Spore inhibition-based enzyme substrate assay for

Toxicological & Environmental Chemistry

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Spore inhibition-based enzyme substrate assay for monitoring of aflatoxin M1 in milk N.A. Singh, N. Kumar, H.V. Raghu, P.K. Sharma, V.K. Singh, Alia Khan, & N. Raghav To cite this article: N.A. Singh, N. Kumar, H.V. Raghu, P.K. Sharma, V.K. Singh, Alia Khan, & N. Raghav (2013) Spore inhibition-based enzyme substrate assay for monitoring of aflatoxin M1 in milk, Toxicological & Environmental Chemistry, 95:5, 765-777, DOI: 10.1080/02772248.2013.807540 To link to this article: http://dx.doi.org/10.1080/02772248.2013.807540

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Date: 05 March 2016, At: 22:21

Toxicological & Environmental Chemistry, 2013 Vol. 95, No. 5, 765–777, http://dx.doi.org/10.1080/02772248.2013.807540

Spore inhibition-based enzyme substrate assay for monitoring of aflatoxin M1 in milk

Downloaded by [National Dairy Research Inst - I C A R] at 22:21 05 March 2016

N.A. Singh, N. Kumar*, H.V. Raghu, P.K. Sharma, V.K. Singh, Alia Khan, and N. Raghav Dairy Microbiology Division, National Dairy Research Institute (NDRI), ICAR, Karnal, Haryana, India (Received 15 April 2013; accepted 16 May 2013) A spore germination-based concept and its transformation into a field level prototype for monitoring aflatoxin M1 (AFM1) in milk was developed. Initially, 15 strains of Bacillus spp. procured from different culture collection were screened for AFM1 sensitivity using spot assay and marker strain showing inhibition at 0.5 ppb was selected based upon maximum zone of inhibition. The selected strain B. megaterium 2949 was further screened for different enzymes activities and subsequently its spores were produced to an extent of 73.13% 3.197% in newly developed sporulation medium containing beef extract (0.0075% 0.0004%), yeast extract (0.015% 0.001%), peptone (0.0375% 0.0016%), and sodium chloride (0.0375% 0.0018%). A spore germination-based concept/ assay was optimized by immobilizing spores in eppendorf with pretreated milk (80 C/15 min) containing germinant and chromogenic substrate followed by incubation at 37 C. The appearance of sky blue color within real time of 45 min indicated spores germination and release of specific marker enzyme such as acetyl esterase and its specific action on chromogenic substrate which demonstrates absence of AFM1 in milk. However, if there was no color change, presence of AFM1 at 0.5 ppb MRL was denoted by Codex. The developed concept on 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. The cost effective test has potential application in dairy farms, manufacturing, and R&D units for routine monitoring of AFM1 in milk. Keywords: spore germination; marker enzyme; AFM1; chromogenic substrate; milk

Introduction Aflatoxins are well-known hepatocarcinogens, mutagens, and immunosuppressive agents produced as secondary metabolites by Aspergillus flavus or A. parasiticus (Prandini et al. 2009). Aflatoxin M1 (AFM1) is hydroxylated metabolite of AFB1 and exhibits a high level of genotoxic activity with serious health risk in dairy food chain because of this mycotoxin accumulates and damages DNA (Shundo and Sabino 2006; Viegas et al. 2012). According to International Agency for Research on Cancer (IARC 2002), aflatoxin is classified as Group-1 of 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 Commission (2001). European Union (EU) specified an AFM1 limit of 0.05 ppb in milk for all EU Member States, and 25 ppt for baby food (Cucci et al. 2007).

*Corresponding author. Email: [email protected] Ó 2013 Taylor & Francis


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Various methods used for detection and quantification of aflatoxins in milk have been developed and few commercial systems like enzyme-linked immunosorbent assay (Mohammadian et al. 2010; Omar 2012), radio-immune-based charm assay (Offiah and Adesiyun 2007), HPLC with fluorescence detection are available (Komarova 2000), electrochemical immunosensor using screen-printed electrodes (Micheli et al. 2005), chemiluminescent enzyme immunoassay, long-range surface plasmon-enhanced fluorescence spectroscopy (Wanga, Dostalek, and Knalla 2008), and impedimetric system based on DNA probe and gold nanoparticles (Din¸c kaya et al. 2011) have been developed. 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. The dormant bacterial spores possess a unique ability to sense environmental changes in response to specific “germinant” and their transformation into growing vegetative cells in real time have enormous scope for their application in biosensing of contaminants in food products. The spore germination concept involves the release of DPA or marker enzymes and their action on specific chromogenic or fluorogenic substrates as a mean for detection of contaminants in milk systems. Attempts were undertaken to develop sporebased analytical devices for detection of broad spectrum antibiotic and b-lactam antibiotic residues in milk which are cost effective, efficient, and reliable (Kumar et al. 2006; Kumar, Das, and Manju 2009; Kumar et al. 2012; Das et al. 2011; Gaare et al. 2012). One such product was commercialized as MDR test to target antibiotic residues in milk on farms or manufacturing stage. Bacillus spp. was reported to be highly sensitive to aflatoxin (Madhyastha et al. 1994) and inhibition at 30 ppm of crude preparation was found in 12 species of genus Bacillus, Clostridium, and Streptomyces (Burmeister and Hesseltine 1966). In view of sensitivity of Bacillus spp. to aflatoxin an attempt was made in this investigation to develop spore inhibition-based enzyme substrate assay (SIB-ESA) as an innovative approach for monitoring AFM1 in milk at dairy farms, manufacturing units, and R&D centers. Materials and methods Procurement and screening of Bacillus megaterium for AFM1 sensitivity Fifteen strains of Bacillus megaterium (MTCC 2444, 2412, 428, 453, 1684, 3165, 4911, 6129, 6130, 6131, 7163, 7349, 2949, ATCC 9885, 14581) were obtained from microbial type culture collection (MTCC), IMTECH Chandigarh, India, and from American type culture collection (ATCC). The procured strains were screened for minimal inhibitory concentrations (MICs) against AFM1 using disc assay (IDF 1991). Growth and sporulation in B. megaterium 2949 Indicator strain of B. megaterium 2949 was streaked on nutrient agar plates and incubated at 37 C for 24 h. The pure colony was transferred into 5 mL propagation medium consisting tryptone, glucose, yeast extract followed by transfer at the rate of 1 mL. One hundred mL tryptone glucose yeast extract (TGY) broth (0.5% tryptone, 0.25% yeast extract, 0.1% dextrose at pH 7), incubated in Innova 42 (Incubator shaker) at 37 C for different incubation periods of 24, 48, or 72 h and analyzed for total viable count (TVC) and spore counts (SC) (Downes and Ito 2001). B. megaterium 2949 was further transferred in sporulation medium. This sporulation medium was optimized by using different concentrations


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of original nutrient broth in gradually decreasing concentration of 100% to 50%, 25%, 10%, and 7.5% of components by weight at pH 6.5, incubated (Innova 42 Incubator shaker) at 37 C for up to 42 h and analyzed for TVC and SC at different intervals 24, 36 or 42 h. The spores were centrifuged at 9000 g/10 min and washed twice. The spore suspension obtained was analyzed for TVC and SC (Downes and Ito 2001) and % of spores was calculated at different time intervals. Growth and sporulation experiments on B. megaterium 2949 were designed using CRD (Critical Random Design) with six replicates (Snedecor and Cochran 1980). Further B. megaterium 2949 spores were also analyzed for AFM1 sensitivity by disc assay (IDF 1991). Screening of spore for de novo production of indicator enzyme The spores from B. megaterium 2949 strain was screened for different enzymes, namely acetyl esterase, tryptophanase, esculinase, b-galactosidase, b-lactamase, a-amylase, and lipase enzymes using discs and strips of Fluka (Sigma-Aldrich Company). Indicator enzyme activity was measured either by wiping colonies of indicator strain with disc containing substrate or making suspension of strain in saline and dipping disk in suspension. Further indicator organism was analyzed for de novo enzyme activity by adding 100 mL spore suspension in 0.5 mL broth containing growth factors followed by addition of 50 mL chromogenic substrate and incubation at 37 C for 20–25 min. Color changes from colorless to colored complex were taken as criteria for de novo enzyme activity for further development of assay for detection of AFM1 in milk. Development of spore inhibition-based assay and its kit prototype The milk was heat treated at 70 C–90 C range for 5–15 min period to minimize background interference contributed by other sources. The optimal conditions for the development of SIB-ESA kit prototype for the detection of AFM1 in milk was optimized, i.e., quantity of spore suspension, milk, and chromogenic substrate (indoxyl acetate). The SIB-ESA was optimized by lyophilization of B. megaterium 2949 spores at optimized condition of 900 g at 20 C under 670 mm pressure. Samples were inoculated with 1 mL pretreated reconstituted skimmed milk (10 g of SMP in 9 mL distilled water at 40 C) at 80 C for 15 min spiked with AFM1 at different levels from 0.1 to 1 ppb for evaluating limit of detection (LOD) of SIB-ESA. The results of spiked samples were compared with results of AOAC approved Charm 6602 analyzer (Kumar et al. 2012; Das et al. 2011) purchased from Charm Sci. Inc. Evaluation of SIB-ESA The performance of SIB-ESA was evaluated by analyzing 185 samples of raw, pasteurized, and dried milk obtained from different dairies in the area of Saharanpur, villages near by Delhi, Haryana, and Rajasthan in India. Samples were analyzed for the presence of AFM1 by using developed SIB-ESA, AOAC-approved Charm 6602 analyzer (Kumar et al. 2012; Das et al. 2011). Effect of contaminants other than AFM1 on working performance of assay The inhibitory effect of commonly used dairy detergents and sanitizers, namely sodium hydroxide (RANKEM, RFCL, New Delhi, India), sodium hypochlorite (Qualigens fine chemicals, Mumbai, India), iodophor and benzalkonium chloride (CDH, New Delhi,

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India), formalin and hydrogen peroxide (SD Fine Chemicals, Mumbai, India), antibiotic residues such as b-lactam, tetracycline, aminoglycoside-N, aminoglycoside-ST, sulpha drugs, macrolide (Hi-media), and other miscellaneous groups, detergents and sanitizers were evaluated for their impact on working performance of SIB-ESA by using a method described by Kumar et al. (2012) and Raghu (2007).


Results Screening and selection of indicator Three strains of B. megaterium ATCC 14581, 9885, and MTCC 2949 out of 15 screened for AFM1 showed sensitivity against AFM1. MTCC 2949 displayed higher sensitivity at 0.5–0.0005 ppm (500 ppb–0.5 ppb) with an inhibition zone of 13, 11, 10, 9 mm, respectively. MIC of ATCC 14581 was 5 ppb while 9885 showed sensitivity up to 0.5 ppb, i.e., codex limit with an inhibition zone of 5 mm. With comparison of inhibition zone against AFM1, MTCC 2949 was found to be more sensitive than ATCC 14581 and 9885 strains. The other 12 strains were found resistant against AFM1 showing no inhibition zone (Table 1). Madhyastha et al. (1994) studied the sensitivity of Bacillus brevis to eight mycotoxins including AFB1, ochratoxin A, citrinin, patulin, penicillic acid, cyclopiazonic acid, penitrem A, and zearalenone. In another study conducted by Burmeister (1967) on invention on microbiological screening test for aflatoxin cultures of B. megaterium NRRL B-1370 and B. brevis NRRL B-1874 were inhibited in presence of aflatoxin. Burmeister and Hesseltine (1966) found that strain of Bacillus brevis and two of B. megaterium were most sensitive to aflatoxin, being inhibited at 10 and 15 ug/mL, respectively. Most sensitive organisms were inhibited by as low as 7.5 mg aflatoxins/mL using B. megaterium giving an inhibition zone of 10.5 mm (Refai et al. 1993). From the screening study, it was concluded that B. megaterium 2949 is most sensitive among all the strains investigated and was selected for further investigation. Table 1. Zone of inhibition (mm) results for different strains of B. megaterium against different concentration of AFM1. Zone of inhibition (mm) against different concentration of aflatoxins (mg/mL) S. no.

Strain no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

MTCC2444 MTCC428 MTCC 453 MTCC 2412 MTCC 1684 MTCC 3165 MTCC4911 MTCC 6129 MTCC6130 MTCC 6131 MTCC7163 MTCC7349 ATCC 14581 ATCC 9885 MTCC 2949

Average of six trials (n ¼ 6).

10

5

0.5

0.05

0.005

0.0005

ve ve ve ve ve ve ve ve ve ve ve ve 9 11 16





ve ve ve ve ve ve ve ve ve ve ve ve 5 9



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Sporulation in B. megaterium 2949 Cultures from 5 mL TGY broth were inoculated and incubated at 37 C on shaker at 150 rpm overnight. After 24 h incubation in 5 mL TGY broth only 0.46% of spores were observed with a TVC and SC of 7.24 0.33 and 4.47 0.39 log CFU, respectively. Further, 1% of the overnight grown culture was inoculated into 100 mL TGY broth and incubated at 37 C for 0, 24 or 48 h. In 100 mL TGY broth, there was no increase of spores observed from 0 to 24 h. With further incubation for 48 h increase of spores (63%) was observed with a TVC and SC of 7.97 0.39 and 7.77 0.19 log CFU/mL, respectively (Figure 1). Since, there was a significant rise in % spores in a broth medium at 37 C for 48 h, this was selected as optimal time period for growth of B. megaterium 2949. After 48 h incubation at 37 C, the grown culture was transferred at a rate of 7.5 mL per 100 mL sporulation medium followed by incubation at 37 C for up to 42 h. After 42-h incubation in different concentration of nutrient broth 100%, 50%, 25%, 10%, and 7.5% of components by weight 50%, 53%, 56%, 59%, and 70% spores were observed, respectively as depicted in Figure 2. The final sporulation medium, i.e., 7.5% nutrient broth was selected with a final composition of beef extract (0.0075% 0.0004%), yeast extract (0.015% 0.001%), peptone (0.0375% 0.0016%), and sodium chloride (0.0375% 0.0018%) based on maximum number of spores. It is well known that nutrient starvation conditions induce the cells to get converted into spores, and this factor was supported by the acidic pH of the medium. During sporulation, it was observed that after 24-h incubation in newly formulated sporulation medium, there was decrease in sporulation (51%) with a TVC and SC of 7.41 0.21 and 7.02 0.2 log CFU/mL, and it was maximal (71%) at the end of 42 h where spore count reached to 8.12 0.08 log CFU/mL, as shown in Figure 3. This extent of sporulation achieved at the end of sporulation process was 73% in washed suspension. A broth medium containing tryptone and manganese sulfate supported heavy sporulation of Bacillus stearothermophilus ATCC 7953 (Thompson and Thames 1967). Efficient sporulation (107 spores per mL Bacillus larvae) occurred in presence of 1.5% to 2.25% yeast extract (Dingman and Stahly 1983).

Figure 1. Growth pattern of B. megaterium 2949 in TGY medium at 37 C under different incubation period (h). Overnight grown 5 mL TGY broth inoculated into 100 mL TGY broth and incubated at 37 C for up to 24 h. Growth was monitored by measuring total viable count (TVC) and spore count (SC). Symbol: (^) TVC, ( ) SC.


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Figure 2. Optimization of sporulation media measuring spores (%) level at different concentration of nutrient broth. The maximum sporulation was observed at 7.5% nutrient broth concentration.

Screening of selected spore for de novo production of indicator enzyme A significant acetyl esterase or esculinase activity was established in spores for application in biosensing of specific analyte in milk and milk products. Indicator strain was streaked on nutrient agar plate and incubated at 37 C for 24 h. Wiped 2–3 colonies were used from the paper zone of diagnostic strip containing 3-acetoxy indol, which is a

Figure 3. Sporulation pattern of B. megaterium 2949 in sporulation medium at 37 C for different time period (h). B. megaterium cells were inoculated into sporulation medium at the rate of 7.5 mL, 100 mL, and incubated at 37 C for up to 48 h. The sporulation was monitored by measuring TVC and SC. Symbol: (^) TVC, ( ) SC.


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Table 2. Screening of B. megaterium 2949 for different enzyme activity by using respective chromogenic disk/strip. S. no

Name of disk/ strip

Enzyme

Substrate

B. megaterium

1 2 3

Indoxyl strips Tributyrin-strips DMACA indole disk

Acetate esterase Lipase Tryptophanase

Positive Positive (weak) Negative

4 5

ß-lactamase strips ONPG Disks

ß-lactamase ß-galactosidase

6

Bile esculin disks

Esculinase

Indoxyl acetate Tributyrin p-dimethyl-aminocinnamaldehyde Benzyl penicillin o-nitrophenyl-ßD-galactopyranose Esculin

Negative Negative Negative

Average of six trials (n ¼ 6).

substrate for acetyl esterase produced by indicator strain and read after 3–5 min. Color changes from colorless to sky blue due to the enzyme produced by micro-organisms were followed by action on chromogenic substrate. The acetyl esterase enzyme activity of B. megaterium indicator strain was compared with E. coli. Further, indicator organism was tested for esterase activity using liquid medium color changes from colorless to sky blue due to the enzyme produced by spores that act on chromogenic substrate was observed within 20 min. The weak lipase activity was observed in selected indicator strain on tributyrin-strips. Other enzymes, such as tryptophanase, b-lactamase, b-galactosidase, and esculinase activities were not observed in selected strains (Table 2). Ruiz et al. (2006) studied the lipolytic system of Bacillus megaterium 370 showing presence of at least two secreted lipases and a cell-bound esterase. Zhang et al. (2011) isolated the Bacillus kribbensis DSM 17871, a novel spore possessing valine arylamidase, b-glucuronidase, and a-glucosidase enzyme activity. Based on these observations, acetyl esterase enzyme was selected as a marker enzyme for development of assay.

Optimization of assay and its kit prototype Milk was pre-heat-treated at 70 C–90 C/5–20 min to avoid background interference. The background interference of milk at 80 C and 90 C heating for 10–20 min was minimal compared to 70 C. Therefore, heating at 80 C for 15 min was selected for optimization of SIB-ESA and its kit prototype. After optimization of pretreatment of milk, SIB-ESA was optimized for different parameters such as quantity of spore suspension, milk, substrate, and incubation time. Based on the intensity of color developed at different levels of spore suspension, milk, substrate, and incubation time periods with positive and negative milk SIB-ESA and its prototype was optimized. In spore suspension at 100, 150, and 200 mL in positive milk samples, there was no color development observed and in case of negative samples color intensity was 0.7. The color development in spore suspension at a level of more than 200 mL was observed in positive samples was 0.25 and for negative sample was higher (Figure 3a). The color intensity developed for the chromogenic substrate at a level of 25 mL and 35 mL was 0.75 for negative milk, and for positive sample there was no color development. The chromogenic substrate above 35 mL color intensity development increased gradually for both positive and negative samples (Figure 3b). In case of optimization of volume of milk, the intensity of color development was higher at 1 mL and color intensity fell gradually as the milk samples rose (Figure 3c). Based on the above observation, the spore suspension of 150 mL, 1 mL, and 25 mL milk was


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Figure 4. Optimization of SIB-ESA for enhances acetyl esterase enzyme activity using B. megaterium 2949. Assay was optimized for spore suspension (a), substrate (b), milk sample (c), and incubation time at 37 C (d) by inoculating positive and negative milk samples. Optimization study of the assay was monitored by checking the intensity of color change and the grading 0.7 is taken as criteria for selection for prototype development.

selected for assembly of spore inhibition-based enzyme substrate-based analytical system for detection of AFM1 and its prototype, as shown in Figure 4. Limit of detection The limit of detection (LOD) for SIB-ESA was established by screening 12 samples of aflatoxin-free milk spiked with different levels of AFM1 residues ranging from 0.1–1 ppb level, as depicted in Figure 5. Concentration of AFM1 up to 0.3 ppb showed no significant inhibition in color due to enzyme activity. Further increase of AFM1 concentration 0.4 ppb onward, there was significant decrease in color with slight blue color and no color change observed at 0. 5 ppb codex MRL limits. The spore germination of B. megaterium 2949 was inhibited at 0.5 ppb of AFM1 level in the form of no color development. Results of this assay were also correlated with Charm 6602 analyzer, as shown in Figure 6. Thus 0.5 ppb was taken as LOD of AFM1 in milk for SIB-ESA. In contrast, higher sensitivity was observed by Bognanno et al. (2006) using HPLC with fluorescence detector and LC-MS an LOD of 250 ng/L (0.25 ppb) was reported. Real foods sample test and validation Different lots of SIB-ESA prototype kit was evaluated for the presence of AFM1 in 185 samples of milk including 128 raw milk, 32 pasteurized milk, and 25 dried milk



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Figure 5. Stepwise performance of SIB-ESA kit prototype for detection of AFM1 in milk based on color change from colorless to sky blue color.

Figure 6. LOD of SIB-ESA. Different concentration of AFM1 was spiked in negative milk and analyzed by using developed assay and percentage inhibition in color change was observed. Where, 50% inhibition was observed at 0.5 ppb, hence, 0.5 ppb of AFM1 was fixed as LOD for the developed assay.


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Figure 7. Evaluation of SIB-ESA with Charm 6602 assay and ELISA-based method with natural samples such as raw, pasteurized, and dried milk.

samples procured from different industrial or private dairies in the area of Saharanpur, villages near by Delhi, Haryana, and Rajasthan in India. With SIB-ESA kit the comparative incidence depicted an incidence of AFM1 in 30% raw milk, 22% pasteurized milk, and 8% samples of dried milk at Codex MRL limit (0.5 ppb). These samples were also analyzed by using AOAC approved microbial receptor assay (CHARM), which showed incidence of AFM1 in 31% raw milk, 16% pasteurized milk, and 16% in dried milk products. Same samples were also analyzed by ELISA-based method and 31% of raw milk, 16% pasteurized milk, and 16% of dried milk samples were found positive 0.5 ppb AFM1. By comparing the result of SIB-ESA with Charm 6602 assay, SIB-ESA was found to yield false negative results in 2.7% samples contributed 1.62% by raw milk and 1.08% by dried milk, and false positive in 2.16% samples contributed 1.08% by raw milk and 1.08% by pasteurized milk samples. When the SIB-ESA results were compared with ELISA-based method, SIB-ESA-generated false negative results in 3.24% samples contributed 2.16% by raw milk and 1.08% by dried milk, and false positive in 2.7% samples contributed 1.62% by raw milk and 1.08% by pasteurized milk samples. However, the SIB-ESA assay was found to correlate 0.97 with Charm and ELISA assay along with 2.7% false-negative and 2.16 false-positive results in case of Charm and 3.24% false-negative and 2.7% false-positive in case of ELISA at Codex MRL for AFM1 in milk (Figure 7). Duarte et al. (2013) investigated 40 samples of pasteurized and UHT milk for AFM1 and found that 27.5% samples were above the cutoff limit of 0.05 ppb of EU MRL using ELISA. Cross reactivity test Commonly occurring inhibitors such as veterinary drug residues, detergents, sanitizers investigated in our current study exerted no marked effect on performance of the assay (Kumar et al. 2010). The veterinary drug residues used for treatment of diseases in dairy animals in India were b-lactam, tetracycline, aminoglycoside-N, aminoglycoside–ST, sulfa drugs, and macrolides. To protect the safety of the consumers, the European Union (EU) has strictly regulated the use of antimicrobials, particularly in food-producing animals, by issuing several regulations and directives (Bogialli and Corcia 2009). In current



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investigation, veterinary drug residues at a concentration of MRL level prescribed by EU/CAC in milk exerted no inhibitory effect on the performance of SIB-ESA. Detergent and sanitizers are other inhibitors mostly present in milk used for cleaning and sanitization of dairy utensils or equipment used for processing, exerted no inhibitory effect on the performance of the SIB-ESA. A proper sanitizer used in milk plant at recommended dosage of iodine is usually 12.5–25 ppm for 1 min and QAC is 150–200 ppm. However, under practical situation, a maximal residual level of 0.5 ppm could be traced in milk when treated at 10 ppm level for sanitation purpose (Thomas 2001). In current investigation an iodine (0.25–2.5 ppm) and QAC (0.5–5 ppm) (Schmidt 1997) were found to exert no inhibitory effect on functional performance of SIB-ESA. The performance of the assay was also not affected in presence of detergents such as NaOH (0.5%–1%) and sodium hypochlorite (0.5–5 ppm). Present investigation on effect of detergents and sanitizers on functional performance of the spore-based assay were supported by Kumar et al. (2012) who demonstrated no negative impact of detergents and sanitizers on functional performance of spore-based assay. The present investigation revealed that 15% inhibition was observed at 3 ppm concentration of concentration of hydrogen peroxide as evidenced by 12.5% inhibition at 0.025% concentration of hydrogen peroxide (H2O2) (Raghu 2007; Kumar et al. 2012) which is lower than the recommended dose in LP system. A recommended level of formalin or formaldehyde, commonly used under field condition, is 0.03% displaying no inhibitory effect on the performance of developed assay.

Conclusions Bacterial endospores possess a unique ability to sense environmental changes in response to specific “germinants” and transform into rapidly growing vegetative cells in real time. This feature can be effectively utilized for the sensing of nonbacterial contaminants in dairy supply chain. Among 15 strains of B. megaterium screened for sensitivity to AFM1, B. megaterium MTCC 2949 showed higher sensitivity at Codex MRL limit and thus selected for development of SIB-ESA. The spore production in B. megaterium in sporulation medium reached a maximum sporulation level of 73% at 37 C for 42 h. Among the enzymes screened in indicator strain for de novo enzyme activity, acetyl esterase enzyme was selected in spores for its application in biosensing of specific analyte in milk and dried milks. An SBA, which involves sporulation of dormant spores of B. megaterium in sporulation medium and their germination and outgrowth in presence of selective germinants, was developed and assembled into a portable field-level kit prototype. The limit of detection (LOD) for SIB-ESA was established at 0.5 ppb of AFM1 with no de novo acetyl esterase enzyme activity observed. Performance of the assay was unaffected in presence of veterinary drugs, detergents, sanitizers, and preservative residues in milk and dried milk.

Acknowledgments National Agricultural Innovative Programme (NAIP) is greatly acknowledged for supporting this research work. The Director NDRI is thankfully acknowledged.

References Bogialli, S., and A.D. Corcia. 2009. “Recent Applications of Liquid Chromatography–Mass Spectrometry to Residue Analysis of Antimicrobials in Food of Animal Origin.” Analytical and Bioanalytical Chemistry 395: 947–966.


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