb. Immobilized spores on 96-well microtitre plates and sensor disks have shown better reproducibility after storage at 4 °C for 6 months. Chromogenic assay showed 1.38% false-negative and 2.77% false-positive results while fluorogenic assay showed 4.16% false-positive and 2.77% false-negative results when analysed for AFM1 using 72 milk samples containing raw, pasteurized, and dried milk. Immobilization of spores makes these chromogenic and fluorogenic assays portable, selective, cost-effective for real-time detection of AFM1 in milk at the dairy farm, reception dock, and manufacturing units of the dairy industry. Key words: spore, immobilization, chromogenic, fluorogenic, aflatoxin M1. Résumé : On a eu recours a` l’immobilisation de spores de Bacillus megaterium sur des tubes Eppendorf par adsorption physique aux fins de détection de l’aflatoxine M1 dans le lait, réalisée par observation visuelle en temps réel de la conversion d’un substrat chromogène sur une période de 45 ± 5 minutes. L’apparition d’une couleur bleu ciel signale l’absence d’AFM1 dans le lait, tandis que l’absence de changement de couleur signale la présence d’AFM1 dans le lait au-dela` de la LMR (limite maximale de résidu) de 0,5 ppb du Codex. La performance fonctionnelle des spores immobilisées a perduré jusqu’a` 6 mois. De plus, on a également immobilisé des spores sur une plaque de microtitration noire de 96 puits par adsorption physique et par piégeage sur disque capteur. Cette approche a entraîné une baisse de la sensibilité de détection qui est descendue a` 0,25 ppb sur une période de 20 ± 5 min telle que mesurée par analyse de la fluorescence a` l’aide d’un lecteur de plaques microbiologiques après l’ajout de lait et d’un substrat fluorogène. Le rapport de fluorescence élevé témoigne d’une hydrolyse de substrat plus prononcée en raison de la libération d’enzymes révélatrices issues de la germination des spores en l’absence d’AFM1 dans le lait. En contrepartie, un rapport de fluorescence plus bas signale la présence d’AFM1 a` une concentration de 0,25 ppb. Les spores immobilisées sur plaques de microtitration de 96 puits et sur disques capteurs affichaient une meilleure reproductibilité après 6 mois de remisage a` 4 °C. L’épreuve chromogène a présenté un taux de résultats faussement positifs de 1,38 % et un taux de résultats faussement négatifs de 2,77 % tandis que l’épreuve fluorogène s’est soldée par 4,16 % de faux positifs et 2,77 % de faux négatifs sur la foi d’une analyse de la présence d’AFM1 dans 72 échantillons de lait cru, pasteurisé ou déshydraté. L’immobilisation de spores permet a` ces épreuves chromogènes et fluorogènes de devenir portatives, sélectives et économiquement efficientes eu égard a` la détection en temps réel d’AFM1 dans le lait, qu’on se trouve a` la ferme laitière, au quai de réception ou dans les usines de transformation de l’industrie laitière. [Traduit par la Rédaction] Mots-clés : spore, immobilisation, chromogène, fluorogène, aflatoxine M1.
Introduction The practical utility of whole-cell biosensors in field applications requires that bacteria be formulated at a lower cost in a way that maintains their sustainability and recognition competency while enabling their long-term storage and transport (Belkin 2003). Several techniques have been reported in recent years for preservation of cells, including immobilization in different types of matrices, freeze-drying, and formation of spores. Among these, the use of Bacillus spores has emerged as a promising approach for
long-term storage of whole-cell biosensing systems for arsenic, zinc (Date et al. 2007), and bacitracin antibiotic (Fantino et al. 2009) at ambient temperature as well as in extreme environmental conditions. Immobilization is a general term used for a wide variety cell attachment or entrapment methods (Lopez et al. 1997). Cell immobilization has been defined as the physical localization of viable microbial cells to a defined region in such a way as to limit their free movement while retaining their catalytic activities (Covizzi et al. 2007). Various techniques used for immobilization
Received 17 July 2014. Revision received 16 September 2014. Accepted 17 September 2014. V.K. Singh, N.A. Singh, N. Kumar, H.V. Raghu, P.K. Sharma, and A. Yadav. Microbial Biosensors and Food Safety Laboratory, Dairy Microbiology Division, National Dairy Research Institute, Indian Council of Agricultural Research, Karnal 132001, Haryana, India. K.P. Singh. Department of Biotechnology, Shri Ram College, Muzaffarnagar, Uttar Pradesh, India. Corresponding author: H.V. Raghu (e-mail: [email protected]). Can. J. Microbiol. 60: 793–798 (2014) dx.doi.org/10.1139/cjm-2014-0465
Published at www.nrcresearchpress.com/cjm on 22 September 2014.
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of microorganisms include gel bead entrapment, carrier binding, adsorption to solid surface, encapsulation, and biofilm development (Chien et al. 2012). Adsorption is the simplest method of reversible immobilization based on van der Waals forces, ionic binding, hydrophobic interactions, and hydrogen bonds. However, it is based on weak forces but still facilitates an efficient binding process (Flickinger and Drew 1999). The immobilization of enzymes could also be done by reversible physical adsorption of natural and synthetic organic polymers. Natural polymers include polysaccharides (cellulose, agar, agarose, and alginate), proteins (collagen, albumin), and carbon, whereas synthetic polymers contain polystyrenes, polyacrylamides, polyamides, and vinyl and allyl polymers (Brena and Batista-Viera 2006). Covalent bonding is one of the most widely used methods for enzyme immobilization and comes under the irreversible immobilization category. Despite all advantages that this method presents when applied to enzymes, it is rarely applied for immobilization of cells because agents used for covalent bond formation are usually cytotoxic and it is difficult immobilize cells without any damage (Flickinger and Drew 1999). Encapsulation is another irreversible immobilization method, in which biocatalysts are restricted by the membrane walls (usually in a form of a capsule) but are free-floating within the core space (Bickerstaff 1997; Cao 2005). Entrapment is an irreversible method, where immobilized particles or cells are entrapped in a support matrix or inside fibres (Górecka and Jastrzêbska 2011). An advantage of using immobilized spores as biocatalysts is that there is an increase in the provision of higher cell density to increase overall substrate conversion rates and shelf life (Chien et al. 2012). Aflatoxin M1 (AFM1) is the 4-hydroxy derivative of aflatoxin B1 and is secreted in the milk of mammals that consume feed containing aflatoxin B1 (Tajik et al. 2007). AFM1 exhibits a high level of genotoxic activity with serious health risk in the dairy food chain; the accumulation of this mycotoxin damages DNA (Shundo and Sabino 2006; Viegas et al. 2012). AFM1 induces DNA adducts, leading to genetic changes in the target cells, which then cause DNA strand breakage, DNA base damage, and oxidative damage that may ultimately lead to cancer (Hamid et al. 2013). According to the International Agency for Research on Cancer (IARC 2002), aflatoxin is classified as a Group 1 human carcinogens, and its actionable level 15–20 ppb in animal feed and 0.5 ppb (AFM1) in dairy products has been established by Codex Alimentarius Commissions (2001). The European Union specified an AFM1 limit of 0.05 ppb in milk for all European Union member states, and 25 ppt for baby food (Cucci et al. 2007). Various biosensor methods used for detection and quantification of AFM1 in milk have been developed (Micheli et al. 2005; Wanga et al. 2009; Dinckaya et al. 2011), and few commercial systems like enzyme-linked immunosorbent assay (Mohammadian et al. 2010; Omar 2012), radio-immune-based Charm assay (Offiah and Adesiyun 2007), high-performance liquid chromatography (HPLC) with fluorescence detection are available. But all of these methods have inherent limitations for being costly and require huge infrastructure and experienced personnel while processing samples. These innovations have limited scope in their application at field level where milk is being produced, collected, chilled, and transported further to dairy plants for their processing (Singh et al. 2013). Crude aflatoxin at 30 ppm was found to inhibit 12 species of the genera Bacillus, Clostridium, and Streptomyces (Burmeister and Hesseltine 1965). Bacillus spp. were reported to be highly sensitive to aflatoxin (Madhyastha et al. 1994). Escherichia coli, Bacillus subtilis, and Enterobacter aerogenes were found to have moderate sensitivity towards a new aflatoxin, namely aflatoxin B2b (Wang et al. 2012). Whole-cell-based sensing systems have been applied in environmental monitoring (not for AFM1), but on-site monitoring of analytes is restricted because of their limited shelf life and transportability (Date et al. 2010). From our previous studies, we have
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developed a spore-inhibition-based enzyme substrate assay for monitoring of AFM1 in milk using Bacillus megaterium 2949 spores (Kumar et al. 2010; Singh et al. 2013). These spores possess a unique ability to sense environmental changes in response to specific a “germinant”, and their transformation into growing vegetative cells in real time has an enormous scope for their application in biosensing of contaminants in food products. The spore germination concept involves the release of marker enzymes and their action on specific chromogenic or fluorogenic substrates as a means for the detection of AFM1 in milk systems (Singh et al. 2013). In our present study, attempts were made to immobilize the same B. megaterium 2949 spores in different formats to increase its shelf life for the detection of AFM1 in milk using chromogenic and fluorogenic assay. These assays owing to their portability and costeffectiveness can be used in dairy plants as well as in research and development.
Materials and methods Chemicals and instrumentation Eppendorf tubes (1.5 mL) were procured from Axygen, USA, and 96-well microtitre plates were procured from Greiner, Germany. The sensor disks were prepared by using glass fibre paper purchased from Millipore (India), which was cut into 6.5-mmdiameter size. AFM1 was procured from Sigma Aldrich (India) and acetone from Rankem (India). Diacetyl fluorescein (DAF) obtained from SigmaAldrich (India). Distilled water used during the experiments was produced in Bioage-Labpure ultra plus (BIO-AGE, Punjab, India). Incubation was done in an Eppendorf Innova 42R shaker (Germany). For spore immobilization, lyophilizer procured from Hanil Science Co., Ltd., Korea, and a vacuum drier from Thermo Scientific, USA, were used. For absorbance of spores and fluorescence measurement, a Victor X3 2030 microbiological plate reader from PerkinElmer (USA) was used. Validation work was done by radioimmunoassay, i.e., Charm system 6602 (Charm Science Inc., USA). The data processing was done by using Microsoft excel (Window 7) data analysis. Spore preparation Fifteen strains of B. megaterium (MTCC 2444, 2412, 428, 453, 1684, 3165, 4911, 6129, 6130, 6131, 7163, 7349, 2949, and ATCC 9885, 14581) were screened for minimal inhibitory concentrations against AFM1 using a disc assay (Singh et al. 2013). Bacillus megaterium MTCC 2949 was streaked on nutrient agar plates for activation overnight at 37 °C followed by transfer into 5.0 mL propagation medium, i.e., tryptone glucose yeast extract (TGYE) broth. Further, indicator B. megaterium 2949 strain was propagated in the TGYE broth medium and spore production was done in the newly developed sporulation medium, as per the protocol explained by Singh et al. (2013). The final pellet obtained was analysed for total viable count and spore count (Downes and Ito 2001). The spore suspension was refrigerated at 2–8 °C and immobilized for its application. Immobilization of spores by different techniques By physical adsorption in Eppendorf tubes Bacillus megaterium spores were immobilized in Eppendorf tubes at a concentration of 107 spores/mL (Allam et al. 2011) and were then covered with parafilm and punctured with a needle. These Eppendorf tubes containing spores were lyophilized at 900g 20 °C, under 670 mm pressure for 1 ± 0.15 h. By physical adsorption in 96-well microtitre plates Bacillus megaterium spores were immobilized in 96-well microtitre plates at a concentration of 108 spores/mL (Allam et al. 2011) by vacuum drying for 1.30 h. Since 96-well microtitre plates are Published by NRC Research Press
Singh et al.
made up of polystyrene, the spores were physically adsorbed by this synthetic polymer.
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By entrapment in sensor disks Sensor disks (size 6.5 mm diameter) were placed in sterile glass Petri plates. Spores at a concentration of 108 spores/mL (Allam et al. 2011) were immobilized by entrapment in sensor disks by air-drying in an incubator at 37 °C for 4 h. Disks without spores and having the same quantity of buffer were labelled as control disks and were dried under similar conditions (Rotman and Cote 2003). Monitoring of analytical performance of immobilized spores Out of different sets of immobilized spores, refrigerated at 4 °C, one set was taken at different intervals from each of the experimental settings, and spores were germinated to vegetative cells in the presence of milk by the reaction of released enzyme, i.e., acetyl esterase, on chromogenic or fluorogenic substrate without any exogenous germinating agents, and the signal was measured visually or by a microbiological plate reader (Victor X3). Chromogenic assay Three batches (A, B, C) of immobilized spores were analysed for their analytical performance after storage at different time intervals. Four Eppendorf tubes were taken for checking immobilized spore performance by chromogenic assay. Raw milk without AFM1 was taken as negative sample; the same milk spiked with AFM1 at 0.5 ppb (Codex limit) was the positive sample and its recovery (94%–100%) was checked by a reference method, i.e., radioimmunoassay, as depicted in Fig. 2 (Offiah and Adesiyun 2007). In radioimmunoassay, 300 L of AF solution (provided by Charm Sciences, USA) was added in a test tube. Then 3/4 of the tube was filled with the milk sample (cooled at 4 ± 2 °C), mixing followed by centrifugation at 3150g for 5 min. The fat layer of the milk sample was removed and a White (receptor) tablet was added to the test tube, followed by 300 ± 100 L of distilled water. Mixing was done for 10 s, and 5.0 mL of the centrifuged milk sample was added. A purple tablet was added immediately and mixed by swirling milk up and down for 15 s. The sample was incubated at 35 ± 2 °C for 3 min followed by centrifugation for 5 min at 3150g. The sample was removed from the centrifuge, and the milk poured off. The fat ring was removed and wiped dry with swabs. A 300 ± 100 L volume of water was added and mixed thoroughly to break up the tablet. Scintillation fluid (300 ± 0.5 mL) was added and mixed for uniform appearance. Counts per minute was taken in the Charm system for 60 s on a [3H] channel. Negative and positive milk samples were pretreated at 80 °C for 15 min, followed by cooling (room temperature) under tap water in a test tube. Negative and positive milk samples (1 mL) and chromogenic substrate (25 mL) were added to Eppendorf tubes containing immobilized spores, followed by incubation at 37 °C for 45 min, and were observed for colour change along with the control without spores, i.e., milk and substrate (Singh et al. 2013). Intertesting of 6 different batches of immobilized spores was performed by chromogenic assay after 1 month of storage. Fluorogenic assay using 96-well microtitre plates One milligram of fluorogenic substrate, i.e., DAF, was dissolved in 2.5 mL of acetone; a second stock was prepared by the addition of 50 L of first stock to 475 L of 10 mmol/L potassium phosphate buffer. Further stock of DAF (11.2 nmol/L) was prepared in milk. A fluorogenic assay was performed in microwell plates containing spores by the addition of 100 L of pretreated milk with DAF, (C of equation 1), along with the controls, i.e., spores with DAF (A of equation 1) and milk with DAF (B of equation 1). After incubation, fluorescence was measured at specific excitation/emission spectra of 485 nm / 535 nm by using a Victor X3 2030 microbiological plate reader.
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A control point for the fluorogenic assay for detection of AFM1 was established by negative milk (without AFM1) and positive milk (AFM1 spiked at 0.25 ppb, as fluorogenic assay was found sensitive up to ≥0.25 ppb). Fluorogenic enzyme assays are more sensitive than chromogenic assays (Zhou et al. 1997) owing to the higher interaction of spores with substrate in a small quantity of milk. The results obtained were analysed for enzyme activity in terms of fluorescence using the following equation: (1)
Enzyme activity (ratio) ⫽ C/A ⫹ B
where A is enzyme activity (photon count per 0.1 s) of spores, B is enzyme activity of milk (photon count per 0.1 s), and C is enzyme activity of spores with milk (photon count per 0.1 s), Control Point of fluorogenic assay at 0.25–0.35 ppb. An enzyme activity (ratio) of ≥0.35 indicates the sample is negative for the presence of AFM1, while ≤0.35 indicates the presence of AFM1 in milk. Fluorogenic assay using sensor disk The spore-sensor disks were placed in 96-well microtitre plates. A mixture of milk and DAF at the rate of 100 L per well was added to microwell plates, followed by incubation at 37 ± 2 °C for 20 ± 5 min. After incubation, fluorescence was measured at specific excitation/emission spectra of 485 nm / 535 nm by using a Victor X3 2030 microbiological plate reader. The results obtained were analysed for enzyme activity in terms of fluorescence using equation 1 above. Experimental design and statistical analysis Experiments were designed using CRD (Critical Random Design) with 3 replicates. The data were analysed statistically and expressed as the mean (±SD) of 3 replicates, and correlation coefficient was calculated according to Snedecor and Cochran (1980).
Results and discussion Spore preparation Out of 15 strains of B. megaterium, strain MTCC 2949 was found to be the most sensitive against AFM1 at 0.25–0.5 ppb, so this strain was used in our assays. In TGYE broth medium, 75% sporulation has been achieved (Singh et al. 2013). Chromogenic assay Three different batches of immobilized spores were analysed monthly. Negative milk samples (i.e., without AFM1) turned skyblue colour while positive milk samples (i.e., with AFM1) showed no colour change. These 3 batches gave a distinguishable colour difference between positive (≥0.5 ppb) and negative samples for up to 5.5–6 months (Fig. 1a), but after 6 months the intensity of the sky-blue colour decreased, resulting in less differentiation between positive and negative samples. After 1 month of storage, 6 batches of immobilized spores showed similar results by chromogenic assay (Fig. 1b), indicating no significant variation in colour intensity was found in the different batches. Negative milk (without AFM1) and positive milk (with AFM1) samples used for analytical performance of chromogenic and fluorogenic assays were selected based on Charm assay results. A control point of AFM1 at 0.5 ppb was set by running a 0.5 ppb positive sample and a 0 ppb control standard (aflatoxin-free) provided by Charm Sciences (3 replicates). Readings at the maximum residue limit (MRL) dose and 0 ppb control were 317 ± 7 and 1100 ± 20, (mean ± SE, n = 3), respectively. So any reading ≤317 was considered positive for AFM1, and a reading between 318 and 1100 or above was considered negative for AFM1. The Charm reading was found inversely proportional to aflatoxin concentration, meaning that the greater the Charm reading the lower the aflatoxin conPublished by NRC Research Press
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Fig. 1. Analytical performance of immobilized spores by chromogenic assay. (a) Working performance of 3 batches. (b) Intertesting of different batches for chromogenic assay (n = 3).
centration in the sample, as depicted in the calibration graph (Fig. 2). Fluorogenic assay using 96-well micro-titre plates Milk (positive or negative) and DAF were added to 96-well microtitre plates containing spores, followed by fluorescence measurement at 485 nm / 535 nm excitation/emission spectra by using a microbiological plate reader (Victor X3 2030). Fluorescence, based on the acetyl esterase enzymatic activity of B. megaterium, was increased after spore germination in the presence of negative milk, but a decreasing trend was observed with positive milk, as the spores were being inhibited by AFM1 (Singh et al. 2013). Analytical performance of immobilized spores in 96-well microtitre plates was analysed for up to 6 months. These spores worked well with a negative sample, with a high fluorescence ratio, ratio decrease with an increase of AFM1 level in milk with 0.25 ppb detection limit up to 6 months, as depicted in Fig. 3. Although spores were immobilized in many wells of the microtitre plates, during the monthly analysis, only 3 microwells were used to perform the fluorogenic assay. Although the whole microtitre plate was incubated, only those spores treated with milk and DAF germinated, while other spores were unaffected. Fluorogenic assay using sensor disks A fluorogenic assay was performed by the addition of 100 L of pretreated milk plus DAF (11.2 nmol/L) on sensor disk immobilized spores (C), along with the controls, i.e., disks containing spores with DAF only (A) and disks containing spores with milk and DAF (B). After incubation for 20 ± 5 min at 37 ± 2 °C, fluorescence was measured at 485 nm / 535 nm excitation/emission spectra. The results obtained were analysed for enzyme activity in terms of fluorescence (ratio) = C/A+B. No significant differences (positive milk critical difference = 0.00234, and negative milk critical difference = 0.00154, ␣ ≤ 0.05, n = 3) were found in the viability and sensing ability of the vegetative cells upon storage of the spores at 4 °C in refrigerated conditions. During their fluorogenic performances, they retain the difference of high and low ratio between negative and positive samples respectively (Fig. 4). These results support that of Date et al. (2007), who demonstrated that a sporebased biosensing mechanism was stable for its analytical performance for a period of 6–8 months when stored at room temperature.
Fig. 2. Charm reading against aflatoxin M1 concentration.
Fig. 3. Fluorescence ratio after storage of immobilized spores, as determined by fluorogenic assay using microwell plates (n = 3).
Fig. 4. Fluorescence ratio after storage of immobilized spores, as determined by fluorogenic assay using sensor disks (n = 3).
Comparative evaluation with real food samples The above 3 immobilization methods are compared in Table 1 in terms of sensitivity, shelf life, limitations, etc. During analytical performance of chromogenic and fluorogenic assays using 72 milk samples containing raw, pasteurized, and dried milk it was observed that the chromogenic assay showed an incidence of AFM1 in 20% of the milk samples at the 0.5 ppb Codex MRL limit, while the fluorogenic assay showed an incidence of AFM1 in 23.6% Published by NRC Research Press
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Table 1. Comparison of spore immobilization methods. Property
In Eppendorf tubes
96-well microtitre plates
Sensor disks
Mechanism
Physical adsorption on polypropylene synthetic polymer 6 months 0.5 ppb Qualitative —
Physical adsorption on polystyrene synthetic polymer 6 months 0.25 ppb Semiquantitative For 1 trial, 3 wells containing spores are needed, but the whole plate is incubated because we cannot separate wells
Entrapment in sensor disk
Shelf life Sensitivity* Detection Limitations
6 months 0.25 ppb Semiquantitative —
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*ppb, parts per billion.
Fig. 5. Comparative results of fluorogenic and chromogenic assays with radioimmunoassay.
Table 2. Comparison of reference methods available for aflatoxin M1 (AFM1) analysis with our developed assays. Detection method
Mechanism
HPLC
Immuno-affinity columns containing antibodies against AFM1 ELISA Antigen–antibody reaction Radioimmunoassay Binding of radio-labeled aflatoxin with microbial receptor sites Charm ROSA Lateral flow assay Chromogenic assay Spore-inhibition-based enzyme (present work) substrate reaction Fluorogenic assay Spore-inhibition-based enzyme (present work) substrate reaction
Assay time (min) Sensitivity* 60
Limitations
6 ppt
75 14
Problem in extraction, costly, laboratory-bound, need experienced person 50 ppt Costly, laboratory-bound, need experienced person 0.5 and 0.25 ppb Costly, laboratory-bound, need experienced person
11 45
0.5 ppb 0.5 ppb
Costly, laboratory-bound, need experienced person —
20
0.25 ppb
—
*ppt, parts per trillion; ppb, parts per billion.
of the samples at ≤0.25 ppb (≤Codex MRL limit). The same samples were also analysed by the Charm-based method, and 22.2% of the samples of raw milk, pasteurized milk, and dried milk were found positive at 0.5 ppb AFM1. By comparing the results of the chromogenic-based assay with the radioimmunoassay (Charm assay), the chromogenic assay was found to yield 1.38% falsenegative and 2.77% false-positive results. When the fluorogenic assay was compared with the radioimmunoassay (Charm assay), the fluorogenic assay generated false-positive results in 4.16% of the samples and false-negative results in 2.77% of the samples (Fig. 5). However, the chromogenic assay and fluorogenic assay were found to correlate 0.997 and 0.999, respectively, with the radioimmunoassay (Charm assay). Duarte et al. (2013) investigated 40 samples of pasteurized and ultra-high temperature processed milk for AFM1 and found that 27.5% of the samples were above the cutoff limit of 0.05 ppb of European Union MRL, using ELISA. The developed concept of chromogenic assay for detection of AFM1 detection was validated and a correlation of 0.97 was established with AOAC approved Charm 6602 and ELISA at Codex MRL, with minimal false-positive and -negative results (Singh et al. 2013). After a comparative analysis of all the reference methods avail-
able on the market for AFM1 analysis with our chromogenic and fluorogenic assays, it can be concluded that our developed methods can be used in dairy, research and development, and milk chilling centres because of their portability, cost-effectiveness, sensitivity, etc. (Table 2).
Conclusion The present study reports spore immobilization through different formats, i.e., by physical adsorption in Eppendorf tubes and in 96-well black microtitre plates and by entrapment in sensor disk. These immobilized spores were germinated in the presence of milk, followed by enzyme release and its action on the substrate, i.e., chromogenic or fluorogenic. Analytical performance of these spores through analysis by chromogenic and fluorogenic assays on a monthly basis showed that in addition to being sensitive, reproducible, robust, easy to use, and cost-effective, the spores also maintain their viability and sensing capability during storage in refrigerated conditions. Spore immobilization provides portability to these assays, making it suitable for the detection of AFM1 in milk at the dairy farm, reception dock, manufacturing unit, Published by NRC Research Press
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and research and development centres. Overall, the analytical performance of chromogenic and fluorogenic assays in terms of the detection limit and reproducibility did not change significantly during the 6 months of storage in refrigerated conditions.
Acknowledgements The National Agricultural Innovative Program (NAIP) is greatly acknowledged for supporting this research work. The Director of the NDRI is thankfully acknowledged for providing facilities at NDRI, Karnal.
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