Characterization of lipase from lactic acid bacteria isolated from fish processing waste.

Vrinda, R. (2013) Characterization of lipase from lactic acid bacteria isolated from fish processing waste. PhD thesis, University of Mysore.

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Abstract

Lactic acid bacteria (LAB) are capable of producing a variety of metabolites which are of utmost importance to the food industry. In the present study, 78 LAB isolates were screened for lipolytic activity from fish processing waste (FPW) of which 30 most potent lipase producing cultures were selected for further characterization. FPW was chosen as the source for isolation of these lipolytic LAB, as it is rich in lipids and there is a greater possibility of occurrence of lipase producers enabling convenient screening and isolation of potent lipolytic LAB isolates. Phylogenetic analysis based on 16S rRNA gene sequences revealed that most of these cultures belonged to the genus Enterococcus whereas a few belonged to the genus Pediococcus. Genetic diversity of LAB isolates was analyzed by RAPD and Simpson index was calculated in order to quantitatively determine the degree of occurrence of different genotypes in a dataset. Consequently, two most potent isolates with respect to lipolytic and antibacterial activity; namely- Enterococcus faecium MTCC5695 (MTCC5695; also deposited as E. faecium NCIM5363) and Enterococcus durans NCIM5427 (ED-27) were selected for further studies. The lipases produced by these isolates were found to be intracellular in nature. Lipases (triacylglycerol acylhydrolases; EC 3.1.1.3) are inducible enzymes whose production depends upon several process variables, such as pH, temperature, incubation period, substrate concentration, inoculum level and inducer concentration. Therefore, the conditions for lipase production by MTCC5695 and ED-27 were optimized by Response Surface Methodology (RSM). The optimized conditions were found to be 5% v/v FWO, 0.15mg/ml FWPH at 24 h of fermentation time for MTCC5695, and 5% v/v FWO, 0.2 mg/ml FWPH and 48h of fermentation time for ED-27, which were further validated. MTCC5695 showed a 3.15 fold increase (543.63 to 1715U/ml) whereas ED-27 showed a fold increase of 3.0 (207.6 U/ml to 612.53 U/ml) in lipase production under optimized conditions in comparison to the unoptimized conditions. Additionally, unstructured mathematical models were used to describe the fermentation kinetics of growth and lipase production of MTCC5695 and ED-27. Logistic model for cell growth and Luedeking-Piret model for lipase production predicted the fermentation profile accurately and the kinetic model parameters α and β evidently implied that the lipase produced by MTCC5695 and ED- 27 to be growth- associated. Intracellular lipases obtained from the bacterial cultures MTCC5695 and ED- 27 upon cultivation at optimized conditions was subjected to partial purification and concentration using aqueous two-phase systems (ATPS). The optimum conditions for MTCC5695 lipase were found to be Disodium Hydrogen phosphate-Polyethylene glycol (Na2HPO4 /PEG8000) at 41.24% tie line length and 0.25 phase volume ratio whereas for ED-27 lipase it was (NH4)2SO4 /PEG 6000 at 41.24% tie line length and 0.5 phase volume ratio. The integration of ultra filtration with ATPE facilitated the selective removal of PEG from the partially purified lipase. The molecular weight of the lipases was found to be around 19.2 kDa and 21.6 kDa for MTCC5695 and ED- 27, respectively. MTCC5695 lipase showed optimal activity at pH 10.8 and temperature 40°C indicating that it was of alkaline nature whereas ED-27 produced an acidic lipase that showed maximal activity at pH 4.6 and 30°C. Characterization of lipases enabled the selective application of these cultures for two different purposes suited to its specific property. MTCC5695 was used as a starter culture for the preparation of curd owing to its lipolytic, antibacterial and probiotic properties. The conditions for curdling were optimized by RSM and were found to be 26.48 h and 2.17%v/v inoculum level for which the second order model was validated. Further co-cultivation studies were performed which indicated that MTCC5695 was effective against a wide spectrum of Gram-negative bacteria and thereby possesses the ability to prevent the spoilage caused by food borne pathogens which in turn increases the shelf-life of the product. This finding holds significance in the fact that MTCC5695 lipase besides its organoleptic properties was also active against Gram-negative bacteria and works synergistically with the enterocin resulting in their complete destruction. This is an added advantage for the food industry as Gram-negative bacteria pose serious threat as they are the major food borne pathogens associated with food spoilage leading to diarrhoea and chronic gastroenteritis on their ingestion. Usually, most of the bacteriocins from LAB show activity only towards Gram-positive bacteria and not Gram-negative pathogens due to the presence of the LPS layer on their cell wall. In this study, it was proven that the synergistic effect of enterocin with lipase had a lethal effect on Gram-negative bacteria. On the contrary, ED-27 exhibited poor curdling properties and the lipase extracted from it was subjected to the degradation of lipids present in the slaughter house effluent in view of its ability to produce an acidic lipase. The rate of enzymatic hydrolysis was optimized for the parameters namely; concentration of lipase used, rate of agitation and time of reaction. The optimal enzyme concentration was found to be 600 U/ml; at agitation rate of 25 rpm for 48 h of reaction time. These results were found to be significant as they help to overcome the problems related to free radical generation when mineral acids are being used. In addition, chemicals like polyelectrolytes are often expensive and their utilization is not feasible both in terms of costs and environmental balance. Thus the enzymatic hydrolysis method demonstrated in this study proved to be noteworthy in terms of pollution control and effective waste management.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Lactic acid bacteria, Enterococcus, lipolytic activity, fish processing waste
Subjects:	600 Technology > 08 Food technology > 16 Nutritive value > 05 Enzymes 600 Technology > 08 Food technology > 16 Nutritive value > 07 Waste utilization 600 Technology > 08 Food technology > 28 Meat, Fish & Poultry > Fish
Divisions:	Food Microbiology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	20 Mar 2015 10:10
Last Modified:	20 Mar 2015 10:10
URI:	http://ir.cftri.res.in/id/eprint/11769

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