Studies on Luciferase Enzymes for their Application in Assessing Hygiene State of Selected Food Samples.

Rajeev, Ranjan (2014) Studies on Luciferase Enzymes for their Application in Assessing Hygiene State of Selected Food Samples. PhD thesis, University of Mysore.

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Abstract

Assurance of public health through the accessibility of safe food and water is an absolute requirement. Food hygiene is one of the major components of food safety which ensure prevention of all hazards that make food injurious to health. These hazards include contamination of food commodities with foodborne pathogens and their toxins, heavy metals and pesticides. Therefore, rapid and on–site monitoring of food commodities and water is of prime importance to ensure safe consumption of food and water. Firefly luciferase enzyme and in vivo bacterial luciferase enzyme based detection methods for monitoring food hygiene have been considered as the most sensitive and rapid biosensing tool for hygiene assessment. In the present study, a novel firefly luciferase enzyme from locally available Asymmetricata humeralis walker having broad pH optimum and high specific activity (pH optima: 7.4–8.2; specific activity: 8.15*109 RLU/mg; fold purification: 362.2 and yield: 0.52%) was extracted and purified to homogeneity. Firefly luciferase enzyme purification was carried out in three steps viz. ammonium sulphate precipitation (35–85%), DEAE sepharose fast flow column and blue sepharose column. A novel bioluminescent probe was designed for the detection of staphylococcal enterotoxin B (SEB) for which ATP was conjugated to SEB using 1– Ethyl–3–(3–dimethylaminopropyl)carbodiimide (EDC) and N–hydroxysulfosuccinimide (Sulpho–NHS) method. ATP–SEB conjugate was purified using G–25 sephadex size exclusion column which was used to develop a dipstick based bioluminescent probe for the detection of SEB toxin based on competitive Immunoassay. Further, the purified enzyme was used for rapid enumeration of microbes upto 50–100 CFU in real food samples such as milk, fruit juice and flavoured milk through ATP assay for which a novel ATPase inhibitor based bacterial ATP extraction buffer (Ethanol, benzalkonium chloride and oligomycin A) was formulated. A similar approach for the evaluation of post mortem time in raw fish as freshness indicator was developed for which somatic cell ATP extraction buffer (Sodium orthovanadate and EDTA) was optimized. The somatic cell ATP extraction buffer developed in the present study was found to enhance the assay sensitivity on an average of 35 fold with respect to control. In vivo bacterial luciferase activity of bioluminescent bacteria (CFTRI–BIOPHOTO–1; GenBank: KC617878.1; Photobacterium leiognathi) isolated from marine mussels was applied as whole cell biosensing element in form of immobilized biophotonic beads for the prescreening of hazardous materials such as heavy metals [Hg(II), As(V), Cd(II)] and pesticide (2,4–D) in spiked water samples after submerged cultivation. Using the present approaches; rapid and sensitive assessment of hygiene can be carried out in food and water.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	firefly luciferase enzyme, Asymmetricata humeralis walker, food safety
Subjects:	600 Technology > 08 Food technology > 13 Quality control > 02 Safety 600 Technology > 08 Food technology > 16 Nutritive value > 05 Enzymes
Divisions:	Fermentation Technology and Bioengineering
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	06 Nov 2015 08:09
Last Modified:	06 Nov 2015 08:09
URI:	http://ir.cftri.res.in/id/eprint/11945

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