Spore immobilization and its analytical performance for monitoring of aflatoxin M1 in milk.

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.

Assessment of developed paper strip based sensor with pesticide residues in different dairy environmental samples.

According to the results of this study, the paper strip biosensor can detect pesticide at very low concentration like fungicide, organochlorine, organophosphate, carbamate, and herbicide group ranges from 1 to 10, 1-50, 250-500, 1-50, and 1 µg/L, respectively in animal feed, water, milk and soil. This is a significant improvement from the previous study, which found that the paper strip biosensor could only detect pesticide levels of up to 500 or 1000 µg/L. A total of 436 samples were collected from the dairy farm, including 58 samples of green feed, 54 samples of dry feed, 45 samples of concentrated feed, 41 samples of fermented feed, 49 samples of manure, 54 samples of soil, and 86 samples of milk. PSA (Primary Secondary Amine) and MgSO4 (1:2 ratio) were used to remove pigments from dairy farm samples to prevent the enzyme-pesticide interaction leading to colour development on the strip, which was successfully achieved. Using a strip-based test and an optimized extraction protocol, pesticides were detected in 38.49% in the samples. Limit of Detection of 15 pesticides from the organochlorine, organophosphate, carbamate, neonicotinoid, pyrethroid, ryanoid, strobilurins, and triazole groups recommended for use in dairy farms were evaluated in feed/fodder. Pesticides were being detected in various dairy farm matrices using the newly developed test. The developed technology can be used as a semi-quantitative test for pesticides monitoring in the dairy farm as well as for screening of primary produce under field condition for organic certification of various food/feed commodities.

Polyaniline-Pectin nanoparticles immobilized paper based colorimetric sensor for detection of Escherichia coli in milk and milk products.

In the food quality and safety arena, there is a need to develop novel and sustainable methodologies that can help in the prevention of foodborne diseases. Herein, we report the development of a rapid conducting polymer strip-based sensor using Polyaniline-pectin (PANI-PEC) for the detection of Escherichia coli in milk and milk products. Polyaniline-pectin nanoparticles stabilized with biopolymer pectin were synthesized and its characterization studies such as FTIR, UV-Vis spectroscopy, electrical conductivity and particle size analysis were done. The assay parameters were optimized for the selective detection of E. coli in milk and milk products. The concentration of PANI-PEC solution immobilized/strip was optimized to be 3 mg/mL as it exhibited good sensitivity and colour intensity. Based on acid production and selectivity for E.coli, concentrations of media components like lactose, tryptophan, yeast extract, chondroitin sulphate, sodium lauryl sulphate, potassium chloride, tergitol-7, gentamycin sulphate and ampicillin trihydrate were optimized as 0.9, 0.1, 0.45, 0.015, 0.1, 2, 0.0125, 0.00016 and 0.015 respectively and sample volume was optimized to 500 µL. The developed PANI-PEC colorimetric strip-based sensor detects 0.52 ± 0.17 log CFU/mL E. coli within 10: 21 h (h). Further shelf-life study revealed that the developed PANI-PEC colorimetric sensor strips are stable at room temperature up to six months exhibiting the same sensitivity. The results obtained here indicate that this novel and simple paper based colorimetric sensor holds potential for application in food industries as a reliable and rapid method for detection of E. coli in milk and milk products at various stages of production and processing.

Rapid detection of pesticide in milk, cereal and cereal based food and fruit juices using paper strip-based sensor.

The study was aimed to validate paper strip sensors for the detection of pesticide residues in milk, cereal-based food, and fruit juices in comparison with GC-MS/MS under field conditions. The detection limit of pesticide using rapid paper strip sensor for organophosphate, carbamate, organochlorine, fungicide, and herbicide group ranges from 1 to 10, 1-50, 250-500, 1-50, and 1 ppb, respectively in milk and milk product, cereal-based food and fruit juices. Among 125 samples of milk samples collected from the market 33 milk samples comprising 31 raw milk and 2 pasteurized milk found positive for pesticide using the strip-based sensor. In cereal based food and fruit juice samples, 6 cereal flours and 4 fruit juices were found positive for pesticide residues. The pesticide positive samples were further evaluated quantitatively using GC-MS/MS wherein 7 samples comprised of raw milk, pasteurized milk, rice flour, wheat flour, maize flour, apple juice, and pomegranate juice have shown the presence of chlorpyrifos, chlorpyrifos-methyl, α-endosulfan, ß-endosulfan DDD and DDT at trace level as well as at above MRL level. It is envisaged that the developed paper strip sensor can be a potential tool in the rapid and cost-effective screening of a large number of food samples for pesticide residues.

Role of stereospecific nature of germinants in Bacillus megaterium spores germination.

The present study was undertaken with the objective to assess the effect of distinct stereoisomeric forms of nutrient germinants (selected sugars and amino acids) on the process of germination onset in dormant spores of Bacillus megaterium MTCC 2949. In this respect, epimers of glucose and enantiomers of alanine were employed in current work. When supplemented with these stereoisomers, spores were found germinated only with d-glucose and d-mannose among epimers of glucose and only with l-alanine among enantiomers of alanine. Interestingly, germination in spores was observed to negligible extent with d-galactose and d-alanine. These findings were obtained on the basis of four type of germination assays, namely reduction in absorbance measured at 600 nm (≤5 to ≥30%), refractility examination (phase bright and dark), esterase assay [fluorescence units 0.455-94.62 (×103)] and fluorescent staining (fluorescent/non-fluorescent signals). Understanding of spores germination process and efficacy of different forms of germinants to trigger germination is of immense importance. It aids in development of sensing and sterilization indicating tools employing chiefly spores as biorecognition elements and in uncovering the mechanism of diseases, food contamination and spoilages resulting from the germination of spores. The findings of current work support the possibility to explore such germination mechanism by significantly giving the clue for potential existence of stereospecific receptor sites on the surface of B. megaterium spores. Perhaps, these sites can specifically differentiate and recognize stereoisomerically diverse forms of germinants for induction of germination.